Formation Mechanism Of Different Acetoin Optical Isomers During The Fermentation Process Of Bacillus Subtilis

Acetoin（AC）is one of the main products of overflow metabolism in microbial glucose metabolism pathway.It has two optical isomers of 3R-AC and 3S-AC and has high market value and important industrial significance in pharmaceutical and chemical synthesis.Microbial fermentation of 3-hydroxybutanone is often accompanied by the formation of by-product 2,3-butanediol（BD）.To improve the production of 3hydroxybutanone in B.subtilis,the gene bdhA encoding Butanediol dehydrogenase（BDH）,which is related to the synthesis of 2,3-butanediol,was deleted in the early stage of the project,but the accumulation of meso-BD was still detected in the bdhA deletion strain.At the same time,it was also found that the ratio of two optical isomers of AC produced by bdhA deletion strain changed greatly,and the amount of 3S-AC decreased significantly.Therefore,this paper focuses on the source of B.subtilis meso-BD and the reduction of 3S-AC.Through the analysis of the fermentation behavior of the strain,the excavation and verification of the new BDH gene,and the catalytic characteristics of the bdhA gene encoding protein,the main research results are as follows:（1）Through literature mining and bioinformatics analysis,a target sequence（named bdhB）that may be related to meso-BD synthesis was found in the genome of B.subtilis 168.The accession number of bdhB in NCBI was NP₃89260.1,which was annotated as oxidoreductase with a length of 747 bp and 98%homology with mesoBDH gene in Bacillus sp.The gene sequence encodes a protein with a theoretical molecular weight of 26.2 KDa.It is a non-secretory protein without transmembrane region and signal peptide.The sequence contains adh_short_C2 domain（Accession:PF13561）,cell wall synthesis protein CwsA family（PF10814）and short-chain dehydrogenase domain（Accession:c135484）.In this paper,the function of bdhB gene was first verified by gene knockout,but the transformants were not obtained through various conditions.According to the prediction of the amino acid sequence of the gene,it is speculated that the enzyme may be related to cell division and cell morphology maintenance,and knockout of the gene may lead to cell death.Therefore,this paper changed the strategy and used qRT-PCR technology to analyze the difference of bdhB gene expression in different fermentation cycles between the original strain and the bdhA deletion strain.Combined with the fermentation behavior of the strain,it was found that the expression of bdhB gene in the bdhA deletion strain was positively correlated with the yield of meso-BD at the same culture time,and the bdhB gene was preliminarily determined to be related to the synthesis of meso-BD.（2）The secretory expression vector pHT43-bdhA of Bacillus subtilis and the expression vectors pET28a-bdhA and pGEX-bdhA of Escherichia coli were successfully constructed.The expression of B.subtilis pHT43-bdhA may be due to the low expression level.The target protein band was not detected by conventional purification methods such as ammonium sulfate precipitation.The protein was expressed by E.coli pET28a expression vector（His-Tag）,and the protein purification effect was not ideal.The R-BDH protein was successfully obtained by using the E.coli pGEX-bdhA expression vector（purified tag as GST tag）.The oxidation products and reduction products of R-BDH were detected by using 3-hydroxybutanone and 2,3-butanediol as substrates,respectively.The results showed that R-BDH could catalyze the mutual conversion between 3R-AC and（2R,3R）-2,3-BD,as well as the mutual conversion between 3S-AC and meso-2,3-BD.（3）BDH sequences from different sources and substrate characteristics were collected based on GenBank,Brenda,Uniport and other databases.Sequence homology and protein structure were compared by bioinformatics methods.It was found that RBDH from different sources were closely related,while meso-BDH,S-BDH and DAR classification characteristics were not obvious.DAR may also belong to meso-BDH or S-BDH.Domain analysis showed that R-BDH contained an alcohol dehydrogenase GroES-like domain and a zinc-binding dehydrogenase domain,while S-BDH,mesoBDH and DAR had a short-chain dehydrogenase domain,an enoyl-（acyl carrier protein）reductase domain and a cytoreductive domain.（4）Based on the changes in the content of different optical isomers of AC and BD in the fermentation products of B.subtilis strains 168 and 168D and the catalytic characteristics of two BDHs in their bodies,it can be inferred that the source of 3S-AC in B.subtilis 168 strains in the case of diacetyl deficiency is 3R-AC catalyzed by double enzymes（R-BDH and meso-BDH）.