GapN诱导型表达小白链霉菌的构建及其对ε-聚赖氨酸生产的影响

Construction of a Streptomyces albulus strain with inducible expression of GapN and its effect onε-polylysine production

目的构建NADP依赖型三磷酸甘油醛脱氢酶(GapN)诱导型表达的小白链霉菌基因工程菌,研究GapN不同表达水平对ε-聚赖氨酸(ε-PL)生产的影响。方法化学合成来自变异链球菌的gapN基因,利用DNA无缝克隆技术构建GapN诱导型表达载体pSET152-Cel-RS2-gapN,再通过接合转移得到GapN诱导型表达的小白链霉菌基因工程菌Q-gapN-Cel;利用实时荧光定量PCR和摇瓶发酵比较等方法研究纤维二糖添加量对gapN基因表达水平、胞内NADPH浓度和ε-PL产量的影响。结果gapN基因的表达水平与纤维二糖的添加量呈正相关;gapN基因的表达能够显著提高胞内NADPH浓度;随着gapN基因表达水平的提高,ε-PL的产量先增加后降低,添加0.03 mM纤维二糖时产量最高,比对照菌株Q-152提高152.6%。结论GapN表达水平对小白链霉菌生产ε-PL有显著影响,通过控制GapN的表达水平能够有效提高小白链霉菌发酵生产ε-PL的能力。

Objective To construct a genetically engineered strain of Streptomyces albulus with inducible expression of NADP-dependent glyceraldehyde triphosphate dehydrogenase(GapN)and study the effects of different GapN expression levels onε-polylysine(ε-PL)production.Methods The gapN gene from Streptococcus mutans was synthesized chemically.DNA seamless cloning technique was used to construct the cellobiose inducible expression vector of pSET152-Cel-RS2-gapN.Then,conjugation transfer was applied to obtain the genetically engineered strain of S.albulus Q-gapN-Cel with inducible expression of GapN.Real-time fluorescence quantitative PCR and shaking flask fermentation were performed to investigate the effects of the supplemental level of cellobiose on gapN gene expression,intracellular NADPH concentration andε-PL production.Results The expression level of gapN gene was positively correlated with the supplemental level of cellobiose.The intracellular NADPH concentrations were significantly increased by gapN gene expression.With the increase of gapN gene expression level,the production ofε-PL firstly increased and then decreased.The highest concentration ofε-PL was obtained when 0.03 mM cellobiose was added,which was 152.6%higher than that of the control strain Q-152.Conclusion The expression level of GapN has a significant effect onε-PL production of S.albulus.The fermentation ability of S.albulus to produceε-PL can be effectively improved by controlling the expression level of GapN.