Ming Wang , Haomiao Wang , Cong Gao , Wanqing Wei , Jia Liu , Xiulai Chen , Guipeng Hu , Wei Song , Jing Wu , Fan Zhang , Liming Liu
{"title":"利用大肠杆菌系统工程高效生产原儿茶酸。","authors":"Ming Wang , Haomiao Wang , Cong Gao , Wanqing Wei , Jia Liu , Xiulai Chen , Guipeng Hu , Wei Song , Jing Wu , Fan Zhang , Liming Liu","doi":"10.1016/j.ymben.2024.02.003","DOIUrl":null,"url":null,"abstract":"<div><p>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 <em>Escherichia coli</em>. In this study, a high-level strain of 3-dehydroshikimate, <em>E. coli</em> DHS01, was developed by blocking the carbon flow from the shikimate-overproducing strain <em>E. coli</em> SA09. Additionally, the PCA biosynthetic pathway was established in DHS01 by introducing the high-activity <em>Ap</em>AroZ. Subsequently, the protein structure and catalytic mechanism of 3-dehydroshikimate dehydratase from <em>Acinetobacter pittii</em> PHEA-2 (<em>Ap</em>AroZ) were clarified. The variant <em>Ap</em>AroZ<sup>R363A</sup>, achieved by modulating the conformational dynamics of <em>Ap</em>AroZ, effectively relieved product inhibition. Additionally, the tolerance of the strain <em>E. coli</em> 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.</p></div>","PeriodicalId":18483,"journal":{"name":"Metabolic engineering","volume":"82 ","pages":"Pages 134-146"},"PeriodicalIF":6.8000,"publicationDate":"2024-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Efficient production of protocatechuic acid using systems engineering of Escherichia coli\",\"authors\":\"Ming Wang , Haomiao Wang , Cong Gao , Wanqing Wei , Jia Liu , Xiulai Chen , Guipeng Hu , Wei Song , Jing Wu , Fan Zhang , Liming Liu\",\"doi\":\"10.1016/j.ymben.2024.02.003\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>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 <em>Escherichia coli</em>. In this study, a high-level strain of 3-dehydroshikimate, <em>E. coli</em> DHS01, was developed by blocking the carbon flow from the shikimate-overproducing strain <em>E. coli</em> SA09. Additionally, the PCA biosynthetic pathway was established in DHS01 by introducing the high-activity <em>Ap</em>AroZ. Subsequently, the protein structure and catalytic mechanism of 3-dehydroshikimate dehydratase from <em>Acinetobacter pittii</em> PHEA-2 (<em>Ap</em>AroZ) were clarified. The variant <em>Ap</em>AroZ<sup>R363A</sup>, achieved by modulating the conformational dynamics of <em>Ap</em>AroZ, effectively relieved product inhibition. Additionally, the tolerance of the strain <em>E. coli</em> 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.</p></div>\",\"PeriodicalId\":18483,\"journal\":{\"name\":\"Metabolic engineering\",\"volume\":\"82 \",\"pages\":\"Pages 134-146\"},\"PeriodicalIF\":6.8000,\"publicationDate\":\"2024-02-16\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Metabolic engineering\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S1096717624000193\",\"RegionNum\":1,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Metabolic engineering","FirstCategoryId":"5","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S1096717624000193","RegionNum":1,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
Efficient production of protocatechuic acid using systems engineering of Escherichia coli
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.
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.