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Title: Expression of cocoa genes in Saccharomyces cerevisiae improves cocoa butter production

Abstract

Cocoa butter (CB) extracted from cocoa beans (Theobroma cacao) is the main raw material for chocolate production, but CB supply is insufficient due to the increased chocolate demand and limited CB production. CB is mainly composed of three different kinds of triacylglycerols (TAGs), 1,3-dipalmitoyl-2-oleoyl-glycerol (POP, C16:0-C18:1-C16:0), 1-palmitoyl-3-stearoyl-2-oleoyl-glycerol (POS, C16:0-C18:1-C18:0) and 1,3-distearoyl-2-oleoyl-glycerol (SOS, C18:0-C18:1-C18:0). In general, Saccharomyces cerevisiae produces TAGs as storage lipids, which consist of C16 and C18 fatty acids. However, cocoa butter-like lipids (CBL, which are composed of POP, POS and SOS) are not among the major TAG forms in yeast. TAG biosynthesis is mainly catalyzed by three enzymes: glycerol-3-phosphate acyltransferase (GPAT), lysophospholipid acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT), and it is essential to modulate the yeast TAG biosynthetic pathway for higher CBL production. We cloned seven GPAT genes and three LPAT genes from cocoa cDNA, in order to screen for CBL biosynthetic gene candidates. By expressing these cloned cocoa genes and two synthesized cocoa DGAT genes in S. cerevisiae, we successfully increased total fatty acid production, TAG production and CBL production in some of the strains. In the best producer, the potential CBL content was eightfold higher than the control strain, suggesting the cocoa genes expressed in thismore » strain were functional and might be responsible for CBL biosynthesis. Moreover, the potential CBL content increased 134-fold over the control Y29-TcD1 (IMX581 sct1Δ ale1Δ lro1Δ dga1Δ with TcDGAT1 expression) in strain Y29-441 (IMX581 sct1Δ ale1Δ lro1Δ dga1Δ with TcGPAT4, TcLPAT4 and TcDGAT1 expression) further suggesting cocoa GPAT and LPAT genes functioned in yeast. We demonstrated that cocoa TAG biosynthetic genes functioned in S. cerevisiae and identified cocoa genes that may be involved in CBL production. Moreover, we found that expression of some cocoa CBL biosynthetic genes improved potential CBL production in S. cerevisiae, showing that metabolic engineering of yeast for cocoa butter production can be realized by manipulating the key enzymes GPAT, LPAT and DGAT in the TAG biosynthetic pathway.« less

Authors:
 [1];  [2];  [2];  [2];  [3]
  1. Chalmers Univ of Tech., Gothenburg (Sweden); Chinese Academy of Sciences, Shanghai (China)
  2. Chalmers Univ of Tech., Gothenburg (Sweden)
  3. Chalmers Univ of Tech., Gothenburg (Sweden); Technical Univ. of Denmark, Lyngby (Denmark)
Publication Date:
Research Org.:
Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1510820
Grant/Contract Number:  
SC0008744
Resource Type:
Accepted Manuscript
Journal Name:
Microbial Cell Factories
Additional Journal Information:
Journal Volume: 17; Journal Issue: 1; Journal ID: ISSN 1475-2859
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Yeast cell factories; Cocoa butter-like lipid; Theobroma cacao; TAG biosynthetic genes; Metabolic engineering; Synthetic biology

Citation Formats

Wei, Yongjun, Bergenholm, David, Gossing, Michael, Siewers, Verena, and Nielsen, Jens. Expression of cocoa genes in Saccharomyces cerevisiae improves cocoa butter production. United States: N. p., 2018. Web. https://doi.org/10.1186/s12934-018-0866-2.
Wei, Yongjun, Bergenholm, David, Gossing, Michael, Siewers, Verena, & Nielsen, Jens. Expression of cocoa genes in Saccharomyces cerevisiae improves cocoa butter production. United States. https://doi.org/10.1186/s12934-018-0866-2
Wei, Yongjun, Bergenholm, David, Gossing, Michael, Siewers, Verena, and Nielsen, Jens. Thu . "Expression of cocoa genes in Saccharomyces cerevisiae improves cocoa butter production". United States. https://doi.org/10.1186/s12934-018-0866-2. https://www.osti.gov/servlets/purl/1510820.
@article{osti_1510820,
title = {Expression of cocoa genes in Saccharomyces cerevisiae improves cocoa butter production},
author = {Wei, Yongjun and Bergenholm, David and Gossing, Michael and Siewers, Verena and Nielsen, Jens},
abstractNote = {Cocoa butter (CB) extracted from cocoa beans (Theobroma cacao) is the main raw material for chocolate production, but CB supply is insufficient due to the increased chocolate demand and limited CB production. CB is mainly composed of three different kinds of triacylglycerols (TAGs), 1,3-dipalmitoyl-2-oleoyl-glycerol (POP, C16:0-C18:1-C16:0), 1-palmitoyl-3-stearoyl-2-oleoyl-glycerol (POS, C16:0-C18:1-C18:0) and 1,3-distearoyl-2-oleoyl-glycerol (SOS, C18:0-C18:1-C18:0). In general, Saccharomyces cerevisiae produces TAGs as storage lipids, which consist of C16 and C18 fatty acids. However, cocoa butter-like lipids (CBL, which are composed of POP, POS and SOS) are not among the major TAG forms in yeast. TAG biosynthesis is mainly catalyzed by three enzymes: glycerol-3-phosphate acyltransferase (GPAT), lysophospholipid acyltransferase (LPAT) and diacylglycerol acyltransferase (DGAT), and it is essential to modulate the yeast TAG biosynthetic pathway for higher CBL production. We cloned seven GPAT genes and three LPAT genes from cocoa cDNA, in order to screen for CBL biosynthetic gene candidates. By expressing these cloned cocoa genes and two synthesized cocoa DGAT genes in S. cerevisiae, we successfully increased total fatty acid production, TAG production and CBL production in some of the strains. In the best producer, the potential CBL content was eightfold higher than the control strain, suggesting the cocoa genes expressed in this strain were functional and might be responsible for CBL biosynthesis. Moreover, the potential CBL content increased 134-fold over the control Y29-TcD1 (IMX581 sct1Δ ale1Δ lro1Δ dga1Δ with TcDGAT1 expression) in strain Y29-441 (IMX581 sct1Δ ale1Δ lro1Δ dga1Δ with TcGPAT4, TcLPAT4 and TcDGAT1 expression) further suggesting cocoa GPAT and LPAT genes functioned in yeast. We demonstrated that cocoa TAG biosynthetic genes functioned in S. cerevisiae and identified cocoa genes that may be involved in CBL production. Moreover, we found that expression of some cocoa CBL biosynthetic genes improved potential CBL production in S. cerevisiae, showing that metabolic engineering of yeast for cocoa butter production can be realized by manipulating the key enzymes GPAT, LPAT and DGAT in the TAG biosynthetic pathway.},
doi = {10.1186/s12934-018-0866-2},
journal = {Microbial Cell Factories},
number = 1,
volume = 17,
place = {United States},
year = {2018},
month = {1}
}

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Figures / Tables:

Fig. 1 Fig. 1: Three Enzymes, GPAT, LPAT and DGAT, determine the TAG structure in the TAG biosynthetic pathway. G3P glycerol-3-phosphate, LPA lysophosphatidic acid, PA phosphatidic acid, DAG diacylglycerol, TAG triacylglycerol, GPAT glycerol-3-phosphate acyltransferase, LPAT lysophosphatidic acid acyltransferase, DGAT acyl-CoA:diacylglycerol acyltransferase, PDAT phospholipid:diacylglycerol acyltransferase. SCT1 and GPT2 are GPAT genes, SLC1 andmore » ALE1 are LPAT genes, DGA1 is the DGAT gene, LRO1 is the PDAT gene. The genes in red were deleted in some of the yeast strains used in this study« less

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