Bo Taidong, Wu Chen, Wang Zeting, Jiang Hao, Wang Feiao, Chen Ning, Li Yanjun
College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China.
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin University of Science and Technology, Tianjin 300457, China.
Metabolites. 2023 Jun 12;13(6):747. doi: 10.3390/metabo13060747.
Shikimate is a valuable chiral precursor for synthesizing oseltamivir (Tamiflu) and other chemicals. High production of shikimate via microbial fermentation has attracted increasing attention to overcome the unstable and expensive supply of shikimate extracted from plant resources. The current cost of microbial production of shikimate via engineered strains is still unsatisfactory, and thus more metabolic strategies need to be investigated to further increase the production efficiency. In this study, we first constructed a shikimate producer through the application of the non-phosphoenolpyruvate: carbohydrate phosphotransferase system (non-PTS) glucose uptake pathway, the attenuation of the shikimate degradation metabolism, and the introduction of a mutant of feedback-resistant 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase. Inspired by the natural presence of bifunctional 3-dehydroquinate dehydratase (DHD)-shikimate dehydrogenase (SDH) enzyme in plants, we then designed an artificial fusion protein of DHD-SDH to decrease the accumulation of the byproduct 3-dehydroshikimate (DHS). Subsequently, a repressed shikimate kinase (SK) mutant was selected to promote shikimate accumulation without the supplementation of expensive aromatic substances. Furthermore, EsaR-based quorum sensing (QS) circuits were employed to regulate the metabolic flux distribution between cell growth and product synthesis. The final engineered strain dSA10 produced 60.31 g/L shikimate with a yield of 0.30 g/g glucose in a 5 L bioreactor.
莽草酸是合成奥司他韦(达菲)和其他化学品的重要手性前体。通过微生物发酵高产莽草酸已引起越来越多的关注,以克服从植物资源中提取莽草酸供应不稳定且昂贵的问题。目前利用工程菌株微生物生产莽草酸的成本仍不尽人意,因此需要研究更多代谢策略以进一步提高生产效率。在本研究中,我们首先通过应用非磷酸烯醇式丙酮酸:碳水化合物磷酸转移酶系统(非PTS)葡萄糖摄取途径、减弱莽草酸降解代谢以及引入抗反馈3-脱氧-D-阿拉伯庚酮糖酸7-磷酸(DAHP)合酶突变体来构建莽草酸生产菌株。受植物中双功能3-脱氢奎尼酸脱水酶(DHD)-莽草酸脱氢酶(SDH)天然存在的启发,我们随后设计了一种DHD-SDH人工融合蛋白以减少副产物3-脱氢莽草酸(DHS)的积累。随后,选择了一种受抑制的莽草酸激酶(SK)突变体,无需添加昂贵的芳香族物质即可促进莽草酸积累。此外,基于EsaR的群体感应(QS)电路被用于调节细胞生长和产物合成之间的代谢通量分布。最终的工程菌株dSA10在5 L生物反应器中产生了60.31 g/L的莽草酸,葡萄糖产率为0.30 g/g。