Modification of PapA5 acyltransferase substrate selectivity for optimization of short-chain alcohol-derived multimethyl-
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BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Modification of PapA5 acyltransferase substrate selectivity for optimization of short-chain alcohol-derived multimethyl-branched ester production in Escherichia coli Julia Roulet 1,2 & Virginia Galván 1,2 & Julia Lara 1,2 & Mario O. Salazar 2 & Valeria Cholich 2 & Hugo Gramajo 1,2 & Ana Arabolaza 1,2 Received: 11 May 2020 / Revised: 10 August 2020 / Accepted: 31 August 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Plant waxes are interesting substitutes of fossil-derived compounds; however, their limited sources and narrow structural diversity prompted the development of microbial platforms to produce esters with novel chemical structures and properties. One successful strategy was the heterologous expression of the mycocerosic polyketide synthase-based biosynthetic pathway (MAS-PKS, PapA5 and FadD28 enzymes) from Mycobacterium tuberculosis in Escherichia coli. This recombinant strain has the ability to produce a broad spectrum of multimethyl-branched long-chain esters (MBE) with novel chemical structures and high oxidation stability. However, one limitation of this microbial platform was the low yields obtained for MBE derived of short-chain alcohols. In an attempt to improve the titers of the short-chain alcohol-derived MBE, we focused on the PapA5 acyltransferase—enzyme that catalyzes the ester formation reaction. Specific amino acid residues located in the two-substrate recognition channels of this enzyme were identified, rationally mutated, and the corresponding mutants characterized both in vivo and in vitro. The phenylalanine located at 331 position in PapA5 (F331) was found to be a key residue that when substituted by other bulky and aromatic or bulky and polar amino acid residues (F331W, F331Y or F331H), gave rise to PapA5 mutants with improved bioconversion efficiency; showing in average, 2.5 higher yields of short-chain alcohol-derived MBE compared with the wild-type enzyme. Furthermore, two alternative pathways for synthetizing ethanol were engineered into the MBE producer microorganism, allowing de novo production of ethanol-derived MBE at levels comparable with those obtained by the external supply of this alcohol. Key points • Mutation in channel 2 changes PapA5 acyltransferase bioconversion efficiency. • Improved production of short-chain alcohol derived multimethyl-branched esters. • Establishing ethanologenic pathways for de novo production of ethanol derived MBE. • Characterization of a novel phenylethanol-derived MBE. Keywords Acyltransferase . Polyketide-associated protein . Substrate selectivity . Mutant variants
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00253-020-10872-w) contains supplementary material, which is available to authorized users. * Hugo Gramajo [email protected] * Ana Arabolaza [email protected]
1
IBR (Instituto de Biología Molecular y Celular de Rosario), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET),
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