Metabolic engineering of Saccharomyces cerevisiae to produce a reduced viscosity oil from lignocellulose

Abstract

BackgroundAcetyl-triacylglycerols (acetyl-TAGs) are unusual triacylglycerol (TAG) molecules that contain an sn-3 acetate group. Compared to typical triacylglycerol molecules (here referred to as long chain TAGs; lcTAGs), acetyl-TAGs possess reduced viscosity and improved cold temperature properties, which may allow direct use as a drop-in diesel fuel. Their different chemical and physical properties also make acetyl-TAGs useful for other applications such as lubricants and plasticizers. Acetyl-TAGs can be synthesized by EaDAcT, a diacylglycerol acetyltransferase enzyme originally isolated from Euonymus alatus (Burning Bush). The heterologous expression of EaDAcT in different organisms, including Saccharomyces cerevisiae, resulted in the accumulation of acetyl-TAGs in storage lipids. Microbial conversion of lignocellulose into acetyl-TAGs could allow biorefinery production of versatile molecules for biofuel and bioproducts.ResultsIn order to produce acetyl-TAGs from abundant lignocellulose feedstocks, we expressed EaDAcT in S. cerevisiae previously engineered to utilize xylose as a carbon source. The resulting strains were capable of producing acetyl-TAGs when grown on different media. The highest levels of acetyl-TAG production were observed with growth on synthetic lab media containing glucose or xylose. Importantly, acetyl-TAGs were also synthesized by this strain in ammonia fiber expansion (AFEX)-pretreated corn stover hydrolysate (ACSH) at higher volumetric titers than previously published strains. The deletion of the four endogenous enzymes known to contribute to lcTAG production increased the proportion of acetyl-TAGs in the total storage lipids beyond that in existing strains, which will make purification of these useful lipids easier. Surprisingly, the strains containing the four deletions were still capable of synthesizing lcTAG, suggesting that the particular strain used in this study possesses additional undetermined diacylglycerol acyltransferase activity. Additionally, the carbon source used for growth influenced the accumulation of these residual lcTAGs, with higher levels in strains cultured on xylose containing media.ConclusionOur results demonstrate that S. cerevisiae can be metabolically engineered to produce acetyl-TAGs when grown on different carbon sources, including hydrolysate derived from lignocellulose. Deletion of four endogenous acyltransferases enabled a higher purity of acetyl-TAGs to be achieved, but lcTAGs were still synthesized. Longer incubation times also decreased the levels of acetyl-TAGs produced. Therefore, additional work is needed to further manipulate acetyl-TAG production in this strain of S. cerevisiae, including the identification of other TAG biosynthetic and lipolytic enzymes and a better understanding of the regulation of the synthesis and degradation of storage lipids.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0751-y) contains supplementary material, which is available to authorized users.

Overexpression of diacylglycerol acetyltransferase from Euonymus europaeus in Yarrowia lipolytica leads to the production of single‐cell oil enriched with 3‐acetyl‐1,2‐diacylglycerols

“…However, lipid accumulation was not significantly increased by the insertion of DAcT activity in Y. lipolytica . Similar results have been reported by Tran, Breuer, et al (), who transformed S. cerevisiae H1246 using EaDAcT isolated from Euonymus alatus . It was previously observed that the insertion of native DGA1 or DGA2 acyltransferase genes (Gajdoš, Ledesma‐Amaro, Nicaud, Čertík, & Rossignol, ) and DGAT2 gene from mouse (Ledesma‐Amaro, unpublished data) into the Q4 mutant of Y. lipolytica under the control of a strong promoter was able to trigger lipid accumulation at an even higher rate than the wild type.…”

“…However, lipid accumulation was not significantly increased by the insertion of DAcT activity in Y. lipolytica. Similar results have been reported by Tran, Breuer, et al (2017), who transformed S. cerevisiae H1246 using…”

Xylose Assimilation for the Efficient Production of Biofuels and Chemicals by Engineered Saccharomyces cerevisiae

“…In a recent study, Tran et al aimed to produce acetyl-triacylglycerols, which possess reduced viscosity and improved cold temperature properties over typical long-chain triacylglycerol molecules (lcTAGs). [76] A diacylglycerol acetyltransferase from Euonymus alatus (EaDAcT) was expressed in S. cerevisiae strains engineered to ferment xylose, enabling acetyl-TAGs accumulation using xylose as a carbon source. The deletion of four genes coding for enzymes involved in lcTAG production improved the proportion of acetyl-TAGs within total lipids.…”

“…Interestingly, xylose cultivation was found to increase the accumulation of lcTAGs but decreased the production of acetyl‐TAGs compared to glucose cultivation, indicating different metabolic patterns of the two sugars in lipids biosynthesis. [ 76 ]…”

Developments and opportunities in fungal strain engineering for the production of novel enzymes and enzyme cocktails for plant biomass degradation

“…To solve this problem, strain engineering has generated industrial fungal strains that have superior performance compared to wild type strains. Genetic engineering approaches have been developed for industrially used fungal species and strains in order to increase their enzyme production (Ha et al, 2011;Tran et al, 2017). However, most of these methods are available for only a limited number of model or industrial fungi.…”