(569d) Biosynthesis of Wax Esters (Industry Candidate)

Microbial synthesis of oleochemicals is a promising path for the cost-effective production of wax esters (WE) from renewable and sustainable feedstocks. In this study, we explored the WE metabolic pathway in both Escherichia coli and Yarrowia lipolytica for biosynthesis of WE from both sugar and lipid substrates. First, the ScFAR from jojoba plant (Simmondsia chinensis), the MmFAR from house mouse (Mus musculus), and the MhFAR from Marinobacter hydrocarbonoclasticus strain VT8 together with the AbWS from Acinetobactor baylyi ADP1 were chosen to study the enzyme efficiency and the substrate specificity in the oleaginous yeast Y. lipolytica via ARS18-based plasmid expression. All enzyme combinations led to the successful WE production. Co-expression of cytoplasmic MhAFR and AbWS resulted in the high accumulation of intracellular WE. Second, the wild type Y. lipolytica ATCC20362 was engineered for WE production by heterologous expression of the MhFAR and AbWS via random genome integration. The E. coli BL21(DE3) was also used to overexpress two genes via a T7-pET plasmid expression system. To improve WE production, both hydrophilic and hydrophobic carbon sources were tested and optimized in shake flask experiments. Our results indicated that in the presence of exogenous oleic acid or oil-based substrates in the culture medium, the efficiency of WE synthesis in both Y. lipolytica and E. coli strains rely on external source of free fatty acids rather than endogenous de novo fatty acid biosynthesis. Switching from glucose to a fresh or waste plant oil improved the WE titer by 70-fold for the engineered Y. lipolytica, which led to a maximum of 7.6 g/L WE within 120hr of cultivation in flasks. The produced intracellular WE contributed to 57% of the yeast biomass, which is so far the highest level reported on microbial production of WE. The predominant intracellular WE in the presence of hydrophobic substrate as sole carbon source were C₃₆, C₃₄ and C₃₂, in order of decreasing abundance, with a large proportion being unsaturated. Co-feeding the glucose with oleic acid, the WE titer of 3.95 g/L and DCW of 19.47 g/L were achieved in engineered E. coli after 40hr fed-batch fermentation, which is also the highest titer reported so far for WE produced by a bacterium. Our research results paved the way for further commercial production of WE from a low-cost renewable feedstock.