Development of a vitamin B5 hyperproducer in Escherichia coli by multiple metabolic engineering

IF 6.8 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Fuqiang Song , Zhijie Qin , Kun Qiu , Zhongshi Huang , Lian Wang , Heng Zhang , Xiaoyu Shan , Hao Meng , Xirong Liu , Jingwen Zhou
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Abstract

Vitamin B5 [D-pantothenic acid (D-PA)] is an essential water-soluble vitamin that is widely used in the food and feed industries. Currently, the relatively low fermentation efficiency limits the industrial application of D-PA. Here, a plasmid-free D-PA hyperproducer was constructed using systematic metabolic engineering strategies. First, pyruvate was enriched by deleting the non-phosphotransferase system, inhibiting pyruvate competitive branches, and dynamically controlling the TCA cycle. Next, the (R)-pantoate pathway was enhanced by screening the rate-limiting enzyme PanBC and regulating the other enzymes of this pathway one by one. Then, to enhance NADPH sustainability, NADPH regeneration was achieved through the novel “PEACES” system by (1) expressing the NAD + kinase gene ppnk from Clostridium glutamicum and the NADP + -dependent gapCcae from Clostridium acetobutyricum and (2) knocking-out the endogenous sthA gene, which interacts with ilvC and panE in the D-PA biosynthesis pathway. Combined with transcriptome analysis, it was found that the membrane proteins OmpC and TolR promoted D-PA efflux by increasing membrane fluidity. Strain PA132 produced a D-PA titer of 83.26 g/L by two-stage fed-batch fermentation, which is the highest D-PA titer reported so far. This work established competitive producers for the industrial production of D-PA and provided an effective strategy for the production of related products.

Abstract Image

Abstract Image

通过多重代谢工程在大肠杆菌中开发维生素 B5 高产菌。
维生素 B5 [D-泛酸 (D-PA)]是一种必需的水溶性维生素,广泛应用于食品和饲料行业。目前,相对较低的发酵效率限制了 D-PA 的工业应用。在这里,我们采用系统的代谢工程策略构建了一种无质粒的 D-PA 超级生产者。首先,通过删除非磷酸转移酶系统、抑制丙酮酸竞争性分支和动态控制 TCA 循环来富集丙酮酸。其次,通过筛选限速酶 PanBC 和逐一调节该途径的其他酶来增强 (R)- 泛酸途径。然后,为了增强 NADPH 的可持续性,通过新型 "PEACES "系统实现了 NADPH 的再生:(1)表达来自谷氨酸梭菌的 NAD+ 激酶基因 ppnk 和来自乙酰丁酸梭菌的 NADP+ 依赖性 gapCcae;(2)敲除内源性 sthA 基因,该基因与 D-PA 生物合成途径中的 ilvC 和 panE 相互作用。结合转录组分析发现,膜蛋白 OmpC 和 TolR 通过增加膜的流动性促进了 D-PA 的外流。菌株 PA132 通过两级饲料批量发酵产生了 83.26 克/升的 D-PA 滴度,这是迄今为止报道的最高 D-PA 滴度。这项工作为 D-PA 的工业化生产建立了有竞争力的生产商,并为相关产品的生产提供了有效的策略。
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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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