Title: Construction of a novel dual-inducible duet-expression system for gene (over)expression in Pseudomonas putida

Abstract

Pseudomonas putida is a widely used host for metabolic engineering and synthetic biology. However, the use of P. putida has been hampered by the availability of a limited set of expression vectors for producing heterologous proteins. To widen the scope of expression vectors for gene co-expression studies, a previously established dual-inducible expression vector pRG_Duet2 developed for Corynebacterium glutamicum has been modified for use in P. putida. This expression vector, named pRGPDuo2, harbors two origins of replication, colE1 for replication in E. coli and pRO1600 for replication in P. putida. Two multiple cloning sites (MCS1 and MCS2) in pRGPDuo2 are individually controlled by inducible promoters Ptac or PtetR/tetA. Functional validation of pRGPDuo2 was confirmed by the co-expression of genes for the fluorescent proteins namely, superfolder green fluorescent protein (sfGFP), and red fluorescent protein (RFP). Moreover, the strength of the fluorescence signal was dependent on the inducer concentrations present in the culture medium. The expression vector pRGPDuo2 is an attractive addition to the existing repertoire of expression plasmids for expression profiling and adds to the tools available for P. putida metabolic engineering.

Authors:

Gauttam, Rahul; Mukhopadhyay, Aindrila; Singer, Steven W.

Publication Date:

Wed Jul 01 00:00:00 EDT 2020

Research Org.:

Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Org.:

USDOE Office of Science (SC)

OSTI Identifier:

1632983

Alternate Identifier(s):

OSTI ID: 1756343

Grant/Contract Number:  

AC02-05CH11231

Resource Type:

Published Article

Journal Name:

Plasmid

Additional Journal Information:

Journal Name: Plasmid Journal Volume: 110 Journal Issue: C; Journal ID: ISSN 0147-619X

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES