Examinando por Autor "Gutierrez-Urrutia, I."

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    Channelling carbon flux through the meta-cleavage route for improved poly(3-hydroxyalkanoate) production from benzoate and lignin-based aromatics in Pseudomonas putida H
    (Wiley Open Access [University Publisher] Society for Applied Microbiology [Associate Organisation], 2020) Borrero-de Acuna, J.; Gutierrez-Urrutia, I.; Hidalgo-Dumont, C.; Aravena-Carrasco, C.; Orellana-Saez, M.; Palominos-Gonzalez, N.; Duuren, J.; Wagner, V.; Glaser, L.; Becker, J.; Kohlstedt, M.; Zacconi, F.
    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.
  • Miniatura
    Ítem
    Enhanced synthesis of medium-chain-length poly(3-hydroxyalkanoates) by inactivating the tricarboxylate transport system of Pseudomonas putida KT2440 and process development using waste vegetable oil
    (Elsevier, 2019-02) Borrero-de Acuña, J.M.; Aravena-Carrasco, C.; Gutierrez-Urrutia, I.; Duchens, D.; Poblete-Castro, I.
    The use of waste materials as feedstock for biosynthesis of valuable compounds has been an intensive area of research aiming at diminishing the consumption of non-renewable materials. In this study, P. putida KT2440 was employed as a cell factory for the bioconversion of waste vegetable oil into medium-chain-length Polyhydroxyalkanoates. In the presence of the waste oil this environmental strain is capable of secreting enzymes with lipase activities that enhance the bioavailability of this hydrophobic carbon substrate. It was also found that the oxygen transfer coefficient is directly correlated with high PHA levels in KT2440 cells when metabolizing the waste frying oil. By knocking out the tctA gene, encoding for an enzyme of the tripartite carboxylate transport system, an enhanced intracellular level of mcl-PHA was found in the engineered strain when grown on fatty acids. Batch bioreactors showed that the KT2440 strain produced 1.01 (g⋅L −1 ) of PHA whereas the engineered ΔtctA P. putida strain synthesized 1.91 (g⋅L −1 ) after 72 h cultivation on 20 (g⋅L −1 ) of waste oil, resulting in a nearly 2-fold increment in the PHA volumetric productivity. Taken together, this work contributes to accelerate the pace of development for efficient bioconversion of waste vegetable oils into sustainable biopolymers. © 2018 Elsevier Ltd.