Study Of3-hydroxy-butanone Production In Bacillus Subtilis From Glucose And Xylose Mixtures

3-hydroxy-butanone (acetoin) is a vital flavor compound, widely used in foodand beverage industry. Focusing on the xylose transport and metabolic pathways,Bacillus subtilis168Δupp was engineered metabolically to overexpress the xylosetransport protein AraE and the xylose isomerase as well as xylulokinase fromEscherichia coli, heterologously. Thus, the xylose metabolic pathway was transcribedconstitutively, which conferred the recombinant B. subtilis to assimilate glucose andxylose simultaneously. Under microaerobic conditions, the engineered B. subtilis wasable to metabolize glucose and xylose in minimal medium without preference. Thisstudy was the first report about glucose derepression for acetoin production byrational design in B. subtilis.Firstly, the xylose transport protein coding gene araE was engineered genetically.Gene araE was placed under the control of promoter P43from B. subtilis168genome.In this study, the AraE expression cassette was constructed to consist of the strongpromoter P43, the lac operator from E. coli, and gene araE coding sequence withribosomal binding site sequentially. In minimal medium,10g/L xylose was exhaustedin32hours under aerobic conditions by the resulting strain BSUL11, in which theutilization of xylose was improved significantly and the xylose transport bottleneckwas ameliorated noticeablely.Further, so as to make B. subtilis glucose derepression, the xylose isomerase andxylulokinase encoded by xylAB operon from E. coli were introduced to BSUL11, andengineered as a constitutive metabolic pathway free of repression from the regulatoryfactors XylR and CcpA. Compared to the control strains assimilating glucosepreferentially, the engineered BSUL12metabolized glucose and xylose withoutpreference. The sugar utilization profiles, as well as the specific activities of theheterologous xylose isomerase and xylulokinase, corroborated that the introduction ofgenes xylA and xylB from E. coli under the control of a constitutive promoter was anefficient strategy to engineer a functional xylose catabolism pathway that was in favorof the release of catabolite repression.Further overexpression of the pentose phosphate pathway in B. subtilis made thespecific growth rate of mutant strains decreased unexpectedly, suggesting the metabolic burden was generated in B. subtilis by overexpression of the pentosephosphate pathway genes. According our previous work, insertional inactivation ofgenes bdhA, acoA, pta which encoded the enzymes for acetoin degradation andbyproduct generation pathways was in favor of improving the yield of the targetproduct acetoin. By introducing the overexpressed xylose transport and heterologousassimilating pathway to the engineered strain BSF3, the optimum strain BSUL13foracetoin production, was obtained. Under microaerobic conditions, batch cultures inminimal medium with10g/L glucose and5g/L xylose as the co-existing carbonsource showed that the main metabolite was acetoin, and the titer of5.5g/L with ayield of0.70mol/(mol·sugar),75%of the theoretical yield was obtained (For10g/Lglucose and5g/L xylose, the theoretical yield is0.937mol/(mol·sugar)). This studyoffered a metabolic engineering strategy on engineering microbe as cell factory forthe production of high-valued chemicals from renewable resource.