强化表达PEPC促进谷氨酸棒状杆菌合成4-羟基异亮氨酸

Enhanced Expression of PEPC Promotes the Synthesis of 4-Hydroxyisoleucine by Corynebacterium glutamicum

摘要:

【目的】实现在谷氨酸棒状杆菌（Corynebacterium glutamicum，C. glutamicum）中高效合成4-羟基异亮氨酸（4-hydroxyisoleucine，4-HIL）。【方法】用不同强度的异亮氨酸（isoleucine，Ile）激活型启动子（P_brnFEN）动态上调异亮氨酸双加氧酶（isoleucine dioxygenase，IDO）基因ido和透明颤菌血红蛋白（VHb）基因vgb的表达。在此基础上，使用不同强度的组成型启动子加强磷酸烯醇式丙酮酸羧化酶（PEPC）基因ppc和丙酮酸羧化酶基因pyc的表达。【结果】在携带不同P_brnFEN启动子的重组菌株中筛选得到菌株C. glutamicum SN-I₇V₇ （SN-I₇V₇），发酵144 h后，其4-HIL浓度达到了（109.14±1.03） mmol/L，比对照菌株C. glutamicum SN02（SN02）提高了46.69%。再利用强启动子P_tacM过表达ppc基因后，最终得到一株生产4-HIL的优势菌株C. glutamicum RS-MP （RS-MP）。该菌株的4-HIL浓度达到了（145.76±6.04） mmol/L，比对照菌株SN-I₇V₇提高了33.55%。【结论】筛选得到的P_brnFEN组合和ppc过表达策略为4-HIL在C. glutamicum中高效合成提供了理论依据。

Abstract:

[Objective] This study aims to achieve the efficient biosynthesis of 4-hydroxyisoleucine (4-HIL) in Corynebacterium glutamicum (C. glutamicum). [Method] The isoleucine (Ile) inducible promoters (P_brnFEN) of varying strengths were used to dynamically upregulate the expression of the isoleucine dioxygenase (IDO) gene ido and the Vitreoscilla hemoglobin (VHb) gene vgb. On this basis, constitutive promoters of different strengths were employed to enhance the expression of the phosphoenolpyruvate carboxylase (PEPC) gene ppc and the pyruvate carboxylase gene pyc. [Result] Among the recombinant strains harboring different P_brnFEN promoters, strain C. glutamicum SN-I₇V₇ (SN-I₇V₇) was selected. After 144 h of fermentation with this strain, the concentration of 4-HIL reached (109.14±1.03) mmol/L, representing a 46.69% increase compared with the control strain C. glutamicum SN02 (SN02). Subsequently, overexpression of ppc with the strong promoter P_tacM led to the development of strain C. glutamicum RS-MP (RS-MP), an elite 4-HIL producer. This strain achieved a 4-HIL concentration of (145.76±6.04) mmol/L, a 33.55% improvement over the control strain SN-I₇V₇. [Conclusion] The screened P_brnFEN combinations and the ppc overexpression strategy provide a theoretical basis for the efficient biosynthesis of 4-HIL in C. glutamicum.