Multiplexed Promoter Engineering for Improving Thaxtomin A Production in Heterologous <i>Streptomyces</i> Hosts
Thaxtomin A is a potent bioherbicide in both organic and conventional agriculture; however, its low yield hinders its wide application. Here, we report the direct cloning and heterologous expression of the thaxtomin A gene cluster in three well-characterized <i>Streptomyces</i> hosts. Th...
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MDPI AG
2022-05-01
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Online Access: | https://www.mdpi.com/2075-1729/12/5/689 |
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author | Xuejin Zhao Yeqing Zong Weijia Wei Chunbo Lou |
author_facet | Xuejin Zhao Yeqing Zong Weijia Wei Chunbo Lou |
author_sort | Xuejin Zhao |
collection | DOAJ |
description | Thaxtomin A is a potent bioherbicide in both organic and conventional agriculture; however, its low yield hinders its wide application. Here, we report the direct cloning and heterologous expression of the thaxtomin A gene cluster in three well-characterized <i>Streptomyces</i> hosts. Then, we present an efficient, markerless and multiplex large gene cluster editing method based on in vitro CRISPR/Cas9 digestion and yeast homologous recombination. With this method, we successfully engineered the thaxtomin A cluster by simultaneously replacing the native promoters of the <i>txtED</i> operon, <i>txtABH</i> operon and <i>txtC</i> gene with strong constitutive promoters, and the yield of thaxtomin A improved to 289.5 µg/mL in heterologous <i>Streptomyces coelicolor</i> M1154. To further optimize the biosynthetic pathway, we used constraint-based combinatorial design to build 27 refactored gene clusters by varying the promoter strength of every operon, and the highest titer of thaxtomin A production reached 504.6 μg/mL. Taken altogether, this work puts forward a multiplexed promoter engineering strategy to engineer secondary metabolism gene clusters for efficiently improving fermentation titers. |
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institution | Directory Open Access Journal |
issn | 2075-1729 |
language | English |
last_indexed | 2024-03-10T03:33:49Z |
publishDate | 2022-05-01 |
publisher | MDPI AG |
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spelling | doaj.art-722bdf7178724c3199ba07c2716ea7312023-11-23T11:50:01ZengMDPI AGLife2075-17292022-05-0112568910.3390/life12050689Multiplexed Promoter Engineering for Improving Thaxtomin A Production in Heterologous <i>Streptomyces</i> HostsXuejin Zhao0Yeqing Zong1Weijia Wei2Chunbo Lou3CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, ChinaCAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, ChinaCAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, ChinaCollege of Life Sciences, University of Chinese Academy of Sciences, Beijing 100149, ChinaThaxtomin A is a potent bioherbicide in both organic and conventional agriculture; however, its low yield hinders its wide application. Here, we report the direct cloning and heterologous expression of the thaxtomin A gene cluster in three well-characterized <i>Streptomyces</i> hosts. Then, we present an efficient, markerless and multiplex large gene cluster editing method based on in vitro CRISPR/Cas9 digestion and yeast homologous recombination. With this method, we successfully engineered the thaxtomin A cluster by simultaneously replacing the native promoters of the <i>txtED</i> operon, <i>txtABH</i> operon and <i>txtC</i> gene with strong constitutive promoters, and the yield of thaxtomin A improved to 289.5 µg/mL in heterologous <i>Streptomyces coelicolor</i> M1154. To further optimize the biosynthetic pathway, we used constraint-based combinatorial design to build 27 refactored gene clusters by varying the promoter strength of every operon, and the highest titer of thaxtomin A production reached 504.6 μg/mL. Taken altogether, this work puts forward a multiplexed promoter engineering strategy to engineer secondary metabolism gene clusters for efficiently improving fermentation titers.https://www.mdpi.com/2075-1729/12/5/689thaxtomin Agene clusterheterologous expressionmultiplexed promoter engineering |
spellingShingle | Xuejin Zhao Yeqing Zong Weijia Wei Chunbo Lou Multiplexed Promoter Engineering for Improving Thaxtomin A Production in Heterologous <i>Streptomyces</i> Hosts Life thaxtomin A gene cluster heterologous expression multiplexed promoter engineering |
title | Multiplexed Promoter Engineering for Improving Thaxtomin A Production in Heterologous <i>Streptomyces</i> Hosts |
title_full | Multiplexed Promoter Engineering for Improving Thaxtomin A Production in Heterologous <i>Streptomyces</i> Hosts |
title_fullStr | Multiplexed Promoter Engineering for Improving Thaxtomin A Production in Heterologous <i>Streptomyces</i> Hosts |
title_full_unstemmed | Multiplexed Promoter Engineering for Improving Thaxtomin A Production in Heterologous <i>Streptomyces</i> Hosts |
title_short | Multiplexed Promoter Engineering for Improving Thaxtomin A Production in Heterologous <i>Streptomyces</i> Hosts |
title_sort | multiplexed promoter engineering for improving thaxtomin a production in heterologous i streptomyces i hosts |
topic | thaxtomin A gene cluster heterologous expression multiplexed promoter engineering |
url | https://www.mdpi.com/2075-1729/12/5/689 |
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