Zi-Xu Zhang, Yu-Zhou Wang, Fang-Tong Nong, Yan Xu, Chao Ye, Yang Gu, Xiao-Man Sun, He Huang
{"title":"在大肠杆菌中建立动态平衡系统以提高重组蛋白的生产","authors":"Zi-Xu Zhang, Yu-Zhou Wang, Fang-Tong Nong, Yan Xu, Chao Ye, Yang Gu, Xiao-Man Sun, He Huang","doi":"10.1007/s00253-022-12145-0","DOIUrl":null,"url":null,"abstract":"<p>The combination of <i>Escherichia coli</i> BL21 (DE3) and the pET expression system is used extensively for the expression of various recombinant proteins (RPs). However, RP overexpression often introduces a growth burden for the host, especially in the case of toxic proteins. The key to solving this problem is to reduce the host burden associated with protein overproduction, which is often achieved by regulating the expression or activity of T7 RNAP or growth-decoupled systems. However, these strategies mainly relieve or interrupt the robbing of host resources, and do not eliminate other types of host burdens in the production process. In this study, we constructed a production system based on a dynamic equilibrium to precisely relieve the host burden and increase the RP production. The system is composed of three modules, including the overexpression of basic growth-related genes (rRNA, RNAP core enzyme, sigma factors), prediction and overexpression of key proteins using the enzyme-constrained model ec_iECBD_1354, and dynamic regulation of growth-related and key protein expression intensity based on a burden-driven promoter. Using this system, the production of many high-burden proteins, including autolysis protein and <i>E. coli</i> membrane proteins, was increased to varying degrees. Among them, the cytosine transporter protein (CodB) was most significantly improved, with a 4.02-fold higher production compared to the wild strain. This system can effectively reduce the optimizing costs, and is suitable for developing various types of RP expression hosts rapidly.</p>","PeriodicalId":8342,"journal":{"name":"Applied Microbiology and Biotechnology","volume":"106 18","pages":"6125 - 6137"},"PeriodicalIF":3.9000,"publicationDate":"2022-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"4","resultStr":"{\"title\":\"Developing a dynamic equilibrium system in Escherichia coli to improve the production of recombinant proteins\",\"authors\":\"Zi-Xu Zhang, Yu-Zhou Wang, Fang-Tong Nong, Yan Xu, Chao Ye, Yang Gu, Xiao-Man Sun, He Huang\",\"doi\":\"10.1007/s00253-022-12145-0\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>The combination of <i>Escherichia coli</i> BL21 (DE3) and the pET expression system is used extensively for the expression of various recombinant proteins (RPs). However, RP overexpression often introduces a growth burden for the host, especially in the case of toxic proteins. The key to solving this problem is to reduce the host burden associated with protein overproduction, which is often achieved by regulating the expression or activity of T7 RNAP or growth-decoupled systems. However, these strategies mainly relieve or interrupt the robbing of host resources, and do not eliminate other types of host burdens in the production process. In this study, we constructed a production system based on a dynamic equilibrium to precisely relieve the host burden and increase the RP production. The system is composed of three modules, including the overexpression of basic growth-related genes (rRNA, RNAP core enzyme, sigma factors), prediction and overexpression of key proteins using the enzyme-constrained model ec_iECBD_1354, and dynamic regulation of growth-related and key protein expression intensity based on a burden-driven promoter. Using this system, the production of many high-burden proteins, including autolysis protein and <i>E. coli</i> membrane proteins, was increased to varying degrees. Among them, the cytosine transporter protein (CodB) was most significantly improved, with a 4.02-fold higher production compared to the wild strain. This system can effectively reduce the optimizing costs, and is suitable for developing various types of RP expression hosts rapidly.</p>\",\"PeriodicalId\":8342,\"journal\":{\"name\":\"Applied Microbiology and Biotechnology\",\"volume\":\"106 18\",\"pages\":\"6125 - 6137\"},\"PeriodicalIF\":3.9000,\"publicationDate\":\"2022-09-03\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"4\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Applied Microbiology and Biotechnology\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://link.springer.com/article/10.1007/s00253-022-12145-0\",\"RegionNum\":3,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Applied Microbiology and Biotechnology","FirstCategoryId":"5","ListUrlMain":"https://link.springer.com/article/10.1007/s00253-022-12145-0","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
Developing a dynamic equilibrium system in Escherichia coli to improve the production of recombinant proteins
The combination of Escherichia coli BL21 (DE3) and the pET expression system is used extensively for the expression of various recombinant proteins (RPs). However, RP overexpression often introduces a growth burden for the host, especially in the case of toxic proteins. The key to solving this problem is to reduce the host burden associated with protein overproduction, which is often achieved by regulating the expression or activity of T7 RNAP or growth-decoupled systems. However, these strategies mainly relieve or interrupt the robbing of host resources, and do not eliminate other types of host burdens in the production process. In this study, we constructed a production system based on a dynamic equilibrium to precisely relieve the host burden and increase the RP production. The system is composed of three modules, including the overexpression of basic growth-related genes (rRNA, RNAP core enzyme, sigma factors), prediction and overexpression of key proteins using the enzyme-constrained model ec_iECBD_1354, and dynamic regulation of growth-related and key protein expression intensity based on a burden-driven promoter. Using this system, the production of many high-burden proteins, including autolysis protein and E. coli membrane proteins, was increased to varying degrees. Among them, the cytosine transporter protein (CodB) was most significantly improved, with a 4.02-fold higher production compared to the wild strain. This system can effectively reduce the optimizing costs, and is suitable for developing various types of RP expression hosts rapidly.
期刊介绍:
Applied Microbiology and Biotechnology focusses on prokaryotic or eukaryotic cells, relevant enzymes and proteins; applied genetics and molecular biotechnology; genomics and proteomics; applied microbial and cell physiology; environmental biotechnology; process and products and more. The journal welcomes full-length papers and mini-reviews of new and emerging products, processes and technologies.