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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Main Authors: Xuejin Zhao, Yeqing Zong, Weijia Wei, Chunbo Lou
Format: Article
Language:English
Published: MDPI AG 2022-05-01
Series:Life
Subjects:
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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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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AT yeqingzong multiplexedpromoterengineeringforimprovingthaxtominaproductioninheterologousistreptomycesihosts
AT weijiawei multiplexedpromoterengineeringforimprovingthaxtominaproductioninheterologousistreptomycesihosts
AT chunbolou multiplexedpromoterengineeringforimprovingthaxtominaproductioninheterologousistreptomycesihosts