Methanol-free biosynthesis of fatty acid methyl ester (FAME) in Synechocystis sp. PCC 6803

Abstract

To meet the increasing global demand of biodiesel over the next decades, alternative methods for producing one of the key constituents of biodiesel (e.g. fatty acid methyl esters (FAMEs)) are needed. Algal biodiesel has been a long-term target compromised by excessive costs for harvesting and processing. In this work, we engineered cyanobacteria to convert carbon dioxide into excreted FAME, without requiring methanol as a methyl donor. To produce FAME, acyl-ACP, a product of the fatty acid biosynthesis pathway, was first converted into free fatty acid (FFA) by a thioesterase, namely 'UcFatB1 from Umbellularia californica. Next, by employing a juvenile hormone acid O-methyltransferase (DmJHAMT) from Drosophila melanogaster and S-adenosylmethionine (SAM) as a methyl donor, FFAs were converted into corresponding FAMEs. The esters were naturally secreted extracellularly, allowing simple product separation by solvent overlay as opposed to conventional algae biodiesel production where the algae biomass must first be harvested and processed for transesterification of extracted triacylglycerols (TAGs). By optimizing both the promoter and RBS elements, up to 120 mg/L of FAMEs were produced in 10 days. Quantification of key proteins and metabolites, together with constructs over-expressing SAM synthetase (MetK), indicated that 'UcFatB1, MetK, and DmJHAMT were the main factors limiting pathway flux. In order to solve the latter limitation, two reconstructed ancestral sequences of DmJHAMT were also tried, resulting in strains showing a broader methyl ester chain-length profile in comparison to the native DmJHAMT. Altogether, this work demonstrates a promising pathway for direct sunlight-driven conversion of CO₂ into excreted FAME.

Keywords: Ancestral sequence reconstruction; Biodiesel; Cyanobacteria; Dodecanoic acid; FAME; Juvenile hormone acid O-methyl transferase; Metabolic engineering; Methyl laurate; SAM-Dependent methyl transferase.