{"title":"在重组大肠杆菌 BL21 中设计生产 5-氨基乙酰丙酸。","authors":"Zhiwen Zhu, Bing Fu, Jiajie Lu, Peize Wang, Chuyang Yan, Fuyao Guan, Jianying Huang, Ping Yu","doi":"10.1080/10826068.2024.2423644","DOIUrl":null,"url":null,"abstract":"<p><p>5-aminolevulinic acid (ALA) is a non-protein amino acid that has been widely used in the fields of medicine and agriculture. This study aims to engineer the C5 pathway of the ALA biosynthesis in <i>Escherichia coli</i> BL21 to enhance ALA production. The ALA synthase genes <i>gltX</i>, <i>hemA,</i> and <i>hemL</i> were overexpressed in <i>E. coli</i> BL21 to lead to the increase in the production of ALA. The sRNA RyhB was also overexpressed to downregulate the expression of ALA dehydratase to reduce the downstream bioconversion of ALA to porphobilinogen. Next, the gene <i>arcA</i> was knocked out by CRISPR-Cas9 technology to open the TCA cycle to promote the respiratory metabolism of the strain to reduce the feedback inhibition of heme to ALA. The fermentation conditions of the engineered strain were optimized by response surface experiments. The time-course analysis of the ALA production was carried out in a 1 L shake flask. Through these efforts, the production of ALA in engineered strain reached 2953 mg/L in a 1 L shake flask. This study contributes to the industrial production of ALA by the engineered <i>E. coli</i> in the future.</p>","PeriodicalId":20401,"journal":{"name":"Preparative Biochemistry & Biotechnology","volume":null,"pages":null},"PeriodicalIF":2.0000,"publicationDate":"2024-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Engineered production of 5-aminolevulinic acid in recombinant <i>Escherichia coli</i> BL21.\",\"authors\":\"Zhiwen Zhu, Bing Fu, Jiajie Lu, Peize Wang, Chuyang Yan, Fuyao Guan, Jianying Huang, Ping Yu\",\"doi\":\"10.1080/10826068.2024.2423644\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>5-aminolevulinic acid (ALA) is a non-protein amino acid that has been widely used in the fields of medicine and agriculture. This study aims to engineer the C5 pathway of the ALA biosynthesis in <i>Escherichia coli</i> BL21 to enhance ALA production. The ALA synthase genes <i>gltX</i>, <i>hemA,</i> and <i>hemL</i> were overexpressed in <i>E. coli</i> BL21 to lead to the increase in the production of ALA. The sRNA RyhB was also overexpressed to downregulate the expression of ALA dehydratase to reduce the downstream bioconversion of ALA to porphobilinogen. Next, the gene <i>arcA</i> was knocked out by CRISPR-Cas9 technology to open the TCA cycle to promote the respiratory metabolism of the strain to reduce the feedback inhibition of heme to ALA. The fermentation conditions of the engineered strain were optimized by response surface experiments. The time-course analysis of the ALA production was carried out in a 1 L shake flask. Through these efforts, the production of ALA in engineered strain reached 2953 mg/L in a 1 L shake flask. This study contributes to the industrial production of ALA by the engineered <i>E. coli</i> in the future.</p>\",\"PeriodicalId\":20401,\"journal\":{\"name\":\"Preparative Biochemistry & Biotechnology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.0000,\"publicationDate\":\"2024-11-05\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Preparative Biochemistry & Biotechnology\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://doi.org/10.1080/10826068.2024.2423644\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOCHEMICAL RESEARCH METHODS\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Preparative Biochemistry & Biotechnology","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1080/10826068.2024.2423644","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
摘要
5-氨基乙酰丙酸(ALA)是一种非蛋白氨基酸,已被广泛应用于医药和农业领域。本研究旨在设计大肠杆菌 BL21 中 ALA 生物合成的 C5 途径,以提高 ALA 的产量。在大肠杆菌 BL21 中过表达 ALA 合成酶基因 gltX、hemA 和 hemL,以提高 ALA 的产量。sRNA RyhB 也被过量表达,以下调 ALA 脱水酶的表达,从而减少 ALA 向卟啉原的下游生物转化。接着,通过 CRISPR-Cas9 技术敲除基因 arcA,以打开 TCA 循环,促进菌株的呼吸代谢,从而减少血红素对 ALA 的反馈抑制。通过响应面实验优化了工程菌株的发酵条件。在 1 升摇瓶中对 ALA 的产量进行了时程分析。通过这些努力,工程菌株在 1 L 摇瓶中的 ALA 产量达到了 2953 mg/L。这项研究有助于未来利用工程大肠杆菌进行 ALA 的工业化生产。
Engineered production of 5-aminolevulinic acid in recombinant Escherichia coli BL21.
5-aminolevulinic acid (ALA) is a non-protein amino acid that has been widely used in the fields of medicine and agriculture. This study aims to engineer the C5 pathway of the ALA biosynthesis in Escherichia coli BL21 to enhance ALA production. The ALA synthase genes gltX, hemA, and hemL were overexpressed in E. coli BL21 to lead to the increase in the production of ALA. The sRNA RyhB was also overexpressed to downregulate the expression of ALA dehydratase to reduce the downstream bioconversion of ALA to porphobilinogen. Next, the gene arcA was knocked out by CRISPR-Cas9 technology to open the TCA cycle to promote the respiratory metabolism of the strain to reduce the feedback inhibition of heme to ALA. The fermentation conditions of the engineered strain were optimized by response surface experiments. The time-course analysis of the ALA production was carried out in a 1 L shake flask. Through these efforts, the production of ALA in engineered strain reached 2953 mg/L in a 1 L shake flask. This study contributes to the industrial production of ALA by the engineered E. coli in the future.
期刊介绍:
Preparative Biochemistry & Biotechnology is an international forum for rapid dissemination of high quality research results dealing with all aspects of preparative techniques in biochemistry, biotechnology and other life science disciplines.