Designing glucose utilization "highway" for recombinant biosynthesis.
Designing glucose utilization "highway" for recombinant biosynthesis.
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DOI：
10.1016/j.ymben.2023.06.016
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发表时间：
2023-06
影响因子：
8.4
通讯作者：
Xuanxuan Zhang;Yufeng Cao;Y. Liu;Yanyan Lei;Ruixue Zhai;Wei Chen;Guizhi Shi;Jian-Ming Jin;Chaoning Liang;Shuang-Yan Tang
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文献类型：
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作者：
Xuanxuan Zhang;Yufeng Cao;Y. Liu;Yanyan Lei;Ruixue Zhai;Wei Chen;Guizhi Shi;Jian-Ming Jin;Chaoning Liang;Shuang-Yan Tang
cAMP受体蛋白（CRP）是一种主要介导碳源分解代谢的全球性调节因子。在此，我们成功地设计了CRP，在葡萄糖作为单碳源的最小培养基中开发出具有改进的重组生物合成能力的微生物底盘细胞。获得的最佳表现的camp独立的crpmu9突变体与crpwild型相比，在2%葡萄糖的存在下，细胞生长速度更快，启动子的表达水平提高了133倍。没有“葡萄糖抑制”的启动子有利于重组表达，因为葡萄糖是高密度发酵中经常使用的廉价碳源。转录组分析表明，CRP突变体在整体上改变了细胞代谢，显示出三羧酸循环活性升高；减少醋酸酯形成；核苷酸生物合成增加；并改善ATP合成、耐受性和抗应激活性。代谢物分析证实糖酵解和乙醛-三羧酸循环的上调促进了葡萄糖的利用。正如预期的那样，在受CRPmu9调控的菌株中，香兰素、柚皮素和咖啡酸的生物合成能力得到了提高。本研究将CRP优化的意义扩展到葡萄糖利用和重组生物合成，超越了常规指定的葡萄糖以外的碳源利用。由crpmu9调控的大肠杆菌细胞可以潜在地作为重组生物合成的有益基础。
cAMP receptor protein (CRP) is known as a global regulatory factor mainly mediating carbon source catabolism. Herein, we successfully engineered CRP to develop microbial chassis cells with improved recombinant biosynthetic capability in minimal medium with glucose as single carbon source. The obtained best-performing cAMP-independent CRPmu9mutant conferred both faster cell growth and a 133-fold improvement in expression level oflacpromoter in presence of 2% glucose, compared with strain under regulation of CRPwild-type. Promoters free from “glucose repression” are advantageous for recombinant expression, as glucose is a frequently used inexpensive carbon source in high-cell-density fermentations. Transcriptome analysis demonstrated that the CRP mutant globally rewired cell metabolism, displaying elevated tricarboxylic acid cycle activity; reduced acetate formation; increased nucleotide biosynthesis; and improved ATP synthesis, tolerance, and stress-resistance activity. Metabolites analysis confirmed the enhancement of glucose utilization with the upregulation of glycolysis and glyoxylate-tricarboxylic acid cycle. As expected, an elevated biosynthetic capability was demonstrated with vanillin, naringenin and caffeic acid biosynthesis in strains regulated by CRPmu9. This study has expanded the significance of CRP optimization into glucose utilization and recombinant biosynthesis, beyond the conventionally designated carbon source utilization other than glucose. TheEscherichiacolicell regulated by CRPmu9can be potentially used as a beneficial chassis for recombinant biosynthesis.