Comparison of wild-type KT2440 and genome-reduced EM42 Pseudomonas putida strains for muconate production from aromatic compounds and glucose

• Published in: Metabolic engineering 2024-01, Vol.81, p.88-99
• Main Authors: Amendola, Caroline R., Cordell, William T., Kneucker, Colin M., Szostkiewicz, Caralyn J., Ingraham, Morgan A., Monninger, Michela, Wilton, Rosemarie, Pfleger, Brian F., Salvachúa, Davinia, Johnson, Christopher W., Beckham, Gregg T.
• Format: Article
• Language: English
• Subjects: Aromatic catabolism; Bioreactors; genome reduction; Glucose - metabolism; Muconic acid; Pseudomonas putida - genetics; Pseudomonas putida - metabolism; Pseudomonas putida EM42; Pseudomonas putida KT2440
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2023.11.004

Pseudomonas putida KT2440 is a robust, aromatic catabolic bacterium that has been widely engineered to convert bio-based and waste-based feedstocks to target products. Towards industrial domestication of P. putida KT2440, rational genome reduction has been previously conducted, resulting in P. putida strain EM42, which exhibited characteristics that could be advantageous for production strains. Here, we compared P. putida KT2440- and EM42-derived strains for cis,cis-muconic acid production from an aromatic compound, p-coumarate, and in separate strains, from glucose. To our surprise, the EM42-derived strains did not outperform the KT2440-derived strains in muconate production from either substrate. In bioreactor cultivations, KT2440- and EM42-derived strains produced muconate from p-coumarate at titers of 45 g/L and 37 g/L, respectively, and from glucose at 20 g/L and 13 g/L, respectively. To provide additional insights about the differences in the parent strains, we analyzed growth profiles of KT2440 and EM42 on aromatic compounds as the sole carbon and energy sources. In general, the EM42 strain exhibited reduced growth rates but shorter growth lags than KT2440. We also observed that EM42-derived strains resulted in higher growth rates on glucose compared to KT2440-derived strains, but only at the lowest glucose concentrations tested. Transcriptomics revealed that genome reduction in EM42 had global effects on transcript levels and showed that the EM42-derived strains that produce muconate from glucose exhibit reduced modulation of gene expression in response to changes in glucose concentrations. Overall, our results highlight that additional studies are warranted to understand the effects of genome reduction on microbial metabolism and physiology, especially when intended for use in production strains. •Comparison of genome-reduced EM42-and KT2440-derived strains to produce muconate.•EM42-derived strains did not show improvements relative to KT2440-derived strains.•Transcriptomics showed that EM42 has global changes in gene expression.•Genome reduction is a promising way to improve strains but is not universally effective.