Combinatorial optimization of CO2 transport and fixation to improve succinate production by promoter engineering
Combinatorial optimization of CO2 transport and fixation to improve succinate production by promoter engineering
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通过启动子工程组合优化 CO2 运输和固定以提高琥珀酸产量
DOI：
10.1002/bit.25927
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发表时间：
2016
影响因子：
3.8
通讯作者：
Tang Ya-Jie
中科院分区：
文献类型：
--
作者：
Yu Jun-Han;Zhu Li-Wen;Xia Shi-Tao;Li Hong-Mei;Tang Ya-Ling;Liang Xin-Hua;Chen Tao;Tang Ya-Jie
平衡代谢工程途径的流量以获得高产的目标产物是代谢工程的主要挑战。在先前的工作中，研究了在大肠杆菌中，在trc启动子下共过表达调节CO2运输（sbtAandbicA）和PEP羧化（磷酸烯醇式丙酮酸（PEP）羧化酶（ppc）和羧激酶（pck））的外源基因以用于琥珀酸生物合成的协同调节CO2运输和固定。为了平衡代谢通量以最大化琥珀酸滴度，本研究通过启动子工程实施了一种微调CO2转运和固定过程的组合优化策略。首先，基于能量矩阵，合成了一个包含20个合理设计的启动子的合成启动子库，与广泛使用的trc启动子相比，其强度从0.8%到100%不等。对rfpandcatreporter基因的评价表明，所合成的启动子稳定，具有一定的适用性。第二，设计了四种不同强度的启动子，分别组装了单个CO2转运基因（sbtAorbicA）和单个CO2固定基因（ppcorpck）的表达。在生物反应器中筛选了三种组合，如Tang 1519（P4-bicA+ pP 19-pck）、Tang 1522（P4-sbtA+ P4-ppc）、Tang 1523（P4-sbtA+ P17-ppc），其琥珀酸产量增加超过10%。最后，基于上述结果，进一步研究了四种运输和固定基因的共表达。在所有组合中，sbtA、bicA和ppc与弱启动子P4以及pck与强启动子P19的共表达（AFP 111/pT-P4-bicA-P4-sbtA+ pACYC-P19-pck-P4-ppc）提供了最好的琥珀酸生产。最高琥珀酸产量为89.4 g/L，比空载体对照高37.5%。这项工作显着提高琥珀酸生产通过组合优化CO2运输和固定。本文使用的启动子工程和组合优化策略代表了一种为生产其他工业重要化学品定制代谢途径的强大方法。Biotechnol. Bioeng. 2016;113：1531-1541.© 2016 Wiley Periodicals，Inc.
To balance the flux of an engineered metabolic pathway to achieve high yield of target product is a major challenge in metabolic engineering. In previous work, the collaborative regulation of CO2transport and fixation was investigated with co‐overexpressing exogenous genes regulating both CO2transport (sbtAandbicA) and PEP carboxylation (phosphoenolpyruvate (PEP) carboxylase (ppc) and carboxykinase (pck)) under trc promoter inEscherichia colifor succinate biosynthesis. For balancing metabolic flux to maximize succinate titer, a combinatorial optimization strategy to fine‐tuning CO2transport and fixation process was implemented by promoter engineering in this study. Firstly, based on the energy matrix a synthetic promoter library containing 20 rationally designed promoters with strengths ranging from 0.8% to 100% compared with the widely used trc promoter was generated. Evaluations ofrfpandcatreporter genes provided evidence that the synthetic promoters were stably and had certain applicability. Secondly, four designed promoters with different strengths were used for combinatorial assembly of single CO2transport gene (sbtAorbicA) and single CO2fixation gene (ppcorpck) expression. Three combinations, such as Tang1519 (P4‐bicA+ pP19‐pck), Tang1522 (P4‐sbtA+ P4‐ppc), Tang1523 (P4‐sbtA+ P17‐ppc) with a more than 10% increase in succinate production were screened in bioreactor. Finally, based on the above results, co‐expression of the four transport and fixation genes were further investigated. Co‐expression ofsbtA,bicA, andppcwith weak promoter P4andpckwith strong promoter P19(AFP111/pT‐P4‐bicA‐P4‐sbtA+ pACYC‐P19‐pck‐P4‐ppc) provided the best succinate production among all the combinations. The highest succinate production of 89.4 g/L was 37.5% higher than that obtained with empty vector control. This work significantly enhanced succinate production through combinatorial optimization of CO2transport and fixation. The promoter engineering and combinatorial optimization strategies used herein represents a powerful approach to tailor‐making metabolic pathways for the production of other industrially important chemicals. Biotechnol. Bioeng. 2016;113: 1531–1541. © 2016 Wiley Periodicals, Inc.