黄色短杆菌ilvN基因定点突变和ilvBN、ilvC串联表达对L-缬氨酸产量的影响

Effects of Brevibacterium flavum with directed mutagenesis of ilvN and co-expression of ilvBNC cluster on L-valine production

由ilv BN、ilv C基因编码的乙酰羟酸合成酶(AHAS)和乙酰羟酸异构还原酶(AHAIR)是L-缬氨酸合成途径的两个关键酶。本实验以黄色短杆菌Brevibacterium flavum MD515为出发菌株,通过PCR技术扩增其ilv BN和ilv C基因,对调节亚基ilv N进行定点突变,获得抗反馈抑制突变型编码基因ilv BNrC;然后将其插入穿梭表达载体p Z8-1中,构建串联表达质粒p Z8-1-ilv BNrC并转化出发菌株,筛选获得工程菌株B.flavum MD515/p Z8-1-ilv BNrC。摇瓶发酵该工程菌株L-缬氨酸产量达29.5 g·L-1,较出发菌株提高27.7%,同时生长速度和生物量也比出发菌株有所提高,丙氨酸含量降低,L-亮氨酸及L-异亮氨酸含量提高。在30 L发酵罐连续补料发酵60 h后L-缬氨酸产量达61.7 g·L-1,糖酸转化率为39.2%。菌株MD515/p Z8-1-ilv BNrC发酵液透光率较出发菌株高且蛋白含量低,这些特性有利于发酵液后期的分离提取。

Acetohydroxy acid synthase（AHAS） and acetohydroxy acid isomeroreductase（AHAIR） encoded by ilv BN and ilv C are two key enzymes which play important roles in the biosynthetic pathway of L-valine.Brevibacterium flavum MD515 was used as the origin strain and site-specific mutagenesis was performed in its ilv N gene which coded for the regulatory subunit of AHAS,resulting in the obtainment of an anti-feedback inhibition gene,named ilv BN＇C.Then,the ilv BNrC gene was ligased to plasmid p Z8-1 for construction of the recombinant plasmid p Z8-1-ilv BNrC,which was subsequently transfored into B.flavum MD515.With this method,the targeted transformant B.flavum MD515/p Z8-1-ilv BNrC showed better L-valine producing capacity of 29.5 g·L-1,27.7% increase than that of original strain when it was cultured in 250 ml shake flasks.Meanwhile,the yield of leucine and isoleucine also increased while the alanine decreased.The biomass and growth rate were also increased.Moreover,fermentation experiments was performed in a 30 L fermentor and the results indicated that the L-valine productivity of B.flavum MD515/p Z8-1-ilv BNrC reached to 61.7 g·L-1 while the conversion rate of glucose/valine was up to 39.2%.The work finally made some simple investigations about the purification of L-valine in B.flavum MD515/p Z8-1-ilv BNrC and B.flavum MD515.The results showed that the light transmittance of strain B.flavum MD515/p Z8-1-ilv BNrC was higher while the protein content was lower than that of B.flavum MD515,which was beneficial for subsequent separation process.