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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.

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-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.

SUBMITTER: Borrero-de Acuna JM

PROVIDER: S-EPMC8601166 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The synthesis and isolation of a novel bimetallic species formed by reacting two equivalents of TMPLi with CuCl in the presence of Et2O are reported. X-ray crystallography reveals the Et2O-free tetranuclear aggregate (TMPCu)2(TMPLi)2 1, which formally results from the catenation of dimers of TMPLi and TMPCu. NMR spectroscopy confirms that, upon dissolution in hydrocarbon media, the crystals fail to form a conventional Gilman cuprate dimer. Instead they exhibit a spectrum which is consistent with that recently proposed for an isomer of dimeric Gilman cuprate. Moreover, while pre-isolated Gilman cuprate is inert to benzene solvent, this new isomer smoothly affects aromatic deprotonation to give mainly Ph(TMP)3Cu2Li2 3, which is formally a heterodimer of Gilman cuprate TMPCu(?-TMP)Li 2 and PhCu(?-TMP)Li 4. Attempts to synthesise 3 through explicit combination of pre-isolated 2 and 4 were successful; additionally, this permitted the preparation of Ph(TMP)3Cu3Li 5 and Ph(TMP)3CuLi3 7 when 4 was combined in 1?:?2 ratios with TMPCu or TMPLi, respectively. 5 was characterised as metallacyclic in the solid-state, its structural features resembling those in 3 but with reduced Li-? interactions. It also proved possible to perform Cu/Li exchange on 5 (using t BuOCu) to give a novel mixed organo(amido)copper species Ph(TMP)3Cu4 6. Remarkably, the unprecedented reactivity of 1 towards benzene is reproduced by heating a 1?:?1 mixture of TMPLi and TMPCu in the same solvent; this gives predominantly 3. On the other hand, mixtures which are rich in either Cu or Li can lead to the selective in situ formation of 5 or 7. Though crystallographic data on 7 could not be obtained, DFT calculations accurately corroborated the observed structures of 3 and 5 and could be used to support 7 having the same structure type, albeit with enhanced Li-? interactions. This was consistent with NMR spectroscopic data. However, in contrast to 3 and 5, for which 2D NMR spectroscopy indicated only conformational changes, 7 was additionally found to exhibit fluxionality in a manner consistent with a dissociative process.

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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.

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