Modification of PapA5 acyltransferase substrate selectivity for optimization of short-chain alcohol-derived multimethyl-branched ester production in Escherichia coli

JULIA ROULET; GALVAN V; LARA J; SALAZAR M; CHOLICH J; GRAMAJO H; ARABOLAZA A

APPLIED MICROBIOLOGY AND BIOTECHNOLOGY

SPRINGER

Lugar: Berlin; Año: 2020

0175-7598

Plant waxes are interesting substitutes of fossil-derived compounds; however, their limited sources and narrow structuraldiversity 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 hasthe ability to produce a broad spectrum of multimethyl-branched long-chain esters (MBE) with novel chemical structures andhigh oxidation stability. However, one limitation of this microbial platform was the low yields obtained for MBE derived ofshort-chain alcohols. In an attempt to improve the titers of the short-chain alcohol-derived MBE, we focused on the PapA5acyltransferase?enzyme that catalyzes the ester formation reaction. Specific amino acid residues located in the two-substraterecognition channels of this enzyme were identified, rationally mutated, and the corresponding mutants characterized bothin vivo and in vitro. The phenylalanine located at 331 position in PapA5 (F331) was found to be a key residue that whensubstituted by other bulky and aromatic or bulky and polar amino acid residues (F331W, F331Y or F331H), gave rise to PapA5mutants with improved bioconversion efficiency; showing in average, 2.5 higher yields of short-chain alcohol-derived MBEcompared with the wild-type enzyme. Furthermore, two alternative pathways for synthetizing ethanol were engineered into theMBE producer microorganism, allowing de novo production of ethanol-derived MBE at levels comparable with those obtainedby the external supply of this alcoh