Improving acetate metabolism of Pseudomonas putida KT2440 by evolutionary and rational engineering
- 1Systems Biotechnology, Technical Faculty, Bielefeld University, Universitaetsstrasse 25, 33615 Bielefeld, Germany
Abstract
Acetate is a promising carbon source for microbial biotechnology as it can be produced sustainably from lignocellulosic biomass or C1 gases. Since acetate is directly activated to acetyl-CoA, it is especially suitable for producing acetyl-CoA-derived products, showcased here with the production of 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAAs). P. putida KT2440 can natively metabolize acetate, but the weak acid has also inhibitory effects on microbial growth. We present an in-depth study on the physiology of P. putida KT2440 using acetate as carbon and energy source and evaluate acetate as feedstock for the biosynthesis of HAAs. Initially, a rational engineering approach to overexpress acetyl-CoA synthetase for acetate activation resulted in an improved growth rate of 16% and reduced lag phase by six hours. To further increase the performance of P. putida KT2440 on acetate, adaptive laboratory evolution was performed. This resulted in an improvement in the growth rate from 0.4 h-1 to 0.6 h-1 and enabled growth on up to 12.5 g L-1 acetate with a shortened lag phase compared to the wild type. Whole-genome sequencing revealed mutations in proteins involved in gene expression regulation and signal transduction. This evolutionary engineering approach informed the deletions of gacS and crc, which resulted in a reduction in the lag phase from seven hours to one hour and an improvement of the growth rate by 25 %, matching the growth properties of the evolved clones. Using the evolved strains for the production of HAAs resulted in faster biomass and product formation with product titers reaching up to 94 % of that of the wild type. In conclusion, we identified mechanisms in the acetate metabolism of P. putida KT2440 and improved the growth performance of the strain by rational and evolutionary engineering, demonstrating the potential of the promising, but challenging 3rd generation feedstock acetate.
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BibTeX
@misc{filbig2026improving,
title = {{Improving acetate metabolism of Pseudomonas putida KT2440 by evolutionary and rational engineering}},
author = {M. Filbig and L. Wachtendonk and L. Hampe and I. Bator and J. Johnsen and E. T. Mohamed and N. Gurdo and J. Parschau and P. I. Nikel and A. M. Feist and T. Tiso and L. M. Blank},
howpublished = {bioRxiv},
year = {2026},
month = aug,
doi = {10.64898/2026.08.21.746131},
url = {https://www.biorxiv.org/content/10.64898/2026.08.21.746131v2},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - Improving acetate metabolism of Pseudomonas putida KT2440 by evolutionary and rational engineering
AU - M. Filbig
AU - L. Wachtendonk
AU - L. Hampe
AU - I. Bator
AU - J. Johnsen
AU - E. T. Mohamed
AU - N. Gurdo
AU - J. Parschau
AU - P. I. Nikel
AU - A. M. Feist
AU - T. Tiso
AU - L. M. Blank
PY - 2026
DA - 2026/08/21
PB - bioRxiv
DO - 10.64898/2026.08.21.746131
UR - https://www.biorxiv.org/content/10.64898/2026.08.21.746131v2
AB - Acetate is a promising carbon source for microbial biotechnology as it can be produced sustainably from lignocellulosic biomass or C1 gases. Since acetate is directly activated to acetyl-CoA, it is especially suitable for producing acetyl-CoA-derived products, showcased here with the production of 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAAs). P. putida KT2440 can natively metabolize acetate, but the weak acid has also inhibitory effects on microbial growth. We present an in-depth study on the physiology of P. putida KT2440 using acetate as carbon and energy source and evaluate acetate as feedstock for the biosynthesis of HAAs. Initially, a rational engineering approach to overexpress acetyl-CoA synthetase for acetate activation resulted in an improved growth rate of 16% and reduced lag phase by six hours. To further increase the performance of P. putida KT2440 on acetate, adaptive laboratory evolution was performed. This resulted in an improvement in the growth rate from 0.4 h-1 to 0.6 h-1 and enabled growth on up to 12.5 g L-1 acetate with a shortened lag phase compared to the wild type. Whole-genome sequencing revealed mutations in proteins involved in gene expression regulation and signal transduction. This evolutionary engineering approach informed the deletions of gacS and crc, which resulted in a reduction in the lag phase from seven hours to one hour and an improvement of the growth rate by 25 %, matching the growth properties of the evolved clones. Using the evolved strains for the production of HAAs resulted in faster biomass and product formation with product titers reaching up to 94 % of that of the wild type. In conclusion, we identified mechanisms in the acetate metabolism of P. putida KT2440 and improved the growth performance of the strain by rational and evolutionary engineering, demonstrating the potential of the promising, but challenging 3rd generation feedstock acetate.
ER -