转录调控因子ALsR表达强度对枯草芽孢杆菌合成乙偶姻和2,3-丁二醇的影响

The transcriptional level effect of ALsR on acetoin and 2,3-butanediol production in Bacillus subtilis

转录调控因子ALs R是枯草芽孢杆菌中赖氨酸家族的转录调控因子,负责调控丙酮酸到乙偶姻和2,3-丁二醇合成途径中乙酰乳酸合成酶和乙酰乳酸脱羧酶的表达.为探究枯草芽孢杆菌生产乙偶姻和2,3-丁二醇过程中ALs R的最适表达强度,选取5个不同强度的启动子来调控ALs R的表达,首先以绿色荧光蛋白(GFP)作为报告基因表征了不同强度启动子的转录活性,然后使用这些不同强度的启动子来调控ALs R的表达,研究ALs R的表达强度对枯草芽孢杆菌乙偶姻和2,3-丁二醇发酵的影响.结果发现,在使用表达水平较强的组成型启动子Pals SD调控ALs R表达时,细胞的发酵周期延长8 h,细胞密度有所下降,乙偶姻和2,3-丁二醇产量也有一定程度的下降.当使用较弱的启动子Psrf A、Pbdh A、Pzwf、Pals R调控ALs R表达时,ALS和ALDC酶活分别提高1.15-2.25倍和2.4-4.8倍,此时比较适于乙偶姻和2,3-丁二醇的合成,乙偶姻和2,3-丁二醇的产量分别提高了9.24%-19.63%和7.16%-14.91%,同时副产物乳酸和乙酸的产量分别降低了5.45%-18.18%和19.46%-31.21%,其中在启动子Pbdh A调控ALs R表达时,即ALS和ALDC酶活分别提高1.9倍和4.1倍,此时最适于乙偶姻和2,3-丁二醇的发酵生产,乙偶姻和2,3-丁二醇的产量分别提高了19.6%和14.9%,副产物乳酸和乙酸的产量也显著下降.本研究发现过量地表达转录调控因子ALs R会对细胞的生长造成影响,不利于乙偶姻和2,3-丁二醇的发酵,而适度强化表达转录调控因子ALs R不会对细胞的生长造成影响,可以有效地提高乙偶姻和2,3-丁二醇的产量,降低发酵过程中的副产物乙酸和乳酸的积累.(图5表4参34)

ALs R,an Lys R-type transcriptional regulator,is essential for the expression of a-acetolactate synthase(ALS)and a-acetolactate decarboxylase(ALDC),which are the key enzymes involved in the production pathway of acetoin and 2,3-butanediol from pyruvate.In this study,we selected five promoters(Pals SD,Psrf A,Pbdh A,Pzwf,and Pals R)to regulate the expression levels of ALs R as well as regulate ALS and ALDC activities.This was performed to observe the Als R effects on the acetoin and 2,3-butanediol production in Bacillus subtilis168.Firstly,green fluorescent protein(GFP),was used as a report gene to characterize the transcriptional activities of different promoters,then ALs R was overexpressed under these different promoters in Bacillus subtilis168.Next,the activities of ALS and ALDC as well as the accumulation of the products and by-products of the recombinant strains were determined to explore the effects on acetoin and 2,3-butanediol production.When ALs R was regulated by the constitutive promoter Pals SD,the fermentation was extended to 8 hours,while cell density as well as acetoin and 2,3-butanediol yields were decreased.However,when ALs R was regulated under the promoters Psrf A,Pbdh A,Pzwf,and Pals R the ALS and ALDC activities were increased by 1.10 to 2.25 folds and 2.25 to3.56folds,respectively,which was more suitable for the production of acetoin and 2,3-butanediol.In addition,the production of acetoin and 2,3-butanediol increased by 9.24%to 19.63%and 7.16%to 14.91%,respectively,while the yield of the by-products lactate and acetate decreased by 5.45%to 18.18%and 19.46%to 31.21%,respectively.Moreover,when ALs R was regulated by the promoter Pbdh A,the enzyme activities of ALS and ALDC experienced 1.9 and 4.1 fold increases,respectively,while the yields of acetoin and 2,3-butanediol were increased by 19.6%and 14.9%respectively,and the production of the by-products of lactate and acetate were decreased.Hence,in this study,we found that the excessive expression of ALs R decreased cell density and the production of acetoin and 2,3-butanediol,while moderate overexpression of ALs R increased acetoin and2,3-butanediol production,but decreased acetate and lactate accumulation.