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

Autor: Borrero-de Acuña JM; Biosystems Engineering Laboratory, Center for Bioinformatics and Integrative Biology (CBIB), Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile., Gutierrez-Urrutia I; Biosystems Engineering Laboratory, Center for Bioinformatics and Integrative Biology (CBIB), Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile.; Institute of Systems Biotechnology, Saarland University, Saarbrücken, Germany., Hidalgo-Dumont C; Biosystems Engineering Laboratory, Center for Bioinformatics and Integrative Biology (CBIB), Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile., Aravena-Carrasco C; Biosystems Engineering Laboratory, Center for Bioinformatics and Integrative Biology (CBIB), Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile., Orellana-Saez M; Biosystems Engineering Laboratory, Center for Bioinformatics and Integrative Biology (CBIB), Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile., Palominos-Gonzalez N; Biosystems Engineering Laboratory, Center for Bioinformatics and Integrative Biology (CBIB), Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile., van Duuren JBJH; Institute of Systems Biotechnology, Saarland University, Saarbrücken, Germany., Wagner V; Institute of Systems Biotechnology, Saarland University, Saarbrücken, Germany., Gläser L; Institute of Systems Biotechnology, Saarland University, Saarbrücken, Germany., Becker J; Institute of Systems Biotechnology, Saarland University, Saarbrücken, Germany., Kohlstedt M; Institute of Systems Biotechnology, Saarland University, Saarbrücken, Germany., Zacconi FC; Facultad de Química y de Farmacia, Pontificia Universidad Católica de Chile, Santiago, Chile.; Institute for Biological and Medical Engineering, Schools of Engineering, Medicine and Biological Sciences, Pontificia Universidad Católica de Chile, Santiago, Chile., Wittmann C; Institute of Systems Biotechnology, Saarland University, Saarbrücken, Germany., Poblete-Castro I; Biosystems Engineering Laboratory, Center for Bioinformatics and Integrative Biology (CBIB), Faculty of Life Sciences, Universidad Andres Bello, Santiago, Chile.
Jazyk: angličtina
Zdroj: Microbial biotechnology [Microb Biotechnol] 2021 Nov; Vol. 14 (6), pp. 2385-2402. Date of Electronic Publication: 2020 Nov 10.
DOI: 10.1111/1751-7915.13705
Abstrakt: 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.
(© 2020 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)
Databáze: MEDLINE
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