Construction Of A Cofactor Self-sufficient Whole-cell Biocatalyst System For Production Of 1,2-amino Alcohols From Epoxides

1,2-amino alcohols are important pharmaceutical intermediates and are widely used in the synthesis of various physiologically active drugs such as neurotransmitters and antiviral agents.Therefore,the exploration of efficient synthetic methods of 1,2-amino alcohols is of great significance for the development of such drugs.Compared with chemical methods,biological methods have attracted wide attention due to their high conversion rate,strong stereoselectivity and mild reaction conditions.Based on the newly identified Pseudomonas aeruginosa-derived?-transaminase?PAK?-TA?,a multi-enzyme cascade catalytic system combining epoxide hydrolase?SpEH?and alcohol dehydrogenase?MnADH?was designed.Thus,a relatively inexpensive epoxy compound is used to synthesize a 1,2-amino alcohol compound with high added value in one step;in order to solve the rate-limiting step in the reaction process,RBS optimization is used to improve the expression of alcohol dehydrogenase in the recombinant strain;in addition,in this study,E.coli-derived glutamate dehydrogenase?GluDH?was introduced into the cascaded catalytic system to construct a closed coenzyme self-circulation system,thereby achieving simultaneous regeneration of the auxiliary substrate L-Glu and the coenzyme NADP⁺.?1?BLAST homology alignment was performed in the gene database using the protein sequence of Chromobacterium violaceum?-aminotransferase as a gene probe.The P.aeruginosa PAK-derived"?-eliminating lyase family protein"and C.violaceum?-transaminase have 64.0%protein sequence identity.Compared with other sources,the?-aminotransferase sequence showed the same enzyme catalytic activity site.So we cloned and expressed the enzyme successfully.The enzyme activity assay showed that the recombinant enzyme exhibited high enzyme activity against?S?-hydroxyphenylacetaldehyde?1c?.Considered that the enzyme has high?-transaminase catalytic properties,it was renamed as PAK?-TA and purified.The enzymatic properties of PAK?-TA showed that the optimum pH and temperature were 8.0 and 37?,respectively,which were non-metal ions dependent enzyme,the K_m value of the substrate?S?-hydroxyphenylacetaldehyde is 0.82 mM.?2?Cloned and expressed SpEH and MnADH,a cascade catalytic system for the one-step synthesis of 1,2-amino alcohols by epoxidation of three enzymes combined with PAK?-TA was designed.After enzyme catalysis,SpEH can catalyze the synthesis of?S?-phenyl-1,2-ethanediol?1b?by 50 mM precursor substrate?S?-epoxybenzene ethane?1a?,and then MnADH catalyzes the synthesis of?S?-hydroxyphenylacetaldehyde?1c?by?S?-1b,and finally PAK?-TA catalyzes the synthesis of?S?-2-amino-1-phenylethanol by?S?-1c.During the conversion process,it was found that MnADH catalyzes?S?-1b to?S?-1c as a rate limiting step.?3?By determining the product formation rate when MnADH and PAK?-TA are added at different ratios,the optimum ratio of the key enzyme MnADH:PAK?-TA to the optimum catalytic reaction rate was determined to be 3:1.Using the RBS analysis site,RBS sequences of different intensities were selected and assembled into the recombinant plasmid pETDuet-mnadh,and serialized with pak?-ta to obtain different RBS-strength alcohol dehydrogenaseandtransaminaseco-expressingrecombinantstrain E.coli/pETDuet-rbs_1-5mnadh-pak?-ta.The enzyme activity assay was used to finally obtain the recombinant strain E.coli/pETDuet-rbs₂mnadh-pak?-ta with MnADH:PAK?-TA activity ratio of 3:1,thus solving the effect of the rate-limiting step.?4?E.coli-derived glutamate dehydrogenase?GluDH?was introduced into the whole catalytic system.This strategy established a"bridge"between MnADH and PAK?-TA to solve the coenzyme NADP⁺supply of MnADH and the cosubstrate L-Glu supply of PAK?-TA.The speh and gludh were successfully tandemly ligated into the pACYCDuet plasmid and transferred into E.coli/pETDuet-rbs2mnadh-pak?-ta to obtain the recombinant strain E.coli BL21?SGMP?which co-expressing four enzymes on the two plasmids.Whole cell transformation,it can catalyze the production of 49.6 mM?S?-2-amino-1-phenylethanol by 50 mM?S?-epoxybenzeneethane in one step,and the conversion rate reaches 99.6%.?5?Expanding the application of the transformation system,and found that the recombinant strain E.coli BL21?SGMP?can also catalyze?S?-propylene oxide?2a?,as well as aliphatic epoxides such as?S?-epichlorohydrin?3a?,?S?-butylene oxide?4a?and?S?-epoxypentane?5a?to form the corresponding chiral 1,2-amino alcohol compounds with a conversion of 65.0%-96.4%.Compared with the chemical synthesis of 1,2-amino alcohol compounds,this method has the characteristics of less environmental pollution and high product purity,and provides an effective method for the production of 1,2-amino alcohol compounds in the pharmaceutical industry.