Efficient production of protocatechuic acid using systems engineering of Escherichia coli

IF 6.8 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Metabolic engineering Pub Date : 2024-02-16 DOI:10.1016/j.ymben.2024.02.003
Ming Wang , Haomiao Wang , Cong Gao , Wanqing Wei , Jia Liu , Xiulai Chen , Guipeng Hu , Wei Song , Jing Wu , Fan Zhang , Liming Liu
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Abstract

Protocatechuic acid (3, 4-dihydroxybenzoic acid, PCA) is widely used in the pharmaceuticals, health food, and cosmetics industries owing to its diverse biological activities. However, the inhibition of 3-dehydroshikimate dehydratase (AroZ) by PCA and its toxicity to cells limit the efficient production of PCA in Escherichia coli. In this study, a high-level strain of 3-dehydroshikimate, E. coli DHS01, was developed by blocking the carbon flow from the shikimate-overproducing strain E. coli SA09. Additionally, the PCA biosynthetic pathway was established in DHS01 by introducing the high-activity ApAroZ. Subsequently, the protein structure and catalytic mechanism of 3-dehydroshikimate dehydratase from Acinetobacter pittii PHEA-2 (ApAroZ) were clarified. The variant ApAroZR363A, achieved by modulating the conformational dynamics of ApAroZ, effectively relieved product inhibition. Additionally, the tolerance of the strain E. coli PCA04 to PCA was enhanced by adaptive laboratory evolution, and a biosensor-assisted high-throughput screening method was designed and implemented to expedite the identification of high-performance PCA-producing strains. Finally, in a 5 L bioreactor, the final strain PCA05 achieved the highest PCA titer of 46.65 g/L, a yield of 0.23 g/g, and a productivity of 1.46 g/L/h for PCA synthesis from glucose using normal fed-batch fermentation. The strategies described herein serve as valuable guidelines for the production of other high-value and toxic products.

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利用大肠杆菌系统工程高效生产原儿茶酸。
原儿茶酸(3, 4-二羟基苯甲酸,PCA)具有多种生物活性,被广泛应用于制药、保健食品和化妆品行业。然而,PCA 对 3-脱氢莽草酸脱水酶(AroZ)的抑制作用及其对细胞的毒性限制了大肠杆菌生产 PCA 的效率。在本研究中,通过阻断莽草酸过量产生菌株大肠杆菌 SA09 的碳流,培育出了 3-脱氢莽草酸高产菌株大肠杆菌 DHS01。此外,通过引入高活性 ApAroZ,在 DHS01 中建立了 PCA 生物合成途径。随后,阐明了皮氏不动杆菌 PHEA-2 的 3-脱氢莽草酸脱水酶(ApAroZ)的蛋白质结构和催化机理。通过调节 ApAroZ 的构象动力学而获得的变体 ApAroZR363A 能有效缓解产物抑制。此外,通过实验室适应性进化,提高了大肠杆菌 PCA04 菌株对 PCA 的耐受性,并设计和实施了一种生物传感器辅助的高通量筛选方法,以加快鉴定高性能 PCA 生产菌株。最后,在一个 5 L 的生物反应器中,最终菌株 PCA05 通过正常的喂料批次发酵,从葡萄糖合成 PCA 的最高滴度为 46.65 g/L,产量为 0.23 g/g,生产率为 1.46 g/L/h。本文所述的策略可作为生产其他高价值和有毒产品的宝贵指南。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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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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