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Rewiring the Streptomyces cell factory for cost-effective production of biomolecules

Rewiring the Streptomyces cell factory for cost-effective production of biomolecules

Final Report Summary - STREPSYNTH (Rewiring the Streptomyces cell factory for cost-effective production of biomolecules)

Executive Summary:
STREPSYNTH aimed to set-up a Streptomyces-based new industrial production platform (SNIP) for high value added biomolecules. Streptomyces lividans was chosen as a bacterial host cell because it has been already shown to be highly efficient for the extracellular production of a number of heterologous molecules that vary chemically; has a robust tradition of industrial fermentation and is fully accessible to genetic intervention. To develop SNIP our strategy had two components: firstly, we have constructed of a collection S. lividans strains with physically or functionally reduced-genomes, termed RedStrep. This reduction is aimed at metabolically streamlining the cell and riding it of agents (e.g. proteases, secondary metabolites) that may potentially hamper the production yield or purification of the aimed heterologous molecules or lessen the metabolic burden. Secondly, we have engineered synthetic parts and cassettes, i.e. reshuffled, rewired and repurposed genetic elements either indigenous to S. lividans or clusters of heterologous genes organized in artificial operons. These elements serve three aims: transcriptional and translational optimization, sophisticated on-demand transcriptional regulation that provide unique fermentation control and metabolic engineering of complete cellular pathways that will channel biomolecules to extracellular secretion. Synthetic parts and cassettes are currently hosted either in the form of plasmids or directly incorporated into the genome so that genetically stable host strains are generated. Optimal combinations of plasmid born and integrated genes were decided ad hoc on a per case basis for optimal production. Several cassettes have been developed and tested. Several genetic engineering interventions were implemented and removed antibiotic production, pigment generation, sigma factor and protease genes. This first generation of reduced genome strain, termed RedStrep1 has been fully sequenced and initial micro-fermentation studies showed that the interventions do not incur any penalty in strain fitness in most cases. They also revealed, however, that fitness in a traditional sense that relates to growth is less relevant in our system. This was because in most instances, reduced growth correlated with enhanced native and heterologous protein secretion. Several systems biology tools (multi-omics) were used to characterize the metabolism, proteome, transcriptome of the wild type and generated reduced genome host cells and mathematical modelling and integration of these results has begun. We used such models to guide fine-tuning rounds of cell factory engineering and fermentation optimization in the later phases of the project. This omics-based approach is hoped to help rationally design in the long run our future genetic interventions, e.g. by targeting over-expressed enzymes. To set up this platform, we focused our attention on two classes of pilot biomolecules with obvious immediate industrial value and application to the industrial partners of the consortium: heterologous proteins (industrial enzymes, biopharmaceuticals, diagnostics) and small molecules (lantipeptides and indolocarbozoles) useful for multiple industrial purposes (biopharmaceuticals, additives, food technology, bioenergy). In addition, we developed pilot fluorescent proteins used as development pilots. These biomolecules are of immediate interest to SMEs that participate and guide the industrial relevance of STREPSYNTH. Feedback from the industrial partners played an important role in further optimizing the host strains in iterative rounds of development. SNIP is a modular platform that can be repurposed for diverse future applications. The project has shown remarkable progress in all its aims that validated its initial objectives with successful multi-fold overproduction of mithramycin and industrial enzymes by participating SMEs. As expected for a project of this complexity we also encountered bottle necks in optimally developing all aspects of our pipeline. Nevertheless, even this experience allowed us to prioritize the issues that need to be addressed in future efforts to maximally tackle the issue of further optimizing the SNIP.
Project Context and Objectives:
STREPSYNTH set-up a Streptomyces-based new industrial production platform (SNIP) for high value-added biomolecules. Our strategy had three components: firstly, construction of a collection of reduced-genome S. lividans TK24 bacterial strains that are metabolically streamlined for the production of heterologous biomolecules. Secondly, engineering of synthetic parts and cassettes, i.e. reshuffled, rewired and repurposed genetic elements and/or genes either taken from S. lividans or from heterologous donors. These elements allowed sophisticated on-demand transcriptional regulation and introduced complete cellular pathways that channel high-value added biomolecules to extracellular secretion. Thirdly, integrated systems biology tools guided metabolic understanding of the engineered cells and rationally fine-tuning rounds of strain genetic engineering for yield improvement, stability and fermentation optimization. Two classes of biomolecules of obviously immediate industrial value to the participating SMEs were analyzed: heterologous proteins (industrial enzymes, biopharmaceuticals, biofuel enzymes, proteins for vaccine development) and small molecules (lantipeptides and indolocarbozoles) useful for multiple industrial purposes (biopharmaceuticals, additives, food technology, bioenergy). These biomolecules were produced by optimized strains under validated industrial scale fermentation as part of STREPSYNTH. The project will deploy cutting-edge research and innovation by some of the leading EU labs. This is a broad multi-disciplinary effort with academic labs contributing to the platform various expertises including microbiology, gene regulation, genomics, protein secretion and small molecule biosynthesis, multi-omics technologies and systems data integration. These specialized tools are rarely available under one roof and thus demanded an EU-wide effort. Participating SMEs exploited these skills to develop high-value added molecules of critical interest to their product portfolios and benefited from gaining access to unique resources and know-how.
Background and scientific context
The biopharmaceuticals and industrial enzymes market has come along way since the early eighties when the first biopharmaceutical product, recombinant human insulin, was launched. Hundreds such products are currently being marketed around the world including thirteen blockbuster drugs. To produce these important heterologous proteins, different prokaryotic and eukaryotic expression systems are used. All have some advantages as well as some disadvantages that should be considered in selecting the appropriate one. This requires evaluating the available options – from yield to the requirement for post-translational modifications, to proper folding, to economics of scale-up and bio-processing, to removal of toxic contaminants (e.g. lipopolysaccharides). Finally, as we seek increasing sustainable, environmentally friendly production methods, treatment of waste products or toxic chemicals used in the production line become also an important point of consideration. No single host cell has emerged so far from which all target proteins can be universally and optimally expressed in large quantities. Therefore, it is important for the biotechnology industry to possess a variety of host-vector expression systems in order to increase the opportunities to screen for the most suitable expression conditions or host cell that delivers a viable industrial option.
With high-level expression and relatively inexpensive culture systems, when possible, microbial hosts are commonly used for the production of recombinant proteins. They grow easily in simple media and are therefore excellent candidate hosts for the production of recombinant proteins. A number of host systems are currently used in industrial processes and several others are under investigation. For the production of recombinant proteins Escherichia coli is often the host of choice, but many heterologous proteins expressed in E. coli are produced as insoluble protein aggregates in the cytoplasm or periplasm, hampering downstream processing and introducing expensive and environmentally-challenging toxic chemicals for the unfolding process. As a consequence, other expression systems, especially systems that secrete the desired protein directly in the culture medium are used or are under intense investigation. If soluble active proteins can be isolated in sufficient quantities from the extracellular fermentation broth, this greatly facilitates the protein recovery and purification work carried out downstream from the fermentation process.
Apart from protein production, Streptomycetes are an important source for drugs, such as antibacterials and anticancer compounds. Such compounds are assembled by dedicated biosynthesis pathways. Important classes include those built on peptidic structures (Amison et al. 2013; Strieker et al. 2010) and polyketides (Weissman and Leadlay 2005). These peptidic compounds show enormous structural diversity and are comparatively easy to alter structurally by genetic methods, e.g. site directed mutagenesis. Structural alterations of these peptides are still mainly limited to the 20 amino acids incorporated in polypeptides or the manipulation of post-translationally modifying enzymes.
In this project we focused on Streptomyces, a Gram-positive bacterium with a proven excellence in secretion capacity, as a host for heterologous protein production/secretion and biosynthesis of small molecules with an emphasis on the incorporation of non-natural amino acids in peptidic compounds. There are several sound reasons to choose Streptomyces. (1) Streptomycetes exhibit unique metabolic diversity and enzymatic capabilities (Hranuel et al., 2005; Chater et al., 2010). (2) They produce secondary metabolites that are valuable for industrial and pharmaceutical purposes (e.g. antibiotics, antitumour agents), and thus have a strong tradition of fermentation know-how. An increasing number of studies over the past years have reported Streptomyces as an ideal host for the production of several secreted heterologous proteins. Recombinant Human Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF) LEUCOTROPIN™, produced in Streptomyces lividans by Cangene, a Canadian biopharmaceutical company, was successfully used to complete Phase III trials and found to be an effective and safe treatment in myeloid reconstitution in subjects with Hodgkin’s and non-Hodgkin’s lymphoma. STREPSYNTH partners have demonstrated that Streptomyces is a good choice for heterologous protein production of eukaryotic proteins of medical importance and for industrial enzymes. Some of these heterologous proteins are secreted as biologically active compounds in a commercially significant yield, they can be purified and have the same characteristics as the native proteins. As a result, these heterologous proteins are now in industrial production using expression/secretion signals we have isolated (Anné et al., 2012).
With the availability of complete genomic sequences Streptomycetes have entered the post-genomic era. One powerful possibility that has thus been materialized is the use of synthetic biology that allows the reshuffling and minimization of genomes, the standardization of synthetic exchangeable parts and the abstraction of genetic functions. Another important capability brought by post-genomic biology is the elucidation of the fundamental logic and constraints that determine the systemic behaviour of the cell and its performance under different (stress) conditions. Systems-level analysis can be investigated using several advanced and extremely powerful techniques including e.g. microarrays and deep sequencing, multidimensional proteomics, metabolic and flux analyses and integrated bioinformatics allowing to gain detailed insight into the functions of gene and protein networks under different conditions. Systems biology thus rationalizes and revolutionizes protein biotechnology and genetic engineering. Transcriptomics examines the global pattern of gene expression at the mRNA level. Since the S. lividans genome has been sequenced, DNA micro arrays for this organism have become available from different sources and complements deep transcriptome sequencing methods. Proteomics has become an integral part of protein analysis. Complex mixtures of proteins can be separated by various means, proteolytic peptides are generated, their mass is determined by high resolution mass spectrometry and then these peptides are matched to their theoretical masses in a database, thus leading to identifying the individual proteins. Finally, the study of the metabolome, the complete set of metabolic reactions of a cell, in different physiological and non-physiological states and differentiation states, provides useful information regarding metabolism and regulation. Metabolic regulation is a central issue in metabolic engineering. Metabolic regulation phenomena depend on intracellular compounds such as enzymes, metabolites and cofactors. A complete understanding of metabolic regulation requires quantitative information about these compounds under in vivo conditions. This quantitative knowledge in combination with the known network of metabolic pathways allows the construction of mathematical models that describe the dynamic changes in metabolite concentrations over time. Metabolic Flux Analysis of biomass may be used as a tool for physiological characterization of a strain that can then be genetically manipulated to introduce a new or altered property to the cell, e.g. an optimized pathway for biomass production, or to more metabolic energy diverted to the synthesis of (heterologous) enzymes, to protein secretion work or to specific metabolites and small molecules of interest such as lantipeptides and indolocarbozoles. MFA has shown its strength already for the production of secondary metabolites, but it has so far never been tested to modulate and optimize protein secretion in any organism. Since carbon sources, fermentation conditions and differentiation stage are known to affect Streptomyces protein secretion and enzyme synthesis (Anné et al., 2012) metabolome engineering promises to have a significant impact on Streptomyces protein secretion and small molecule biotechnology.
Introduction of societal and industrial context
Within the pharmaceutical industry, recombinant proteins are key to the discovery of new drugs and are the basis of the high throughput compound screens. Consequently, pharmaceutical companies invest many millions of euros annually in generating these reagents. Recombinant proteins belong to a drug class known as biopharmaceuticals or biologics. Biopharmaceuticals are becoming increasingly common growing from 0.5% of worldwide pharmaceutical sales in 1989 to over 9% in 2002 with worldwide sales totalling $32 to $53 billion in 2010. Biologics have become important for innovation, being among the most commercially successful new products. In 2016 the world’s top 100 pharmaceutical products, 45% of sales came from biologics. This compares with only 33% in 2010 and 15% in 2002. Improving R&D productivity is critical for the pharmaceutical industry. Global investment in pharmaceutical R&D by the top 500 pharmaceutical and biotech companies reached an estimated $133 billion in 2011, a 93% increase from 2002. To ensure it delivers sustainable returns on its R&D investment, the industry aims to increase its probability of success in developing commercially viable new drugs and moving to a lower, more flexible cost base. Despite remarkable progress in biopharmaceuticals production in eukaryotic cells, microbial hosts still remain the most cost-effective solution for proteins that do not need excessive post-translational modifications.
These figures also include market expectation for antitumour and antiviral compounds, two extremely important global markets. The global anti-infectives market is valued at US$66.5 billion with antiviral agents accounting for 24% of sales (excluding vaccines which target viral infectious diseases). According to the “The Antivirals Market: R&D Pipelines, Market Analysis and Competitive Landscape” report, the antiviral market will be driven by the uptake of newer antiretroviral agents in combination therapy and the launch of ten new products for the treatment of HIV and hepatitis which will address treatment-resistant patients. Also, cancer is a global health care priority, one with rising and commercial importance with an increasing number of cases. Anticancer drug sales exceeded US$50 billion in 2009, and grew exponentially thereafter. Production of such compounds was materialized in STREPSYNTH.
Depletion of non-renewable fossil fuels makes biofuels preferred fuels of tomorrow. Enzymes are important to make this process less energy intensive and more environmentally friendly, such as use of enzymes for the hydrolysis of cellulose to produces fermentable sugars for bioethanol production. There are obstacles, however, facing the use of enzymes for biofuel production, including high cost. This can certainly be lowered with efficient production systems. To this end, STREPSYNTH aimed for high and cost-effective production of cellulases and chitinases. The enzyme market as a whole is big, with estimates for Biofuel enzymes exceeding US$900 million by the year 2017.
Concept and main innovations
Microbes and particularly bacteria are well established hosts for the production of biopharmaceuticals and small molecules. For many of these processes, although alternative hosts exist, commonly financial benefits, manipulation know-how and stability of processes favour the continued use of bacteria as host. In many cases, traditional host strains such as Escherichia coli are used with great success. Nevertheless, in several cases heterologous proteins may not be synthesized properly in E. coli or products may not be produced in such a way as to be enough or stable or functional. Alternative bacterial hosts provide an immense treasure-trove of possibilities as alternative hosts. The full potential of these approaches has not yet been materialized and Streptomyces bacteria are one of the most promising options as a heterologous host. Being Gram-positive bacteria, these cells secrete proteins and secondary metabolites, like antibiotics, directly in the surrounding medium. Laboratory scale experiments and some large scale efforts have also demonstrated that several proteins can be produced by Streptomyces at high levels. Many of these proteins are heterologous coming from human, bacterial or plant sources. Despite significant breakthroughs, progress in enhancing production yields for these biomolecules has relied to a large extent on empirical decisions due to the absence of a lot of background biology. Moreover, secretory production of some proteins using S. lividans was found to be inefficient so far and/or recalcitrant to yield optimization during fermentation. This is presumably due to a number of unidentified production 'bottlenecks' and “checkpoints”, incompatibility with the host secretory pathway or interaction with non-essential host pathways. To develop S. lividans into a robust host for protein secretion biotechnology and the production of small molecules, STREPSYNTH proposed a radically different approach. It first generated reduced genome strains with minimal interference from non-essential genes (e.g. late phase secondary metabolite synthesizing proteins, proteases and sigma factors). It then completely by-passed potential incompatibilities with indigenous cell machineries by bringing into S. lividans cells tailor-made heterologous gene cassettes of whole metabolic or secretion pathways or pathways that incorporate non-canonical amino acids into lantipeptides. STREPSYNTH was inspired by important breakthroughs in synthetic biology, -omics technologies and systems biology, the molecular understanding of protein secretion, genome manipulation tools and the availability of complete genomes including that of S. lividans TK24 used here. Collectively, these tools empower us to design strategies that can lead to deep molecular understanding of these bacteria and to integrate their multi-layered biologies into networks with predictive value. As a result, we have come close to the possibility of rational design of new industrial strains, with regulated production of heterologous biomolecules and robust behaviour under a large-scale bio-processing setting.
STREPSYNTH explored these exciting developments and brought the following innovations:
a. Minimal genome strains. A centerpiece of our effort is the surgical removal of genomic fragments expected to lighten the metabolic burden going into processes that are undesirable or of no use to bio-processing regimes. These reduced genome (RedStrep) strains are expected to increase the stability of the cells in long-term fermentation and to channel more metabolic potential to the biosynthesis of the heterologous biomolecule. In addition, our experience was that even strins that showed reduced fitness secreted more heterologous proteins. Minimal cells have been engineered in other Streptomyces species mainly focusing on the production of antibiotics.
b. Transcriptional/translational regulatory circuits. Genomic interventions in a fine-tuned, advanced cell are expected to disrupt existing regulatory networks that control transcription and various metabolic processes. We intervened in these regulatory processes by altering the expression of specific transcription factors (e.g. sigma factors). Also, we developed regulatory circuits that can act as on-off switches for the production of heterologous biomolecules. These tools included modified promoters and can open a vast array of opportunities in building tight control during fermentation growth and thus increasing the uniformity and reproducibility of the process.
c. Portable protein secretion cassettes and optimally matched substrates. Our understanding of the molecular mechanism of protein secretion has made significant breakthroughs in recent years. We are now in a position to know all of the components of a secretory pathway and have structural understanding for many of them. Moreover, we are recently becoming aware of the molecular basis of the recognition of secreted preproteins by the export machineries. STREPSYNTH built on these developments and constructed two groups of novel tools: portable cassettes with secretory pathway components and heterologous proteins that are rendered secretory by fusion to signal peptides. STREPSYNTH also made use and developed further advanced algorithms that allow for the first time prediction of optimal matching of signal peptide and mature domain information with the secretion system. Currently, secretion of biopharmaceuticals and industrial enzymes relies on a few empirically derived signal peptides and ignore mature domain information. The system described here is not yet available for any bacterial host.
d. Small molecule biosynthesis. STREPSYNTH brought to Streptomyces cells tools for the incorporation of non-canonical amino acids preiovusly established only for E. coli. To generate S. lividans into a Synthetic Biology host platform for various peptide/protein synthesis we established, adapted and optimized the supplementation-based and the amber-stop codon suppression methods.
e. Systems biology for optimization of biomolecule producing strains. STREPSYNTH advanced systems-level understanding of S. lividans biology where little information was previously available. We integrated 4 levels of organism-wide information: metabolomics, fluxomics, transcriptomics and proteomics. Data collection in these -omics approaches used robust methodologies and applied them for the first time to reduced genome RedStrep strains. This system's view of the cell contributed not only to the industrial applicability but also to basic understanding of bacterial cells.
f. Predictive modelling of molecular cell outcomes. Data integration of the –omics approaches in "e." above lead to the formulation of multi-parametric networks that describe the producing strain in a mathematical model. The power of these models lies in their ability to provide hints for rational genomic intervention to the producing strains. Such tools are expected to revolutionize strain development and open the possibility for application-customizable tailoring.
Project Results:
Scientific and technical objectives
Synthetic and systems biology offer unprecedented opportunities for rational understanding of cell complexity and allowing the diversion and exploitation of their biochemical pathways and the introduction of new ones. The goal of STREPSYNTH is to combine synthetic biology (reduced genome strains, novel transcriptional regulator networks, transplanted heterologous biosynthetic and protein trafficking pathways) with systems biology (metabolomics, fluxomics transcriptomics and proteomics and data integration) with industrial strength fermentation to decipher the cellular processes that stimulate or hinder overproduction and extracellular release of biomolecules of value to the SME partners. As a host organism we chose Streptomyces, an efficient secretor of heterologous proteins and producer of small molecules. A basic understanding of the mechanisms underlying protein production and the effect it has on the fitness of the host cell will allow us to re-engineer it with targeted genetic intervention. The work lead to a major contribution towards a firm foundation for the rational engineering of strains for the production of proteins. STREPSYNTH materialized its goals through a multi-disciplinary intertwined approach with the following discrete foci:
Major deliverables achieved for the different work packages are briefly summarized below and related to the milestones of the project.
1. Genomics toolbox: reduced-genome S. lividans strains: We generated fast and reliable gene deletion system for S. lividans, construct an S. lividans strain with a reduced genome/improved metabolic properties and test its fitness.
ML1, ML2
2. Transcription toolbox: synthetic parts and regulatory circuits: We generated and verified synthetic promoters, Ribosome Binding Sites and “insulators” in S. lividans, construct biological parts for Synthetic Regulatory Circuits (SRCs) in S. lividans and construct SRCs to control production and secretion of target molecules.
ML3, ML4
3. Metabolomics and fluxomics toolbox: We established standard operating procedures for robust metabolomics and standard operating procedures for 13C- and 15N-based fluxomics using S. lividans TK24.
ML5
4. Synthetic cassettes for protein secretion: We established functional expression of secretory pathway cassettes in S. lividans RedStrep and establish expression and guided secretion of heterologous proteins of industrial interest.
ML6
5. Synthetic cassettes for non-natural amino acids incorporation: We generated an S. lividans prototype platform for the incorporation of non-canonical amino acids (ncAA) into ribosomally synthesized peptides and proteins, will generate novel peptide structures by ncAA incorporation and bioprofiling, will extend the concept to other ribosomally synthesized peptides from Actinomycetes.
ML7
6. Multi-omics of industrial producer strains: We perform comparative multi-omics analysis (metabolomics, fluxomics, transcriptomics, proteomics) on RedStrep1 strains with improved expression of products of interests, and/or with synthetic parts and/or expression cassettes and generate informative multi-omics data for computational modelling.
ML8
7. Systems models guiding rational strain optimization: We created a database for all omics data generated in STREPSYNTH, apply statistical analysis and data mining methods to elicit systems level changes in RedStrep1 strains, will develop a protein-protein interaction network for RedStrep1 and develop a computational model predicting genetic interventions in RedStrep1 to further increase product yield and/or production rate.
ML9, ML10
8. Industrially optimized Streptomyces factories: We expressed Products of Interest (PoIs; Table 1.1.1) in RedStrep1 and -2 strains, performed preliminary evaluation of production performance of RedStrep1 strains for Products of Interest, construct RedStrep2 strains from RedStrep1 based on systems biology-based rational strain design, will optimize heterologous secretion cassettes for industrial robustness, optimize small molecule synthesizing cassettes for industrial robustness, will establish a “forced evolution” approach targeting production of specific bioactive secondary metabolites.
ML11-ML14
Scientific objectives
1. S. lividans reduced genome strains with improved metabolic behaviour, growth and stability (ML1,ML2).
2. Library of S. lividans genetic regulatory switches, networks and manipulated sigma factors for altered gene expression (ML3).
3. Metabolic/Fluxomics models of S. lividans (ML5)
4. Systems biology understanding of S. lividans (ML8)
5. Peptide production with incorporated non-natural amino acids (ML7)
6. Functional reconstitution of E.coli Sec genes in S. lividans (ML6).
7. Mutagenesis of key components of the secretion pathway and heterologous preprotein for optimal matching to improve heterologous protein secretion (ML6, ML10)
Technological objectives
1. S. lividans strains that secrete in sufficient amount proteins of interest for the SME partners. (ML1, ML2,ML6)
2. S. lividans strains that secrete in sufficient amount small molecules of interest for the SME partners. (ML7)
3. Improved S. lividans RedStrep1 and RedStrep2 strains with a series of plasmids or chromosomal alterations allowing improved heterologous protein secretion and/or small molecules (ML1, ML2).
4. Metabolomix/Fluxomix system for analysis of the S. lividans cell (ML5).
5. Multi-omics data for analysis of the S. lividans cell (ML8).
6. Genetic switches and parts for S. lividans gene regulation (ML3, ML4).
7. Mathematical Networks and predictive models for optimal secretion of a set of industrially important heterologous proteins (ML9)
8. Improved fermentation and downstream processing methods using micro-fermentors (ML14)

STREPSYNTH aimed to set up a Streptomyces-based new industrial production platform (SNIP) for high value added biomolecules. Our experimental strategy had three main components: first, we constructed RedStrep, the generic name for a collection of reduced-genome bacterial strains based on Streptomyces lividans. S. lividans (strain TK24, and derivatives thereof) was chosen because it is highly efficient for the extracellular production of a number of biomolecules has robust foundations of industrial fermentation. Second, we develop synthetic parts and cassettes, from indigenous to S. lividans or heterologous genes and genetic elements such that we can manipulate transcription and develop complete cellular pathways for dedicated production output. Thirdly, we will use systems biology as a means of guiding further strain optimization for increased yield under industrial fermentation regimes. As a first exploration of SNIP we chose two classes of industrially relevant biomolecules: heterologous proteins and small molecules (lantipeptides and indolocarbozoles) useful for a variety of purposes (biopharmaceuticals, additives, food technology, bioenergy).
RedStrep1, is a stable host strain with improved fermentation behaviour during high yield production of heterologous biomolecules. RedStrep1 will result from surgical deletion of several biosynthetic gene clusters as well as of the protein degrading machinery (WP1) and gave rise to a panel of strains with reduced genomes. These deletions optimized metabolic fluxes and improved stability of heterologous proteins. To improve rational transcriptional control, RedStrep1 was further engineered by removing late-exponential growth sigma factors, possibly lowering maintenance efforts and increasing product yields (WP1). In addition, synthetic promoters and regulatory networks were introduced (WP2) to provide temporal and product-saving control of gene expression. To gain metabolic understanding of RedStrep1, advanced metabolomics and fluxomics methods and Standard Operating Procedures (SOPs) for their use in S. lividans (WP3). One class of biomolecules produced will be heterologous proteins, after engineering them in such a way as to direct them to become secreted via a heterologous secretion pathway (WP4) containing various components (chaperones, motors, channels, processing factors) of the bacterium E. coli cloned as artificial operons or cassettes. This complete secretory pathway will work in parallel to the Streptomyces indigenous one. The cassettes will be mutagenized so as to optimize them for secreting specific heterologous proteins (e.g. a biopharmaceutical). The latter proteins will be fused to optimized E. coli signal peptides so that they follow exclusively the heterologous secretory pathway. A similar approach will be followed for small biomolecules (WP5). Biosynthesis gene clusters will be adapted by means of synthetic genes for the incorporation of non-natural amino acids into peptides of major clinical importance, e.g. labyrinthopeptins, with antiallodynic and antiviral properties. A systems-level multi-pronged approach to understand RedStrep1 strains hosting the necessary cassettes will include transcriptomics, proteomics, metabolomics and fluxomics analysis of RedStrep1+cassette strains (WP6). Aliquots of the same biomass material will be used for all –omics studies. These data-sets feed into WP7 that analyzes and integrates the -omics datasets and then builds predictive models that incorporate the detected metabolites, fluxes, proteins and transcripts. These models aim to identify potential rate-limiting steps in metabolic network models describing the optimal function of RedStrep1 as a cell factory. WP7 will propose specific chromosomal gene targets that will be modified in WP8 so as to lead to RedStrep2, strains with potentially optimized metabolism and bio-process behaviour, while at the same time delivering the highest possible yields of the biomolecules of interest. At the same time, the machineries themselves for protein secretion or small molecule biosynthesis and export will be modified through mutagenesis of their respective gene cassettes so as to yield optimal combinations of machineries and secreted proteins on one hand and enzymatic machineries and chemical modification variability and amount of small molecule product, on the other. All of the preparatory work performed in WP8 feeds into WP9 which aims to take lab-scale strain growth from pilot to industrial scale robustness in 50-100 Lt fermentors and then to efficient downstream processing (WP9). WP9 is an "end-user" WP that requires close cross-talk with WP1, WP8. WP9 imposes its own requirements for fermentation performance and downstream processing and thus guides WP8 on the genetic engineering optimization of strains. To strengthen the innovation dimension of STREPSYNTH and to increase the likelihood of market uptake all WPs support the development of detailed Standard Operating Procedures (SOPs); reproducibility testing, statistically sound verification of results. STREPSYNTH would like to communicate the merits of synthetic biology in facilitating sustainable growth in an environmentally friendly manner. To this end training and seminar courses and dissemination activities will be set up (WP10). STREPSYNTH results will be disseminated widely throughout the EU and will be particularly relevant for industries and institutes active in fermentation and products derived from it. The involvement of partners specifically devoted to training & education will be exploited to increase public awareness of the research results, e.g. in a form of public demonstrations and other similar activities by Consortium partners. Project results will be disseminated via different channels. The existing marketing channels of the industrial partners will be used to disseminate their technology. A web-site will be set-up and links will be provided from the different partners and from other important sites of use to industry, including to SMEs.
During the four reporting periods we have made significant inroads in several aspects of the STREPSYNTH project. This included: numerous genetic tools, purified proteins and antibodies, protocols for protein expression, secretion and purification, software, labeling for metabolomics and fluxomics, defined media, synthetic genes and promoters, vectors, multiple reduced genome strains, proteomics methodologies and quantitative analysis of the secretome, an annotated database of Streptomyces protein topology, transcriptomics analysis of gene expression at various stages of growth, pilot scale production in an industrial setting, integration of omics results in an interactive online tool (Slivdb), contruction of S. lividans strains that harbor heterologous protein secretion cassettes. In this bustling activity, 14 milestones, 42 deliverables and 64 rferred publications have been satisfied and completed. We report on a final collection of improved RedStrep 1 production strains derived from two different methodological approaches and their corresponding genome sequences (D1.5); we characterized biological “parts” for construction of synthetic circuits and these have included multiple combinations of protomoters, ribosome binding sites, intitator sequences and regulatory switches (D2.2); we have made significant progress in developing cellular “memory” regulatory circuits for sustainable production of target molecules (D2.3) and have been applying and constantly optimizing them for application in expression/secretion controlling units for controllable protein expression and heterologous protein and small molecule production (D2.4); We have developed protocols for targeted and untargeted metabolomics for S. lividans strains (D3.1; D3.3) and a mathematical framework for optimal design and analysis of isotope tracer experiments and Experimental setup for fluxomics validated for S. lividans (D3.2); We have also developed synthetic biology approaches to introduce a heterologous secretion machinery cassettes functional in RedStrep1 (D4.1 and have a collection of heterologous proteins functionally secreted from RedStrep1 derivative strains, including industrially relevant enzymes such as thermostable cellulase (D4.2); We have developed S. lividans strains with a codon optimized labyrinthopeptin biosynthesis for supplementation based incorporation of Met, Pro and Trp analogues (D5.1) and strains incorporating Pyl and analogs in defined positions of labyrinthopeptin by amber stop codon-suppression (D5.2); We have expanded supplementation and amber suppression to other ncAAs and one other RiPP biosynthesis gene cluster (D5.3); we have reported on the generation of multi-omics data of RedStrep 1 strains expressing products of interest delivered for computational modelling and rational strain design (D6.1) and on multi-omics data of RedStrep 1 strains expressing products of interest delivered for computational modelling and rational strain design (D6.1); we have a validated genome-scale metabolic network model for RedStrep1 (D7.1) and have generated a bioinformatics platform for integrated analysis of omics data (D7.2); we have delivered a computational framework for metabolic engineering of RedStrep strains (D7.3) and a prototype protein-protein interaction network model for a particular RedStrep 1 (D7.4). Predicted sets of genetic modifications for increased production yield and/or rate in RedStrep 1 have been proposed based on our models and constructed (D7.5); we have developed strains that carry optimized heterologous secretion cassettes (D8.1) and have developed a similar cassette small molecule cassette for the enhanced production of an antitumor drug (D8.2); Using forced evolution circuits we optimized bioactive molecule production (D8.3) and genetically engineered RedStrep 2.0 strains resulting from metabolic engineering and other multi-omics input (D8.4). This significant body of work validated the Streptomyces-based new industrial production platform and provided insight for its further future development.

Below we provide a brief description of our main ST results.
1. A BAC library consisting of 15-60 kb fragments from the Streptomyces lividans TK24 genome was generated. A BAC vector harboring the gusA gene encoding β-glucuronidase was used for the library construction. Following ligation and transformation, a total of 4465 BAC clones were analyzed on agarose gels with regard to insert size. 2877 clones were subjected to sequencing. Sequence information from both ends of 15-100 kb inserts in 1889 clones was obtained. 1630 clones covering 96.4% of the genome were stored in 17 x 96 well plates. The plates were accessible to other partners of STREPSYNTH for use in downstream tasks involving engineering of improved host strain for production of high value added biomolecules.
2. To support the STREPSYNTH project partners, JUELICH(10) developed an elaborate set of in-depth S.lividans strain characterization procedures. This toolset is based on an intensively optimized and verified workflow, covering working-cell-bench, pre- and main-culture procedures for parallel and reliable cultivation of these filamentous organisms. High reproducibility, for technical and biological replicates, has been shown not only in a lab-scale bioreactor reference system, but also in a microtiter-plate based cultivation device at elevated throughput. The latter one provides the capability of 48 parallel cultivations, with online measurement of cell density (backscatter), dissolved oxygen, pH and fluorescence for each well separately. With the option for easy access to a high number of replicates, statistical methods can be utilized to strengthen the scientific conclusions. For the S.lividans TK24 wild-type strain, the highly standardized cultivation process has been studied in detail in both, the MTP-based and the lab-scale bioreactor system. Comparable results were obtained, including final biomass concentration, substrate consumption, byproduct formation kinetics and morphology of the culture. These results underline the robustness of the cultivation procedure. As a first example for phenotypic characterization the developed MTP toolset was applied to investigate the effects of Thiostrepton on growth and mRFP protein production (by fluorescence) behavior of a plasmid carrying derivative of S.lividans TK24 in comparison to the wild-type strain. Additionally, first deletion mutants of S.lividans have been compared towards their growth behavior using the small scale system. Automation approaches for sampling and data analysis have been implemented and are ready to use to further accelerate the characterization process of S.lividans derivatives. The toolset for S.lividans strain characterization developed by partner 10 (JUELICH) provided an important key technology.
3. Two complementary systems for efficient and reliable gene deletions in Streptomyces lividans were generated in order to develop RedStrep1 strain. The systems are based on positive selection for double crossover recombination using the blue-pigment indigoidine bpsA or gusA β-glucoronidase reporter genes. Using the bpsA system, four basic and critical antibiotic clusters (act, encoding the blue-pigmented actinorhodin; red, encoding the red-pigmented undecylprodigiosin; cda, coding for the peptide Ca2+-dependent antibiotic; coel, encoding the yellow pigmented colimycin) were deleted in the genome of the S. lividans TK24 strain. Using this system, the derivative S. lividans Δact Δred strain with deleted both coloured antibiotic clusters (act and red) was prepared. It represents the first starting RedStrep1 strain, in which all other useful deletions (e.g. other antibiotic clusters, sigma factor genes, protease genes and gamma-butyrolactone system genes), will be introduced after their fitness and other testing. In addition, several genes (for gamma-butyrolactone system, sigma factors SigH and SigB, and highly-expressed sRNA) were already deleted and characterised. Using gusA and a BAC library-based system 9 individual genes encoding RNA polymerase sigma factors and proteinases were also deleted within the chromosome of S. lividans TK24. Each approach has its own strengths: the bpsA-based gene deletion system proved to be highly effective for deletions of large portions of the chromosome, while the gusA strategy proved to be more appropriate for a high throughput genome reduction. Development of these two complementary strategies insured the generation of platform strains RedStrep1 and RedStrep2.
4. Partner 4 (IMB SAS) constructed a S. lividans TK24 Δact Δred strain in which both coloured antibiotic clusters (actinorhodin, cluster 14 and undecylprodigiosin, cluster 10) have been deleted, and removed both of the antibiotic resistance markers as reported for deliverable 1.3. The exact positions of actinorhodin (act) and undecylprodigiosin (red) deletions are: 1. act cluster: Deletion from pos. 2961047 (downstream of TK24_2650, SLIV_12920, fwd. strand) to pos. 2982325 (3’-end of TK24_2672, SLIV_13030, rev. strand, start pos. 2981887, stop pos. 2982327). The FRT sites remained between these nucleotides. 2. red cluster: Deletion from pos. 2081767 (3’-end of TK24_1901, SLIV_09115, rev. strand, start pos. 2082438, stop pos. 2081758) to pos. 2113444 (upstream of TK24_1922 (redD), SLIV_09220, rev. strand). The B-CC and P-GG sites remained between these nucleotides. This strain represents the starting S. lividans TK24 deletion strain designated RedStrep 1, where all other useful future deletions (other antibiotic clusters, sigma factor genes, protease genes and gamma-butyrolactone system genes), will be introduced and afterwards tested for fitness and other parameters. The strain was sent to partner 6 (UniBi) for next generation sequencing (NGS).
5. A final collection of six strains was constructed based on the RedStrep1 strain, the derivative of Streptomyces lividans TK24, in which two prominent secondary metabolite clusters (actinorhodin: act; undecylprodigiosin: red) had been removed. The RedStrep1 strain was the deliverable 1.3 and its genome sequence was deliverable 1.4. Based on RedStrep 1, the genomic deletion systems developed by the project partners 2 (HZI) and 4 (IMB-SAS) were employed further to introduce specific mutations in genes that proved the most useful in a broad prior study in which more than 40 mutant strains were constructed and their physiology assessed. The constructed mutant strains were delivered to other members of consortium for further studies, including fitness testing by partner 10 (JUELICH), genome sequencing by partner 6 (UNIBI), and proteomics studies by partner 1 (KUL). The RedStrep 1 strains containing additional combinations of mutations are designated as: RedStrep 1.X. Fitness testing showed that RedStrep 1 and its derivatives were not different in growth behaviour. Therefore, only RedStrep 1 and one of its derivatives (RedStrep 1.3) were analysed by genomic sequencing and no significant functional genomic difference was found.
6. There is an urgent need for synthetic controlling elements that give an opportunity to rationally engineer gene circuits and cell factories. To fulfil this goal several inducible systems based on a cumate or a resorcinol switch have been developed. This genetic tool allows selection of an optimal ribosomal binding site for any gene of interest and uses a library of terminators. We successfully developed a new cumate (p-isopropylbenzoic acid)-inducible gene switch in S. lividans TK24 that is based on the CymR regulator, the operator sequence (cmt) from the Pseudomonas putida cumate degradation operon and the P21 synthetic promoter. A resorcinol-inducible expression system is also functional and is composed of the RolR regulator and the PA3 promoter fused to the operator (rolO) from the resorcinol catabolic operon of Corynebacterium glutamicum. Using the gusA (β-glucuronidase) gene as a reporter, it was shown that the newly generated expression systems are tightly regulated and hyper-inducible. The basal activity of the non-induced promoters is negligible in both cases. The systems are also dose-dependent, which allows the modulation of gene expression even from a single promoter. Finally, these systems are non-toxic and inexpensive, due to the use of cumate and resorcinol, and they are easy to use because the inducers are water soluble and are easily taken up by cells. Therefore, the P21-cmt-CymR and PA3-rolO-RolR systems are powerful tools for rational engineering S. lividans and other actinobacteria. In addition, we succeeded in creating a genetic tool that gives an opportunity for the selection of efficient RBSs for any gene of interest and as a result rational improvement of the expression of gene on translational level. A library of efficient rho factor-independent terminators that allow controlling of transcription in different operons is developed for S. lividans as well.
7. The genome of Streptomyces lividans TK24 accommodates ca. 27 gene clusters for secondary metabolites and thus is a potentially good producer of bioactive molecules and a suitable host for heterologous expression of different compounds. Nevertheless, it is a known fact, that the compound of interest or its intermediates can be toxic for the host, and often this is the serious limitation for heterologous expression of valuable metabolites. That’s why the ability to switch the biosynthesis on and off at a desired growth stage is an important task. To control biosynthetic processes in the model strain Streptomyces lividans, and adjust them to our needs, we aimed to create robust regulatory systems, so-called Synthetic Regulatory Circuits (SRCs). SRCs consist from synthetic parts, including regulatory genes from S. lividans and other organisms. They will be tested in combination with specific promoters, their cognate operators and reporter genes. Toggle Switch or the Cellular Memory SRC (Fig.1) could function as a synthetic regulatory loop, where CymR repressor blocks the expression of both: activator ActII-ORF4 and repressor RolR. At the same time, expression of CymR can be blocked by RolR which is expressed later in the growth phase, and thus at the beginning CymR dominates. When the inducer (cumate) is added, the expression of RolR should be switched on, thus shutting down the expression of CymR. The latter event ensures the continuous expression of repressor RolR, that blocks repressor CymR and thus allows expression of ActII-ORF4, which activates the expression of gene of interest. Addition of resorcinol, the RolR effector, will inactivate RolR, thus returning the whole system into the original (off) state.
8. To control biosynthetic processes in the model strain Streptomyces lividans, and to adjust them to our needs, we aimed to create robust regulatory systems, so-called Synthetic Regulatory Circuits (SRCs). SRCs consist of synthetic parts, including regulatory genes from S. lividans and other organisms. They will be tested in combination with specific promoters, their cognate operators and reporter genes. Toggle Switch or the Cellular Memory SRC (Figure 1) could function as a synthetic regulatory loop, where CymR repressor blocks the expression of both: activator ActII-ORF4 and repressor RolR. At the same time, expression of CymR can be blocked by RolR which is expressed from a weaker promoter, and thus at the beginning CymR dominates.
9. Traditionally, heterologous proteins produced in Streptomyces are usually fused to signal peptides of strongly expressed/secreted endogenous Streptomyces proteins, which are then taken through the native secretion pathway. STREPSYNTH aims to re-design the heterologous protein secretion in Streptomyces in such a way, that it will be taken through an orthogonal secretion pathway from another bacterium, and consequently secreted heterologous proteins will be fused to heterologous signal peptides. Thus, the orthogonal secretion pathway will work in parallel to the indigenous one. As an orthogonal secretion system, the well-characterized Sec system of E. coli has been chosen. All secretory proteins that use the Sec pathway are synthesized as "pre-proteins" with amino-terminal signal sequences that are proteolytically removed in a post-translocation step. Signal peptides act as "ribosome to membrane" address tags and once at the membrane also activate the channel comprising the SecY, SecE and SecG proteins. The Sec minimal secretion machinery, in addition to SecYEG include cytosolic "holdase" SecB and peripheral ATPase SecA. For building an orthologous secretion system for Streptomyces, the five proteins of Sec pathway, SecA, B, Y, Y and G have been chosen that comprise “minimal” functional secretion machinery. Expression of these genes should ideally be controlled in Streptomyces, and also coordinated with the expression of the protein targeted for secretion. Initially, we have designed as expression/secretion controlling Synthetic Regulatory Circuit (SRC) based on the regulatory system of Lactococcus lactis that responds to nisin. However, after careful review of recent literature, this idea was discarded at too risky. Instead, a more streamlined SCR was designed based on the resorcinol-responsive repressor RolR.
10. A general Standard Operation Protocol (SOP) for metabolomics for Streptomyces (Resulting from Deliverable 3.1: Protocol for targeted and untargeted metabolomics for S. lividans strains; Deliverable 3.3: Protocol for extended targeted and untargeted metabolomics for S. lividans strains)
11. KULeuven (Partner 1) (with support from UMCR (Partner 7)) and ITT (Partner 9) were successful in setting up the experimental protocol and mathematical framework of 13C-MFA for S. lividans and to introduce some new approaches in the mathematical approaches. To our knowledge, no publication on 13C-based flux maps for S. lividans exists. Further work included the following aspects. Transfer of methods to WP6, where 13C-MFA was used to analyze metabolic fluxes in other strains, i.e. genome-reduced S. lividans with and without heterologous expression and metabolically engineered strains. Replicate and parallel experiments increase the parameter estimation quality, in particular, the confidence intervals on the parameter estimates. LC-MS/MS analysis was performed by ITT on the samples generated by KULeuven-CIT. LC-MS/MS chromatograms were analyzed. LC-MS/MS has the advantages of generation more informative tandem MS/MS data, returns free mass isotopomer data for free metabolites and is able to also measure metabolites that cannot be identified on GC-MS (e.g. metabolites from the pentose phosphate pathway). This additional information further improved flux estimation quality.
12. To develop Strepromyces lividans TK24 as a specialized secretor of polypeptides that do not interfere with the house-keeping secretion process and that can be individually targeted for rational optimization, we grafted the Sec pathway from E.coli. previously SecA proteins from the two organism had been shown to be incompatible and E.coli uses Sec proteins like SecB that are absent from Streptomyces. The engineered Sec cassette contains the secY, secE, secG, secA and secB genes that has been designed to be optimized in terms of its codon usage and has been made synthetically. The synthetic genes were integrated in the chromosome of TK24 and of RedStrep 1.3 one of the genome reduced strains that show optimized secretion of mCherry (constructed by partner IMB-SAS) and expression was tested using a series of antibodies raised against the five Sec proteins. Heterologous proteins were synthesized on plasmids using E.coli signal peptides. One example of these proteins is Cellulase A from Rodothermus marinus that was successfully expressed in S. lividans with a reduced genome as an example for heterologous protein expression/secretion using this system and was purified to homogeneity using a simple method applicable for industrial applications.
13. To develop Strepromyces lividans TK24 hosts that are more suitable for heterologous protein secretion it is desirable to secrete the product of interest using the secretion casettes in media that do not contain any pigmented antibiotics and dyes. Towards this goal 5 secondary metabolite gene clusters, extracellular glycan encoded genes matA and matB, and region encoding transcription factor HrdD were deleted from the chromosome. The secreted protein profiles vary measurably between the strains compared to the secretome of the wild type. This demonstrated a direct effect of the deleted clusters on secretome biogenesis. The engineered strains showed similar fitness compared with the wild type. Some of the mutated strains produce more than double the native secretome of S. lividans TK24 (WT). These results resemble a preliminary study for formation of a robust strain of S. lividans TK24 fit for long time fermentation and excessive heterologous production. Using these strains, we could then develop fluorescent protein reporters that are secreted by either the Tat or the Sec system of the cells. We characterized in detail the properties of these secreted proteins and they can now be used as platforms for the secretion of other heterologous proteins, either using the technology that we developed either alone or as fusions with the fluorescent proteins.
14. By means of synthetic genes and site directed mutagenesis we performed site specific substitutions of Met, Trp, and Pro codons in the labyrinthopeptin precursor peptide. Expression of peptides was verified in corresponding auxotrophic S. lividans mutants deficient in the biosynthesis of Met, Trp and Pro, which have been be delivered by partner 2 (HZI). The SPI approach was assessed for a set of ncAAs with the following amino acids: Met surrogates: l-azidohomoalanine (Aha) and l-homopropargylglycine (Hpg). Trp surrogate: l 5 hydroxytryptophan (OH-Trp), l-4-fluorotryptophan (F Trp) and l 7 azatryptophan (Aza-Trp). Pro surrogates: l-cis-4-fluoroproline(S-F-Pro), l-trans-4-fluoroproline (R-F-Pro) and l trans-4-hydroxyproline (OH-Pro). All tested isostructural analogs were incorporated successfully in LabA1 and LabA2. All requirements of task 5.1 were achieved, and the S. lividans auxotrophic mutants of Pro, Met and Trp that harbour the corresponding plasmids of labyrinthopeptin for SPI approach application are ready and available. However, future work will optimize and increase the production yield of the newly generated congeners for biological activity assays (i.e.: the anti-retroviral and the antiallodynic).
15. We generated a new strain of S. lividans containing the stop codon suppression system in its chromosomal DNA under the control of the strong promoter ermEp*. Lab gene cluster was analysed and modified by exchanging amber stop codons (TAG) in the unrequired positions into opal stop codon (TGA). Labyrinthopeptin structural genes were cloned into another vector where an amber stop codon (TAG) was installed in the open reading frame of structural genes by means of site directed mutagenesis and/or synthetic genes. All required plasmids were constructed and expressed for the incorporation of bulky ncAAs into labyrinthopeptins by expanding the genetic code through SCS approach in the developed S. lividans / pylT:PylRS strain. The SCS approach was assessed for a set of following orthogonal ncAAs: N-Alloc-L-Lys (Alk), N-Boc-L-Lys (Bok), Nε-5-norbornene-2-yloxycarbonyl-L-Lys (Nbk) and S-allyl-L-Cys (Sac). Only Alk was successfully incorporated and detected at two positions of LabA1 (Asn2 and Glu7), while the incorporation of the other ncAAs at the chosen positions was not possible. Based on peak area
integration and the calibration curves established in this study, the yield of the congeneric labyrinthopeptins that contain Alk was very low or even close to trace amounts (48 μg/L for LabA1/Asn2::Alk and 29 μg/L for LabA1/Glu7::Alk). Thus antiviral activity assays were not possible. Tuning the expression of pylT and pylRS under the control of different promoters, or the insertion of an ermEp promoter before the synthetic genes SG2 and SG6 to increase LabA1 and LabA2 production did not improve the efficiency of this approach for the incorporation of ncAAs into labyrinthopeptins. However, all requirements of task 5.2 were achieved, and the S. lividans /PylRS strain that harbour the corresponding plasmids of labyrinthopeptin for SCS approach application are ready and available.
16. To expand the range of ncAAs modified antibiotics, the cinnamycin biosynthesis gene cluster
(biologically active RiPP) was cloned from the chromosome of Streptomyces cinnamoneus
using transformation-associated recombination (TAR) cloning technique. The resulting construct pCinCatInt was introduced into S. lividans TK24 and S. albus J1074. The production of cinnamycin was assayed by antibacterial test and LC/MS. As result, the S. albus strain was superior in cinnamycin production over the S. lividans TK24 (3-fold difference in production
level). Thus, all further manipulations were performed in S. albus. Both S. albus and S.
lividans carrying pCinCatInt were accumulating cinnamycin and deoxycinnamycin, lacking
hydroxyl group at Aps15. In order to simplify further work the gene cinX, encoding Asp15
hydroxylase, was deleted from the expression construct. Expression of mutated construct
resulted in production of deoxycinnamycin solely. Two positions in deoxycinnamycin were
chosen for non-canonical amino acids incorporation: Arg2 and Phe10. The codons encoding
Arg2 and Phe10 were replaced with the TAG amber stop codon within expression construct.
Co-expression of mutated constructs with the Pyl system accompanied with
supplementation of corresponding cultures with 20 mM of Alk, Cyc and Boc ncAAs resulted
in production of new derivatives of deoxycinnamycin. Phe10 was successfully replaced with
all three ncAA variants tested. The replacement of Arg2 resulted in much lower production
level and only two derivatives were obtained carrying Alk and Cyc, but not Boc.
Incorporation of Alk was the most efficient resulting in accumulation of 8 mg/L of Phe10Alk
and 0.75 mg/L of Arg2Alk deoxycinnamycins. Production of other deoxycinnamycin
derivatives was lower: Phe10Cyc – 1.75 mg/L; Phe10Boc – 0.31 mg/L; Arg2Cyc – 0.032 mg/L.
The concentrations were estimated by LC/MS using standards curve for pure cinnamycin.
17. RedStrep 1 strains derived from S. lividans TK24. S. lividans TK24 was characterized as a reference background for the subsequently derived RedStrep strains. Effects of medium, growth phase and the presence of heterologous proteins are examined via omics analyses and are the reference (nominal) state for systems modeling in WP7. In addition, these results served as a means for the development and validation of omics protocols. The project is now at a stage at which sufficient information has been collected to finalize the choice of RedStrep strains that are optimal for subsequent specific applications. We have robust backbones in the main RedStrep series 1.3-1.5 which are now further optimized to help the participating SMEs with the removal of melanin production capabilities to reduce small molecule contamination. In addition, we have a detailed collection of more than 20 strains with engineered deletions that have been shown to improve secretion of proteins. So, strain engineering is being performed to optimally combine features of interest in a few super-strains optimized in each case.
18. Using multi-omics experiemnts we determined that in S. lividans TK24, heterologous production and secretion of CelA leads to a reduced growth rate, a distinct shift in the central carbon metabolism towards NADPH-production, and clear gene expression changes in subsets of genes correlating to CelA-production and the OsdR regulon. Transcriptomics data uncovered stress responses in the recombinant CelA-producing strain mostly related to secretory stress and DNA damage. The cause of this (perceived) DNA damage and secretory stress is unclear and requires further study. The latter could indicate unknown bottlenecks in secretion, but identification of specific targets for strain improvement again requires additional research. Isoenzymes linked to secondary metabolism are co-expressed with the OsdR regulon, and could be (partially) responsible for the measured flux increase through the PPP. Increased fluxes through both the PPP and the TCA lead to higher NADPH generation in a CelA-producing strain, which exceeds the amount needed for protein production. Redox balancing in the heterologous protein producing S. lividans fails and alternative routes are not yet fully understood. Further studies to the contribution of PPP isoenzymes and understanding transhydrogenase activity are required. The findings presented here help build a foundation for strain improvement of the industrially important organism S. lividans.
19. We determined the first working genome-scale metabolic network model of S.lividans. A preliminary validation was established in the sense that the predicted growth capabilities are realistic. The genome-scale metabolic network (GSMN) model formed the basis for developing of an expanded and further validated S. lividans TK24 GSMN model, deriving validated GSMN models for RedStrep strains, and identifying genetic targets for metabolic engineering in S. lividans and RedStrep strains. Model expansion embedded gap filling, insertion of biosynthetic pathways identified from omics data from WP6 (i.e. transcriptomics, metabolomics, proteomics) and importing information from recent literature data on other Streptomyces strains. For model validation, bioreactor data (e.g. phenotypes on difference substrates, phenotypes of genome-reduced strains, phenotypes for metabolically engineered strains) as well as omics (metabolomics, transcriptomics, proteomics and fluxomics) data were used.
20. We developed a WWW platform fully compliant with the STREPSYNTH Project specifications. It allowed all project’s partners to successfully achieve their information and data sharing needs. By doing so, they will help us building SLivDB which intends to become the Reference Database for Streptomyces lividans. As large amount of data are not yet available for testing, this platform may evolve during next months based on feed-back that we will get from the other STREPSYNTH partners. WP partners collaboration will lead to establishing naming conventions for all omics data that will be integrated in SLivDB. Based on this, the other partners had a strong tool for integrating all their SLivDB results.
21. Systems level modeling (i.e. integrated models of genes, proteins, fluxes, among others) can help identify gene targets for optimization of recombinant production of Products of Interest in Streptomyces lividans and its genome-reduced RedStrep 1 strains. Computational prediction of genetic modifications for improving product yield and rate in RedStrep1 was implemented by using computational frameworks and was applied to rationally predict genetic targets for strain optimization. Two frameworks focus more on strain fitness, namely, the genome reduction approach based on FBA (flux balance analysis) and the gene targets derived from Tn-Seq analysis. Two frameworks focus on optimization production of a product of interest. On the hand, production of secondary metabolites or alike molecules is envisaged. Since the pathway closely connects to the metabolic network (in a limited number of precursors), this approach closely leans to common metabolic engineering approaches. On the other hand, heterologous protein is envisaged, which is less obvious because the pool of amino acids is addressed and heterologous protein biosynthesis has to compete with the native protein biosynthesis. Besides computational frameworks for rational gene target identification, improvements are made to (i) the genome-scale metabolic network reconstruction for S. lividans TK24, and (ii) the 13C-based metabolic flux estimation method.
22. The potential PPI network of S. lividans was reconstructed in silico based on available information from experimentally determined protein interactions of S. lividans orthologues in other bacteria and bioinformatics resources. We were able to increase the experimentally supported PPI information for S. lividans from 1 PPI (homodimer) available in PPI databases to 1064 PPIs between 783 proteins. Integrating the predicted PPIs from the EcoliNet and STRING bioinformatics resources, we increased the size of the PPI network to 8982 PPIs involving 1001 proteins. This is a very significant result, because it provides a useful resource to the scientific community for a microorganism of industrial interest to further our understanding of the physiology of this organism and to identify potential targets for useful genetic modifications. Indeed, within the project, we anticipate that this network will help in the integrated analysis of omic data, be connected with the metabolic network of the organism that has been reconstructed within the project and be used to investigate the impact of certain genetic modifications to the physiology of certain strains and their yield in desired products.
23. To identify gene targets for optimization of recombinant production of Products of Interest in Streptomyces lividans and its genome-reduced RedStrep 1 strains (WP1) we applied the computational frameworks we established to rationally predicting genetic targets for strain optimization. A number of interesting target for further genetic manipulation were identified predicted to yield improved heterologous protein production by metabolic engineering for the model proteins: CelA, hTNFα and mCherry. CelA is an industrially relevant protein, for which good secretion is already observed in the wildtype. hTNFα is an important protein for biopharmaceutical development. mCherry is a robust model protein with easy quantitative read-out of its export. Medium: 21 metabolic media (MMGLC, MMGLC + 1 of 20 amino acids). In one Methodology (Constraint-based modeling) gene knockouts were identified that increase protein production while keeping the metabolic flux distribution in the knockout mutant close to the wild-type flux (minimal adjustment). Targets that scored consistently well on all criteria: Transaldolase (SLIV_28035), Transketolase (SLIV_28040) and Glucose-6-phosphate isomerase (SLIV_28005). In the second Methodology (Constraint-based modeling) rections were identified whose removal decrease biomass growth capabilities, while only having a limited impact on heterologous production potential. The rationale is that by reducing growth, more resources will be routed to the product. Targets that score consistently well on all criteria: Succinyl-CoA synthetase (SLIV_14320 + SLIV_14325), Malate dehydrogenase (SLIV_14235) and Glycine hydroxymethyltransferase (SLIV_11155 and SLIV_14185).
24. To develop Strepromyces lividans TK24 as a specialized secretor of polypeptides that do not interfere with the house-keeping secretion process and that can be individually targeted for rational optimization of the secretory pathway, we grafted core components of the Sec pathway from E.coli into Streptomyces lividans TK24 both the wild type and Redstrep derivative strains generated in Strepsynth. Previously SecA proteins from the two organisms had been shown to be incompatible in reciprocal genetic complementation experiments and E.coli uses chaperones like SecB that are absent from Streptomyces. The engineered Sec cassette contains the secY, secE, secG, secA and secB genes that has been designed to be optimized in terms of its codon usage and has been made as synthetic DNA fragments. The synthetic genes were integrated in the chromosome of TK24 or carried in plasmids and expression was tested using a series of antibodies that were raised against the five Sec proteins. Heterologous proteins were integrated into the chromosome of TK24 using E.coli signal peptides. One example of these proteins is the thermostable Cellulase A from Rodothermus marinus that was successfully expressed in S. lividans TK24 as an example for heterologous protein expression/secretion using this system and was purified to homogeneity using a simple method applicable for industrial applications.
25. Compound EC-70124 is the product of a combinatorial biosynthesis project and is biosynthesized using the genes rebODCP involved in the biosynthesis of the precursor arcyriaflavin A from the rebeccamycin biosynthetic pathway expressed under the control of ermE* promoter (Sánchez et al. 2002). The sugar component of this molecule, L-olivose is biosynthesized using the genes from Streptomyces antibioticus from the oleandomycin biosynthetic gene cluster expressed by their own promoters (Aguirrezabalaga et al. 2000). The genes staG and staN come from the staurosporine biosynthetic pathway and are expressed under the control of ermE* promoter (Salas et al. 2005). In order to increase production of EC-70124 several expression cassettes were constructed. The first construction was a cassette containing the operon rebODCP controlled by its own promoter, this construct included the gene rebR which induces expression of the rebODCP operon. This cassette was introduced in integrative plasmid pSET152 and in multicopy plasmid pWHM4. These plasmids were introduced into S. lividans TK24 and production of arcyriaflavin A was evaluated. Production using integrative plasmid pSES4 was 10-15 mg/L and production driven by multicopy plasmid pWBE3 was 350-450 mg/L. The strain containing pWBE3 was selected. The cassette containing staGN under the control of ermE* promoter was introduced into pWBE3 upstream the rebODCP operon in both orientations and production of EC-70124 was evaluated. The genetic organization corresponding to divergent operons was more productive (pWBGN2). New cassettes containing staGN under the control of rebO promoter and under the control of synthetic promoter P21 (Siegl et al. 2013), the latest with and without a transcription terminator were constructed. Another cassette containing staGN under control of kasO*p promoter (Wang et al. 2013) was constructed as well. These cassettes were cloned into pWBE3 diverging from operon rebODCP and production was compared to pWBGN2. Production of EC-70124 was abolished when cassettes containing P21 promoter with and without transcription terminators were used. It was noted that during the cloning process, copy number of these plasmids in E. coli DH10B were considerably lower than parent plasmid pWBE3 and growth of the exconjugant S. lividans clones was slower than S. lividans containing pWBE3. Production using cassettes containing staGN under kasO*p promoter did not improve when compared to ermE*p promoter. The main improvement in EC-70124 production was achieved by changing ermE*p promoter on operon rebODCP to rebO promoter. Production of EC-70124, after culture medium optimization, went from 40 mg/L up to 120 mg/L.
26. To increase the yield of bioactive secondary metabolites, which are very often produced at very low level, or activate silent biosynthetic gene clusters, random mutagenesis has been used in the past to obtain over-producers. However, screening for high producer is very laborious. Linking the production of the target molecule to the regulatory circuit modulating spontaneous mutation frequency and simultaneously to a reporter system capable of detection production of a target metabolite provides an attractive alternative that may revolutionize strain development programs. The Phenotype-Linked Forced Evolution circuit (PLFEC) to be constructed consists of two parts: (1) The controllable expression of the “mutator” gene, which shall drastically increase spontaneous mutation frequency; (2) The reporter gene under the control of promoter-operator of divergently oriented genes of TetR-like repressors in the same clusters allowing selection of mutants with increased production of a specific molecule. Such TetR-like repressor controls expression of the reporter, and upon binding the molecule of interest the TetR-like repressor is released from the operator and thus the reporter is expressed. Upon switching ON the “mutator” part, the mutations start to accumulate, and cells expressing the reporter (and thus producing the target molecule) can be easily identified.
27. For the improved expression of products of interest (PoIs; both proteins and secondary metabolites) various combinations of S. lividans RedStrep strains were exploited together with expression vectors with various expression cassettes. For the expression of selected recombinant proteins, both replicative and integrative expression vectors with various promoters and expression cassettes, were used. For the expression of some difficult to express proteins constructs containing fusion proteins between well-expressed and secreted mRFP protein and the relevant PoIs were introduced. Among recombinant proteins analyzed for the expression under various expression conditions belong in addition to model mRFP protein various enzymes with a biotechnological use, recombinant allergens and agrobodies. To the secondary metabolites considered as PoIs belong mainly potential anti-tumor drugs from the mithramycin family, which production was analyzed in various RedStrep strains from already prepared vectors pMTMF and pMTMF-W containing relevant gene clusters. For the recombinant proteins expression among a number of various set-ups the optimal one was use of a strong p21, ermA or vsi promoter together with S. venezualea RBS/signaling peptide in a replicating (pUWL-based shuttle vector, pIJ486 – native S. lividans vector) or integrating (pMU1- or pSET152-based) vectors.
28. We provided proof of principle that SNIP enables cost-effective production of Products of Interest at labscale for high value added biomolecules. The improved production efficiency of SNIP is illustrated for a small molecule produced in REDSTREP 1 strains. In particular, the efficient and improved lab protocol for production of Mithramycin A (MTM) and the MTM analogues, MTM SK and EC-8042, has been established using the collection of S. lividans RedStrep strains generated in STREPSYNTH. Mithramycin A (MTM) is an antitumor compound used in the clinic for treatment of chronic and acute myeloid leukemia, testicular carcinoma as well as hypercalcemia and PagetÅLs disease Although, the clinical use of MTM was limited due to its hepatoxicity, an MTM phase I/II clinical trial was recently performed for the treatment of refractory Ewing Sarcoma. Structurally, MTM consists of a tricyclic aglycone with two aliphatic side chains attached at C3 and C7, and a trisaccharide (D-olivose-D-oliose-D-mycarose) and a disaccharide (D-olivose-D-olivose) chains attached at positions two and six of the aglycone, respectively (Figure 1). Modifications of this molecule by combinatorial biosynthesis and/or biocatalysis allowed to generate MTM analogues with improved properties: higher potency and/or lower toxicity. So, the increased demand of MTM and its analogues highlights the need for more efficient production procedures.
29. The thermostable cellulase CelA enzyme from Rhodothermus marinus was used as a test/model protein for developing optimal growth media and conditions for heterologous protein production and secretion in bioreactors applying the Streptomyces lividans cell factory system. CelA was produced with a N-terminal signal peptide derived from the subtilisin inhibitor protein from S. venezuelae and secreted into the growth medium. Optimal growth media and conditions were developed that stall biomass production but promote excessive CelA secretion in the fermentor medium. Under optimal growth conditions in nutrient broth medium, significant amounts of mature CelA (50–90 mg/L or 100–120 mg/g of dry cell weight) are secreted in the spent growth media after 7 days. With reference to the optimized conditions determined, a protocol for Lab-scale fermentation for optimal production of Products of Interest is provided. Further, a protocol to rapidly purify CelA to homogeneity from culture supernatants was developed. The established production protocol will be applied on other enzymes or proteins of interest applying different RedStrep production strains. The CelA production system is not optimal for comparing the production/secretion efficiency of the different RedStrep strains, as the encoding gene is cloned and expressed in a proliferating vector. Therefore, production/secretion efficiency of two selected Streptomyces RedStrep strains was evaluated and compared to the TK24 wild type strain by analyzing production of highly thermostable amylase Amo228 isolated from a metagenome, encoded by genes cloned into an insertion vector and inserted into the strains’ genomes. The production was tested in lab-scale fermentation and enzyme activity in secreted proteins assessed, for the RedStrep strains and the wild type. Specific amylase activity in the medium was highest in RedStrep 1.17 with 0,96U/mL in spent culture medium, then in RedStrep 1.3 with 0,96U/mL and lowest in S. lividans TK24 with 0,37U/mL. The difference between strains was even more pronounced when amounts of total proteins produced is taken into account or 2,1U/mg in RedStrep 1.17 compared with 0,3U/mg in RedStrep 1.3 and 0,2U/mg in S. lividans TK24, confirming RedStrep 1.17 as the best producer and/or secretor of products of interest.
30. A common problem with biopharmaceuticals and other industrial proteins (enzymes) is their aggregation. This can occur over the entire manufacturing process and strongly impair the final product quality and purity. To ensure the safety of patients and to monitor the quality of the industrial proteins during the entire manufacturing process, it is important to recognize protein aggregates early in order to minimize it or even completely prevent. This process should go fast and if possible, be economically feasible. The aim of this task was to separate monomers and aggregates of several model proteins and monoclonal antibodies (mAbs) using well-established method of Size Exclusion Chromatography. Afterwards we performed the measurements of their secondary structures using the FT-IR (Fourier transform infrared Spectroscopy). The significant changes of the IR spectra have been observed between monomers and their aggregates. Using FTIR it is possible to distinguish between monomers and their aggregates. Especially the proteins that in their native form are predominantly α-Helices (range: 1660-1650 cm-1), one could find additional bands in the β-sheet folds (intramolecular: 1695-1683 cm—1 and 1644-1620 cm -1 ; intermolecular: 1620-1595 cm -1 ). We have artificially generated the aggregates of the model protein Bovine serum albumin (BSA), as well as the protein therapeutics cetuximab and infliximab using gradually increased temperature. The FTIR measurements could confirm the changes in the secondary structures of these proteins. Having this proof of concept in hand, it is possible to use FTIR spectroscopy for monitoring the protein purification process in the laboratories and factories in order to avoid the protein aggregation and ensure the protein quality and functionality.
31. For the large, industrial scale fermentation were selected two model systems: a/ mithramycins producing S. lividans RedStrep 1.3 strain containing the expression plasmid pMTMF with 34 ORFs needed for the heterologous production of mithramycins and b/ S. lividans TK24 strain harboring the expression plasmid pIJ486_vsi-celA driving the production and extracellular secretion of the thermostable cellulase CelA enzyme from Rhodothermus marinus. Both mithramycins and CelA expressing strain were initially cultivated in flask cultures and then taking in account data from lab fermentations (D9.1 and D9.2) their cultivation was upscaled to larger 20-40 L fermentations. Several fermentations (three for the mithramycins production and two for CelA production) were run to optimize large-scale production of either heterologous metabolite or protein pinpointing the fermentation conditions suited for the optimal production of these heterologous products of interest (POI). The mithramycin-producing RedStrep 1.3 strain was cultivated of for 144 hrs in the optimal SM17QK medium in 30 L or 75 L fermenters (the running volume 20 or 40 L), and the secreted mithramycins were then adsorbed to Diaion HP20 resin. The CelA-producing S. lividans strain was grown in 75 L fermenters (40 L running volume) in nutrient broth (NB) without NaCl for up to 96 hrs, cells were removed by centrifugation and the CelA-containing medium was filtrated and then 20-fold concentrated by ultrafiltration. Both final product mithramycins bound to Diaion HP20 resin and the CelA-containing concentrate were provided to Partner 13 (Entrechem - mithramycins) and Partner 15 (MATIS - CelA) for further analysis.
32. A multi-step process for purification of mithramycin SK was developed allowing its isolation as an individual peak with >95% purity. In this process an Streptomyces lividans RedStrep1.3/pMTMFDW recombinant strain was grown in a 20L-fermenter containing 16 liters of SM17QK optimised production medium. After 6 days of fermentation, the whole culture was centrifuged. For the purification of mithramycin SK, the supernatant (Figure 1) was extracted with resin. A solution containing mithramycin SK and two related compounds (RS: mithramycin SA and mithramycin SDK) was obtained and semi-purified by reverse phase flash chromatography eliminating one of the RS (mithramycin SA). Mithramycin SK was further isolated from the other RS (mithramycin SDK) by low pressure chromatography followed by preparative HPLC. Pure fractions of mithramycin SK (>95% purity) were collected and lyophilized.
Potential Impact:
Synthetic biology has reached the stage that in synergy with systems biology, metabolic engineering and molecular biology has significant potential to transform (micro)organisms into efficient cell factories able to produce compounds important for human welfare and favourable for the environment in a cost-effective manner. Integration of multiple disciplines and merging of different technological and scientific tools including (bio)informatics, chemistry, metabolic and bioprocess engineering, molecular and systems biology, mathematics and computer modelling, empowers synthetic biology to propel industrial microbiology and to provide future solutions beyond the state of the art. STREPSYNTH aims to set-up a Streptomyces-based new industrial production platform (SNIP) for high value-added biomolecules. To evaluate the concept as widely as possible, two classes of biomolecules, with obviously immediate industrial value and application, were chosen for this industrial expression/production platform: on the one hand heterologous proteins (e.g. industrial enzymes, biofuel enzymes, proteins for vaccine development), and on the other hand, small molecules (lantipeptides and indolocarbozoles of medical relevance). STREPSYNTH is industry-driven, and therefore focused on compounds that can be immediately commercialized by the SME partners. The project advanced research in the field of synthetic biology and generated innovative tools and methods for biotechnology. The approach that STREPSYNTH took resulted in solutions that are hoped to accelerate process design and reduced time-to-market for the biomolecules of interest. Furthermore, it resulted in scientific and technological breakthroughs, which will increase the industrial competitiveness of European SMEs and create new economic opportunities. This has the important benefit of investing in a platform for future potential use as a production line of completely different types of molecules. Biotech SMEs are often important intermediaries between academia and industry for the purpose of developing and disseminating technology. As an example of this we present new consortium-generated strains that over-produce up to three-times more of an anti-cancer drug for a Spanish SME and new enzymes for industrial biotechnology and biofuels for an Icelandic SME. SME biotechnology companies also transfer knowledge from academia to their customers using their strong networks. The purpose of these networks is to identify frontline research suitable for commercialization. Their products may also consist of the licensing of patented research findings. Our improved scientific understanding of the genetic and molecular circuits behind metabolite production and protein secretion processes is an important driver of technological innovation and a foundation for new growth. The development and dynamics of the biotechnology industry are heavily dependent on academic research findings. The STREPSYNTH project merged research and applications that lead to new production processes important for the participating industries. Within the Pharmaceutical industry, recombinant proteins are key tools in the discovery of new drugs and are the basis of the high throughput compound screens which are characteristic of today’s drug discovery process. The use of biologics has become an important source of innovation. To ensure it delivers a sustainable return on its R&D investment, the industry is working to increase its probability of success in developing commercially viable new drugs and moving to a lower, more flexible cost base. Despite remarkable progress in the production of biopharmaceuticals in eukaryotic cells and cell lines, microbial hosts still remain the most cost-effective solution for proteins. High market expectation exists for antitumour and antiviral compounds, two extremely important global markets. Production of such anticancer and antiviral compounds is also envisaged in STREPSYNTH. With the inevitable depletion of the nonrenewable resources of fossil fuels and due to their favorable environmental features, biofuels promise to be the preferred fuels of tomorrow. Towards this end, STREPSYNTH aimed for high and cost-effective production of cellulases and chitinases.

Synthetic biology has matured in recent years to the stage that in synergy with systems biology, metabolic engineering and molecular biology tools it has significant potential to transform (micro)organisms into efficient cell factories able to produce compounds important for human welfare and favourable for the environment in a cost-effective manner. Integration of multiple disciplines and merging of different technological and scientific tools including (bio)informatics, chemistry, metabolic and bioprocess engineering, molecular and systems biology, mathematics and computer modelling, empowers synthetic biology to propel industrial microbiology and to provide future solutions beyond the state of the art. As a field, synthetic biology is defined by three main advances: (1) automated DNA synthesis and assembly; (2) abstraction of genetic functions, and (3) standardization of genetic parts. STREPSYNTH aims to set-up a Streptomyces-based new industrial production platform (SNIP) for high value added biomolecules. To develop SNIP our strategy has two components: first, we constructed RedStrep, the generic name for a collection of reduced-genome bacterial strains based on Streptomyces lividans. S. lividans was chosen because it has been already shown to be highly efficient for the extracellular production of a number of heterologous molecules of various nature including proteins, metabolites such as lantibiotics and other biomolecules and can exploit the robust foundations of a more than 60 year old tradition of streptomycetes industrial fermentation. Synthetic biology advances are underdeveloped in Streptomyces and STREPSYNTH brought these bacteria to the forefront. Second, we employed synthetic parts and cassettes, i.e. reshuffled and repurposed genetic elements indigenous to S. lividans or heterologous genes organized in artificial operon clusters with three aims: transcriptional and translational optimization, sophisticated on-demand transcriptional regulation that will provide unique fermentation control and metabolic engineering of complete cellular pathways. Synthetic parts and cassettes will be either directly incorporated into the genome or will be hosted in the form of plasmids. As a first application of this ambitious platform we chose two classes of biomolecules with obvious immediate industrial value and application: heterologous proteins (industrial enzymes, biopharmaceuticals, biofuel enzymes, diagnostics) and small molecules (lantipeptides and indolocarbozoles) useful for a variety of purposes (biopharmaceuticals, additives, food technology, bioenergy). STREPSYNTH is an industry-driven project, and therefore focuses on industrially relevant compounds that can be immediately commercialized by the SME partners of the consortium, such as Mithramycin by the Spanish SME. Moreover, the participating industrial partners will guide the development of the project towards maintaining a sharp focus on industrial relevance in all of the decision-making steps. STREPSYNTH is the first step in establishing SNIP. It is important to note that SNIP's modular cassette design does not limit its use to the production of the specific biomolecules chosen here but permits adaptation of the SNIP platform to any number of future industrial production problems.
Because of its demonstrated potential and already existing industrial use, S. lividans (TK24, and derivatives thereof, is the strain used throughout STREPSYNTH) has become a new favourite cloning/expression host cell for many groups working in the area of heterologous expression and industrial production. This strain possesses an excellent intrinsic capacity to produce and secrete proteins at high levels and is one of the best hosts for the heterologous expression of biosynthetic gene clusters. Moreover, the whole genome sequence of S. lividans has recently become available thanks to the FP6-granted EU project Streptomics, in which some members of the current consortium were involved. However, the full potential of the organism is far from been materialized. Synthetic and systems biology and the maturation of fundamental biological knowledge now empower us to make serious inroads towards this industrial production platform. This is primarily because of four fundamental tools: metabolic optimization, regulated fluxomics, transcriptional control and regulation on-demand and complete pathway bio-engineering.
STREPSYNTH will create a stable host strain with improved fermentation behaviour during high yield production of heterologous biomolecules. To this end RedStrep1, a panel of strains with a reduced genome, will be constructed in STREPSYNTH. RedStrep1 will result from surgical deletion of several biosynthetic gene clusters as well as of the protein degrading machinery (WP1). These deletions will remove metabolic burden and misdirection of fluxes into unnecessary metabolites and will improve stability of heterologous protein products. To improve rational transcriptional control, RedStrep1 will be further engineered by manipulating sigma factors. As many sigma factor genes are dispensable and product secretion is often associated with late-exponential growth, non-essential sigma factors that drive transcription of genes at the entrance to stationary phase will be removed, possibly lowering maintenance efforts and increasing product yields (WP1). In addition, synthetic promoters and regulatory networks were introduced (WP2) to provide temporal and product-saving control of gene expression. To gain metabolic understanding of RedStrep1, advanced metabolomics and fluxomics methods and Standard Operating Procedures (SOPs) for their use in S. lividans were established ranging from metabolic labelling, chromatographic methods to optimal data analysis (WP3). The first class of biomolecules produced were heterologous proteins, after engineering them in such a way as to direct them to become secreted via a heterologous secretion pathway (WP4). For this, groups of genes that encode different moieties of the secretion pathway (chaperones, motors, channels, processing factors) of the bacterium E. coli were cloned as artificial operons or cassettes (taking into account Streptomyces' codon preference) in RedStrep1. This provided a complete secretory pathway that works in parallel to and does not interfere with the indigenous one of Streptomyces. Physical independence allows now these heterologous cassettes to be mutagenized at will so as to optimize them for groups of proteins or even for a single high value added heterologous protein (e.g. a given biopharmaceutical). The latter proteins will be fused to optimized E. coli signal peptides so that they follow exclusively the heterologous E.coli grafted secretory pathway. A similar approach was followed for the second class of small biomolecules (WP5). Biosynthesis gene clusters were adapted by means of synthetic genes for the incorporation of non-natural amino acids into peptides of major clinical importance, e.g. labyrinthopeptins, with antiallodynic and antiviral properties. The availability of RedStrep1 strains hosting the necessary cassettes for the production of biomolecules, and transcriptomics and metabolomics/fluxomics tools set the stage for systems level understanding of the engineered cell factories. A systems level multi-pronged approach will include advanced transcriptomics, proteomics, metabolomics and fluxomics analysis of RedStrep1+cassette strains (WP6). To ensure biological uniformity across the application of these complex tools, aliquots of the same biomass material was used for all –omics studies. This was an intense WP that generated large amounts of data that provided organism-level signatures for the cell as it grows to produce tailor-made biomolecules. These data-sets fed into WP7 that at a first level analyzed and integrated the -omics datasets and at a second level builds predictive models that incorporate the detected metabolites, fluxes, proteins and transcripts. These models aimed to identify potential rate-limiting steps in metabolic network models describing the optimal function of RedStrep1 as a cell factory. WP7 proposed specific gene targets from the RedStrep1 chromosome that can be modified either through deletion or over-expression so as to lead to strains with potentially optimized metabolism and bio-process behaviour, while at the same time delivering the highest possible yields of the biomolecules of interest. Strain optimization was the focus of WP8. Genetic engineering intervention on RedStrep1yielded the optimized RedStrep2. WP9 took lab-scale strain growth from pilot to industrial scale robustness in 50-100 Lt fermentors and then to efficient downstream processing (WP9). WP9 was an "end-user" WP that required close cross-talk with WP1, WP8. WP9 imposed its own requirements for fermentation performance and downstream processing and thus guided WP8 on the genetic engineering optimization of strains. To strengthen the innovation dimension of STREPSYNTH and to increase the likelihood of market uptake all WPs support the development of detailed Standard Operating Procedures (SOPs); reproducibility testing, statistically sound verification of results. STREPSYNTH would like to contribute to a dialogue between science and the public, particularly in what concerns the merits of synthetic biology in facilitating sustainable growth in an environmentally friendly manner. To this end training courses were set up and much attention was paid to dissemination (WP10) via a set of seminars to communicate and amplify the positive role of dialogue between science and society. The effort of the STREPSYNTH partners is geared towards achieving significant research results and at the same time remaining sensitive to society's viewpoint. STREPSYNTH would like to offer an example of an extensively co-operating scientific and technological community, aimed at developing the tools for positively challenging societal needs. The results of the project have been and will be disseminated widely throughout the EU and will be particularly relevant for the industries and research institutes active in the fermentation field and products derived from it. The involvement of partners specifically devoted to training & education will be exploited to increase public awareness of the research results, e.g. in a form of public demonstrations and other similar activities by Consortium partners. This is believed to provide a positive picture of the Community research to citizens and to improve the attractiveness of this science field. The project results will be disseminated via different channels. The existing marketing channels of the industrial partners will be used to disseminate their technology. Furthermore, technical and scientific publications, more than 564 in peer reviewed journals, made in all kinds of journals and magazines and presentations were given in scientific conferences. A web-site was set-up.
STREPSYNTH advanced research in the field of synthetic biology and generated innovative tools and methods for biotechnology applications. Here our focus is on S. lividans as a host but we anticipate that technical solutions, methodological breakthroughs and scientific discoveries will benefit the wider community of synthetic biology and the industrial microbiology of other microorganisms. Synthetic biology is enhanced here through its synergy and combination with omics-technologies, systems biology and metabolic engineering that will be used for the development and engineering of S. lividans for the efficient and heterologous production of industrial enzymes such as cellulases, chitinases, laccases, lyases and transferases, and proteins of biomedical importance. STREPSYNTH is thus important for the participating SMEs with an interest in applications in the Bioenergy market as some of the industrial enzymes will be aimed at the emerging biofuels market. Additionally, STREPSYNTH produced clinically relevant metabolites such as non-natural amino acids contained in lantipeptides, with proven antiviral activity against a range of highly virulent viruses including HIV, and mithramycin indolocarbozoles -with antitumoral and anti-inflammatory activity - alongside other clinically relevant compounds.

Key approaches are the engineering of the minimized cells, de novo design of robust and sustainable biomolecular circuits, independent secretion modules, synthetic pathways, and more robust metabolism in an industrial fermentation setting. The approach that STREPSYNTH used is expected to result in accelerated process design and reduced time-to-market for the biomolecules of interest. Furthermore, it is expected to result in scientific breakthroughs, which will increase the industrial competitiveness of European SMEs and create new economic opportunities. An important aspect of this is the systems level understanding of Streptomyces after incorporation of several layers of molecular detail. Another important aim of the project was the development of the SNIP as a future-proof platform rather than as a specialized route to a single strain and bioprocess development of today. This has the important benefit of investing in a platform for future potential use as a production line of completely different types of molecules. As five SMEs were involved in the project, and their products are the main focus of strain development, the project was without doubt of interest and benefit to SMEs. Also, a Scientific Advisory Board with members from Academia and SMEs and larger companies familiar with the topic was installed, as such this project is fully industry-driven and will help to contribute to the realisation of that benefit and guide its execution.

The sheer complexity, scope and ambition of the project made it imperative that for a successful end goal, a productive interaction needs to be established between disciplines: microbiology, proteomics, transcriptomics, metabolomics, bioinformatics, physico-chemistry and industrial biotechnology. To implement this, a multi-disciplinary team of appropriately trained leading scientists at all levels is required. Such a goal cannot be attained at a national level, but needs inter-EU cooperation, since no country on its own has all the expertise required carrying out such a multidisciplinary research.

This SME-focused IP project brought together 16 highly competent research teams (5 SMEs, 4 research organization and 7 from Academia) and secures trans-national resources from 8 EU and 2 Associated countries. In this RTD area large efforts are spent, especially in the USA, Canada and Japan. None of the partners, obviously, would be able to establish an equally competent consortium within their own country alone. The Consortium constituted the necessary and sufficient resources to be able to realise this complex project, assembling new partnerships and building on the foundation of previous EU networks to get sufficient critical mass to deliver cutting-edge performance.

To be successful in this domain, we have therefore put together this multidisciplinary consortium aiming to meet the following objectives:
1. to unify top researchers each experienced in the research of the selected work packages. Each of these WPs was designed with input from the industrial partners and guided by their interests. We consider this academia-industry cross-talk as vital for the success of the project.
2. to reduce the genome size of S. lividans (RedStrep strains) to make it a more robust host organism pporpriate for industrial fermentation (with new optimal properties such as reduced energy consumption, growing to higher cell density, less protein product degradation etc).
3. to evaluate S. lividans with a reduced genome as a robust host organism for the production of heterologous proteins and other biomolecules of interest to the participating SMEs and research centres. Proteins of different origin with economical relevance in the field of bioenergy, industrial biotechnology and biopharmaceuticals will be tested as well as clinically relevant peptides with anti-tumor and anti-inflammatory activity.
4. to engineer S. lividans with novel transcriptional capabilities and tight regulatory control to modulate growth and improve production capacities with rational intervention
5. to establish an advanced systems biology platform for the analysis of Streptomyces as a cell factory with in-built capacity for prediction of road-blocks for industrial performance optimization
6. to engineer the bacterial secretory pathway as a series of transferable cassettes, matched with optimal pairs of signal peptides with fused proteins of interest.
7. to engineer biosynthetic cassettes for the production of peptides and small molecules with unconventional amino acids to improve their biological activity
8. to train junior scientists and engineers to a common language of industrially-relevant modern biology
9. to prepare junior scientists for market awareness, as industry is likely to be a future employer
10. to build bridges between research and industry and keep the project end-product-focused
11. to train scientists and SMEs in knowledge transfer methods (If ASTP joins)
12. to generate and search for patenting opportunities to protect the knowledge gained in this project

Reinforcing competitiveness of the European biotechnology industry
Europe has a long-standing tradition of excellence in research and innovation, and European teams continue to lead progress in many fields of science and technology, including biotechnology. However, Europe’s centres of excellence are scattered across the Members States and their efforts often fail to add up in the absence of adequate networking and co-operation. European bench to market performance lacks ambition at times and academics do not always aggressively pursue the industrial fruition of their important research findings. Also, frequent times European companies allocate less resources to research and development compared to their American counterparts. STREPSYNTH aims to bring together several important research groups and companies in the field of biotechnology, bioinformatics, engineering research and fermentation to build a common research and innovation structure. We will be able to establish a productive interaction between microbiology, engineering and industrial biotechnology, and pharma-industry. STREPSYNTH will bring together molecular biologists, (bio)chemists, engineers, bioinformatics specialists, protein identification experts, fermentation process and metabolomic experts. These specialists are members of research institutes, universities and SMEs who have a dedicated and expressed interest in the molecular biology, biochemistry and application of bacterial production systems for heterologous compounds to be used for different purposes. Moreover, a central aim is to investigate in this framework the correlations of biochemical information with genetic and/or molecular data for gaining better insight in the cellular network that determines the production process of cell factories. The consortium aspires and is excited to exploit the increasing infusion of rational design afforded by modern –omics biology that plays an increasingly more important role as a complement to traditional strain development programmes.

Nowadays the biotechnology industry is internationally still dominated by US companies. The European SMEs are relatively small companies, whereas the US biotechnology industry started earlier, produces more than three times the revenues of European biotech, employs many more people (162.000 against 61.000) is much more strongly capitalised and in particular has many more products in the pipeline. The involvement of SMEs in Europe applying improved heterologous protein secretion systems for different purposes are under-explored when compared to the US, Canada and Japan.
The use of synthetic biology in combination with metabolomic/proteomic/transcriptomic/fluxomic technologies and appropriate fermentation technology proposed here will bring the following contributions to the evolution of EU policies:
• A competitive situation for European companies including the participating SMEs. Using innovative technologies, this project aimed to get deeper insight in the cellular behaviour during protein secretion and to identify major bottlenecks during the heterologous protein secretion process. In addition, strains will be constructed that allow the production of natural and modified (heterologous) compounds on demand. The overall knowledge gained, and tools developed in STREPSYNTH will allow constructing better producer strains and optimized fermentation conditions, and as a consequence strengthen the international market position for European SMEs and other bigger companies (e.g. pharma, biofuels, industrial enzymes). One goal of our efforts is that a model platform is developed that will allow European companies to produce molecules at industrial scale in relevant yields which cannot be produced by current expression/fermentation system. Consequently, the participating companies will gain a competitive advantage in this field.
• Clean production and sustainable development. Production strains that produce the desired biomolecules in larger amounts in a cost-effective manner are a more environment-friendly approach. This is because the production process will be more efficient as such requiring less energy input and smaller volumes of culture media. Moreover, the downstream process for correctly folded proteins that are secreted rather than produced as intra-cellular inclusion bodies is less complicated from a bio-pressing perspective. Secretion biotechnology therefore avoids the use of toxic compounds for protein denaturation and refolding that subsequently need to be disposed of in an environmentally friendly manner.
• Creation of jobs for highly skilled professionals. Important employment opportunities for highly skilled scientists and technical experts will be created by the implementation of the innovative production process for therapeutically and biotechnologically important compounds. Our project will develop and exploit technologies that will create new employment opportunities for scientists from the EU regions and will give those scientists who left the opportunity to return and strengthen the European industry by their experience. The creation of a European Research Area is a central point of the endeavour to transfer Europe into the world’s most competitive and dynamic knowledge-based economy and as such helping to reach the goal of Europe 2020. In this context, STREPSYNTH will contribute to offer attractive employment for highly skilled scientist and professionals. As more than 95% of our currently trained PhDs are not expected to get tenured jobs in academia their exposure to alternative, industry-centred career paths is an important part of the career-awareness training through STREPSYNTH.

Market potential of gained knowledge and of deliverables obtained
Life sciences and biotechnology are widely regarded as one of the most promising frontier technologies for the coming decades, since they offer opportunities to address many of the global needs relating to health, ageing, food and the environment, and to sustainable development. Small and medium size companies are important players in biobusiness, a largely SME-driven market. This industry contributes significantly to saving lives and improving the quality of life of the citizens of Europe.
Biotech SMEs are often important intermediaries between academia and industry for the purpose of developing and disseminating technology. They are suppliers of technology platforms, knowledge, services, and product embryos to larger companies, such as international pharmaceutical companies or large companies in for example biocatalysis and bioenergy. SME biotechnology companies also transfer knowledge from academia to their customers using their strong networks. The purpose of these networks is to identify frontline research suitable for commercialization. Their products may also consist of the licensing of patented research findings. Our improving scientific understanding of the genetic and molecular circuits behind metabolite production and protein secretion processes is an important driver of technological innovation and a foundation for new growth. The development and dynamics of the biotechnology industry are heavily dependent on academic research findings. The STREPSYNTH project merges research and applications leading to new production processes important for the participating industries.
Within the Pharmaceutical industry, recombinant proteins are key tools in the discovery of new drugs and are the basis of the high throughput compound screens which are characteristic of today’s drug discovery process. Consequently, all pharmaceutical companies invest many millions of euros worth of resources every year in the generation of these reagents. Recombinant proteins are themselves drugs belonging to a class known as biopharmaceuticals. Biopharmaceuticals are becoming increasingly common and have grown from 0.5% of worldwide pharmaceutical sales in 1989 to over 9% in 2002 with worldwide sales totalling $32 to $53 billion in 2010. The world pharmaceutical market grew by 4.5% in 2011. The use of biologics has become an important source of innovation, with biologics among the most commercially successful new products. Forecasts for 2016 predict that of the world’s top 100 pharmaceutical products, 45% of sales will come from biologics. This compares with only 33% in 2010 and 15% in 2002. Improving R&D productivity is a critical constant challenge for the pharmaceutical industry. Global investment in pharmaceutical R&D by the top 500 pharmaceutical and biotech companies reached an estimated $133 billion in 2011, a 93% increase from $69 billion in 2002. To ensure it delivers a sustainable return on its R&D investment, the industry is working to increase its probability of success in developing commercially viable new drugs and moving to a lower, more flexible cost base. Despite remarkable progress in the production of biopharmaceuticals in eukaryotic cells and cell lines, microbial hosts still remain the most cost-effective solution for proteins that do not need excessive post-translational modifications.
These figures also include market expectation for antitumour and antiviral compounds, two extremely important global markets. The global anti-infective market is currently valued at US$66.5 billion with antiviral agents accounting for 24% of sales (excluding vaccines which target viral infectious diseases). According to a new report “The Antivirals Market: R&D Pipelines, Market Analysis and Competitive Landscape”, the anti-viral market will be driven by the uptake of newer anti-retroviral agents in combination therapy and the launch of ten new products for the treatment of HIV and hepatitis which will address the need of treatment-resistant patients. This will help to stem the tide of this prevalent and often lethal killer in our society today. Also cancer is a global health care priority, one with rising and commercial importance with an increasing number of cases. The anticancer drug sales exceeded US$50 billion in 2009, which will grow exponentially in the coming years. Production of such anticancer and antiviral compounds are also envisaged in STREPSYNTH.
With the inevitable depletion of the nonrenewable resources of fossil fuels and due to their favorable environmental features, biofuels promise to be the preferred fuels of tomorrow. Enzymes are important catalysts to make this process less energy intensive and environmentally friendly. The use of enzymes for the hydrolysis of cellulose to produce fermentable sugars for bioethanol production for example makes the utility cost of enzymatic hydrolysis much lower compared to the alternative methods of acidic hydrolysis because it is carried out at mild conditions and does not require a subsequent treatment step. There are several obstacles, however, facing the use of enzymes as catalysts for the production of biofuels, most importantly is their high costs. This cost can certainly be lowered when efficient production systems are available for the required enzymes. Towards this end, STREPSYNTH aims for high and cost-effective production of cellulases, chitinases and laccases. The enzyme market as a whole is very big, with estimates for the global market for Biofuel enzymes exceeding US$900 million by the year 2017.
It is expected that the SNIP platform developed in STREPSYNTH will be very valuable as a robust and efficient production system for such important compounds with high market value, and as such will be very helpful to contribute in these bio-industrial developments of tomorrow.

Input from basic research carried out during STREPSYNTH was directed towards training & education initiatives and dissemination activities. Besides protecting the project results via patenting, dissemination of STREPSYNTH knowledge and results was planned to occur via a number of activities: through the normal channels i.e. participation in symposia, conferences, workshops and publications in relevant journals. However, care was also devoted by all partners to ensure that the potential for patenting the results will not in any way be jeopardised by a premature release of the results.
For publication of the results the Steering committee decided the manner of publication of data arising from this project. No Party published data which included Knowledge of another Party, Pre-Existing know-how of another Party or confidential information of another Party even where such data is amalgamated with such first Party’s Knowledge, Pre-existing Know-How or other information, document or material. Data from any particular laboratory was included in publications by another laboratory with its explicit permission. Thus, although it is expected that all the data produced under this project appeared and will appear in joint publications, any individual group were at liberty to withdraw its own data from such a publication. Publication of data obtained in some, but not all the participating laboratories were under the authorship of the active scientists in those laboratories. The first author will normally be the person who writes the first complete draft. Laboratories that do not provide data for that publication need not to be included as co-author, but it should be clearly indicated in the text that the paper emanated in whole or part from this joint project. In all publications, proper references to the origin and generator of the information shall be made. All publications will be submitted to the Steering Committee that will formulate any objections within 14 days after receipt.
A set of seminars and workshops to communicate and amplify the positive role of dialogue between science and society. Specific examples are:
o Organization of a workshop on Metabolic and Protein Interaction Network Analysis in 2014 in Patras (FORTH/ICE-HT)
o Organization of a summer school on Metabolomics and Fluxomics in 2015 in Leuven (KU Leuven/U MCR)
o Organization of a summer school on Proteins to Proteomics in 2016 in Leuven (KU Leuven)
Dissemination through the participation in the organisation of International Conferences, to communicate and amplify the positive role of dialogue between science and society. Specific examples are:
o Metabolic Engineering Conference, Computer Applications in Biotechnology (CAB13), Metabolomics, International Conference on Systems Biology (ICSB)
The existing marketing channels of the industrial partners were used to disseminate their technology and products. These dissemination trails mainly include our participation at local and selected international biotechnology-focused fairs as well as contact with business relation-mediating agencies (e.g. CzechTrade or within CzechBio, the cluster of Czech biotech companies). HZI has a strategic collaboration with BASF and Sanofi which are aware and highly interested in the current proposal, the possible licensing of the results of the project will be discussed with these companies. TUB has excellent contacts to many SMEs (e.g. Aicuris, BRAIN) as well as big pharma companies (e.g. Sanofi, Novartis) and big biotech (e.g. Evonics) which were approached for further use of the developed platform or further development of the generated peptide drugs.
A project web-site was set-up and maintained by PROGENUS to be used as one of the dissemination channels. It contains a public section and a project partner section. The restricted section contains all the official documents and reports generated during the project. Access to the restricted site will be granted to all the project participants and to the programme's officer.
At the University setting, it is obvious that results of the ongoing research was discussed with students in courses. Moreover, projects were offered to masters and PhD students to work in the participating labs on the topic of the proposal.
Dissemination to Society and Citizens. Participation to the “science week” for pre-university students (KU Leuven). Participation to open door days, Press releases in newsletters, magazines, science sections of newspapers and other multi-media channels (such as radio, TV) when relevant results were achieved. In Spain we made use of R&D dissemination organizations (like the agency SINC in Spain) to disseminate successful results of the project. SINC, aside from their own website for science and technology news, is the source of this kind of news for mainstream media in Spain. Reaching a broader audience will be done by articles in local media, since both Leuven and Oviedo are cities where University activities are well known and EU programs like FP7 could make an impact in local media and help the community understand the importance of this pan-European R&D program. In Greece corresponding agencies like Praxis and the National Documentation Center were used.
Dissemination was conducted by National organizations and Networks such as The Belgian Society for Microbiology (KU Leuven; J. Anné being the President of the Society), the DECHEMA Working Group Systems and Synthetic Biology (FZ-Jülich; M. Oldiges being a member of the working group). Norwegian Biochemical Society and Center for Biological Design in Trondheim (CenTroN, Prof S. Zotchev, the VAAM (German Association of Applied Microbiology and Molecular Biology) (Prof R. Süssmuth, Prof A. Luzhetskyy), SMEs like Apronex included information about STREPSYNTH on its web page, in professional magazines such as Gate-to-Biotech as well as through press releases in public newsletters. HZI will communicate the results of the project through its monthly journal covering the activity of the Institute.
The following table gives an overview of the dissemination plans of STREPSYNTH:
R & D Results End User Dissemination/Exploitation
Combination of beneficial mutations in a genome-reduced S. lividans Participating companies, basic researchers. Interested biotech and pharma using industrial microbiology Journals, lectures, websites, training and seminars, visits to companies, technology transfer offices
Biological parts and synthetic regulatory circuits that control production and secretion of target molecules. Basic researchers; participating companies
White and red biotechnology industries Journals and conferences; patents and licenses. White and red biotechnology industries. Biotech scientific and producers associations
Validated genome-scale metabolic model network for RedStrep and predicted sets of genetic modifications that are expected to increase compound production yield and/or rate Basic researchers; participating companies
White and red biotechnology industries Journals and conferences; patents and licenses. White and red biotechnology industries. Biotech scientific and producers associations
Analytical tools for systems biology analysis & Fluxomics of Streptomyces and data focussed on protein secretion Researchers; biotech & pharma industry; SMEs, Journals and conferences; patents and licenses. Biotech scientific and producers associations
Engineered S. lividans strains for improved production of proteins and lantipeptides of interest to industrial partners Participating companies
Interested white and red biotech industries Participating companies, other interested companies
Proteins and lantipeptides produced with industrially optimized Streptomyces using synthetic cassettes for non-natural amino acids and heterologous secretion systems Participating companies, biotechnological, pharma & diagnostical industry, SMEs Interested users (Bioindustry, general public)
Patents and results dissemination Participating companies, biotechnological, pharma & diagnostical industry, SMEs Interested users (Bioindustry, general public)
Future Projects National/international public officials EU committees & conferences
Business plan Participating companies VCs, interesting parties

Exploitation strategy
To ensure sustainable growth, more and better jobs, as well as industry competitiveness, the wide use and dissemination of the knowledge generated in STREPSYNTH is of utmost importance and as such helps to strengthen the scientific and technological bases of the EU. In order to co-ordinate the innovation activities, the Consortium appointed Dr Annie Van Broekhoven as project exploitation manager. As a highly experienced person in the field, she will be responsible for monitoring and coordination of the innovation-related activities (knowledge, IPR, exploitation & dissemination issues, set-up of follow up activities). She will receive feedback from appointed exploitation managers in each partner organization. The exploitation manager of STREPSYNTH will make a detailed plan to manage the knowledge and the use of results. She will report to the Steering committee on the innovation related activities on a 6 monthly base. General rules on knowledge management, use of research results and findings and related IPR issues will further be described in detail in the Consortium agreement that will be finalized and signed upon initiation of the project. In addition, it is intended to make use of an independent association with a profile such as the ‘Association of European Science & Technology Transfer Professionals’ to give training courses to professionalize and promote technology and knowledge transfer between the European science base and industry. Therefore, contacts will be sought with an association with a large coverage of technology transfer professionals at public knowledge institutions and from many different countries.
Activities on the use and exploitation of the results
o Necessary agreements between the project partners about patenting and exploitation of network results will be set up within the context of the consortium agreement;
o A careful plan of the dissemination activities and the use to be made of the project’s foreground information should however be seen as more than a simple obligation, but instead an essential step to pave the way for the research from the lab to the market.
o Screening of the project results on possibilities for patenting; the individual partners are obliged to report timely to the project exploitation manager on results that may yield results with patenting potential;
o Identification of results that may commercially be exploited; the partners are obliged to report timely to the project exploitation manager on exploitation possibilities based on results developed by the network;
o Preparation of patenting and exploitation opportunities and plans for the Steering committee
The Steering committee will decide which actions will be further taken with respect to patenting and exploitation. Afterwards, information that can be publicly disclosed will be communicated through the network website.
The following table gives an overview of the exploitation plans of STREPSYNTH:
R & D Results End User Exploitation
Combination of beneficial mutations in a genome-reduced S. lividans Participating companies, basic researchers. Interested biotech and pharma using industrial microbiology Visits to companies, technology transfer offices
Biological parts and synthetic regulatory circuits that control production and secretion of target molecules. Basic researchers; participating companies
White and red biotechnology industries White and red biotechnology industries: patents and licenses; drug, diagnostics and biotech product development and marketing. Biotech scientific and producers associations
Validated genome-scale metabolic model network for RedStrep and predicted sets of genetic modifications that are expected to increase compound production yield and/or rate Basic researchers; participating companies
White and red biotechnology industries White and red biotechnology industries: patents and licenses; drug, diagnostics and biotech product development and marketing. Biotech scientific and producers associations
Analytical tools for systems biology analysis & Fluxomics of Streptomyces and data focussed on protein secretion Researchers; biotech & pharma industry; SMEs, Patents and licenses. Biotech scientific and producers associations
Engineered S. lividans strains for improved production of proteins and lantipeptides of interest to industrial partners Participating companies
Interested white and red biotech industries Participating companies, other interested companies
Proteins and lantipeptides produced with industrially optimized Streptomyces using synthetic cassettes for non-natural amino acids and heterologous secretion systems Participating companies, biotechnological, pharma & diagnostical industry, SMEs Interested users (Bioindustry, general public)
Exploitation routes
RTD results will feed into future RTD directions of the partners involved. One of the aims of STREPSYNTH is to develop a versatile platform rather than a unique, fitted solution. We therefore expect this platform to be open to adaptation to other future needs of the participating or other companies. The cassette approach we follow for the engineering of specific synthetic pathways can be easily replaced by other cassettes in which other enzymes of interest are produced. We also expect to have input in discussion steered by EU committees/policy groups thereby contributing to the generation of new legislation that relates to aspects of synthetic biology, patenting and use of research results. Marketing & socio-economic studies will be inevitably touched upon within the project's duration as in many cases they will serve as guides for exploring the project's potential fully and in setting the foundations for future research. As soon as it became relevant, and also after the end of the project, consortium members will continue their efforts to manufacture & market the products in a cost-effective way using the bacterial strains and the knowledge obtained in this project. To strengthen the innovation dimension of STREPSYNTH and to increase the likelihood of market uptake of our results we implemented five main policies: a. extensive support for the development of Good Laboratory Practice (GLP) technical standards by including in all WPs detailed Standard Operating Procedures (SOPs); b. elaborate reproducibility testing; c. statistically robust verification of results and comparison with other methods; d. identifying and collaborating with potential industrial users from within the project, from the advisory committee and from aggressive dissemination activities towards external industrial concerns; e. identifying potential sources of finance for commercialisation. Support from other funding bodies and local schemes will be considered, encouraging a bottom-up approach to further development and strengthening of the competitive position of the participating companies in the marketplace. This is important also for future job creation. Some of the results are not be ready for direct commercialisation, but will require future projects to bring them to maturation and to fully explore them. Other results are closer to be commercialised in a short time frame after the end of the project. Since it is highly likely that interesting new proteins, biomolecules and biochemical and industrial-scale processes will become available during the course of the project, follow-up projects will be needed.
An alternative aspect of 'exploitation' at the Universities and research institutes is that of forming a fundamental base to stimulate further research and the education and training of a new generation of researchers in this field. The experience and knowledge on synthetic biology, the –omics analyses and modelling and bioinformatics obtained during the course of STREPSYNTH, will be introduced into high level university teaching, increasing the quality of teaching and the technical and scientific knowledge of the students. Also the obvious industrial relevance of this effort and the intimate involvement of interested companies in the project allows us to put together an intense technology-transfer training programme where the relevance of such academia-industry consortia is made obvious and where a future career for the students in life technologies is explained and promoted. The project has intense potential in this direction as synthetic biology-based tools and rational exploitation of omics-scale results are expected to drive future company creation and growth in the European area. The performed in STREPSYNTH is frontier research, that will result in sustainable development, and enhance the ability of the SMEs involved to improve their competitiveness, with an immediately positive effect in their financial growth and in job creation. The effect will not be restricted to the Consortium, but will have a multiplicator effect in the EU Biobusiness, which is mainly an SME-driven market.
The exploitation plans and intentions of the partners
• KU Leuven-REGA. LMB having experience in the field of protein secretion, has expand its know-how on the optimal heterologous protein secretion methodologies allowing LMB to stay at the frontline of basic knowledge in the field, to construct specialized strains allowing more efficient protein production with a potential for future applications, to develop software predictors for heterologous protein secretion optimisation. IP protection and aggressive efforts to identify industrial partners are being sought for all these activities.
o KU Leuven-CIT focuses on model-based optimization and control in industrial biotechnology, biological wastewater treatment and chemical process industry. With this project, CIT has strengthened and expanded its research on metabolic modelling and engineering of microbial cell factories for sustainable production of industry-relevant compounds.
o Helmholtz Center for Infectious (HZI) used the results of the project to generate the rules for synthetic biology of actinobacteria: limits and advantages of the genome reduction; gene expression optimization using synthetic biobricks etc. Current proposal perfectly complements end extends the activities of the group in synthetic biology field.
o Norwegian University of Science and Technology (NTNU) benefited from the proposed project via building up an expertise in construction and characterization of biological parts and synthetic regulatory circuits, and their use in ongoing and future projects on drug discovery.
o Institute of Molecular Biology, Slovak Academy of Sciences (IMB SAS) expects to exploit the proposed results in expanding knowledge in regulation of secondary metabolism in these bacteria and in construction of new Streptomyces strains useful for improved production of new bioactive compounds.
• Technische Universität Berlin (TUB). The project further strengthened the Synthetic Biotechnology and Antibiotics research group; allowed developing new compounds of biomedical interest.
• Universität Bielefeld (UNIBI) gained experience and proof of capabilities by the Technology Platform Genomics confirmed the excellence of the group, and allowed it to attract new joined projects of industrial importance, directly or indirectly strengthening European biotechnology
• The University of Manchester (UMCR) by this project wanted to provide and extend their experience in the field of protein secretion and in particular the role that metabolomics and metabolic flux analysis can assist in synthetic biology.
• Foundation for Research and Technology- Hellas (FORTH). New knowledge was communicated to the scientific community through publications to international peer-reviewed journals and presentations at conferences in the fields of systems - synthetic biology and metabolic engineering.
• Technion – Israel Institute of Technology (IIT) will use the accumulated knowledge and experience on metabolic modelling to address additional metabolic engineering challenges.
• Forschungszentrum Jülich GmbH. Based on this project, IBG-1 wants to foster its position in the emerging field of accelerated bioprocess development and to provide knowledge and technology allowing for more efficient protein production.
• Progenus is interested in STREPSYNTH to develop its high throughput sequencing activities, contacts with academic and industrial partners within the consortium and new bioinformatics facilities to enable our web-based result vizualisation solution to integrate data coming from different sources (proteomics, transcriptomics, metabolomics and fluxomics data).
• PharmBiotec strengthened its unique technology in view of protein analytics by further developing their special methods, e.g. CE-MS, FTIR and general pharmaceutical technology of macromolecular drugs; established its fermentation related skills and technologies: on the one hand getting even more experienced in routine biotech work and on the other hand by direct application of the synthetic biology related results of this project for its fermentation business.
• EntreChem, S.L. – Biotechnology. Its involvement in STREPSYNTH helped position the company as a world leader in Streptomyces genetics and systems biology oriented towards the discovery and development of new bioactive products, especially in the area of oncology. EntreChem used the results of the project to supply new hybrid natural products, a big hurdle that has prevented many new combinatorial biosynthesis products discovered in recent years to progress beyond the early preclinical stage.
• Apronex s.r.o. established and developed contacts with academic and industrial partners within the consortium; enlarged its portfolio of methodological approaches and especially expression systems. This will provide a competitive advantage in the field of effective production of industrial enzymes and other recombinant proteins.
• Matís ltd test different S. lividans strains, engineered within STREPSYNTH for optimized production of recombinant proteins. Based on the results, Matís exploited the respective strains for production of its own propriety enzymes.
• Q-Biologicals profiled itself as an SME capable of producing industrial products, in particular, proteins, enzymes and lantibiotics out of Streptomyces, which is a clear advantage in Europe as only a few SMEs are offering such services. Q-Bio provided industrial interested partners with pilot batches of biological materials developed in this project, and was able to produce these materials under cGMP. Offering a cheap production platform for microbial antigens potential veterinary vaccine candidates will certainly have a clear economic impact to the company.
More specifically the exploitation plans and expected benefits of the SMEs are as follows:
Entrechem (S)
- High yield production of 2 molecules which are tremendously important in oncology.
- Increase of production of molecules already in preclinical – and close to clinical phase.
- Novel proof-of-concept combinatorial compounds.
- Strengthening of proven success of combinatorial biosynthesis.
- Gain unique market position in Europe and Worldwide. This will allow: activity expansion; new job creation for Spain; attraction of new investment.
- Large impact on human health sector (cancer treatment).
- Impact for the pharmaceutical industry in Spain and Europe.
- Entrechem is supported by several Spanish investors (chemical and biotechnological industry, financial institutes, Asturias region) who will also benefit from the success of Entrechem.
Apronex (CZ)
- Extended portfolio of methodological approaches and especially expression systems.
- Lower production costs throught improvements in the properties of recombinant proteins (folding, easier purification) compared to classical E.coli-based expression systems.
- Enhanced competitive advantage in effective production of industrial enzymes/recombinant proteins.
- Establishing and developing collaborations/networking with new academic and industrial partners.
- attracting new customers via networking, new methodologies and new products.
- Improved attractiveness for future national/international, academic/industrial collaborative projects.
- Employment of new skilled personnel – initially 1-2 with projected 3-5 that will have also a direct positive impact on Czech economy and employment.
PharmBioTeC (D)
- Establishment of new techniques, methodologies to strengthen its services portfolio.
Introduction of new analytical techniques like Fourier-transform-infrared spectroscopy in aqueous medium or capillary electrophoresis coupled to mass spectromerty included routinely in analyses. This will open up new markets and enhance competitiveness.
- Networking for future business.
- Participation will ensure the attraction of additional competitive grant monies that will also lead to attraction of new industrial service contracts.
- Employment boost from a staff of 22 to ~30 projected for 2016.
QBiologicals (BE)
- introduction of new cell factory technology for the production of high-value biomolecules: biopharmaceuticals, veterinary vaccines, and industrial enzymes.
- Improved product quality: Enhanced product yields/optimized purification/reduced degradation/no LPS contamination/efficient production of folded proteins.
- Drastic reduction of process costs/competitive advantage.
- Expected synergies boosted by the cGMP production facility (as of Q4 2013).
- Boost of process innovation especially in industrial chiral enzymes. Such chemical reactions impact energy consumption and waste. They will be replaced by low impact, user-friendly enzymatic reactions.
- New production contracts with major pharmaceutical and veterinary companies.
- Will contribute to growth of current staff of 13 (PhD, MSc and BSc-type education) to ~30 by 2018.
- Direct increase in the income stream.
- Inexpensive, effective veterinary vaccines will benefit the European farming industry due to reduced morbidity and will reduce antibiotic use (thus curbing the incidence of antibiotic resistance).
- Benefit to other Belgian companies [e.g. Dupont (Brugge); Puratos (Andenne)] to make use of new technologies and large-scale cGMP facilities.
- Direct positive impact on the Belgian economy, innovation and employment. A cGMP facility producing commercial vaccine lots involves at least 100 to 150 persons (75% of them operators).
Progenus (BE)
- Development of bioinformatics services for new diagnostics for the veterinary and human health markets to drastically reduce process costs/ offer competitive solutions.
- Further development of existing software (BioXpress, GAP); creation of 2 new, IP-protected software.
- Emergence as a reference provider in Next Generation Sequencing services including bioinformatics (e.g. probe design, gene annotation) and molecular biology information in a common platform.
- Development of web-based pipelines to manage, store, analyze and search the huge amount of information created by a company and its subsidiaries or between different partners in a consortium
- Gaining new contracts with major but also SME pharmaceutical and veterinary companies.
- Expansion in EU and ASEAN region; attraction of new investment; from a current 1.3 mil to ~2 mil €.
- Impact on employment: from 10 employees at present to 15 - 20 employees by 2016.

Intellectual property rights
Partners took appropriate action to protect the foreground information that could be used for industrial or commercial applications. The formalisms described in Desca III formed the starting basis for the formulation of a detailed description of treatment of IPR. Inventorship in inventions was be determined on a case-by-case basis taking into account the role and contribution of each party in the conception and/or reduction to practice of such invention. Any Participant shall be entitled to take any action to protect any result created solely by him during the Project, without the help of the other Participants which could be used for industrial or commercial exploitation (by the means of patents, know-how, copyrights or other similar rights). If protection of a patentable invention is sought, the filing will be done in the name and at the expense of this Participant. If an invention results from the contribution of several Participants (Contribution by intellectual participation or in providing biological material), the said Participants will own this invention. The co-owners of the invention shall agree on a case-by-case basis on the best manner of protecting such joint invention. A co-ownership Agreement will be established between all the co-owners to define the arrangement between them as regards the share of property of each co-owner, the share of expenses relative to the filing, prosecuting and maintaining of the patent application. The terms of the license will fairly reflect the nature of the invention, the risks incurred and the costs of subsequent research and development needed to bring the invention to the market place. Ownership of background information by any of the partners shall not be affected by this project. Each of the partners shall, on a royalty-free basis, make available his background information to the other partners where and to the extent that such information is necessary for the execution of the research and the development of the work under this project. A clear and adequate description of how the participants will organise IPR ownership and user rights (e.g. licences, royalties) among themselves was provided in the CA.
Exploitation of this research project could occur via patent disclosures from the RTD performers to the small medium enterprises (SME). A privileged condition should definitely be established for the SMEs participating in the project to exploit the generated know-how. Patents will be taken by the partners whenever possible. Final discussions leading to signing of the CA determined one of two models, i.e. a. whether the participating SMEs / SME Associations retain the full ownership of all project results ("foreground") and the RTD-performers are remunerated accordingly or b. RTD performers keep part ownership or the entire foreground. All were described clearly in the CA. The consortium foresaw that members of the SME Associations and/or Other enterprises and end-users involve resources in the project and receive in return access rights for the dissemination and use of results generated by the project. Access rights to IPR elements "background" and "foreground" to carry out the project will be defined. The table below lists the main items by partner and type of access right granted to other partners of STREPSYNTH. These items were presented in detail in the CA in which access rules were presented.
List of Websites:
http://www.strepsynth.eu

Contact details:
Prof. Tassos Economou
Laboratory of Molecular Bacteriology, Head
Rega Institute, Dpt of Microbiology and Immunology,
KULeuven
Herestraat 49, PO box 1037
3000 Leuven, Belgium
tel. +32 16 37 92 73
www: http://rega.kuleuven.be/bac/economou

Project information

Grant agreement ID: 613877

Status

Closed project

  • Start date

    1 December 2013

  • End date

    30 November 2018

Funded under:

FP7-KBBE

  • Overall budget:

    € 11 330 683,76

  • EU contribution

    € 8 691 070

Coordinated by:

KATHOLIEKE UNIVERSITEIT LEUVEN