Development of genetic tools for heterologous protein expression in a pentose‐utilizing environmental isolate of Pseudomonas putida

Gauttam, Rahul; Eng, Thomas; Zhao, Zhiying; ul ain Rana, Qurrat; Simmons, Blake A.; Yoshikuni, Yasuo; Mukhopadhyay, Aindrila; Singer, Steven W.

doi:10.1111/1751-7915.14205

Title: Development of genetic tools for heterologous protein expression in a pentose‐utilizing environmental isolate of Pseudomonas putida

Journal Article · Tue Jan 24 00:00:00 EST 2023 · Microbial Biotechnology (Online)

DOI:https://doi.org/10.1111/1751-7915.14205· OSTI ID:1915688

Gauttam, Rahul ^[1];

^[1]; Zhao, Zhiying ^[2]; ul ain Rana, Qurrat ^[1]; Simmons, Blake A. ^[1]; Yoshikuni, Yasuo ^[2]; Mukhopadhyay, Aindrila ^[1];

^[1]

The Joint BioEnergy Institute Emeryville California USA, Biological Systems and Engineering Division Lawrence Berkeley National Laboratory Berkeley California USA
Joint Genome Institute Berkeley California USA, Environmental Genomics and Systems Biology Division Lawrence Berkeley National Laboratory Berkeley California USA

Abstract Pseudomonas putida has emerged as a promising host for the conversion of biomass‐derived sugars and aromatic intermediates into commercially relevant biofuels and bioproducts. Most of the strain development studies previously published have focused on P. putida KT2440, which has been engineered to produce a variety of non‐native bioproducts. However, P. putida is not capable of metabolizing pentose sugars, which can constitute up to 25% of biomass hydrolysates. Related P. putida isolates that metabolize a larger fraction of biomass‐derived carbon may be attractive as complementary hosts to P. putida KT2440. Here we describe genetic tool development for P. putida M2, a soil isolate that can metabolize pentose sugars. The functionality of five inducible promoter systems and 12 ribosome binding sites was assessed to regulate gene expression. The utility of these expression systems was confirmed by the production of indigoidine from C6 and C5 sugars. Chromosomal integration and expression of non‐native genes was achieved by using chassis‐independent recombinase‐assisted genome engineering (CRAGE) for single‐step gene integration of biosynthetic pathways directly into the genome of P. putida M2. These genetic tools provide a foundation to develop hosts complementary to P. putida KT2440 and expand the ability of this versatile microbial group to convert biomass to bioproducts.

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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:: DE‐AC02‐05CH11231; AC02-05CH11231

OSTI ID:: 1915688

Alternate ID(s):: OSTI ID: 1958351; OSTI ID: 1958753

Journal Information:: Microbial Biotechnology (Online), Journal Name: Microbial Biotechnology (Online) Vol. 16 Journal Issue: 3; ISSN 1751-7915

Publisher:: Wiley-BlackwellCopyright Statement

Country of Publication:: United Kingdom

Language:: English

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