Enhanced triacylglycerol production with genetically modified Trichosporon oleaginosus

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Abstract

Mitochondrial pyruvate dehydrogenase (PDH) is important in the production of lipids in oleaginous yeast, but other yeast may bypass the mitochondria (PDH bypass), converting pyruvate in the cytosol to acetaldehyde, then acetate and acetyl CoA which is further converted to lipids. Using a metabolic model based on the oleaginous yeast Yarrowia lipolytica, we found that introduction of this bypass to an oleaginous yeast should result in enhanced yield of triacylglycerol (TAG) on substrate. Trichosporon oleaginosus (formerly Cryptococcus curvatus) is an oleaginous yeast which can produce TAGs from both glucose and xylose. Based on the sequenced genome, it lacks at least one of the enzymes needed to complete the PDH bypass, acetaldehyde dehydrogenase (ALD), and may also be deficient in pyruvate decarboxylase and acetyl-CoA synthetase under production conditions. We introduced these genes to T. oleaginosus in various combinations and demonstrated that the yield of TAG on both glucose and xylose was improved, particularly at high C/N ratio. Expression of a phospholipid:diacyltransferase encoding gene in conjunction with the PDH bypass further enhanced lipid production. The yield of TAG on xylose (0.27 g/g) in the engineered strain approached the theoretical maximum yield of 0.289 g/g. Interestingly, TAG production was also enhanced compared to the control in some strains which were given only part of the bypass pathway, suggesting that these genes may contribute to alternative routes to cytoplasmic acetyl CoA. The metabolic model indicated that the improved yield of TAG on substrate in the PDH bypass was dependent on the production of NADPH by ALD. NADPH for lipid synthesis is otherwise primarily supplied by the pentose phosphate pathway (PPP). This would contribute to the greater improvement of TAG production from xylose compared to that observed from glucose when the PDH bypass was introduced, since xylose enters metabolism through the non-oxidative part of the PPP. Yield of TAG from xylose in the engineered strains (0.21-0.27 g/g) was comparable to that obtained from glucose and the highest so far reported for lipid or TAG production from xylose.

Original languageEnglish
Article number1337
JournalFrontiers in Microbiology
Volume9
Issue numberJUN
DOIs
Publication statusPublished - 21 Jun 2018
MoE publication typeA1 Journal article-refereed

Fingerprint

Trichosporon
Pyruvic Acid
Triglycerides
Xylose
Oxidoreductases
Yeasts
Lipids
Pentose Phosphate Pathway
Acetyl Coenzyme A
Acetate-CoA Ligase
Pyruvate Decarboxylase
Yarrowia
Genes
Cryptococcus
NADPH Dehydrogenase
Glucose
Acetaldehyde
NADP
Cytosol
Phospholipids

Keywords

  • Cryptococcus curvatus
  • Enhanced yield
  • Lipid production
  • Pyruvate dehydrogenase bypass
  • Triacylglycerol
  • Trichosporon oleaginosus
  • Yeast

Cite this

@article{822767dc23e24cff80ab9dc662895eb4,
title = "Enhanced triacylglycerol production with genetically modified Trichosporon oleaginosus",
abstract = "Mitochondrial pyruvate dehydrogenase (PDH) is important in the production of lipids in oleaginous yeast, but other yeast may bypass the mitochondria (PDH bypass), converting pyruvate in the cytosol to acetaldehyde, then acetate and acetyl CoA which is further converted to lipids. Using a metabolic model based on the oleaginous yeast Yarrowia lipolytica, we found that introduction of this bypass to an oleaginous yeast should result in enhanced yield of triacylglycerol (TAG) on substrate. Trichosporon oleaginosus (formerly Cryptococcus curvatus) is an oleaginous yeast which can produce TAGs from both glucose and xylose. Based on the sequenced genome, it lacks at least one of the enzymes needed to complete the PDH bypass, acetaldehyde dehydrogenase (ALD), and may also be deficient in pyruvate decarboxylase and acetyl-CoA synthetase under production conditions. We introduced these genes to T. oleaginosus in various combinations and demonstrated that the yield of TAG on both glucose and xylose was improved, particularly at high C/N ratio. Expression of a phospholipid:diacyltransferase encoding gene in conjunction with the PDH bypass further enhanced lipid production. The yield of TAG on xylose (0.27 g/g) in the engineered strain approached the theoretical maximum yield of 0.289 g/g. Interestingly, TAG production was also enhanced compared to the control in some strains which were given only part of the bypass pathway, suggesting that these genes may contribute to alternative routes to cytoplasmic acetyl CoA. The metabolic model indicated that the improved yield of TAG on substrate in the PDH bypass was dependent on the production of NADPH by ALD. NADPH for lipid synthesis is otherwise primarily supplied by the pentose phosphate pathway (PPP). This would contribute to the greater improvement of TAG production from xylose compared to that observed from glucose when the PDH bypass was introduced, since xylose enters metabolism through the non-oxidative part of the PPP. Yield of TAG from xylose in the engineered strains (0.21-0.27 g/g) was comparable to that obtained from glucose and the highest so far reported for lipid or TAG production from xylose.",
keywords = "Cryptococcus curvatus, Enhanced yield, Lipid production, Pyruvate dehydrogenase bypass, Triacylglycerol, Trichosporon oleaginosus, Yeast",
author = "Kari Koivuranta and Sandra Castillo and Paula Jouhten and Laura Ruohonen and Merja Penttil{\"a} and Wiebe, {Marilyn G.}",
year = "2018",
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day = "21",
doi = "10.3389/fmicb.2018.01337",
language = "English",
volume = "9",
journal = "Frontiers in Microbiology",
issn = "1664-302X",
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T1 - Enhanced triacylglycerol production with genetically modified Trichosporon oleaginosus

AU - Koivuranta, Kari

AU - Castillo, Sandra

AU - Jouhten, Paula

AU - Ruohonen, Laura

AU - Penttilä, Merja

AU - Wiebe, Marilyn G.

PY - 2018/6/21

Y1 - 2018/6/21

N2 - Mitochondrial pyruvate dehydrogenase (PDH) is important in the production of lipids in oleaginous yeast, but other yeast may bypass the mitochondria (PDH bypass), converting pyruvate in the cytosol to acetaldehyde, then acetate and acetyl CoA which is further converted to lipids. Using a metabolic model based on the oleaginous yeast Yarrowia lipolytica, we found that introduction of this bypass to an oleaginous yeast should result in enhanced yield of triacylglycerol (TAG) on substrate. Trichosporon oleaginosus (formerly Cryptococcus curvatus) is an oleaginous yeast which can produce TAGs from both glucose and xylose. Based on the sequenced genome, it lacks at least one of the enzymes needed to complete the PDH bypass, acetaldehyde dehydrogenase (ALD), and may also be deficient in pyruvate decarboxylase and acetyl-CoA synthetase under production conditions. We introduced these genes to T. oleaginosus in various combinations and demonstrated that the yield of TAG on both glucose and xylose was improved, particularly at high C/N ratio. Expression of a phospholipid:diacyltransferase encoding gene in conjunction with the PDH bypass further enhanced lipid production. The yield of TAG on xylose (0.27 g/g) in the engineered strain approached the theoretical maximum yield of 0.289 g/g. Interestingly, TAG production was also enhanced compared to the control in some strains which were given only part of the bypass pathway, suggesting that these genes may contribute to alternative routes to cytoplasmic acetyl CoA. The metabolic model indicated that the improved yield of TAG on substrate in the PDH bypass was dependent on the production of NADPH by ALD. NADPH for lipid synthesis is otherwise primarily supplied by the pentose phosphate pathway (PPP). This would contribute to the greater improvement of TAG production from xylose compared to that observed from glucose when the PDH bypass was introduced, since xylose enters metabolism through the non-oxidative part of the PPP. Yield of TAG from xylose in the engineered strains (0.21-0.27 g/g) was comparable to that obtained from glucose and the highest so far reported for lipid or TAG production from xylose.

AB - Mitochondrial pyruvate dehydrogenase (PDH) is important in the production of lipids in oleaginous yeast, but other yeast may bypass the mitochondria (PDH bypass), converting pyruvate in the cytosol to acetaldehyde, then acetate and acetyl CoA which is further converted to lipids. Using a metabolic model based on the oleaginous yeast Yarrowia lipolytica, we found that introduction of this bypass to an oleaginous yeast should result in enhanced yield of triacylglycerol (TAG) on substrate. Trichosporon oleaginosus (formerly Cryptococcus curvatus) is an oleaginous yeast which can produce TAGs from both glucose and xylose. Based on the sequenced genome, it lacks at least one of the enzymes needed to complete the PDH bypass, acetaldehyde dehydrogenase (ALD), and may also be deficient in pyruvate decarboxylase and acetyl-CoA synthetase under production conditions. We introduced these genes to T. oleaginosus in various combinations and demonstrated that the yield of TAG on both glucose and xylose was improved, particularly at high C/N ratio. Expression of a phospholipid:diacyltransferase encoding gene in conjunction with the PDH bypass further enhanced lipid production. The yield of TAG on xylose (0.27 g/g) in the engineered strain approached the theoretical maximum yield of 0.289 g/g. Interestingly, TAG production was also enhanced compared to the control in some strains which were given only part of the bypass pathway, suggesting that these genes may contribute to alternative routes to cytoplasmic acetyl CoA. The metabolic model indicated that the improved yield of TAG on substrate in the PDH bypass was dependent on the production of NADPH by ALD. NADPH for lipid synthesis is otherwise primarily supplied by the pentose phosphate pathway (PPP). This would contribute to the greater improvement of TAG production from xylose compared to that observed from glucose when the PDH bypass was introduced, since xylose enters metabolism through the non-oxidative part of the PPP. Yield of TAG from xylose in the engineered strains (0.21-0.27 g/g) was comparable to that obtained from glucose and the highest so far reported for lipid or TAG production from xylose.

KW - Cryptococcus curvatus

KW - Enhanced yield

KW - Lipid production

KW - Pyruvate dehydrogenase bypass

KW - Triacylglycerol

KW - Trichosporon oleaginosus

KW - Yeast

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U2 - 10.3389/fmicb.2018.01337

DO - 10.3389/fmicb.2018.01337

M3 - Article

AN - SCOPUS:85048936946

VL - 9

JO - Frontiers in Microbiology

JF - Frontiers in Microbiology

SN - 1664-302X

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