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

Abstract

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 (P lac , P tac , P tetR/tetA and P bad ) 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.

Authors:
 [1];  [1];  [1]; ORCiD logo [1]
  1. The Joint BioEnergy Institute Emeryville CA USA, Biological Systems and Engineering Division Lawrence Berkeley National Laboratory Berkeley CA USA
Publication Date:
Research Org.:
Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1786898
Alternate Identifier(s):
OSTI ID: 1786901; OSTI ID: 1798794
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Published Article
Journal Name:
Microbial Biotechnology (Online)
Additional Journal Information:
Journal Name: Microbial Biotechnology (Online) Journal Volume: 14 Journal Issue: 6; Journal ID: ISSN 1751-7915
Publisher:
Wiley-Blackwell
Country of Publication:
United Kingdom
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Gauttam, Rahul, Mukhopadhyay, Aindrila, Simmons, Blake A., and Singer, Steven W. Development of dual‐inducible duet‐expression vectors for tunable gene expression control and CRISPR interference‐based gene repression in Pseudomonas putida KT2440. United Kingdom: N. p., 2021. Web. doi:10.1111/1751-7915.13832.
Gauttam, Rahul, Mukhopadhyay, Aindrila, Simmons, Blake A., & Singer, Steven W. Development of dual‐inducible duet‐expression vectors for tunable gene expression control and CRISPR interference‐based gene repression in Pseudomonas putida KT2440. United Kingdom. https://doi.org/10.1111/1751-7915.13832
Gauttam, Rahul, Mukhopadhyay, Aindrila, Simmons, Blake A., and Singer, Steven W. Wed . "Development of dual‐inducible duet‐expression vectors for tunable gene expression control and CRISPR interference‐based gene repression in Pseudomonas putida KT2440". United Kingdom. https://doi.org/10.1111/1751-7915.13832.
@article{osti_1786898,
title = {Development of dual‐inducible duet‐expression vectors for tunable gene expression control and CRISPR interference‐based gene repression in Pseudomonas putida KT2440},
author = {Gauttam, Rahul and Mukhopadhyay, Aindrila and Simmons, Blake A. and Singer, Steven W.},
abstractNote = {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 (P lac , P tac , P tetR/tetA and P bad ) 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.},
doi = {10.1111/1751-7915.13832},
journal = {Microbial Biotechnology (Online)},
number = 6,
volume = 14,
place = {United Kingdom},
year = {Wed May 19 00:00:00 EDT 2021},
month = {Wed May 19 00:00:00 EDT 2021}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1111/1751-7915.13832

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