Periodic Reporting for period 2 - SHIKIFACTORY100 (Modular cell factories for the production of 100 compounds from the shikimate pathway)
Okres sprawozdawczy: 2020-07-01 do 2021-12-31
In the past decade, the demand for food ingredients from sustainable sources have been gaining momentum. Gradual replacement of artificial ingredients is becoming a strong driver among large companies in an attempt to improve consumer satisfaction. In 2015, a cluster of food brands led by Nestlé vowed to use only natural ingredients in products marketed in North America. Moreover, it becomes clear that natural resources used in these industrial sectors are often limited and climate dependent, leading to price fluctuations. In this and other fields, there is thus a strong industrial commitment towards the replacement of traditional techniques for product synthesis, such as petrochemical transformation or devoting large areas of arable land to grow plants with low yields of the target compounds, while fostering a paradigm shift towards more sustainable biotechnologies.
However, to support this significant market trend, it is important to develop scalable technologies that can offer efficient biological processes for compounds that can be scaled-up respecting environmental and social aspects. In many cases where the chemical synthesis has been put in place, the complexity of the molecules leads to prices that, although lower than most of the ones associated with natural products, are still within the same order of magnitude.
Combining the latest advances on synthetic biology, systems biology and rapid prototyping the project SHIKIFACTORY100 seeks the rapid and cost-effective development of biological alternatives, which can be used to create resource-efficient and cost-effective microbial processes for the production of novel compounds or compounds that are currently only produced by petrochemical processes or limited to plant extraction.
The overall objective of SHIKIFACTORY100 is empowering cost-effective synthesis of >100 compounds derived from shikimate to expand diversity of nature's chemical production. This project proposes a complete pipeline taking advantage of emerging synthetic biology and rapid prototyping techniques combined with knowledge of synthetic chemistry, enzyme engineering, systems biology and bioinformatics.
The main technical objectives (vectors) of the project are (Figure 1):
● DISCOVERY - Collection of natural and non-natural reactions and compounds as well as the discovery and ranking of novel pathways for the production of the target compounds.
● DESIGN - In silico and in vivo design, rapid prototyping for the pathways selected in the previous vector and optimization efforts toward chassis and advancing production strains.
● APPLICATION - Application and initial upscaling for a subset of compounds and sustainability appraisal.
However, to support this significant market trend, it is important to develop scalable technologies that can offer efficient biological processes for compounds that can be scaled-up respecting environmental and social aspects. In many cases where the chemical synthesis has been put in place, the complexity of the molecules leads to prices that, although lower than most of the ones associated with natural products, are still within the same order of magnitude.
Combining the latest advances on synthetic biology, systems biology and rapid prototyping the project SHIKIFACTORY100 seeks the rapid and cost-effective development of biological alternatives, which can be used to create resource-efficient and cost-effective microbial processes for the production of novel compounds or compounds that are currently only produced by petrochemical processes or limited to plant extraction.
The overall objective of SHIKIFACTORY100 is empowering cost-effective synthesis of >100 compounds derived from shikimate to expand diversity of nature's chemical production. This project proposes a complete pipeline taking advantage of emerging synthetic biology and rapid prototyping techniques combined with knowledge of synthetic chemistry, enzyme engineering, systems biology and bioinformatics.
The main technical objectives (vectors) of the project are (Figure 1):
● DISCOVERY - Collection of natural and non-natural reactions and compounds as well as the discovery and ranking of novel pathways for the production of the target compounds.
● DESIGN - In silico and in vivo design, rapid prototyping for the pathways selected in the previous vector and optimization efforts toward chassis and advancing production strains.
● APPLICATION - Application and initial upscaling for a subset of compounds and sustainability appraisal.
From a management perspective, a centralized system to allow monitoring the project progress and results was made available (GitLab) and linked a centralized synthetic biology parts repository (ICE) and pathway navigation system (ShikiAtlas). All platforms are in place and available to all the partners in the consortium. The work on the discovery vector has been focused on the expansion of natural biosynthetic routes for the production of shikimate derivatives as well as on the discovery of novel compounds, specially novel sweeteners with improved features. For the expansion of the natural portfolio of pathways involved in the production of shikimate derivatives, the work has been mainly computational, with the power of three state-of-the-art technologies being integrated and applied with success to 106 compounds. Regarding the discovery of novel sweeteners, the computational partners have been applying a novel machine learning framework to design and evaluate millions of candidate chemical structures. From these, >600 were shared with the experimental partners which synthesized 48, identifying 3 with sweeteness and non-toxic properties. In parallel, the work on the design vector has been focused on the design of the host chassis that will receive the final pathways. Preliminary chassis cells for both hosts (E. coli and S. cerevisiae) are available. For the improvement of bottleneck enzymes a high-throughput gene discovery framework as been established and applied to 100 molecules. Finally, rapid prototyping protocols were established and successfully applied to 53/100 molecules, generating prototype strains with validated production.
In summary:
● common pipeline established for the generation of potential biosynthetic pathways
● generated potential pathways for 106 compounds.
● creation of ShikiAtlas and ARBRE, tools that allow to explore >400k reactions and 62k compounds obtainable from the shikimate pathway.
● developed a deep learning pipeline to design new chemical structures
● synthesis of 48 potential sweeteners, 3 identified as sweet and non-toxic and 2 with potential biosynthetic pathways identified
● established a framework for cell factory design and SDDB - a centralized database for strain designs
● a new large-scale multi-compartment model for S. cerevisiae was developed
● preliminary chassis cells for E. coli and S. cerevisiae are available
● frameworks for gene discovery and computational protein engineering were developed and applied
● >600 synbio parts made available on ICE
● implemented prototype strains for 53 of the 100 compounds
● 1 patent finalized, 1 submitted and potential for at least 9 more is being evaluated.
In summary:
● common pipeline established for the generation of potential biosynthetic pathways
● generated potential pathways for 106 compounds.
● creation of ShikiAtlas and ARBRE, tools that allow to explore >400k reactions and 62k compounds obtainable from the shikimate pathway.
● developed a deep learning pipeline to design new chemical structures
● synthesis of 48 potential sweeteners, 3 identified as sweet and non-toxic and 2 with potential biosynthetic pathways identified
● established a framework for cell factory design and SDDB - a centralized database for strain designs
● a new large-scale multi-compartment model for S. cerevisiae was developed
● preliminary chassis cells for E. coli and S. cerevisiae are available
● frameworks for gene discovery and computational protein engineering were developed and applied
● >600 synbio parts made available on ICE
● implemented prototype strains for 53 of the 100 compounds
● 1 patent finalized, 1 submitted and potential for at least 9 more is being evaluated.
In ShikiFactory100, seamless integration and interoperability of retrosynthesis solutions will provide for the first time a metabolic engineering CAD platform that we expect to produce the largest expansion to the natural biocatalytic portfolio of the shikimate hub so far, two crucial contributions to the advancement of the field. We will apply these approaches to machine-learning designed and chemically synthesized novel structures with sweetening properties. These will be followed by a high-throughput gene discovery/enzyme engineering pipeline using structure-based methods able to deal with hundreds of enzymes with parallelization, originating a novel tool. One will be able to design pathways for metabolites of interest in a semi-automated. Rapid prototyping will be brought to another level by applying them systematically to hundreds of constructs, requiring an efficient integration of design and analysis tools. These will then be merged with chassis strains optimized by means of a novel computational framework. Toxicity of the products will be tackled by evolving strains that are highly tolerant to them via automated ALE. We expect to achieve several scientific and technological breakthroughs within synthetic biology and industrial biotechnology, fostering innovation capacity and industrial competitiveness, including the creation of new economic opportunities for European industries, the reduction of time-to-market of bio-based production solutions for shikimate-related compounds, and the project benefits for the industrial partners in the project consortium. Furthermore, we expect to originate broader aggregated societal impacts, including environmental benefits, as well as the creation of jobs in the short-term and medium term. Finally, we expect to provide health benefits by producing a bio-based sweetener with low effect on the gut microbiota.