Yifan Wang, Dan Shu, Zhemin Li, Di Luo, Jie Yang, Dongbo Chen, Tianfu Li, Xiaonan Hou, Qi Yang, Hong Tan
{"title":"提高灰葡萄孢 1',4'-反式-ABA 二醇产量的工程策略。","authors":"Yifan Wang, Dan Shu, Zhemin Li, Di Luo, Jie Yang, Dongbo Chen, Tianfu Li, Xiaonan Hou, Qi Yang, Hong Tan","doi":"10.1186/s12934-024-02460-8","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>Currently, industrial fermentation of Botrytis cinerea is a significant source of abscisic acid (ABA). The crucial role of ABA in plants and its wide range of applications in agricultural production have resulted in the constant discovery of new derivatives and analogues. While modifying the ABA synthesis pathway of existing strains to produce ABA derivatives is a viable option, it is hindered by the limited synthesis capacity of these strains, which hinders further development and application.</p><p><strong>Results: </strong>In this study, we knocked out the bcaba4 gene of B. cinerea TB-31 to obtain the 1',4'-trans-ABA-diol producing strain ZX2. We then studied the fermentation broth of the batch-fed fermentation of the ZX2 strain using metabolomic analysis. The results showed significant accumulation of 3-hydroxy-3-methylglutaric acid, mevalonic acid, and mevalonolactone during the fermentation process, indicating potential rate-limiting steps in the 1',4'-trans-ABA-diol synthesis pathway. This may be hindering the flow of the synthetic pathway. Additionally, analysis of the transcript levels of terpene synthesis pathway genes in this strain revealed a correlation between the bchmgr, bcerg12, and bcaba1-3 genes and 1',4'-trans-ABA-diol synthesis. To further increase the yield of 1',4'-trans-ABA-diol, we constructed a pCBg418 plasmid suitable for the Agrobacterium tumefaciens-mediated transformation (ATMT) system and transformed it to obtain a single-gene overexpression strain. We found that overexpression of bchmgr, bcerg12, bcaba1, bcaba2, and bcaba3 genes increased the yield of 1',4'-trans-ABA-diol. The highest yielding ZX2 A3 strain was eventually screened, which produced a 1',4'-trans-ABA-diol concentration of 7.96 mg/g DCW (54.4 mg/L) in 144 h of shake flask fermentation. This represents a 2.1-fold increase compared to the ZX2 strain.</p><p><strong>Conclusions: </strong>We utilized metabolic engineering techniques to alter the ABA-synthesizing strain B. cinerea, resulting in the creation of the mutant strain ZX2, which has the ability to produce 1',4'-trans-ABA-diol. By overexpressing the crucial genes involved in the 1',4'-trans-ABA-diol synthesis pathway in ZX2, we observed a substantial increase in the production of 1',4'-trans-ABA-diol.</p>","PeriodicalId":18582,"journal":{"name":"Microbial Cell Factories","volume":null,"pages":null},"PeriodicalIF":4.3000,"publicationDate":"2024-06-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11210036/pdf/","citationCount":"0","resultStr":"{\"title\":\"Engineering strategies for enhanced 1', 4'-trans-ABA diol production by Botrytis cinerea.\",\"authors\":\"Yifan Wang, Dan Shu, Zhemin Li, Di Luo, Jie Yang, Dongbo Chen, Tianfu Li, Xiaonan Hou, Qi Yang, Hong Tan\",\"doi\":\"10.1186/s12934-024-02460-8\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>Currently, industrial fermentation of Botrytis cinerea is a significant source of abscisic acid (ABA). The crucial role of ABA in plants and its wide range of applications in agricultural production have resulted in the constant discovery of new derivatives and analogues. While modifying the ABA synthesis pathway of existing strains to produce ABA derivatives is a viable option, it is hindered by the limited synthesis capacity of these strains, which hinders further development and application.</p><p><strong>Results: </strong>In this study, we knocked out the bcaba4 gene of B. cinerea TB-31 to obtain the 1',4'-trans-ABA-diol producing strain ZX2. We then studied the fermentation broth of the batch-fed fermentation of the ZX2 strain using metabolomic analysis. The results showed significant accumulation of 3-hydroxy-3-methylglutaric acid, mevalonic acid, and mevalonolactone during the fermentation process, indicating potential rate-limiting steps in the 1',4'-trans-ABA-diol synthesis pathway. This may be hindering the flow of the synthetic pathway. Additionally, analysis of the transcript levels of terpene synthesis pathway genes in this strain revealed a correlation between the bchmgr, bcerg12, and bcaba1-3 genes and 1',4'-trans-ABA-diol synthesis. To further increase the yield of 1',4'-trans-ABA-diol, we constructed a pCBg418 plasmid suitable for the Agrobacterium tumefaciens-mediated transformation (ATMT) system and transformed it to obtain a single-gene overexpression strain. We found that overexpression of bchmgr, bcerg12, bcaba1, bcaba2, and bcaba3 genes increased the yield of 1',4'-trans-ABA-diol. The highest yielding ZX2 A3 strain was eventually screened, which produced a 1',4'-trans-ABA-diol concentration of 7.96 mg/g DCW (54.4 mg/L) in 144 h of shake flask fermentation. This represents a 2.1-fold increase compared to the ZX2 strain.</p><p><strong>Conclusions: </strong>We utilized metabolic engineering techniques to alter the ABA-synthesizing strain B. cinerea, resulting in the creation of the mutant strain ZX2, which has the ability to produce 1',4'-trans-ABA-diol. By overexpressing the crucial genes involved in the 1',4'-trans-ABA-diol synthesis pathway in ZX2, we observed a substantial increase in the production of 1',4'-trans-ABA-diol.</p>\",\"PeriodicalId\":18582,\"journal\":{\"name\":\"Microbial Cell Factories\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":4.3000,\"publicationDate\":\"2024-06-26\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11210036/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Microbial Cell Factories\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://doi.org/10.1186/s12934-024-02460-8\",\"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/s12934-024-02460-8","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}
Engineering strategies for enhanced 1', 4'-trans-ABA diol production by Botrytis cinerea.
Background: Currently, industrial fermentation of Botrytis cinerea is a significant source of abscisic acid (ABA). The crucial role of ABA in plants and its wide range of applications in agricultural production have resulted in the constant discovery of new derivatives and analogues. While modifying the ABA synthesis pathway of existing strains to produce ABA derivatives is a viable option, it is hindered by the limited synthesis capacity of these strains, which hinders further development and application.
Results: In this study, we knocked out the bcaba4 gene of B. cinerea TB-31 to obtain the 1',4'-trans-ABA-diol producing strain ZX2. We then studied the fermentation broth of the batch-fed fermentation of the ZX2 strain using metabolomic analysis. The results showed significant accumulation of 3-hydroxy-3-methylglutaric acid, mevalonic acid, and mevalonolactone during the fermentation process, indicating potential rate-limiting steps in the 1',4'-trans-ABA-diol synthesis pathway. This may be hindering the flow of the synthetic pathway. Additionally, analysis of the transcript levels of terpene synthesis pathway genes in this strain revealed a correlation between the bchmgr, bcerg12, and bcaba1-3 genes and 1',4'-trans-ABA-diol synthesis. To further increase the yield of 1',4'-trans-ABA-diol, we constructed a pCBg418 plasmid suitable for the Agrobacterium tumefaciens-mediated transformation (ATMT) system and transformed it to obtain a single-gene overexpression strain. We found that overexpression of bchmgr, bcerg12, bcaba1, bcaba2, and bcaba3 genes increased the yield of 1',4'-trans-ABA-diol. The highest yielding ZX2 A3 strain was eventually screened, which produced a 1',4'-trans-ABA-diol concentration of 7.96 mg/g DCW (54.4 mg/L) in 144 h of shake flask fermentation. This represents a 2.1-fold increase compared to the ZX2 strain.
Conclusions: We utilized metabolic engineering techniques to alter the ABA-synthesizing strain B. cinerea, resulting in the creation of the mutant strain ZX2, which has the ability to produce 1',4'-trans-ABA-diol. By overexpressing the crucial genes involved in the 1',4'-trans-ABA-diol synthesis pathway in ZX2, we observed a substantial increase in the production of 1',4'-trans-ABA-diol.
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