{"title":"利用突变酶模块构建大肠杆菌木质素苷生物合成网络。","authors":"Yuqi Qiao, Doudou Huang, Yajing Li, Songfan Jiang, Xiao Chen, Junfeng Chen, Ying Xiao, Wansheng Chen","doi":"10.1186/s12934-024-02467-1","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Due to the complexity of the metabolic pathway network of active ingredients, precise targeted synthesis of any active ingredient on a synthetic network is a huge challenge. Based on a complete analysis of the active ingredient pathway in a species, this goal can be achieved by elucidating the functional differences of each enzyme in the pathway and achieving this goal through different combinations. Lignans are a class of phytoestrogens that are present abundantly in plants and play a role in various physiological activities of plants due to their structural diversity. In addition, lignans offer various medicinal benefits to humans. Despite their value, the low concentration of lignans in plants limits their extraction and utilization. Recently, synthetic biology approaches have been explored for lignan production, but achieving the synthesis of most lignans, especially the more valuable lignan glycosides, across the entire synthetic network remains incomplete.</p><p><strong>Results: </strong>By evaluating various gene construction methods and sequences, we determined that the pCDF-Duet-Prx02-PsVAO gene construction was the most effective for the production of (+)-pinoresinol, yielding up to 698.9 mg/L after shake-flask fermentation. Based on the stable production of (+)-pinoresinol, we synthesized downstream metabolites in vivo. By comparing different fermentation methods, including \"one-cell, one-pot\" and \"multicellular one-pot\", we determined that the \"multicellular one-pot\" method was more effective for producing (+)-lariciresinol, (-)-secoisolariciresinol, (-)-matairesinol, and their glycoside products. The \"multicellular one-pot\" fermentation yielded 434.08 mg/L of (+)-lariciresinol, 96.81 mg/L of (-)-secoisolariciresinol, and 45.14 mg/L of (-)-matairesinol. Subsequently, ultilizing the strict substrate recognition pecificities of UDP-glycosyltransferase (UGT) incorporating the native uridine diphosphate glucose (UDPG) Module for in vivo synthesis of glycoside products resulted in the following yields: (+)-pinoresinol glucoside: 1.71 mg/L, (+)-lariciresinol-4-O-D-glucopyranoside: 1.3 mg/L, (+)-lariciresinol-4'-O-D-glucopyranoside: 836 µg/L, (-)-secoisolariciresinol monoglucoside: 103.77 µg/L, (-)-matairesinol-4-O-D-glucopyranoside: 86.79 µg/L, and (-)-matairesinol-4'-O-D-glucopyranoside: 74.5 µg/L.</p><p><strong>Conclusions: </strong>By using various construction and fermentation methods, we successfully synthesized 10 products of the lignan pathway in Isatis indigotica Fort in Escherichia coli, with eugenol as substrate. Additionally, we obtained a diverse range of lignan products by combining different modules, setting a foundation for future high-yield lignan production.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":4.3000,"publicationDate":"2024-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11225284/pdf/","citationCount":"0","resultStr":"{\"title\":\"Construction of lignan glycosides biosynthetic network in Escherichia coli using mutltienzyme modules.\",\"authors\":\"Yuqi Qiao, Doudou Huang, Yajing Li, Songfan Jiang, Xiao Chen, Junfeng Chen, Ying Xiao, Wansheng Chen\",\"doi\":\"10.1186/s12934-024-02467-1\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>Due to the complexity of the metabolic pathway network of active ingredients, precise targeted synthesis of any active ingredient on a synthetic network is a huge challenge. Based on a complete analysis of the active ingredient pathway in a species, this goal can be achieved by elucidating the functional differences of each enzyme in the pathway and achieving this goal through different combinations. Lignans are a class of phytoestrogens that are present abundantly in plants and play a role in various physiological activities of plants due to their structural diversity. In addition, lignans offer various medicinal benefits to humans. Despite their value, the low concentration of lignans in plants limits their extraction and utilization. Recently, synthetic biology approaches have been explored for lignan production, but achieving the synthesis of most lignans, especially the more valuable lignan glycosides, across the entire synthetic network remains incomplete.</p><p><strong>Results: </strong>By evaluating various gene construction methods and sequences, we determined that the pCDF-Duet-Prx02-PsVAO gene construction was the most effective for the production of (+)-pinoresinol, yielding up to 698.9 mg/L after shake-flask fermentation. Based on the stable production of (+)-pinoresinol, we synthesized downstream metabolites in vivo. By comparing different fermentation methods, including \\\"one-cell, one-pot\\\" and \\\"multicellular one-pot\\\", we determined that the \\\"multicellular one-pot\\\" method was more effective for producing (+)-lariciresinol, (-)-secoisolariciresinol, (-)-matairesinol, and their glycoside products. The \\\"multicellular one-pot\\\" fermentation yielded 434.08 mg/L of (+)-lariciresinol, 96.81 mg/L of (-)-secoisolariciresinol, and 45.14 mg/L of (-)-matairesinol. Subsequently, ultilizing the strict substrate recognition pecificities of UDP-glycosyltransferase (UGT) incorporating the native uridine diphosphate glucose (UDPG) Module for in vivo synthesis of glycoside products resulted in the following yields: (+)-pinoresinol glucoside: 1.71 mg/L, (+)-lariciresinol-4-O-D-glucopyranoside: 1.3 mg/L, (+)-lariciresinol-4'-O-D-glucopyranoside: 836 µg/L, (-)-secoisolariciresinol monoglucoside: 103.77 µg/L, (-)-matairesinol-4-O-D-glucopyranoside: 86.79 µg/L, and (-)-matairesinol-4'-O-D-glucopyranoside: 74.5 µg/L.</p><p><strong>Conclusions: </strong>By using various construction and fermentation methods, we successfully synthesized 10 products of the lignan pathway in Isatis indigotica Fort in Escherichia coli, with eugenol as substrate. Additionally, we obtained a diverse range of lignan products by combining different modules, setting a foundation for future high-yield lignan production.</p>\",\"PeriodicalId\":18582,\"journal\":{\"name\":\"Microbial Cell Factories\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":4.3000,\"publicationDate\":\"2024-07-05\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11225284/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Microbial Cell Factories\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://doi.org/10.1186/s12934-024-02467-1\",\"RegionNum\":2,\"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":"Microbial Cell Factories","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1186/s12934-024-02467-1","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
Construction of lignan glycosides biosynthetic network in Escherichia coli using mutltienzyme modules.
Background: Due to the complexity of the metabolic pathway network of active ingredients, precise targeted synthesis of any active ingredient on a synthetic network is a huge challenge. Based on a complete analysis of the active ingredient pathway in a species, this goal can be achieved by elucidating the functional differences of each enzyme in the pathway and achieving this goal through different combinations. Lignans are a class of phytoestrogens that are present abundantly in plants and play a role in various physiological activities of plants due to their structural diversity. In addition, lignans offer various medicinal benefits to humans. Despite their value, the low concentration of lignans in plants limits their extraction and utilization. Recently, synthetic biology approaches have been explored for lignan production, but achieving the synthesis of most lignans, especially the more valuable lignan glycosides, across the entire synthetic network remains incomplete.
Results: By evaluating various gene construction methods and sequences, we determined that the pCDF-Duet-Prx02-PsVAO gene construction was the most effective for the production of (+)-pinoresinol, yielding up to 698.9 mg/L after shake-flask fermentation. Based on the stable production of (+)-pinoresinol, we synthesized downstream metabolites in vivo. By comparing different fermentation methods, including "one-cell, one-pot" and "multicellular one-pot", we determined that the "multicellular one-pot" method was more effective for producing (+)-lariciresinol, (-)-secoisolariciresinol, (-)-matairesinol, and their glycoside products. The "multicellular one-pot" fermentation yielded 434.08 mg/L of (+)-lariciresinol, 96.81 mg/L of (-)-secoisolariciresinol, and 45.14 mg/L of (-)-matairesinol. Subsequently, ultilizing the strict substrate recognition pecificities of UDP-glycosyltransferase (UGT) incorporating the native uridine diphosphate glucose (UDPG) Module for in vivo synthesis of glycoside products resulted in the following yields: (+)-pinoresinol glucoside: 1.71 mg/L, (+)-lariciresinol-4-O-D-glucopyranoside: 1.3 mg/L, (+)-lariciresinol-4'-O-D-glucopyranoside: 836 µg/L, (-)-secoisolariciresinol monoglucoside: 103.77 µg/L, (-)-matairesinol-4-O-D-glucopyranoside: 86.79 µg/L, and (-)-matairesinol-4'-O-D-glucopyranoside: 74.5 µg/L.
Conclusions: By using various construction and fermentation methods, we successfully synthesized 10 products of the lignan pathway in Isatis indigotica Fort in Escherichia coli, with eugenol as substrate. Additionally, we obtained a diverse range of lignan products by combining different modules, setting a foundation for future high-yield lignan production.
期刊介绍:
Microbial Cell Factories is an open access peer-reviewed journal that covers any topic related to the development, use and investigation of microbial cells as producers of recombinant proteins and natural products, or as catalyzers of biological transformations of industrial interest. Microbial Cell Factories is the world leading, primary research journal fully focusing on Applied Microbiology.
The journal is divided into the following editorial sections:
-Metabolic engineering
-Synthetic biology
-Whole-cell biocatalysis
-Microbial regulations
-Recombinant protein production/bioprocessing
-Production of natural compounds
-Systems biology of cell factories
-Microbial production processes
-Cell-free systems