The Study On Cascade Biocatalytic Synthesis Of α-functionalized Organic Acids From Glycine

The α-functionalized organic acids,such as α-keto acids,α-amino acids and α-hydroxy acids,are important building blocks because they play prominent roles in chemical synthesis,cosmetics,and pharmaceutical manufacturing.The preparation of α-functionalized organic acids can be greatly simplified by adopting a protocol involving the catalytic assembly of achiral building blocks.However,the enzymatic assembly of small amino acids and aldehydes to form numerous α-functionalized organic acids is highly desired and remains a significant challenge.Herein,we report an artificially designed chiral-group-resetting biocatalytic process,which uses simple achiral glycine and aldehydes to synthesize stereodefined α-functionalized organic acids.The main results were described as follows:(1)Based on cascade biocatalysis,we designed a-OH/-NH2-group resetting process,which is presented as introduction–deletion–reintroduction.This chiral group resetting process facilitates the synthesize of numerous α-functionalized organic acids.The designed modular cascade biocatalysis platform has four modules: a basic module(BM)and three extender modules(EMs).When cascading BM with EM1,we can reset chiral-OH for the synthesis of stereodefined α-hydroxy acid;while cascading BM with EM2 or EM3 can reset chiral-NH2 for the synthesis of stereodefined α-amino acid.Then the enzymes with better properties were screened by literature mining and database mining to construct these modules.It was verified that each module could work normally,but the low activity of Cg TD in BM to large substrates was the speed-limiting step of cascade reaction.(2)The "open gate" strategy was recruited for the protein engineering of CgTD,and the mutant with high activity toward bulky substrate was obtained.Firstly,the structure model of ΔCg TD,which deleted the regulatory domain of Cg TD,was obtained by homology modeling,and then analyzed by molecular docking,pocket analysis,channel analysis,and dynamics simulation.It was found that there was a bottleneck in the substrate channel of Cg TD which existed as a "gate".After that,according to the structural characteristics of the "gate",we proposed an "open gate" strategy to improve the activity of Cg TD toward bulky substrates.By means of alanine scanning,CASTing,iterative saturation mutation,and combination mutation,the gate residues,anchoring residues and hinge residues of the "gate" structure were mutated.As a result,the best mutant Cg TDMu7(V111A/V119N/K123S/V137I/K260S/R261T)was obtained.The activity of mutant Cg TDMu7 toward 3a was increased by 6.8-folds,the Km value was decreased by 4-folds,and the kcat was increased to 136.1 s-1.Furthermore,the mutant Cg TDMu7 also showed high catalytic activity toward 3e.(3)The abundance of different intermediates was visualized by using the intrinsic spectral characteristics of PLP cofactor.Based on the reaction mechanism of PLP dependent enzymes and UV-Vis spectrum,the transition intermediates of Cg TD catalyzed deamination was analyzed.On the basis of the reported reaction mechanism of PLP dependent enzyme,the key amino acid residues D147 and R261 were determined.At last,the mechanism of mutants,with the enhanced activity todard bulk substrates,was analyzed.(4)By assembling different modules,strains with different synthetic functions were constructed and screened,and their synthetic potential was investigated by analytical transformation.Four recombinant E.coli strains(OA01-04),for α-keto acid synthesis,were constructed by assembling Pa TA and Cg TDMu7.The best strain E.coli OA02 was used to transform 1a-i into the corresponding keto acid 4a-i,and the conversion was 68-91%.By assembling BM with EM1,24 recombinant E.coli strains(OA05-28)for α-hydroxy acid synthesis were obtained.Aldehydes 1a-i was transformed into(S)-and(R)-5a-i by using the best strains E.coli(OA15)and E.coli(OA23),respectively,with the conversion of 55-92% and high ee(96-99%).By assembling BM with EM2,12 recombinant E.coli strains(OA29-40)for(S)-α-amino acid synthesis were obtained.Aldehydes 1a-i was transformed into(S)-6a-i by using the best strain E.coli(OA34),resulting in a conversion of 65-93% and ee of 89-98%.By assembling BM with EM3,12 recombinant E.coli strains(OA53-64)for(R)-α-amino acid synthesis were obtained.Aldehydes 1a-i was transformed into(R)-6a-i by using the best strain E.coli(OA59),resulting in a high ee of >98% and conversion of 32-89%.(5)The synthesis of α-functionalized organic acids was scaled up to 100 mL to further evaluate the feasibility of the biocatalytic cascades.Ten representative α-functionalized organic acids were successfully synthesized using the corresponding E.coli whole-cell catalysts.And then purified by extraction,chromatography,and crystallization,with the isolated yield of 45-78%.Finally,the structure and molecular weight of the purified products were analyzed and identified by NMR and HRMS.The results showed that the structure of the purified products was correct,which further proved the accuracy and universal applicability of the cascade system constructed in this study.