Design of Escherichia coli coculture system to produce multi-methyl-branched esters

BRACALENTE FERNANDO; SABATINI MARTÍN; GALVAN VIRGINIA; GRAMAJO HUGO C.; ARABOLAZA ANA L.

Paraná

Congreso; Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2018

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Microbial lipid production represents a potential alternative feedstock for oleochemical industries. In our laboratory, we constructed an E. coli strain capable of producing multi-methyl branched wax esters (MBWE), by heterologous expression of three enzymes involved in the Mycobacterium tuberculosis mycoserosate biosynthesis pathway: FadD28; the PKS Mas, which synthetizes multi-methyl- branched fatty acids; and PapA5, that transfers the acyl group to an alcohol. In order to decrease the toxic effect of high alcohol concentration added to the media, we aimed to develop a coculture system, where the wax producing strain (RQ5 pMB22) is grown with another E. coli strain engineered to produce isobutanol.When RQ5 pMB22 was co-cultured with BL21(DE3) pIAA11 pIAA12, the functionality of this system was confirmed after TLC of total lipid extracts. Next, new alcohol producing strains were generated to increase alcohol titers. The productivity of the new strains, CB1 and CB2, were measured by GC-MS. In batch cultures, the CB1 strain was able to produce 18,75 ± 0,45 mM of isobutanol after 24 hs of induction, which represented an increase of 1800% with respect to BL21. When this strain was cocultivated with RQ5 pMB22 in a minimal M9 medium, the consortium CB1/RQ5, at the best inoculation ratio of 1:2, produced 30.48 ± 2.02 µg MBWE ml-1. This productivity was comparable to the 32.58 ± 1.53 µg MBWE ml-1 exhibited by a monoculture based on the single-cell RQ5 pMB23 with the external add of isobutanol to the growth media.In this work, we demonstrate that it is feasible to optimize the production of industrially relevant bio-esters by the simply coculture of two E. coli strain in a minimal medium.