| Acetoin,an important food additive and platform compound,is extensively used in many fields such as foods,chemical synthesis and agriculture.Bacillus subtilis 168,generally recognized as a safe strain?GRAS?,was engineered by disrupting gene sigF,sigE,bdhA,ldh and ackA,overexpressing ?-acetolactate synthase?ALS?and ?-acetolactate decarboxylase?ALDC?during the stationary phase,to block sporulation,reduce the accumulation of competing compounds and improve the biosynthesis of acetoin.The gene spo0 A,sigF and sigE,coding sporulation master regulator and specific sigma factor respectively,were deleted in B.subtilis 168 by the Cre/lox system respectively.The deletion of spo0 A blocked the synthesis of spores at the initial stage of sporulation successfully,while the deletion of sigF and sigE prevented the synthesis of spores at the asymmetric division stage of sporulation successfully.At the spore formation stage,the cell morphology of strain BSD1 changed that cell turn to an aberrant three-chamber sporangium with two forespore-like compartment at the poles.The cell growth of B.subtilis?spo0A was inhibited to some degree by the deletion of spo0 A.The biomass of B.subtilis?spo0A was decreased by 31.0%,and the yield of acetoin decreased by 13.5%,compared with the the starting strain B.subtilis 168.The deletion of sigF and sigE had a little influence to the biosynthesis of acetoin in BSD1.The results indicated that blocking the sporulation of B.subtilis 168 did not inprove the production of acetoin,but blocking sporulation by a disruption of sigE and sigF is a better way to abtain sporulation-deficient B.subtilis than by a disruption of spo0 A.The biosynthesis of 2,3-butanediol,lactate and acetate were blocked by inactivation of gene bdhA,ldh and ack A respectively.Acetoin reductase,lactate dehydrogenase and acetate kinase are encoded by bdhA,ldh and ackA,respectively.By inactivation of bdhA in BSD1,the bioconversion between acetoin and 2,3-butanediol was restrained,resulted in an shorter fermentation time and acetoin yield was improved to 29.99 g·L-1,while the acetoin productivity was enhanced from 0.24 g·L-1·h-1 to 0.36 g·L-1·h-1.However,the accumulation of lactate,acetate and succinate were increased,and the 2,3-butanediol yield still reached 6.84 g·L-1,which indicated that strain B.subtilis 168 has other minor acetoin reductase;By disruption of ldh in bdhA-deleted strain,the biomass was improved,acetoin yield enhanced to 32.87 g·L-1.Compared with the bdhA-deleted strain,lactate yield was declined by 79.4%,acetate and succinate yield were increased by 52.7% and 12.3% respectively;The deletion of ackA had a little impact to the production of acetoin.Though the enzyme activities of acetate kinase was decreased by 88.9%,acetate yield was almost unchanged.ALS and ALDC were co-expressed in strain BSD4 by autoinducible promoter to improve the production of acetoin.The strength of different promoter candidates measured by the enzyme activites of ALS and ALDC.The promotor PsrfA showed a lower activity during fermentation prophase,but its activity increased rapidly after 36 h of fermentation.Acetoin yield of BSD4/ pMA5-PsrfA-alsSD improved to 39.08 g·L-1,increased by 18.0% compared with strain BSD4,while the mole yield was improved from 0.68 mol·mol-1 to 0.79 mol·mol-1.In addition,the by-product acetate and succinate decreased by 25.0% and 24.3% respectively.Finally,acetoin yield was improved to 46.57 g·L-1 by fed-batch fermentation,while the acetoin productivity and mole yield was improved to 0.49 g·L-1·h-1and 0.74 mol·mol-1 respectively. |
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