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C-C Bond Formation Using Top Performing Enzymes

Periodic Reporting for period 1 - CC-TOP (C-C Bond Formation Using Top Performing Enzymes)

Reporting period: 2021-03-01 to 2023-02-28

Industrial Biotechnology is a Key Enabling Technology under Horizon 2020, expected to boost technological innovation and industrial leadership in the EU. It can improve chemical processes in compliance with the principles of Green Chemistry and effectively address social, environmental and economic challenges of our time. Assembling simple fragments to build larger, more complex products is an essential technology at the heart of industrial organic synthesis. With traditional chemical methods, C-C bond formation or "carboligation", is difficult to control selectively, and often involves the use of toxic solvents and hazardous reagents; whereas a biotechnology approach, using enzymes from a variety of microbial sources and with appropriate “tailoring” when needed, can catalyze carboligation reactions with excellent precision under very mild conditions. I also has the potential to be more cost-effective, often producing high yields of the desired product with fewer steps and less by-product formation.
For unleashing the vast, hidden treasury of enzymatic carboligation, the CC-TOP network has identified and is currently addressing a number of critical needs, technology gaps, practical challenges and synthetic opportunities with a huge potential for innovation. CC-TOP is a European research project involving 9 academic, 3 industrial beneficiaries, and 8 external partners, aiming to harness the power of biocatalysts to make industrial organic synthesis more sustainable; We aim to bridge the knowledge gap in CC-bond forming enzymes starting from enzyme discovery up to industrial process implementation. For the purpose, we combine basic research and applied engineering to optimise biocatalysts that can make the difference in the market, by producing much in demand chiral chemicals and pharmaceutical precursors. The main goal of the CC-TOP ETN is to deliver tailored training and education to a group of 15 top Early-career Stage Researchers on cutting-edge carboligation enzyme technology and its translation into efficient Industrial Biotechnology applications. The overall approach of the CC-TOP research aims to contribute to a more sustainable future, thereby supporting the European Green Deal.
CC-TOP network members have identified critical gaps in enzymatic carboligation workflows that yet remain as global obstacles for broader commercial applications: the lack of a broad range of accessible enzyme platforms; the lack of predictive understanding of carboligase function; the lack of sufficient knowledge on the scalability of carboligation processes; the long process development timelines. To unlock the full power of the bio-based carboligation technology, these shortcomings are being addressed by upgrading the toolbox of carboligases offering sufficient catalytic range and substrate scope. CC-TOP researchers are developing three major classes of biocatalysts presenting unique opportunities for innovation and a vast potential to impact the chemical and pharmaceutical industries: phosphoenolpyruvate (PEP) lyases driven by a high-energy substrate (WP1); thiamine diphosphate (TDP) dependent enzymes with high potential for innovative reactivity (WP2); designer biocatalysts for promiscuous new-to-nature activities (WP3).
Identifying novel or engineering optimized, industrially viable enzymes involves repetitive, labor-intensive and time-consuming procedures. CC-TOP is developing several methodologies to drastically accelerate the discovery and engineering of biocatalysts that cover an expanded catalytic scope for carboligation: high-throughput technologies for carboligation enzyme discovery; structural analysis of biocatalytic transformations; data-driven protein engineering; computational modeling for engineering complex carboligation reactions.
• Two maximum diversity screening panels of about 100 novel PEP lyases with 5 distinct specificities were created, for characterization of their substrate scope, activity, stability, and utility for synthetic applications
• An ultra-high throughput droplet-based cascade assay for screening the natural and artificial diversity of C-C bond forming enzymes was designed and optimized in bulk and is now being adapted to microfluidics
• New colorimetric assay technology was developed and validated for discovering and characterizing the activity of PEP dependent lyases with native and artificial substrates.
• Reaction conditions and kinetic parameters of a synthetically useful PEP-dependent synthase were evaluated for developing and validating mathematical models of complex reaction kinetics of carboligation enzymes
• Putative genes of "split” transketolases were identified from (meta)genomic repositories, 3 of which were expressed and purified for characterization of their biophysical properties
• The thermostable transketolase from Geobacillus stearothermophilus was successfully crystallized and its 3D protein structure elucidated - atomic resolution 2.1 Å .
• A first set of mutants of the transketolase from E. coli was obtained with and without reaction intermediates to improve understanding of the factors determining novel selectivity of evolved variants
• Two site-saturation libraries were designed and constructed to improve transketolase activity against non-natural electrophiles; variants are now screened and kinetically characterized for their substrate scope
• More than 200 enzymes presenting a novel ThDP binding motif were discovered by homology BLAST in a search for enzymes that produce tertiary alcohols, of which 3 enzymes were cloned and purified for characterization of their substrate scope
• Transketolase variants were further engineered for expanded substrate scope towards different donor and acceptor specificities; a set of candidates were expressed, purified and analyzed for stereoselective formation of unusual higher-carbon sugars
• A toolbox of catalysts including TDP dependent enzymes and various aldolases that cover attractive reactions were cloned and expressed and are now being screened for novel reactivity from an industrial perspective
• A multi-step synthesis route to a chiral compound of commercial interest was identified involving carboligation key step - currently under development as a model for exploitation in industrial sectors (pharma,/agrochemical/fragrances)
• A thermophilic transaldolase was crystallized and the structure of the enzyme in complex with different substrates was resolved at 1.9 Å resolution
• Novel one- and dual-substrate reactions were discovered for a transaldolase; a variant was constructed by structure-guided rational redesign to enable it to also accept non-phosphorylated substrates
• A computational docking approach enabled the identification of several aliphatic and aromatic aldehydes as novel substrates for 4-OT, for which further improvement of activity and enantioselectivity is pursued
• A combination of rational design and laboratory evolution was applied to engineer a well-performing Michaelase, which showed a more than 300-fold enhancement in catalytic activity compared to wild-type, outclassing all previously engineered 4-OT variants
• For the construction of novel artificial carboligation catalysts, various heterocyclic ligands were synthesized, complexed with different divalent metal ions and covalently anchored to protein scaffold as artificial cofactors, which are now screened for aldol and Michael reactivity
The CC-TOP research approach structured in three Work Packages (WPs) and 15 individual ESR project