【作者】 李欣；

【导师】 饶志明；

【作者基本信息】 江南大学 ， 发酵工程， 2017， 硕士

【摘要】 乙偶姻作为一种重要的食品添加剂和平台化合物,广泛应用于食品、化工和农业等行业。本论文以食品安全菌株Bacillus subtilis168为研究对象,通过敲除基因sigF,sigE,bdhA,ldh和ackA,并在发酵中后期加强表达α-乙酰乳酸合成酶（ALS）和α-乙酰乳酸脱羧酶（ALDC）,以阻断芽孢的生成和减少副产物的积累,进而加强乙偶姻合成途径代谢流量。利用Cre/lox敲除体系分别敲除芽孢合成关键应答调节蛋白基因spo0A和芽孢特异性σ因子编码基因sigF、sig E,得到重组菌B.subtilis△spo0A和BSD1。spo0A基因的敲除成功的在芽孢合成起始阶段阻断了芽孢的产生;而sigF和sig E基因的共敲除成功的在芽孢合成不对称分裂阶段阻断了芽孢的产生,同时重组菌BSD1的细胞形态在芽孢形成阶段发生了变化,在细胞的两端各形成了一个类似前芽孢的细胞室。spo0A基因的缺失在一定程度上抑制了B.subtilis△spo0A的生长,菌体浓度下降了31.0%,乙偶姻产量降低了13.5%;而sigF和sigE基因的缺失对重组菌BSD1细胞的生长及乙偶姻的合成均无显著的影响。实验结果表明,阻断芽孢的合成不能提高乙偶姻的产量,敲除基因sigF和sigE是阻断芽孢形成的最优策略。通过敲除乙偶姻还原酶基因bdhA、乳酸脱氢酶基因ldh和乙酸激酶基因ackA,分别阻断副产物2,3-丁二醇、乳酸和乙酸的合成。bdhA基因的敲除阻断了乙偶姻与2,3-丁二醇的相互转化,发酵周期缩短,乙偶姻产量达到29.99 g·L^-1,生产效率从0.24 g·L^-1·h^-1上升到0.36 g·L^-1·h^-1。同时乙酸、乳酸和琥珀酸的产量也有所提高,发酵结束时发酵液中仍有6.84 g·L^-1的2,3-丁二醇,表明枯草芽孢杆菌中还存在其他具有乙偶姻还原酶活性的酶。ldh基因的敲除提高了菌体的生物量,乙偶姻产量得到进一步提高,达到32.87g·L^-1,乳酸产量降低了79.4%,乙酸和琥珀酸的产量分别提高了52.7%、12.3%。ackA基因的缺失对菌株合成乙偶姻无显著影响,虽然乙酸激酶的酶活降低了88.9%,但乙酸产量基本没有变化。利用自诱导启动子在发酵中后期共表达ALDC和ALS,加强乙偶姻的合成。比较了几个自诱导启动子在发酵中后期的表达能力,研究发现启动子PsrfA在发酵前期活性较低,在36 h后活性迅速上升,显著的提高了ALDC和ALS的酶活。通过摇瓶发酵,重组菌BSD4/pMA5-PsrfA-alsSD的乙偶姻产量达到39.08 g·L^-1,比菌株BSD4提高了18.0%,摩尔得率达到0.79 mol·mol^-1。乙偶姻合成途径代谢流的加强,使副产物乙酸和琥珀酸的产量分别下降了25.0%、24.3%,而2,3-丁二醇和乳酸的产量基本无变化。对重组菌BSD4/pMA5-Psrf A-alsSD进行分批补料发酵试验,乙偶姻产量提高至46.57 g·L^-1,生产效率达到0.49 g·L^-1·h^-1,摩尔得率为0.74 mol·mol^-1,实现了乙偶姻的高效合成。更多还原

【Abstract】 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 α-acetolactate synthase（ALS）and α-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.更多还原