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

Gauttam, Rahul; Mukhopadhyay, Aindrila; Simmons, Blake A.; Singer, Steven W.

doi:10.1111/1751-7915.13832

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

Journal Article · Wed May 19 00:00:00 EDT 2021 · Microbial Biotechnology (Online)

DOI:https://doi.org/10.1111/1751-7915.13832· OSTI ID:1786898

Gauttam, Rahul ^[1]; Mukhopadhyay, Aindrila ^[1]; Simmons, Blake A. ^[1];

^[1]

The Joint BioEnergy Institute Emeryville CA USA, Biological Systems and Engineering Division Lawrence Berkeley National Laboratory Berkeley CA USA

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.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1786898

Alternate ID(s):: OSTI ID: 1786901; OSTI ID: 1798794

Journal Information:: Microbial Biotechnology (Online), Journal Name: Microbial Biotechnology (Online) Vol. 14 Journal Issue: 6; ISSN 1751-7915

Publisher:: Wiley-BlackwellCopyright Statement

Country of Publication:: United Kingdom

Language:: English

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