Regulation Of Fermentative Production Of Acetoin And (2R,3R)-butanediol By Bacillus Subtilis

2,3-Butanediol（2,3-BD）and its precursor acetoin（AC）are typical fermentation products of microorganisms with mixed acid metabolism pathways,and are used in flavoring agents,fuel additives,solvents,polyesters,rubbers and fuels and other fields,have a wide range of applications.In this study,a strain of Bacillus subtilis that can produce acetoin and2,3-butanediol was obtained by mutagenesis screening,the fermentation medium was optimized,and the oxygen supply of acetoin and 2,3-butanediol was studied.Transformation regulation.The product distribution of the strain is robust,and the product yield is high,which has the prospect of industrial application.First,screening of acetoin and 2,3-butanediol-producing Bacillus was performed.Through the enrichment culture of sea mud samples and the screening of single bacteria,three types of strains were obtained,namely:strain 2-2,which mainly produces 2,3-BD mixture;strain 2-2,which mainly produces（2R,3R）-BD The main strain 10-1;the strain GM3-6mainly produces（3R）-AC,but the concentration of（3R）-AC product is lower.In addition,the Bacillus subtilis DL01 preserved by the previous research group was subjected to compound mutagenesis screening of low temperature plasma+5-bromouracil to obtain mutant strain M9.Second,the fermentation medium is optimized.The effects of different compositions of fermentation medium on the product concentration were compared,and the composition of the fermentation medium was preliminarily determined;the effects of the initial sugar concentration of inorganic nitrogen sources and organic nitrogen sources on bacterial growth and product formation were investigated by single-factor experiments;it is determined that the factors and influences of the components in the fermentation medium that have a significant effect on the total product concentration are:yeast powder>dry corn steep liquor>manganese sulfate;finally,the final composition of the fermentation medium is determined by the steepest climbing experiment:glucose 160 g/L;FM760 Angel yeast extract powder 7g/L;corn steep liquor dry powder 12 g/L;KH₂PO₄ 6 g/L;Mn SO₄ 0.13 g/L.Finally,the effects of different oxygen supply conditions on bacterial growth and metabolism were investigated.Fermented under the conditions of 0.2 vvm and ORP value of-190～-220 m V,the main product was（2R,3R）-BD,and 75.0 g/L of 2,3-BD and acetoin were obtained by batch fermentation for 58 h.The production intensity was 1.39 g/L/h,and the yield was 0.47 g/g sugar,of which（2R,3R）-BD was 53.0 g/L,accounting for 92%of the total concentration of 2,3-butanediol;at 0.25 vvm,When the ORP value was-120～-150 m V,the main product was acetoin,and 65.1 g/L 2,3-BD and acetoin were obtained by batch fermentation for 49 h,and the yield was 0.44 g/g sugar,the production intensity reached 1.33g/L/h,of which 50.3 g/L of acetoin was contained,accounting for 77%of the total product concentration.Transcriptome analysis of samples from these two typical conditions revealed that under conditions of sufficient oxygen supply,the transcription level of 2,3-butanediol dehydrogenase was down-regulated,the carbon flux of the 2,3-butanediol metabolic pathway was reduced,and the product The yield decreased,the expression of genes related to reducing force NADH and energy ATP increased,the expression of genes related to ABC transporter material transport was up-regulated,the expression of genes related to flagellar assembly was down-regulated,and the genes related to nitrogen metabolism showed varying degrees of change.In addition,the expression of genes related to sporulation was also significantly up-regulated,which should be related to the accumulation of acetoin.