Metabolic Engineering And Modification Of Acetoin High Producing Bacillus Subtilis

Acetoin （3-hydroxy-2-butanone）, which naturally exists in certain fruits, is widely usedto add flavor to food and also serves as a precursor in the synthesis of many importantcompounds. The present methods for acetoin production aer divided into chemical synthesis,enzymatic conversion and mocrobiol fermentation. On depletion of fossil resources, microbialfermentation of bulk chemicals using renewable resources is favorable with lowenvironmental stresses.（1） This work isolated a bacterial which can convert150g L-1glucose to42.2g L-1acetoin and15.6g L-12,3-butanediol, with acetoin molar yield of57.5%and productivity of0.29g （L h）^-1. The interconversion between acetoin and2,3-butanediol was observed that theacetoin/2,3-butanediol ratio was switched from1:2to2.7:1in the prophase and latephase offermentation, respectively. By phylogenetic analysis, the strain was identified as Bacillusubtilis and numbered JNA3-10.（2） B. subtilis mutant JNA-UD-6which blocked in2,3-butanediol dehydrogenase （BDH）was screened by compound mutation. Acetoin production was improved to53.9g L-1while2,3-butanediol production was decreased to6.5g L-1. The molar yield of acetoin wasimproved to73.5%, with an acetoin productivity of0.37g （L h）^-1. Sequenceing of the bdhAgene of JNA-UD-6indicated that a nonsense mutation （g.353T>A） that precluded thesynthesis of a full-length functional AR/BDH occurred. The bdhA gene in JNA3-10wasknocked out by homologous integration to prove that blocking of the metabolic flux fromacetoin to2,3-butanediol is a key factor for improving acetoin production. Although therecombinant BM strain produced48.3g L-1acetoin, it had a decreased fermentation durationof96h, with a productivity of0.50g （L h）^-1.（3） For genetic engineering breeding of an acetoin high production B. subtilis, the keyenzymes α-acetolactate synthetase （ALS） and α-acetolacetate decearboxylase （ALDC） onacetoin synthesis pathway were overexpressed. Compared with BM strain, theovere-xpression of ALS, ALDC and ALS+ALDC increased acetoin probuction by5.2%,4.1%and10.8%, respectively, but the biomass was inhibited by overe-xpression of theseenzymes. With a long fermentation duration, the highested acetoin production andproductivity was53.5g L-1and0.50g （L h）^-1.（4） The PbdhApromoter of bdhA gene was cloned and experimentally proved has1/5.7activity of the strong promoter HpaII. Further, acetoin synthesis by BM strain was enhancedby regulating ALsR （regulator of the alsSD operon） expression using either of the twopromoters. Although the HpaII promoter highly enhanced the transcription of the alsSDoperon by overexpression of ALsR, the production of acetoin was not significantly increased.In contrast, moderate enhancement of ALsR expression using PbdhAsignificantly improvedacetoin production to56.5g L-1in84h, with a productivity of0.59g （L h）^-1.（5） When fermented with glucose, B. subtilis produces2,3-butanediol, lactic acid andethanol as NADH-dependent by-products. So acetoin production by B. subtilis can beimproved through weakening the NADH-linked pathways to redistribute the carbon flux from pyruvate to acetoin. For further decreasing the NADH-dependent metabolic flux, wesuccessfully identified an active water-forming NADH oxidase edcoded by yodC gene, whichcatalyzes O2and NADH to harmless H2O. Then we expressed YODC in BM strain with eitherhighly or moderately level under the control of promoters HpaII and PbdhA, respectively. Bymoderate-expression of YODC, the yields of acetoin and acetic acid were increased, while theproduction of2,3-butanediol, lactic acid and ethanol were decreased. The engineered B.subtilis strain produced acetoin of56.7g L-1, with a productivity of0.68g （L h）^-1.（6）6-phosphoric acid fructose kinase （PFKA） and pyruvate kinase （PK） were cloned andovere-xpressed in BM strain. The glycolysis was enhanced that the fermentation time couldbe shortened by this strategy. Simultaneously, the biomass of B. subtilis was greatly improvedby over-expression of PFKA. Thus, we combined over-expression of PFKA andmoderate-expression of AlsR and NOX strategies. Genetic manipulation on the expressionorder of ALsR and NOX was also studied. Finally, by fed-batch fermentation, therecombinant B. subtilis produced85.6g L-1acetoin, with a productivity of0.89g （L h）^-1.Using traditional and genetical breeding methods, compared to B. subtilis JNA3-10,acetoin production and productivity were improved by102.8%and200.7%, respectively.This work proposed metabolic engineering strategies for improving acetoin production thatmay be used in industrial strains.