谷氨酸棒杆菌乙酰羟酸合酶的高效表达调控及应用

Efficient expression regulation of acetohydroxyacid synthase for production of branched-chain amino acids in Corynebacterium glutamicum

摘要:

谷氨酸棒杆菌是支链氨基酸工业生产的主力菌，乙酰羟酸合酶(acetohydroxyacid synthase, AHAS)是支链氨基酸合成的关键酶，强化AHAS的表达是提高菌种水平的关键手段。然而目前还未实现高效调控AHAS，本研究首先基于前期开发的靶基因表达调控报告系统，从6个组成型强启动子中筛选乙酰羟酸合酶编码基因ilvBN的高效表达启动子，成功获得P_gpmA*启动子，表达强度是P_ilvBN天然启动子的23.3倍。其次，在P_gpmA*启动子基础上，构建并通过平板荧光成像初步筛选了3种人工合成核糖体结合位点(ribosome binding site, RBS)文库，发现“R₍₉₎N₍₆₎”为优势的突变文库，通过进一步的孔板复筛，成功获得36个不同的强度增强的RBS突变体，最高强度可达天然启动子P_ilvBN的62.3倍。最后，选择P_gpmA*启动子分别组合3种RBS(野生型、RBS18和RBS36)调控ilvBN^S155F的表达生产L-缬氨酸，L-缬氨酸产量随着表达调控元件强度的增强而提高，分别为1.17、1.38、2.29 g/L。在RBS18调控的基础上进一步组合ilvC过表达，L-缬氨酸产量可达7.57g/L。本研究获得的AHAS表达调控元件库，可为改造AHAS生产L-缬氨酸等支链氨基酸提供丰富元件，并为其他关键酶的表达调控提供思路和方法借鉴。

关键词:谷氨酸棒杆菌;支链氨基酸;乙酰羟酸合酶;组成型强启动子;人工合成核糖体结合位点文库

Abstract:

Corynebacterium glutamicum is a major workhorse in the industrial production of branched-chain amino acids (BCAAs). The acetohydroxyacid synthase (AHAS) encoded by ilvBN is a key enzyme in the biosynthesis of BCAAs. Enhancing AHAS expression is essential for engineering BCAA producers. However, at present, the available studies only used limited promoters to regulate AHAS expression, which is insufficient for achieving efficient regulation. Herein, we first employed a previously developed reporter system to screen out a strong constitutive promoter P_gpmA* from six candidate promoters for expressing ilvBN. P_gpmA* showcased the expression strength 23.3-fold that of the native promoter P_ilvBN. Moreover, three synthetic RBS libraries based on the promoter P_gpmA* were constructed and evaluated by plate fluorescence imaging. The results revealed that “R₍₉₎N₍₆₎” was the best mutant library. A total of 36 RBS mutants with enhanced strength were further screened by evaluation in 96-deep-well plates, and the highest strength reached up to 62.3-fold that of P_ilvBN. Finally, the promoter P_gpmA* was combined with three RBS mutants (WT, RBS18, and RBS36) to fine-tune the expression of ilvBN^S155F for L-valine biosynthesis, respectively. Increased expression strength led to enhanced L-valine production, with titers of 1.17, 1.38, and 2.29 g/L, respectively. The combination of RBS18 strain with the further overexpression of ilvC produced 7.57 g/L L-valine. The regulatory elements obtained in this study can be utilized to modulate AHAS expression for BCAA production in C. glutamicum. Additionally, this strategy can guide the efficient expression regulation of other key enzymes.

Key words:Corynebacterium glutamicum;branched-chain amino acids;acetohydroxyacid synthase;strong constitutive promoter;synthetic ribosome binding site library