PRODUCTION OF NEW OLEOCHEMICHALS IN MICROORGANISMS

PASCUTTI, FEDERICO; GRAMAJO, HUGO; ARABOLAZA, ANA LOREANA; ÁLVAREZ, HECTOR

Los Cocos

Congreso; SAMIGE 2022; 2022

Since the escalating cost of crude oil and the increasing concerns about its finite supply, there is anemerging strategic need to develop additional renewable products from plant, algae ormicroorganisms. In this context, there are considerable environmental and economic drivers todevelop new and improved bio-based chemicals. Particularly, oleochemicals such as free fatty acids (FA), triglycerides (TAG) and wax esters (WE) are considered as viable alternatives, to most of the currently used petrochemicals.On the other hand, oleochemicals generally derived from plants, are comprised mostly of long chainfatty acids (LCFA), whilst medium chain fatty acids are restricted only to a limited number of cropsthat are not suitable for their mass agronomic production. Neutral lipids, such as TAG and WE,comprised of medium chain fatty acids (MCFA) are gaining relevance in the food, jet-fuel, lubricants, plastics and cosmetic industries, given their differential physicochemical characteristics.Furthermore, the lubricant industry also relies on the use of petrochemicals derived components forthe production of synthetic or semi-synthetic WE. These molecules present several advantages overvegetable oils in their use as lubricants, mostly due to their high oxidation stabilities and resistanceto hydrolysis. Nevertheless, due to the high costs of obtaining WE from existing sources, their use islimited to specialized and high value product areas such as cosmetic, pharmaceutical and/orspecialty lubricants. In this context, microbial-cell-factories are an attractive model for theproduction of specific products as it provides the opportunity to convert sustainable biomass into high value chemicals. The assembly of metabolic activities derived from different organisms allowsthe reconstitution of designed biosynthetic pathways for the production of novel molecules withdesired features. With the aim of producing novel lipids, such as TAG and WE containing MCFA wepropose the synthesis of these molecules using Escherichia coli as a platform. To achieve this, we first sequenced the genome of two Rhodococcus strains isolated from contaminated sources, with the ability to synthesize neutral lipids esterified with diverse acyl-chain FA. Wax ester synthase/diacilglycerol: acyltrasnferase (WS/DGAT) are the key enzymes for production of WE andTAG in bacteria. We found 20 different WS/DGAT homologs from the sequenced genomes; 4 of them were selected based in phylogenetic analysis and heterologously expressed in a genetically modified E. coli (ΔdgkA ΔfadE ΔaraBAD) in order to analyze their ability to incorporate MCFA into the neutral lipids. Thin layer chromatography, GC-MS and LC-MS confirmed the synthesis of TAG containing MCFA in the four recombinant strains. Further research is necessary to determine which of these WS/DGAT enzymes is most proficient at incorporating MCFA into neutral lipids.