构建整合型重组枯草芽孢杆菌高效合成胍基乙酸

Efficient biosynthesis of guanidoacetic acid by a recombinant strain of Bacillus subtilis

摘要:

胍基乙酸作为一种能源性物质，在食品、医药和饲料等行业有着广泛的应用前景，但目前尚未实现利用生物法工业化生产胍基乙酸。本研究在食品级安全菌株枯草芽孢杆菌中设计胍基乙酸的合成路线，利用调控关键酶表达、解除反馈抑制、增加膜通透性等技术，实现全细胞催化高效合成胍基乙酸。首先基于进化树挖掘筛选最佳L-精氨酸:甘氨酸脒基转移酶，利用强启动子与基因组整合相结合的策略提高关键酶表达水平；其次，引入谷氨酸棒杆菌中用于L-精氨酸合成的鸟氨酸循环途径，缓解副产物L-鸟氨酸对酶的反馈抑制，并敲除L-精氨酸降解途径，强化底物再生；再次，增强N-乙酰胞壁-L-丙氨酸酰胺酶(N-acetylmuramoyl-L-alanine amidase, LytC)基因表达，改善细胞膜通透性；最后，使用菌株Bs-13在最佳转化条件下，经24 h获得13.1 g/L胍基乙酸，转化速率为0.54 g/(L·h)，底物甘氨酸的转化率为92.7%。以上策略一定程度上提高了胍基乙酸的生产效率，为生物法合成胍基乙酸提供了参考。

Abstract:

Guanidinoacetic acid, as an energetic substance, has a wide range of applications in the food, pharmaceutical, and feed industries. However, the biosynthesis of guanidinoacetic acid has not been applied in industrial production. In this study, we designed the synthetic route of guanidinoacetic acid in a food-grade strain of Bacillus subtilis. By regulating the expression of key enzymes, lifting feedback inhibition, and increasing membrane permeability, we achieved the efficient synthesis of guanidinoacetic acid by whole-cell catalysis. Firstly, the optimal L-arginine:glycine amidinotransferase was screened based on the phylogenetic tree, and the expression of the key enzyme was enhanced by a strategy combining strong promoter and genome integration. Secondly, the ornithine cycle for L-arginine synthesis in Corynebacterium glutamicum was introduced to alleviate the feedback inhibition of the enzyme by the byproduct L-ornithine, and the L-arginine degradation pathway was knocked down to enhance substrate regeneration. Thirdly, the expression of N-acetylmuramoyl-L-alanine amidase (LytC) was up-regulated to increase the cell membrane permeability. Finally, after optimization of whole-cell production conditions, strain Bs-13 achieved guanidinoacetic acid production at a titer of 13.1 g/L after 24 h, with a proudction rate of 0.54 g/(L·h) and a glycine conversion rate of 92.7%. The above strategy improved the production of guanidinoacetic acid and provided a reference for the biosynthesis of guanidinoacetic acid.