Channelling carbon flux through the meta-cleavage route for improved poly(3-hydroxyalkanoate) production from benzoate and lignin-based aromatics in Pseudomonas putida H

Borrero-de Acuna, Jose Manuel; Gutierrez-Urrutia, Izabook; Hidalgo-Dumont, Cristian; Aravena-Carrasco, Carla; Orellana-Saez, Matias; Palominos-Gonzalez, Nestor; van Duuren, Jozef B. J. H.; Wagner, Viktoria; Glaser, Lars; Becker, Judith; Kohlstedt, Michael; Zacconi, Flavia C.; Wittmann, Christoph; Poblete-Castro, Ignacio

Abstract

Lignin-based aromatics are attractive raw materials to derive medium-chain length poly(3-hydroxyalkanoates) (mcl-PHAs), biodegradable polymers of commercial value. So far, this conversion has exclusively used the ortho-cleavage route of Pseudomonas putida KT2440, which results in the secretion of toxic intermediates and limited performance. Pseudomonas putida H exhibits the ortho- and the meta-cleavage pathways where the latter appears promising because it stoichiometrically yields higher levels of acetyl-CoA. Here, we created a double-mutant H-Delta catA Delta A2 that utilizes the meta route exclusively and synthesized 30% more PHA on benzoate than the parental strain but suffered from catechol accumulation. The single deletion of the catA2 gene in the H strain provoked a slight attenuation on the enzymatic capacity of the ortho route (25%) and activation of the meta route by nearly 8-fold, producing twice as much mcl-PHAs compared to the wild type. Inline, the mutant H-Delta catA2 showed a 2-fold increase in the intracellular malonyl-CoA abundance - the main precursor for mcl-PHAs synthesis. As inferred from flux simulation and enzyme activity assays, the superior performance of H-Delta catA2 benefited from reduced flux through the TCA cycle and malic enzyme and diminished by-product formation. In a benzoate-based fed-batch, P. putida H-Delta catA2 achieved a PHA titre of 6.1 g l(-1) and a volumetric productivity of 1.8 g l(-1) day(-1). Using Kraft lignin hydrolysate as feedstock, the engineered strain formed 1.4 g l(- 1) PHA. The balancing of carbon flux between the parallel catechol-degrading routes emerges as an important strategy to prevent intermediate accumulation and elevate mcl-PHA production in P. putida H and, as shown here, sets the next level to derive this sustainable biopolymer from lignin hydrolysates and aromatics.

Más información

Título según WOS: Channelling carbon flux through the meta-cleavage route for improved poly(3-hydroxyalkanoate) production from benzoate and lignin-based aromatics in Pseudomonas putida H
Título de la Revista: MICROBIAL BIOTECHNOLOGY
Número: 6
Editorial: Wiley
Fecha de publicación: 2020
DOI:

10.1111/1751-7915.13705

Notas: ISI