Department of Industrial Microbiology and Biotechnology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
Sci China Life Sci. 2012 Apr;55(4):283-90. doi: 10.1007/s11427-012-4304-0. Epub 2012 May 9.
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 (PGDH(r)). 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 g(CDM) (-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 PGDH(r) 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 C(1) 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.
L-丝氨酸作为细胞生长的重要组成部分，很难直接从葡萄糖发酵生产 L-丝氨酸。在本研究中，通过阻断和削弱 L-丝氨酸向丙酮酸和甘氨酸的转化，并结合 3-磷酸甘油酸激酶（PGK）和反馈抗性 PGDH（PGDH(r)）的共表达，从头设计 Corynebacterium glutamicum ATCC 13032。由此产生的菌株 SER-8 在分批补料发酵中表现出较低的比生长速率和从 Phase I 到 Phase V 的 L-丝氨酸水平的显著差异。细胞内 L-丝氨酸池达到（14.22±1.41）μmol g(CDM)（-1），高于甘氨酸池，与野生型菌株发酵相反。此外，代谢通量分析表明，PGK 的过表达将戊糖磷酸途径（PPP）的通量导向糖酵解途径（EMP），PGDH(r)的表达改善了 L-丝氨酸生物合成途径。此外，L-丝氨酸向甘氨酸的通量下降了 24%，表明激活剂 GlyR 的缺失导致丝氨酸羟甲基转移酶（SHMT）表达下调。总之，我们的研究结果表明，L-丝氨酸池的管理对谷氨酸棒杆菌的生存能力至关重要，通过引入甘氨酸裂解系统（GCV）降解过量甘氨酸来提高 C1 单位的生成是谷氨酸棒杆菌中 L-丝氨酸生产的一个有前途的目标。
