Functional expression and characterization of five wax ester synthases in Saccharomyces cerevisiae and their utility for biodiesel production

Abstract

Background Wax ester synthases (WSs) can synthesize wax esters from alcohols and fatty acyl coenzyme A thioesters. The knowledge of the preferred substrates for each WS allows the use of yeast cells for the production of wax esters that are high-value materials and can be used in a variety of industrial applications. The products of WSs include fatty acid ethyl esters, which can be directly used as biodiesel. Results Here, heterologous WSs derived from five different organisms were successfully expressed and evaluated for their substrate preference in Saccharomyces cerevisiae . We investigated the potential of the different WSs for biodiesel (that is, fatty acid ethyl esters) production in S. cerevisiae . All investigated WSs, from Acinetobacter baylyi ADP1, Marinobacter hydrocarbonoclasticus DSM 8798, Rhodococcus opacus PD630, Mus musculus C57BL/6 and Psychrobacter arcticus 273-4, have different substrate specificities, but they can all lead to the formation of biodiesel. The best biodiesel producing strain was found to be the one expressing WS from M. hydrocarbonoclasticus DSM 8798 that resulted in a biodiesel titer of 6.3 mg/L. To further enhance biodiesel production, acetyl coenzyme A carboxylase was up-regulated, which resulted in a 30% increase in biodiesel production. Conclusions Five WSs from different species were functionally expressed and their substrate preference characterized in S. cerevisiae , thus constructing cell factories for the production of specific kinds of wax ester. WS from M. hydrocarbonoclasticus showed the highest preference for ethanol compared to the other WSs, and could permit the engineered S. cerevisiae to produce biodiesel.

Enhancing fatty acid ethyl ester production in Saccharomyces cerevisiae through metabolic engineering and medium optimization

“…Recalling a final titer of 3.8 ± 0.3 mg/L for ScAT2200, this leads to a slight increase from ScAT2211, harboring the codon optimized atfA* , and a 49% increase from ScAT2212, expressing MhATF1* . These results are consistent with previous reports, which used codon‐optimized wax synthase genes and extensive genetic disruptions without nitrogen limitation (Runguphan and Keasling, ; Shi et al, ). They also show that the dramatic effect of culture conditions alone on FAEE production is comparable to other metabolic engineering strategies.…”

“…The inherent nature of S. cerevisiae to produce large quantities of ethanol directed our metabolic engineering strategy toward increasing the availability of the other substrate in FAEEs synthesis, acyl‐CoAs, in the cytosol by eliminating competing acyl‐CoA utilization pathways. This is in contrast to other reports, which aim to increase acyl‐CoA concentration by overexpressing genes involved in fatty acid synthesis (Runguphan and Keasling, ; Shi et al, ). Our strategies include (1) disrupting the transportation of acyl‐CoAs into the peroxisome for the β‐oxidation (Step 8, Fig.…”

“…Sequences of Acinetobacter sp. ADP1 atfA and Marinobacter hydrocarbonoclasticus DSM 8798 MhATF1 codon‐optimized for expression in S. cerevisiae were previously described (Shi et al, ) and synthesized as gBlock fragments (IDT Inc., Coralville, IA). Expression plasmids were constructed using Gibson Assembly (Gibson et al, ).…”

“…Recently, several strategies have been reported for metabolically engineering S. cerevisiae for enhanced FAEEs production. One strategy was to manipulate the fatty acid biosynthesis pathway by overexpressing or otherwise altering the regulation of acc1 and FAS proteins (Runguphan and Keasling, ; Shi et al, ). With this strategy, the highest titer of FAEEs produced directly from glucose were reported to be about 5.4 mg/L.…”

Rapid ester biosynthesis screening reveals a high activity alcohol‐O‐acyltransferase (AATase) from tomato fruit

“…Purification is also challenging requiring non‐ionic detergents or protein solubility tags to maintain activity . As a result the standard activity assay for volatile esters is headspace gas chromatography (GC) analysis of whole cell lysate reactions , while long chain acyl esters are quantified by thin layer chromatography and by GC‐MS . Such assays are not ideal for the rapid and high throughput screening necessary for effective enzyme discovery and engineering.…”