Metabolic engineering and flux analysis of Corynebacterium glutamicum for L-serine production  

作　　者：LAI ShuJuan ZHANG Yun LIU ShuWen LIANG Yong SHANG XiuLing CHAI Xin WEN TingYi 

机构地区：[1]Department of lndustrial Microbiology and Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China [2]Graduate University of Chinese Academy of Sciences, Beijing 100049, China

出　　处：《Science China(Life Sciences)》2012年第4期283-290,共8页中国科学（生命科学英文版）

基　　金：supported by grants from Ministry of Science and Technology of China (Grant Nos.2008ZX09401-05 and 2010ZX09401-403);the National Natural Science Foundation of China (Grant No. 31100074);Chinese Academy of Sciences (Grant No. XBXA-2011-009)

摘　　要：L-Serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of L-serine from glucose. In this study, Corynebacterium glutamicum ATCC 13032 was engineered de novo by blocking and at- tenuating the conversion of L-serine to pyruvate and glycine, releasing the feedback inhibition by L-serine to 3-phosphoglycerate dehydrogenase （PGDH）, in combination with the co-expression of 3-phosphoglycerate kinase （PGK） and feedback-resistant PGDH （PGDHr）. The resulting strain, SER-8, exhibited a lower specific growth rate and significant differ- ences in L-serine levels from Phase I to Phase V as determined for fed-batch fermentation. The intracellular L-serine pool reached （14.22_＋1.41） ~trnol gcoM-1, which was higher than glycine pool, contrary to fermentation with the wild-type strain. Furthermore, metabolic flux analysis demonstrated that the over-expression of PGK directed the flux of the pentose phosphate pathway （PPP） towards the glycolysis pathway （EMP）, and the expression of PGDHr improved the L-serine biosynthesis pathway. In addition, the flux from L-serine to glycine dropped by 24%, indicating that the deletion of the activator GlyR re- sulted in down-regulation of serine hydroxymethyltransferase （SHMT） expression. Taken together, our findings imply that L-serine pool management is fundamental for sustaining the viability of C. glutamicum, and improvement of C1 units genera- tion by introducing the glycine cleavage system （GCV） to degrade the excessive glycine is a promising target for L-serine pro- duction in C. glutamicum.L-Serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of L-serine from glucose. In this study, Corynebacterium glutamicum ATCC 13032 was engineered de novo by blocking and attenuating the conversion of L-serine to pyruvate and glycine, releasing the feedback inhibition by L-serine to 3-phosphoglycerate dehydrogenase (PGDH), in combination with the co-expression of 3-phosphoglycerate kinase (PGK) and feedback-resistant PGDH (PGDHr). The resulting strain, SER-8, exhibited a lower specific growth rate and significant differences in L-serine levels from Phase I to Phase V as determined for fed-batch fermentation. The intracellular L-serine pool reached (14.22±1.41) μmol gCDM 1, which was higher than glycine pool, contrary to fermentation with the wild-type strain. Furthermore, metabolic flux analysis demonstrated that the over-expression of PGK directed the flux of the pentose phosphate pathway (PPP) towards the glycolysis pathway (EMP), and the expression of PGDHr improved the L-serine biosynthesis pathway. In addition, the flux from L-serine to glycine dropped by 24%, indicating that the deletion of the activator GlyR resulted in down-regulation of serine hydroxymethyltransferase (SHMT) expression. Taken together, our findings imply that L-serine pool management is fundamental for sustaining the viability of C. glutamicum, and improvement of C1 units generation by introducing the glycine cleavage system (GCV) to degrade the excessive glycine is a promising target for L-serine production in C. glutamicum.

关 键 词：Corynebacterium glutamicum L-SERINE intracellular metabolites metabolic engineering elementary mode analysis 

分 类 号：TQ922.9[轻工技术与工程—发酵工程] TQ921.3