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bioRxiv · 10.1101/2021.02.02.429437

High Throughput Fitness Profiling Reveals Loss Of GacS-GacA Regulation Improves Indigoidine Production In Pseudomonas putida

Eng, T. T.·Banerjee, D.·Lau, A. K.·Bowden, E.·Herbert, R. A.·Trinh, J.·Prahl, J.-P.·Deutschbauer, A.·Tanjore, D.·Mukhopadhyay, A.
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Abstract

Pseudomonas putida KT2440 is an emerging industrial microbe amenable for use with renewable carbon streams including aromatics such as para-coumarate (pCA). We examined this microbe under common stirred-tank bioreactor parameters with quantitative fitness assays using a pooled transposon library containing nearly all (4,778) non-essential genes. Assessing differential fitness values by monitoring changes in mutant strain abundance over time identified 31 genes with improved fitness in multiple bioreactor-relevant parameters. Twenty-one genes from this subset were reconstructed, including GacA, a signaling protein, TtgB, an ABC transporter, and PP_0063, a lipid A acyltransferase. Twelve deletion strains with roles in varying cellular functions were evaluated for conversion of pCA, to a heterologous bioproduct, indigoidine. Several mutants, such as the {Delta}gacA strain improved both fitness in a bioreactor and showed an 8-fold improvement in indigoidine production (4.5 g/L, 0.29 g/g pCA, 23% MTY) from pCA as the carbon source.

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Eng, T. T., Banerjee, D., Lau, A. K., Bowden, E., Herbert, R. A., Trinh, J., Prahl, J.-P., Deutschbauer, A., Tanjore, D., Mukhopadhyay, A.. 2021-02-03. High Throughput Fitness Profiling Reveals Loss Of GacS-GacA Regulation Improves Indigoidine Production In Pseudomonas putida. https://doi.org/10.1101/2021.02.02.429437

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