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

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

Resultado de la investigación: Contribución a una revistaArtículorevisión exhaustiva

2 Citas (Scopus)

Resumen

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-ΔcatAΔA2 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-ΔcatA2 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-ΔcatA2 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-ΔcatA2 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.

Idioma originalInglés
PublicaciónMicrobial Biotechnology
DOI
EstadoEn prensa - 2020

Áreas temáticas de ASJC Scopus

  • Biotecnología
  • Bioingeniería
  • Bioquímica
  • Microbiología y biotecnología aplicadas

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