Selective Recognition And Regulation Mechanism Of Alcohol Dehydrogenase For Synthesis Of Chiral Heterocyclic Alcohols And Construction Of Catalytic Systems

Alcohol dehydrogenase（ADH）,also known carbonyl reductase（CR）,can catalyze reversible redox reactions between alcohols and ketones（or aldehydes）with high stereoselectivity,high regioselectivity,and high chemical selectivity.As an efficient and robust biocatalyst,it is used in the synthesis of chiral alcohol block compounds with diverse functions,and also used in the synthesis of fine chemical products and important chiral pharmaceuticals.Alcohol dehydrogenase is divided into short-chain alcohol dehydrogenase（SDR）,medium-chain alcohol dehydrogenase（MDR）,and long-chain alcohol dehydrogenase（LDR）.SDR and MDR are mainly used in the biosynthesis of chiral alcohols due to their functional diversity.At present,alcohol dehydrogenase mainly focused on the asymmetric reduction of ketone compounds with significant differences on both sides of the carbonyl group.However,it exhibits poor stereoselectivity and low reaction activity for asymmetric reduction of cyclic ketone compounds with similar or small differences on both sides of the carbonyl group.At the same time,there are few alcohol dehydrogenase libraries capable of catalyzing difficult-to-reduce cyclic ketones,and their selective recognition and regulatory functions for catalyzing difficult-to-reduce cyclic ketones are unclear.Thus,we applied functional alcohol dehydrogenases as templates to explore more alcohol dehydrogenases from NCBI database and established catalytic characterization system in this study.Based on sequence-structure-function analysis,we explored the selective molecular recognition mechanism and functional regulation of alcohol dehydrogenases in asymmetric reduction of heterocyclic ketones.We applied the regioselectivity regulation mechanism to engineer the stereoselective modification of other homologous alcohol dehydrogenase.Finally,we coupled the asymmetric catalytic system with coenzyme cycle system for the synthesis of important pharmaceutical intermediates containing chiral alcohols.The main research results are listed as follows:（1）76 strains of alcohol dehydrogenase were screened from the NCBI database using template genes and classified as short chain alcohol dehydrogenase-1,short chain alcohol dehydrogenase-2,and medium chain alcohol dehydrogenase.Using short-chain alcohol dehydrogenase Cg KR1（Gen Bank:CAG58832.1）and medium-chain alcohol dehydrogenase CpaCR-2（Gen Bank:ABB97513.1）as the target genes,a total of 76 alcohol dehydrogenases with 25%-90%homology from NCBI database were constructed and expressed.By constructing an evolutionary tree of alcohol dehydrogenases,we divided it into short-chain alcohol dehydrogenase-1（SDR-1）,short-chain alcohol dehydrogenase-2（SDR-2）,and medium-chain alcohol dehydrogenase（MDR）,respectively.Using tetrahydrofuranone（1a）,tetrahydrothienone（2a）,N-Boc-pyrrolidone（3a）,and N-Boc-piperidinone（4a）as template substrates,we characterized the catalytic performance of alcohol dehydrogenases.The alcohol dehydrogenases showed the certain substrate preference,SDR family enzymes mainly exhibiting good catalytic activity and stereoselectivity toward substrate 4a,MDR exhibits good catalytic performance toward substrate 3a.（2）The stereoselective regulation mechanisms of SDR-1 and SDR-2 for the asymmetric synthesis of chiral heterocyclic alcohols were obtained through computational simulation analysis.We predicted the structure of alcohol dehydrogenases with moderate catalytic activity and moderate stereoselectivity through SWISS-MODEL or Alphafold2.Molecular docking between alcohol dehydrogenases and template substrate via Schr（?）dinger software,we mainly investigated amino acid residues within the 5（?）range of active pocket.Then,based on catalytic performance characterization,we predicted possible residues that affect the stereoselectivity of alcohol dehydrogenase through multiple sequence alignment and residue conservation analysis.For the stereoselective regulation mechanism of SDR-1,we found that residues 201 and 205located in the lid structure are key residues that regulate the stereoselectivity of SDR-1,and the phenylalanine at position 205 formed a pi-alkyl interaction with the tert-butyl group,thereby exhibiting R-selectivity,and the phenylalanine at position 201 formed a pi-alkyl interaction with the piperidine ring,thereby exhibiting S-selectivity.residues 91 and 191 have important impact on its catalytic activity.We applied this strategy to engineer the stereoselective modification of RsdCR,CpsCR,and CkoCR of SDR-1 homologous enzymes,and finally flipped their stereoselectivity and achieved ee value of over 90%（R/S）for asymmetric reduction of N-Boc-piperidone.In addition,we found that residues 92 and 93,located in the loop region,were important factors for stereoselectivity change of SDR-2,and residues at positions 145 and 176 acted as switches for large and small pockets to control stereoselectivity.We applied this strategy to engineer the stereoselective modification of CglCR-3,CglCR-2,and VpoCR of SDR-2homologous enzymes,and flipped their stereoselectivity.The ee value of asymmetric reduction of N-Boc-piperidone reached over 90%（R/S）.Finally,we investigated the catalytic performance of SDR-1,SDR-2,and their mutants for 11 heterocyclic ketones.We found that SDR-2 exhibited better catalytic performance than SDR-1.SDR-2 exhibited high catalytic activity and stereoselectivity for different substituents of piperidone,with conversion rates and ee values reaching over 90%.For pyrrolidone and azones with different substituents,as well as furanone and thiophenone compounds,SDR-1 and SDR-2 exhibit general catalytic activity and stereoselectivity.（3）The stereoselective regulation mechanisms of MDR for the asymmetric synthesis of chiral heterocyclic alcohols were obtained through computational simulation analysis.We found the two branches of MDR are extremely similar in protein structures through the evolutionary relationship and structural comparison.We predicted possible residues that affect the stereoselectivity of MDR through multiple sequence alignment and residue conservation analysis.We reshaped the loop-2 region of SpaCR to expand the active pocket cavity and ultimately obtained a stereoselective flipped mutant m125（F284A/W285A）,which exhibited99%conversion rate and 99%ee value for N-Boc-pyrrolidone.At the same time,we applied this strategy to engineer the stereoselective modification of the homologous enzyme CorCR,and obtained stereoselective flipped mutant m135（F314A/W315A）,which exhibited an 84%conversion rate and 90%ee value（R）for N-Boc-pyrrolidone.Finally,we investigated the catalytic performance of SpaCR,CorCR,and their mutants for asymmetric reduction of 12heterocyclic ketones.We found that SpaCR had better catalytic performance than CorCR.SpaCR and exhibited high catalytic activity and high stereoselectivity for pyrrolidones with conversion rates and ee values reaching over 90%.In addition,SpaCR,CorCR,and their mutants exhibit catalytic activity and stereoselectivity for different substituents of piperidones and azaketones,as well as furanone and thiophenone compounds.（4）We coupled alcohol dehydrogenase with the coenzyme cycle system for the synthesis of chiral drug intermediates.We obtained five recombinant strains containing GDH,including CglCR-3-GDH,CglCR-3-m49-GDH,CglCR-2-m72-GDH,SpaCR-m113-GDH,and SpaCR-m125-GDH.All recombinant strains were expressed normally and their catalytic performance was characterized using template substrates 3a and 4a.Simultaneously,the catalytic efficiency of in vivo coupling in the coenzyme circulation system is relatively higher than that of in vitro coupling.Cultivation and reaction conditions of recombinant strains were established at the shaking flask level.The optimal culture conditions of CglCR-3-GDH are listed as follows:Inducer concentration is 0.2 m M,induced temperature is 17℃,induced time is 14 hours.The cultivation conditions of CglCR-3-m49-GDH are listed as follows:Inducer concentration is 0.2m M,induced temperature is 20℃,induced time is 14 hours.The cultivation conditions for CglCR-2-m72-GDH are listed as follows:Inducer concentration is 0.3 m M,induced temperature is 20℃,induced time is 14 hours.The cultivation conditions for SpaCR-m113-GDH and SpaCR-m125-GDH are listed as follows:Inducer concentration is 0.3 m M,induced temperature is 17℃,induced time is 14 hours.The optimal reaction conditions are listed as follows:substrate concentration is 100 m M,cofactor concentration is 0.2 equivalent of substrate concentration,glucose concentration of 1.2 equivalent of substrate concentration,reaction temperature is 30℃ and reaction time is 24 hours.The optimal cultivation conditions from the fermentation tank level（1000 mL）are as follows:When adding inducer,the OD₆₀₀ of cell is 2.0-2.5,the cultivation time is 16-18 hours,and the final OD₆₀₀ of the recombinant strain is about 25-26（60-62 g/L）.The 100 mL reaction conditions are as follows:substrate concentration is 200 m M,biocatalyst concentration is 30-40 g/L,reaction temperature is 30℃ and reaction time is 24 hours.The substrate is completely transformed and the ee value of the product is 99%.