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Periodic Report Summary 3 - STREPSYNTH (Rewiring the Streptomyces cell factory for cost-effective production of biomolecules)

Project Context and Objectives:
STREPSYNTH aims 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 has two components: firstly, we have begun construction 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. 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 in the form of plasmids and once stabilized, will be directly incorporated into the genome so that genetically stable host strains are generated. These planned cassettes are at an advanced state of development and are being tested. Several genetic engineering interventions are also in place and have removed antibiotic production and pigment generation and protease genes. This first generation reduced genome strain, termed RedStrep1 has been fully sequenced and initial micro-fermentation studies show that the interventions do not incur any penalty in its fitness. Additional deletions are being engineered targeting selectively secondary metabolite genes and sigma factors that will block expression of several genes. In addition, several systems biology tools (multi-omics) will be used to characterize the metabolism, proteome, transcriptome of the host cells and mathematical modelling and integration of these results has begun. We will use such models to guide fine-tuning rounds of cell factory engineering and fermentation optimization in next phase of the project. This omics-based boost is hoped to help rationally design our future genetic interventions, e.g. by targeting over-expressed enzymes. To set up this platform, we chose to focus 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). These biomolecules are of immediate interest to SMEs that participate and guide the industrial relevance of STREPSYNTH. Feedback from the industrial partners also plays 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. Up to now the project can show remarkable progress in all its aims that to a large extend validate its initial objectives.
Project Results:
During the first three 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, 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, 13 milestones have been satisfied and several important deiverables 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 (D4.1and have a ollection of heterologous proteins functionally secreted from RedStrep1 derivative strains, including industraially 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 are in the process of developing strains incorporating Pyl and analogs in defined positions of labyrinthopeptin by amber stop codon-suppression (D5.2); We have expanded of 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 are in the progress of developing optimized secretion strains that carry heterologous 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 have optimized bioactive molecule production (D8.3) and are currently genetically engineered RedStrep 2.0 strain resulting from metabolic engineering (D8.4).
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 wide as possible, two classes of biomolecules with obviously immediate industrial value and application are chosen for this expression 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 mecidal relevance). STREPSYNTH is industry-driven, and therefore focuses on compounds that can be immediately commercialized by the SME partners. The project will advance research in the field of synthetic biology and generate innovative tools and methods for biotechnology. The approach that STREPSYNTH uses will 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. 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 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. 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 aims for high and cost-effective production of cellulases, chitinases and laccases.
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