Engineering Pseudomonas putida for efficient aromatic conversion to bioproduct using high throughput screening in a bioreactor

Thomas Eng; Deepanwita Banerjee; Andrew K Lau; Emily Bowden; Robin A Herbert; Jessica Trinh; Jan-Philip Prahl; Adam Deutschbauer; Deepti Tanjore; Aindrila Mukhopadhyay

doi:10.1016/j.ymben.2021.04.015

Engineering Pseudomonas putida for efficient aromatic conversion to bioproduct using high throughput screening in a bioreactor

Metab Eng. 2021 Jul:66:229-238. doi: 10.1016/j.ymben.2021.04.015. Epub 2021 May 6.

Affiliations

¹ Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, 5885, Hollis Street, Emeryville, CA, USA; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Rd, Berkeley, CA, USA.
² Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Rd, Berkeley, CA, USA; Advanced Biofuels and Bioproducts Process Development Unit, Lawrence Berkeley National Laboratory, Hollis Street, Emeryville, CA, 5885, USA.
³ Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Rd, Berkeley, CA, USA.
⁴ Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, 5885, Hollis Street, Emeryville, CA, USA; Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Rd, Berkeley, CA, USA; Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Rd, Berkeley, CA, USA. Electronic address: amukhopadhyay@lbl.gov.

PMID: 33964456
DOI: 10.1016/j.ymben.2021.04.015

Abstract

Pseudomonas putida KT2440 is an emerging biomanufacturing host amenable for use with renewable carbon streams including aromatics such as para-coumarate. We used a pooled transposon library disrupting nearly all (4,778) non-essential genes to characterize this microbe under common stirred-tank bioreactor parameters with quantitative fitness assays. Assessing differential fitness values by monitoring changes in mutant strain abundance identified 33 gene mutants with improved fitness across multiple stirred-tank bioreactor formats. Twenty-one deletion strains from this subset were reconstructed, including GacA, a regulator, TtgB, an ABC transporter, and PP_0063, a lipid A acyltransferase. Thirteen deletion strains with roles in varying cellular functions were evaluated for conversion of para-coumarate, to a heterologous bioproduct, indigoidine. Several mutants, such as the ΔgacA strain improved fitness in a bioreactor by 35 fold and showed an 8-fold improvement in indigoidine production (4.5 g/L, 0.29 g/g, 23% of maximum theoretical yield) from para-coumarate as the carbon source.

Keywords: Bioreactor; Metabolic engineering; Para-coumarate; Pseudomonas putida; RB-TnSeq.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Bioreactors
Carbon
Gene Library
High-Throughput Screening Assays
Pseudomonas putida* / genetics

Substances

Carbon