Development of dual‐inducible duet‐expression vectors for tunable gene expression control and CRISPR interference‐based gene repression in Pseudomonas putida KT2440

Summary The development of P. putida as an industrial host requires a sophisticated molecular toolbox for strain improvement, including vectors for gene expression and repression. To augment existing expression plasmids for metabolic engineering, we developed a series of dual‐inducible duet‐expressi...

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Autores principales:	Rahul Gauttam, Aindrila Mukhopadhyay, Blake A. Simmons, Steven W. Singer
Formato:	article
Lenguaje:	EN
Publicado:	Wiley 2021
Materias:	Biotechnology TP248.13-248.65
Acceso en línea:	https://doaj.org/article/085bd22aa4b04e3fa97dece028780c1a
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spelling	oai:doaj.org-article:085bd22aa4b04e3fa97dece028780c1a2021-11-18T15:39:53ZDevelopment of dual‐inducible duet‐expression vectors for tunable gene expression control and CRISPR interference‐based gene repression in Pseudomonas putida KT24401751-791510.1111/1751-7915.13832https://doaj.org/article/085bd22aa4b04e3fa97dece028780c1a2021-11-01T00:00:00Zhttps://doi.org/10.1111/1751-7915.13832https://doaj.org/toc/1751-7915Summary The development of P. putida as an industrial host requires a sophisticated molecular toolbox for strain improvement, including vectors for gene expression and repression. To augment existing expression plasmids for metabolic engineering, we developed a series of dual‐inducible duet‐expression vectors for P. putida KT2440. A number of inducible promoters (Plac, Ptac, PtetR/tetA and Pbad) were used in different combinations to differentially regulate the expression of individual genes. Protein expression was evaluated by measuring the fluorescence of reporter proteins (GFP and RFP). Our experiments demonstrated the use of compatible plasmids, a useful approach to coexpress multiple genes in P. putida KT2440. These duet vectors were modified to generate a fully inducible CRISPR interference system using two catalytically inactive Cas9 variants from S. pasteurianus (dCas9) and S. pyogenes (spdCas9). The utility of developed CRISPRi system(s) was demonstrated by repressing the expression of nine conditionally essential genes, resulting in growth impairment and prolonged lag phase for P. putida KT2440 growth on glucose. Furthermore, the system was shown to be tightly regulated, tunable and to provide a simple way to identify essential genes with an observable phenotype.Rahul GauttamAindrila MukhopadhyayBlake A. SimmonsSteven W. SingerWileyarticleBiotechnologyTP248.13-248.65ENMicrobial Biotechnology, Vol 14, Iss 6, Pp 2659-2678 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Biotechnology TP248.13-248.65
spellingShingle	Biotechnology TP248.13-248.65 Rahul Gauttam Aindrila Mukhopadhyay Blake A. Simmons Steven W. Singer Development of dual‐inducible duet‐expression vectors for tunable gene expression control and CRISPR interference‐based gene repression in Pseudomonas putida KT2440
description	Summary The development of P. putida as an industrial host requires a sophisticated molecular toolbox for strain improvement, including vectors for gene expression and repression. To augment existing expression plasmids for metabolic engineering, we developed a series of dual‐inducible duet‐expression vectors for P. putida KT2440. A number of inducible promoters (Plac, Ptac, PtetR/tetA and Pbad) were used in different combinations to differentially regulate the expression of individual genes. Protein expression was evaluated by measuring the fluorescence of reporter proteins (GFP and RFP). Our experiments demonstrated the use of compatible plasmids, a useful approach to coexpress multiple genes in P. putida KT2440. These duet vectors were modified to generate a fully inducible CRISPR interference system using two catalytically inactive Cas9 variants from S. pasteurianus (dCas9) and S. pyogenes (spdCas9). The utility of developed CRISPRi system(s) was demonstrated by repressing the expression of nine conditionally essential genes, resulting in growth impairment and prolonged lag phase for P. putida KT2440 growth on glucose. Furthermore, the system was shown to be tightly regulated, tunable and to provide a simple way to identify essential genes with an observable phenotype.
format	article
author	Rahul Gauttam Aindrila Mukhopadhyay Blake A. Simmons Steven W. Singer
author_facet	Rahul Gauttam Aindrila Mukhopadhyay Blake A. Simmons Steven W. Singer
author_sort	Rahul Gauttam
title	Development of dual‐inducible duet‐expression vectors for tunable gene expression control and CRISPR interference‐based gene repression in Pseudomonas putida KT2440
title_short	Development of dual‐inducible duet‐expression vectors for tunable gene expression control and CRISPR interference‐based gene repression in Pseudomonas putida KT2440
title_full	Development of dual‐inducible duet‐expression vectors for tunable gene expression control and CRISPR interference‐based gene repression in Pseudomonas putida KT2440
title_fullStr	Development of dual‐inducible duet‐expression vectors for tunable gene expression control and CRISPR interference‐based gene repression in Pseudomonas putida KT2440
title_full_unstemmed	Development of dual‐inducible duet‐expression vectors for tunable gene expression control and CRISPR interference‐based gene repression in Pseudomonas putida KT2440
title_sort	development of dual‐inducible duet‐expression vectors for tunable gene expression control and crispr interference‐based gene repression in pseudomonas putida kt2440
publisher	Wiley
publishDate	2021
url	https://doaj.org/article/085bd22aa4b04e3fa97dece028780c1a
work_keys_str_mv	AT rahulgauttam developmentofdualinducibleduetexpressionvectorsfortunablegeneexpressioncontrolandcrisprinterferencebasedgenerepressioninpseudomonasputidakt2440 AT aindrilamukhopadhyay developmentofdualinducibleduetexpressionvectorsfortunablegeneexpressioncontrolandcrisprinterferencebasedgenerepressioninpseudomonasputidakt2440 AT blakeasimmons developmentofdualinducibleduetexpressionvectorsfortunablegeneexpressioncontrolandcrisprinterferencebasedgenerepressioninpseudomonasputidakt2440 AT stevenwsinger developmentofdualinducibleduetexpressionvectorsfortunablegeneexpressioncontrolandcrisprinterferencebasedgenerepressioninpseudomonasputidakt2440
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Development of dual‐inducible duet‐expression vectors for tunable gene expression control and CRISPR interference‐based gene repression in Pseudomonas putida KT2440

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