Escherichia coli coculture for de novo production of esters derived of methyl-branched alcohols and multi-methyl branched fatty acids

BRACALENTE, FERNANDO; SABATINI, MARTÍN; ARABOLAZA, ANA (CORRESPONDING); GRAMAJO, HUGO (CORRESPONDING)

MICROBIAL CELL FACTORIES

BIOMED CENTRAL LTD

Año: 2022 vol. 21

1475-2859

Background: A broad diversity of natural and non-natural esters have now been made in bacteria, and in othermicroorganisms, as a result of original metabolic engineering approaches. However, the fact that the properties ofthese molecules, and therefore their applications, are largely defined by the structural features of the fatty acid andalcohol moieties, has driven a persistent interest in generating novel structures of these chemicals.Results: In this research, we engineered Escherichia coli to synthesize de novo esters composed of multi-methyl-branched-chain fatty acids and short branched-chain alcohols (BCA), from glucose and propionate. A coculture engi-neering strategy was developed to avoid metabolic burden generated by the reconstitution of long heterologousbiosynthetic pathways. The cocultures were composed of two independently optimized E. coli strains, one dedicatedto efficiently achieve the biosynthesis and release of the BCA, and the other to synthesize the multi methyl-branchedfatty acid and the corresponding multi-methyl-branched esters (MBE) as the final products. Response surface meth-odology, a cost-efficient multivariate statistical technique, was used to empirical model the BCA-derived MBE produc-tion landscape of the coculture and to optimize its productivity. Compared with the monoculture strategy, the utiliza-tion of the designed coculture improved the BCA-derived MBE production in 45%. Finally, the coculture was scaled upin a high-cell density fed-batch fermentation in a 2 L bioreactor by fine-tuning the inoculation ratio between the twoengineered E. coli strains.Conclusion: Previous work revealed that esters containing multiple methyl branches in their molecule presentfavorable physicochemical properties which are superior to those of linear esters. Here, we have successfully engi-neered an E. coli strain to broaden the diversity of these molecules by incorporating methyl branches also in thealcohol moiety. The limited production of these esters by a monoculture was considerable improved by a design ofa coculture system and its optimization using response surface methodology. The possibility to scale-up this processwas confirmed in high-cell density fed-batch fermentations.