通过系统重构酵母基因组和反义转录调控工具包,实现高滴度天麻素生产。

IF 6.8 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Yang Gu , Yaru Jiang , Changfan Li , Jiang Zhu , Xueyao Lu , Jianyue Ge , Mengchen Hu , Jieying Deng , Jingbo Ma , Zhiliang Yang , Xiaoman Sun , Feng Xue , Guocheng Du , Peng Xu , He Huang
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引用次数: 0

摘要

天麻素是一种酚苷,是天麻的主要成分,具有镇静、催眠、抗惊厥和神经保护作用。在微生物宿主中进行植物天然产品的异源工程生产是一种安全、经济、可扩展的植物提取替代方法。在这里,我们展示了一种可实现高滴度生产天麻素的工程化脂肪溶解酵母。我们通过提高莽草酸途径的通量和优化葡萄糖基转移系统,系统地重构了酵母基因组。我们在酵母基因组中引入了五十多种基因修饰,包括酶筛选、限速步骤的减轻、启动子选择、基因组整合位点优化、竞争途径的下调以及消除天麻素降解。同时,我们开发了铜诱导反义转录调控(CATR)工具。所开发的 CATR 工具包通过在溶脂芽孢杆菌中添加铜,实现了对中脉素合成的动态抑制和激活。这一策略还被进一步用于动态调节丙酮酸激酶节点,以有效地将糖酵解通量转向莽草酸途径,同时保持细胞的正常生长速度。总之,通过这些努力,在振荡絮凝剂中获得了 9477.1 毫克/升的天麻素,在 5 升生物反应器中获得了 13.4 克/升的天麻素,产率为 0.149 克/克葡萄糖,这凸显了利用微生物发酵大规模、可持续生产天麻素的潜力。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
High titer production of gastrodin enabled by systematic refactoring of yeast genome and an antisense-transcriptional regulation toolkit

Gastrodin, a phenolic glycoside, is a prominent component of Gastrodia elata, which is renowned for its sedative, hypnotic, anticonvulsant, and neuroprotective activities. Engineering heterologous production of plant natural products in microbial host represents a safe, cost-effective, and scalable alternative to plant extraction. Here, we present the construction of an engineered Yarrowia lipolytica yeast that achieves a high-titer production of gastrodin. We systematically refactored the yeast genome by enhancing the flux of the shikimate pathway and optimizing the glucosyl transfer system. We introduced more than five dozen of genetic modifications onto the yeast genome, including enzyme screening, alleviation of rate-limiting steps, promoter selection, genomic integration site optimization, downregulation of competing pathways, and elimination of gastrodin degradation. Meanwhile, we developed a Copper-induced Antisense-Transcriptional Regulation (CATR) tool. The developed CATR toolkit achieved dynamic repression and activation of violacein synthesis through the addition of copper in Y. lipolytica. This strategy was further used to dynamically regulate the pyruvate kinase node to effectively redirect glycolytic flux towards the shikimate pathway while maintaining cell growth at proper rate. Taken together, these efforts resulted in 9477.1 mg/L of gastrodin in shaking flaks and 13.4 g/L of gastrodin with a yield of 0.149 g/g glucose in a 5-L bioreactor, highlighting the potential for large-scale and sustainable production of gastrodin from microbial fermentation.

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来源期刊
Metabolic engineering
Metabolic engineering 工程技术-生物工程与应用微生物
CiteScore
15.60
自引率
6.00%
发文量
140
审稿时长
44 days
期刊介绍: Metabolic Engineering (MBE) is a journal that focuses on publishing original research papers on the directed modulation of metabolic pathways for metabolite overproduction or the enhancement of cellular properties. It welcomes papers that describe the engineering of native pathways and the synthesis of heterologous pathways to convert microorganisms into microbial cell factories. The journal covers experimental, computational, and modeling approaches for understanding metabolic pathways and manipulating them through genetic, media, or environmental means. Effective exploration of metabolic pathways necessitates the use of molecular biology and biochemistry methods, as well as engineering techniques for modeling and data analysis. MBE serves as a platform for interdisciplinary research in fields such as biochemistry, molecular biology, applied microbiology, cellular physiology, cellular nutrition in health and disease, and biochemical engineering. The journal publishes various types of papers, including original research papers and review papers. It is indexed and abstracted in databases such as Scopus, Embase, EMBiology, Current Contents - Life Sciences and Clinical Medicine, Science Citation Index, PubMed/Medline, CAS and Biotechnology Citation Index.
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