PROCESS OPTIMIZATION FOR THE PRODUCTION OF A THERMOSTABLE STERYLGLUCOSIDASE IN ESCHERICHIA COLI

EBERHARDT, F.; HAILS, G.; AGUIRRE, A.; CERMINATTI, S.; PEIRU, S.; CASTELLI, M.E.; MENZELLA, H.

Dubrovnik

Conferencia; 9th Conference on Recombinant Protein Production (RPP9); 2017

Biodiesels produced from transesterification of vegetable oils have a major problem in quality due to the presence of precipitates,which are mostly composed of sterylglucosides (SGs). We have recently developed an enzymatic method for the efficient removalof SGs from biodiesel, based on the activity of a thermostable β-glycosidase with sterylglucosidase activity (SGase) fromThermococcus litoralis. However, the viability of the implementation of this process at industrial scale relies on the reduction of theproduction costs of this enzyme. In the present work we describe the development of an Escherichia coli-based manufacturingsystem, and a high cell density fermentation process to improve SGase production. Strain engineering includes the assessment ofdifferent promoters to drive the expression of a codon-optimized gene, and the co-expression of molecular chaperones. A high celldensity fermentation process was developed, where improvements in cost and efficiency of SGase production were achieved bythe optimization of the fed batch protocol and media composition (carbon source, inducer and post-induction nutrient feedingstrategies). A 300-fold increase in the production titers of SGase was achieved, which directly impacts on the costs of the industrialprocess for treating biodiesel.