The Study On C-C Bond-formation Reactions Based On Enzymatic Substrate And Functional Promiscuity

C-C bonds formation is a crucial process in organic synthesis for establishing the carbon backbone of organic molecules.However,only a limited number of enzyme catalyzed C-C bond-forming reactions have been applied in biocatalytic organic synthesis.As a biocatalyst,the biochemical reaction mediated by enzyme has the advantages of mild conditions and green environmental protection.However,compared to chemical catalysts,the limitations of natural enzyme functionality restrict their widespread application in biomanufacturing.In addition to catalyzing specific reactions,enzymes also exhibit promiscuity and can catalyze non-natural reactions under specific conditions.Exploring and developing new functions of existing natural enzymes for catalyzing C-C bond-forming reactions using promiscuous catalysis is an intriguing and challenging scientific problem.In this paper,we focus on pyruvate decarboxylase（PDC）and old yellow enzyme（OYE）as research subjects.By utilizing the substrate promiscuity of PDC for C-C bonds formation,we designed an enzymatic-chemical pathway for synthesizingα-hydroxyketones and further expanded this pathway to synthesize optically pure（S）-1-phenyl-1,2-ethanediol[（S）-PED]and（R）-1-phenyl-1,2-ethanediol[（R）-PED].Additionally,based on substrate similarity,we developed a new function of OYE for catalyzing Morita-Baylis-Hillman（MBH）reaction and significantly improved its performance through functional selectivity regulation and directed evolution.The main research results are as follows:1.An enzymatic-chemical cascade pathway was designed for the synthesis ofα-hydroxyketone compounds based on the substrate promiscuity of pyruvate decarboxylase.The synthetic pathway consisted of a two-step reaction:（i）the reaction of benzaldehyde with the unnatural substrate glyoxylic acid catalyzed by the pyruvate decarboxylase from Candida tropicalis 1798（Ct PDC1）to generate the synthetic pathway of 2-hydroxy-2-phenylacetaldehyde,and（ii）the synthesis of 2-hydroxyacetophenone（2-HAP）from 2-hydroxy-2-phenylacetaldehyde via a spontaneous proton transfer reaction.The putative enzyme-catalyzed reaction mechanism was verified by UV-Vis,and the mechanism of the spontaneous reaction was explored using an HCC model calculation under water molecules,H⁺and Mg²⁺-catalyzed conditions,respectively.Finally,the optimal mutant Ct PDC1^C223E was obtained by protein engineering,and the yield was increased to 79.4%.2.Expanding the application of the enzymatic-chemical cascade pathway for the synthesis ofα-hydroxyketone compounds.By assembling and optimizing the cascade path,the yield reached to 92.7%,and the substrate scope of the reaction was expanded to catalyze the synthesis of 15α-hydroxyketone compounds.In addition,optically pure（S）-or（R）-PED were synthesized by introducing a stereospecific carbonyl reductase（SCR or RCR）at the end of the pathway using a one-pot,two-stage conversion method in yields of 73.7%and 56.0%,respectively.2-HAP,（S）-PED and（R）-PED were synthesized by 100 m L preparation-scale conversion experiments,with total yields of 90%,71.2%and 55.4%,and isolated yields of 81%,63%and 60%,respectively,demonstrating the synthetic potential of the cascade path designed in this study.3.Based on substrate similarity,a new function of C-C bond-forming catalyzed by OYE was developed.By excluding the effect of other components of the reaction system on the MBH reaction,it was determined that 3gr7 has a promiscuous function to catalyze both the reduction reaction and the MBH reaction.It was also verified in four other old yellow enzymes.By modulating the functional selectivity of the reaction,i.e.,removing the cofactor FMN to disrupt the H^-transfer pathway and targeted mutation of tyrosine residues to disrupt the H⁺transfer pathway weakened the natural reductive function of old yellow enzyme and enhanced the C-C formation function,the optimal protein for catalyzing the MBH reaction was 3gr7.8(3gr7^{Q102A-R215A-R308A-Y169F}),and the titer（116.0μM）of MBH product 2-（hydroxy（4-nitrophenyl）methyl）cyclopent-2-en-1-one was 141.4%higher than that of 3gr7.4.The catalytic performance of the MBH reaction was enhanced by protein engineering.Firstly,the crystal structure of 3gr7.8 was obtained through protein crystallization experiments.Based on alanine scanning,MD simulations and saturation mutagenesis,crucial residues C26and E59 involved in catalyzing the MBH reaction were identified.Subsequently,saturation mutagenesis and combinatorial mutations were performed on specific sites that exhibited substantial changes in yield,resulting in the optimal mutant variant 3gr7.11 being obtained.This mutant achieved an impressive yield of 77.8%with an e.r.value of 88:12 during catalysis of the MBH reaction.Furthermore,through MD simulations,it was revealed that the improvement in mutation performance mainly resulted from increased interactions between W62 and I247 with Int D after mutation occurred.Lastly,analysis of the substrate scope for the optimal mutant variant showed high e.r values but generally lower yields（ranging from 2.4%to 22%）.Among pentacyclic products,as electron-withdrawing substituents transitioned from strong to weak（-NO₂>-CN>-Cl>-Br）,there was a gradual decrease in product yield.