系统代谢工程改造谷氨酸棒杆菌高产L-丙氨酸

Systems metabolic engineering of Corynebacterium glutamicum for the efficient production of L-alanine

L-丙氨酸是一种重要的天然氨基酸,广泛应用于医药、食品、饲料等领域。目前,L-丙氨酸主要通过大肠杆菌来生产,开发安全、工业稳定的谷氨酸棒杆菌生产菌株具有重要的意义。该研究筛选了外源丙氨酸脱氢酶,在不以其他氨基酸为氨基供体的条件下高效率合成L-丙氨酸。强化非磷酸转移酶系统(phosphotransferase system,PTS)葡萄糖摄取途径,极大地促进了细胞的生长和L-丙氨酸的合成。引入异源Entner-Dondoroff途径,通过4步反应即可实现糖酵解途径10步反应供应前体物丙酮酸,进一步提高了L-丙氨酸的产量。此外,为了避免抗生素的使用和保证菌株的遗传稳定性,将关键基因整合到基因组上。最终工程菌株在5 L发酵罐发酵48 h产生104 g/L L-丙氨酸。该研究为利用谷氨酸棒杆菌工业化生产L-丙氨酸奠定了基础,同时为其他丙酮酸族产品的代谢工程研究提供了参考。

L-alanine is an important natural amino acid,which is widely used in medicine,food and feed industries.At present,L-alanine is mainly produced by Escherichia coli.It is of great significance to develop safe and industrially robust Corynebacterium glutamicum production strains.In this study,exogenous alanine dehydrogenase was optimized to synthesize L-alanine efficiently without using other amino acids as an amino donor.The non-phosphotransferase system glucose uptake pathway was strengthened by the simultaneous inactivation of the transcriptional repressor IolR and overexpression of the glucose kinase,which drastically promote the cell growth and L-alanine synthesis.The introduction of exogenous Entner-Doudoroff pathway that supply pyruvate only through 4-step reactions,an alternative to the 10-step Embden-Meyerhof-Parnas(EMP)pathway,further increased the production of L-alanine.In addition,in order to avoid the use of antibiotics and guarantee the genetic stability of the producer,essential genes were integrated into the genome of the strain.The final engineered strain produced 104 g/L L-alanine after 48 h fermentation in a 5 L fermenter.The study lays a solid foundation for the industrial production of L-alanine by C.glutamicum,and provides a reference for the metabolic engineering of other microbes to produce products derived from pyruvate.