HIGH CELL DENSITY ESCHERICHIA COLI COCULTURE SYSTEM FOR DE NOVO PRODUCTION OF MULTI-METHYL‑BRANCHED ESTERS

BRACALENTE F, SABATINI M, GALVÁN V, ARABOLAZA, A, GRAMAJO H

Congreso; SAIB PABMB 2019; 2019

BT-P19HIGH CELL DENSITY ESCHERICHIA COLI COCULTURE SYSTEM FOR DE NOVO PRODUCTION OF MULTI-METHYL‑BRANCHED ESTERSBracalente F, Sabatini M, Galván V, Arabolaza, A, Gramajo HIBR-CONICET. Facultad de Ciencias Bioquímicas y Farmacéuticas. UNR.E-mail: gramajo@ibr-conicet.gov.arMicrobial lipid production represents a potential alternative feedstock for oleochemical industries. In our laboratory, we managed to turn E. coli into a biofactory, by the heterologous expression of a polyketide synthase (PKS)-based biosynthetic pathway from Mycobacterium tuberculosis and redefinition of its biological role towards the production of a variety of multi-methyl-branched esters (MBE). The final step of this pathway involves the transesterification of a multi-methyl-branched fatty acid, synthetized by the PKS Mas, to an acceptor alcohol that was supplemented into the culture media. With the aim to develop a whole de novo bioprocess (i.e. without adding exogenous alcohol), an E. coli co-culture system was designed, where the MBE producing strain (RQ5 pMB23) is grown with another E. coli strain engineered to produce the branched alcohol isobutanol (CB1 pIAA11). Previous results indicated that the consortium CB1/RQ5, at intermediate inoculation ratios (~1:1) in batch cocultures of M9 medium, had a very closed productivity to the one exhibited by a monoculture based on the single-cell RQ5 pMB23 with the external addition of isobutanol to the growth media. Based on this evidence, in this work, we decided to scale-up this coculture system for the de novo production of MBE of isobutanol, so we undertook the optimization of a high cell density fed‑batch fermentation process. To set the cultivation conditions, 1 L fed-batch fermentations (BIOFlo115 Bioreactor) were carried out using the consortium CB1/ RQ5. Variables to evaluate were: (1) inoculation ratio between the two cells; (2) time course progression of each constituent strain; (3) alcohol accumulation; and both (4) final cell density and (5) quantity of MBE produced per DCW. Interestingly, we found out that, due to different growth rate of each strain, co-cultures with dominant CB1 ratios (above 4:1) were necessary to reach amounts of MBE comparable to the fermentation of RQ5 pMB23 monoculture. In conclusion, we demonstrate the feasibility and scability of the production of industrially relevant bio-esters by the coculture of two E. coli strain in a minimal medium.