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STREPSYNTH Report Summary

Project ID: 613877
Funded under: FP7-KBBE
Country: Belgium

Periodic Report Summary 2 - 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 of reduced-genome S. lividans strains, 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 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. Many of these planned cassettes are at an advanced state of development. Several genetic engineering interventions are also in place and have removed several antibiotic production and pigment generation and protease genes. This first generation reduced genome strain, termed RedStrep1 has been fully sequenced and initial microfermentation studies show that it does not have 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 phases of the project. This omics-based boost is hoped to help rationally design our future genetic interventions. To set up SNIP, 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, biofuel enzymes, 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 play 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 reporting period we have made significant inroads in several aspects of the StrepSynth project. Work on all relevant WPs has begun at an excellent pace. This includes: numerous genetic tools such as promoters, ribosome binding sites and vectors, purified proteins and antibodies, software methods for omics analysis and integration, labeling for metabolomics and fluxomics, defined media that have been development and standardized for use across the network, synthetic genes and promoters, reduced genome strains with specific genes knocked out, micro-fermenation and larger scale fermentation protocols, BAC libraries and specific gene replacements methods, proteomics methodologies and analysis of the secretome, an annotated database of streptomyces protein topology transcriptomics analysis of gene expression at various stages of growth. In this bustling activity, a number of milestones have been satisfied and several important deiverables completed. Scientifc deliverables completed are:
We have presented a Technology platform for the efficient genome engineering S. lividans (DL1_1); we have reported on a new microbioreactor cultivation setup to characterize S. lividans and its mutants under various experimental conditions (DL1_2). Several different media have been examined and using control fluorescent proteins it has been possible to establish standard operating conditions with which to monitor fitness of various engineered strains. We also presented the first generation RedStrep 1 production strain (DL1_3). Two complementary systems for efficient and reliable gene deletions in Streptomyces lividans were generated in order to develop RedStrep1. The systems are based on positive selection for double crossover recombination using the blue-pigment indigoidine bpsA or gusA β-glucoronidase reporter genes. 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. 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). In addition, several genes (for gamma-butyrolactone system, sigma factors SigH and SigB, and highly-expressed sRNA) were deleted. We also set up a genome sequencing methodology and brought to a completion the characterization of the wild type TK24 strain (Ruckert et al publication in J. Biotechnol) and then also of the first generation RedStrep 1 production strain (DL1_4). We inititated the generation of biobricks to regulate gene expression. For this we presented a library of characterized synthetic constitutive and inducible promoters, ribozyme-containing UTRs and gene-specific RBSs for S. lividans (DL2_1).

Potential Impact:
Synthetic biology has reached the stage that in synergy with systems biology, metabolic engineering and molecular biology 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. 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 (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. 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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