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

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。

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.