Broad-Host-Range Expression Reveals Native and Host Regulatory Elements That Influence Heterologous Antibiotic Production in Gram-Negative Bacteria

ABSTRACT Heterologous expression has become a powerful tool for studying microbial biosynthetic gene clusters (BGCs). Here, we extend the transformation-associated recombination cloning and heterologous expression platform for microbial BGCs to include Gram-negative proteobacterial expression hosts....

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Autores principales:	Jia Jia Zhang, Xiaoyu Tang, Michelle Zhang, Darlene Nguyen, Bradley S. Moore
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	Gram negative heterologous expression Proteobacteria broad-host-range vector violacein Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/748b1cccf587411e8ba73b0dbd938e30
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spelling	oai:doaj.org-article:748b1cccf587411e8ba73b0dbd938e302021-11-15T15:51:51ZBroad-Host-Range Expression Reveals Native and Host Regulatory Elements That Influence Heterologous Antibiotic Production in Gram-Negative Bacteria10.1128/mBio.01291-172150-7511https://doaj.org/article/748b1cccf587411e8ba73b0dbd938e302017-11-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.01291-17https://doaj.org/toc/2150-7511ABSTRACT Heterologous expression has become a powerful tool for studying microbial biosynthetic gene clusters (BGCs). Here, we extend the transformation-associated recombination cloning and heterologous expression platform for microbial BGCs to include Gram-negative proteobacterial expression hosts. Using a broad-host-range expression platform, we test the implicit assumption that biosynthetic pathways are more successfully expressed in more closely related heterologous hosts. Cloning and expression of the violacein BGC from Pseudoalteromonas luteoviolacea 2ta16 revealed robust production in two proteobacterial hosts, Pseudomonas putida KT2440 and Agrobacterium tumefaciens LBA4404, but very little production of the antibiotic in various laboratory strains of Escherichia coli, despite their closer phylogenetic relationship. We identified a nonclustered LuxR-type quorum-sensing receptor from P. luteoviolacea 2ta16, PviR, that increases pathway transcription and violacein production in E. coli by ∼60-fold independently of acyl-homoserine lactone autoinducers. Although E. coli harbors the most similar homolog of PviR identified from all of the hosts tested, overexpression of various E. coli transcription factors did not result in a statistically significant increase in violacein production, while overexpression of two A. tumefaciens PviR homologs significantly increased production. Thus, this work not only introduces a new genetic platform for the heterologous expression of microbial BGCs, it also challenges the assumption that host phylogeny is an accurate predictor of host compatibility. IMPORTANCE Although Gram-positive heterologous hosts such as Streptomyces have been developed and optimized to support diverse secondary metabolic reactions, there has been comparatively less work on Gram-negative hosts, some of which grow faster and are easier to work with. This work presents a new genetic platform for direct cloning and broad-host-range heterologous expression of BGCs in Gram-negative proteobacterial expression hosts, and we leverage this platform to uncover regulatory elements that influence violacein expression from Pseudoalteromonas. Although it is often assumed that BGCs will be more successfully expressed in more closely related hosts, our work suggests that this may not be a general rule of thumb, as heterologous production of natural products can be influenced by specific host regulatory and/or biosynthetic elements, and the identity and effectiveness of those elements are difficult to predict. We argue for the use of a diverse set of heterologous hosts, which may also provide insights into the BGC biosynthetic mechanism and the biological function of BGCs.Jia Jia ZhangXiaoyu TangMichelle ZhangDarlene NguyenBradley S. MooreAmerican Society for MicrobiologyarticleGram negativeheterologous expressionProteobacteriabroad-host-range vectorviolaceinMicrobiologyQR1-502ENmBio, Vol 8, Iss 5 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Gram negative heterologous expression Proteobacteria broad-host-range vector violacein Microbiology QR1-502
spellingShingle	Gram negative heterologous expression Proteobacteria broad-host-range vector violacein Microbiology QR1-502 Jia Jia Zhang Xiaoyu Tang Michelle Zhang Darlene Nguyen Bradley S. Moore Broad-Host-Range Expression Reveals Native and Host Regulatory Elements That Influence Heterologous Antibiotic Production in Gram-Negative Bacteria
description	ABSTRACT Heterologous expression has become a powerful tool for studying microbial biosynthetic gene clusters (BGCs). Here, we extend the transformation-associated recombination cloning and heterologous expression platform for microbial BGCs to include Gram-negative proteobacterial expression hosts. Using a broad-host-range expression platform, we test the implicit assumption that biosynthetic pathways are more successfully expressed in more closely related heterologous hosts. Cloning and expression of the violacein BGC from Pseudoalteromonas luteoviolacea 2ta16 revealed robust production in two proteobacterial hosts, Pseudomonas putida KT2440 and Agrobacterium tumefaciens LBA4404, but very little production of the antibiotic in various laboratory strains of Escherichia coli, despite their closer phylogenetic relationship. We identified a nonclustered LuxR-type quorum-sensing receptor from P. luteoviolacea 2ta16, PviR, that increases pathway transcription and violacein production in E. coli by ∼60-fold independently of acyl-homoserine lactone autoinducers. Although E. coli harbors the most similar homolog of PviR identified from all of the hosts tested, overexpression of various E. coli transcription factors did not result in a statistically significant increase in violacein production, while overexpression of two A. tumefaciens PviR homologs significantly increased production. Thus, this work not only introduces a new genetic platform for the heterologous expression of microbial BGCs, it also challenges the assumption that host phylogeny is an accurate predictor of host compatibility. IMPORTANCE Although Gram-positive heterologous hosts such as Streptomyces have been developed and optimized to support diverse secondary metabolic reactions, there has been comparatively less work on Gram-negative hosts, some of which grow faster and are easier to work with. This work presents a new genetic platform for direct cloning and broad-host-range heterologous expression of BGCs in Gram-negative proteobacterial expression hosts, and we leverage this platform to uncover regulatory elements that influence violacein expression from Pseudoalteromonas. Although it is often assumed that BGCs will be more successfully expressed in more closely related hosts, our work suggests that this may not be a general rule of thumb, as heterologous production of natural products can be influenced by specific host regulatory and/or biosynthetic elements, and the identity and effectiveness of those elements are difficult to predict. We argue for the use of a diverse set of heterologous hosts, which may also provide insights into the BGC biosynthetic mechanism and the biological function of BGCs.
format	article
author	Jia Jia Zhang Xiaoyu Tang Michelle Zhang Darlene Nguyen Bradley S. Moore
author_facet	Jia Jia Zhang Xiaoyu Tang Michelle Zhang Darlene Nguyen Bradley S. Moore
author_sort	Jia Jia Zhang
title	Broad-Host-Range Expression Reveals Native and Host Regulatory Elements That Influence Heterologous Antibiotic Production in Gram-Negative Bacteria
title_short	Broad-Host-Range Expression Reveals Native and Host Regulatory Elements That Influence Heterologous Antibiotic Production in Gram-Negative Bacteria
title_full	Broad-Host-Range Expression Reveals Native and Host Regulatory Elements That Influence Heterologous Antibiotic Production in Gram-Negative Bacteria
title_fullStr	Broad-Host-Range Expression Reveals Native and Host Regulatory Elements That Influence Heterologous Antibiotic Production in Gram-Negative Bacteria
title_full_unstemmed	Broad-Host-Range Expression Reveals Native and Host Regulatory Elements That Influence Heterologous Antibiotic Production in Gram-Negative Bacteria
title_sort	broad-host-range expression reveals native and host regulatory elements that influence heterologous antibiotic production in gram-negative bacteria
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/748b1cccf587411e8ba73b0dbd938e30
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Broad-Host-Range Expression Reveals Native and Host Regulatory Elements That Influence Heterologous Antibiotic Production in Gram-Negative Bacteria

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