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

Abstract

Background: Acetyl-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. Results: In 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. Themore » 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. Conclusion: Our 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.« less

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Univ. of Wisconsin, Madison, WI (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1618672
Alternate Identifier(s):
OSTI ID: 1427618
Grant/Contract Number:  
FC02-07ER64494
Resource Type:
Published Article
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Name: Biotechnology for Biofuels Journal Volume: 10 Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
Springer Science + Business Media
Country of Publication:
Netherlands
Language:
English
Subject:
09 BIOMASS FUELS; 59 BASIC BIOLOGICAL SCIENCES; Acetyl-TAGs; Saccharomyces cerevisiae; AFEX corn stover hydrolysate; Metabolic engineering

Citation Formats

Tran, Tam N. T., Breuer, Rebecca J., Avanasi Narasimhan, Ragothaman, Parreiras, Lucas S., Zhang, Yaoping, Sato, Trey K., and Durrett, Timothy P. Metabolic engineering of Saccharomyces cerevisiae to produce a reduced viscosity oil from lignocellulose. Netherlands: N. p., 2017. Web. doi:10.1186/s13068-017-0751-y.
Tran, Tam N. T., Breuer, Rebecca J., Avanasi Narasimhan, Ragothaman, Parreiras, Lucas S., Zhang, Yaoping, Sato, Trey K., & Durrett, Timothy P. Metabolic engineering of Saccharomyces cerevisiae to produce a reduced viscosity oil from lignocellulose. Netherlands. doi:10.1186/s13068-017-0751-y.
Tran, Tam N. T., Breuer, Rebecca J., Avanasi Narasimhan, Ragothaman, Parreiras, Lucas S., Zhang, Yaoping, Sato, Trey K., and Durrett, Timothy P. Mon . "Metabolic engineering of Saccharomyces cerevisiae to produce a reduced viscosity oil from lignocellulose". Netherlands. doi:10.1186/s13068-017-0751-y.
@article{osti_1618672,
title = {Metabolic engineering of Saccharomyces cerevisiae to produce a reduced viscosity oil from lignocellulose},
author = {Tran, Tam N. T. and Breuer, Rebecca J. and Avanasi Narasimhan, Ragothaman and Parreiras, Lucas S. and Zhang, Yaoping and Sato, Trey K. and Durrett, Timothy P.},
abstractNote = {Background: Acetyl-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. Results: In 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. Conclusion: Our 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.},
doi = {10.1186/s13068-017-0751-y},
journal = {Biotechnology for Biofuels},
number = 1,
volume = 10,
place = {Netherlands},
year = {2017},
month = {3}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1186/s13068-017-0751-y

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Cited by: 2 works
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Figures / Tables:

Fig. 1 Fig. 1: Acetyl-TAGs possess an sn-3 acetate group. Structures of representative acetyl-TAG and long-chain TAG (lcTAG) molecules

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    Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.