Limits...
Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid short- and branched-chain alkyl esters biodiesel.

Teo WS, Ling H, Yu AQ, Chang MW - Biotechnol Biofuels (2015)

Bottom Line: However, while fatty acid methyl or ethyl esters are useful biodiesel produced commercially, fatty acid esters with branched-chain alcohol moieties have superior fuel properties.Crucially, this includes improved cold flow characteristics, as one of the major problems associated with biodiesel use is poor low-temperature flow properties.Both enzymes were found to catalyze the formation of fatty acid esters, with different alcohol preferences.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, Singapore, 117597 Singapore ; NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore, 28 Medical Drive, Singapore, 117456 Singapore.

ABSTRACT

Background: Biodiesel is a mixture of fatty acid short-chain alkyl esters of different fatty acid carbon chain lengths. However, while fatty acid methyl or ethyl esters are useful biodiesel produced commercially, fatty acid esters with branched-chain alcohol moieties have superior fuel properties. Crucially, this includes improved cold flow characteristics, as one of the major problems associated with biodiesel use is poor low-temperature flow properties. Hence, microbial production as a renewable, nontoxic and scalable method to produce fatty acid esters with branched-chain alcohol moieties from biomass is critical.

Results: We engineered Saccharomyces cerevisiae to produce fatty acid short- and branched-chain alkyl esters, including ethyl, isobutyl, isoamyl and active amyl esters using endogenously synthesized fatty acids and alcohols. Two wax ester synthase genes (ws2 and Maqu_0168 from Marinobacter sp.) were cloned and expressed. Both enzymes were found to catalyze the formation of fatty acid esters, with different alcohol preferences. To boost the ability of S. cerevisiae to produce the aforementioned esters, negative regulators of the INO1 gene in phospholipid metabolism, Rpd3 and Opi1, were deleted to increase flux towards fatty acyl-CoAs. In addition, five isobutanol pathway enzymes (Ilv2, Ilv5, Ilv3, Aro10, and Adh7) targeted into the mitochondria were overexpressed to enhance production of alcohol precursors. By combining these engineering strategies with high-cell-density fermentation, over 230 mg/L fatty acid short- and branched-chain alkyl esters were produced, which is the highest titer reported in yeast to date.

Conclusions: In this work, we engineered the metabolism of S. cerevisiae to produce biodiesels in the form of fatty acid short- and branched-chain alkyl esters, including ethyl, isobutyl, isoamyl and active amyl esters. To our knowledge, this is the first report of the production of fatty acid isobutyl and active amyl esters in S. cerevisiae. Our findings will be useful for engineering S. cerevisiae strains toward high-level and sustainable biodiesel production.

No MeSH data available.


Metabolic engineering strategy to produce FASBEs. FASBEs can be produced by expressing a wax ester synthase (ws2 or Maqu_0168). Isobutanol pathway enzymes (Ilv2, Ilv5, Ilv3, Aro10, Adh7) were overexpressed in the mitochondria to accumulate more isobutanol and FABEs. At the same time, production of isoamyl alcohol and active amyl alcohols were also increased, resulting in increased FAIEs and FAAEs production. To boost FASBEs production, negative regulators of INO1 (Opi1, Rpd3) were deleted. Genes overexpressed are shown in green. Red crosses gene deletions
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4634726&req=5

Fig1: Metabolic engineering strategy to produce FASBEs. FASBEs can be produced by expressing a wax ester synthase (ws2 or Maqu_0168). Isobutanol pathway enzymes (Ilv2, Ilv5, Ilv3, Aro10, Adh7) were overexpressed in the mitochondria to accumulate more isobutanol and FABEs. At the same time, production of isoamyl alcohol and active amyl alcohols were also increased, resulting in increased FAIEs and FAAEs production. To boost FASBEs production, negative regulators of INO1 (Opi1, Rpd3) were deleted. Genes overexpressed are shown in green. Red crosses gene deletions

Mentions: On the other hand, only trace amounts of fatty acid isoamyl esters (FAIEs) and FAEEs were produced in an engineered yeast strain expressing wax ester synthase from A. baylyi ADP1 and with ARE1, ARE2, DGA1 and LRO1 disrupted [12, 13]. In addition, the metabolic engineering of yeast to produce and accumulate fatty acid isobutyl esters (FABEs) and fatty acid active amyl esters (FAAEs) has not been reported. Here, we engineered yeast to produce fatty acid short- and branched-chain esters (FASBEs), including ethyl, isobutyl, active amyl and isoamyl esters, using endogenously synthesized fatty acids and alcohols (Fig. 1). First, two wax ester synthase genes (ws2 and Maqu_0168 from Marinobacter sp.) were cloned and expressed. Second, negative regulators of the INO1 gene in phospholipid metabolism, Rpd3 and Opi1, were deleted. INO1 gene encodes for inositol-3-phosphate synthase that makes inositol phosphates and inositol-containing phospholipids. As synthesis of phospholipids requires fatty acyl-CoAs as precursors, the removal of INO1 negative regulators may boost flux towards fatty acyl-CoAs-derived phospholipids and the abovementioned esters [14].The deletion of RPD3 and OPI1 was shown previously to enable simultaneous increase of phospholipids and desired product 1-hexadecanol [15]. Third, isobutanol pathway enzymes (acetolactate synthase Ilv2, ketoacid reductoisomerase Ilv5, dihydroxyacid dehydratase Ilv3, α-ketoacid decarboxylase Aro10, and alcohol dehydrogenase Adh7) targeted into the mitochondria were overexpressed to boost production of alcohol precursors. Ilv2, Ilv5 and Ilv3 are naturally located in the mitochondria, whereas Aro10 and Adh7 were re-targeted to the mitochondria using N-terminal fusion with mitochondria localization signal from subunit IV of the yeast cytochrome c oxidase (encoded by COX4) [16, 17]. Compartmentalization of this pathway into the mitochondria enabled high-level production of branched-chain alcohols. Finally, by combining these engineering strategies with high-cell-density fermentation, over 230 mg/L FASBEs were produced, which represents the highest titer reported in yeast to date.Fig. 1


Metabolic engineering of Saccharomyces cerevisiae for production of fatty acid short- and branched-chain alkyl esters biodiesel.

Teo WS, Ling H, Yu AQ, Chang MW - Biotechnol Biofuels (2015)

Metabolic engineering strategy to produce FASBEs. FASBEs can be produced by expressing a wax ester synthase (ws2 or Maqu_0168). Isobutanol pathway enzymes (Ilv2, Ilv5, Ilv3, Aro10, Adh7) were overexpressed in the mitochondria to accumulate more isobutanol and FABEs. At the same time, production of isoamyl alcohol and active amyl alcohols were also increased, resulting in increased FAIEs and FAAEs production. To boost FASBEs production, negative regulators of INO1 (Opi1, Rpd3) were deleted. Genes overexpressed are shown in green. Red crosses gene deletions
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4634726&req=5

Fig1: Metabolic engineering strategy to produce FASBEs. FASBEs can be produced by expressing a wax ester synthase (ws2 or Maqu_0168). Isobutanol pathway enzymes (Ilv2, Ilv5, Ilv3, Aro10, Adh7) were overexpressed in the mitochondria to accumulate more isobutanol and FABEs. At the same time, production of isoamyl alcohol and active amyl alcohols were also increased, resulting in increased FAIEs and FAAEs production. To boost FASBEs production, negative regulators of INO1 (Opi1, Rpd3) were deleted. Genes overexpressed are shown in green. Red crosses gene deletions
Mentions: On the other hand, only trace amounts of fatty acid isoamyl esters (FAIEs) and FAEEs were produced in an engineered yeast strain expressing wax ester synthase from A. baylyi ADP1 and with ARE1, ARE2, DGA1 and LRO1 disrupted [12, 13]. In addition, the metabolic engineering of yeast to produce and accumulate fatty acid isobutyl esters (FABEs) and fatty acid active amyl esters (FAAEs) has not been reported. Here, we engineered yeast to produce fatty acid short- and branched-chain esters (FASBEs), including ethyl, isobutyl, active amyl and isoamyl esters, using endogenously synthesized fatty acids and alcohols (Fig. 1). First, two wax ester synthase genes (ws2 and Maqu_0168 from Marinobacter sp.) were cloned and expressed. Second, negative regulators of the INO1 gene in phospholipid metabolism, Rpd3 and Opi1, were deleted. INO1 gene encodes for inositol-3-phosphate synthase that makes inositol phosphates and inositol-containing phospholipids. As synthesis of phospholipids requires fatty acyl-CoAs as precursors, the removal of INO1 negative regulators may boost flux towards fatty acyl-CoAs-derived phospholipids and the abovementioned esters [14].The deletion of RPD3 and OPI1 was shown previously to enable simultaneous increase of phospholipids and desired product 1-hexadecanol [15]. Third, isobutanol pathway enzymes (acetolactate synthase Ilv2, ketoacid reductoisomerase Ilv5, dihydroxyacid dehydratase Ilv3, α-ketoacid decarboxylase Aro10, and alcohol dehydrogenase Adh7) targeted into the mitochondria were overexpressed to boost production of alcohol precursors. Ilv2, Ilv5 and Ilv3 are naturally located in the mitochondria, whereas Aro10 and Adh7 were re-targeted to the mitochondria using N-terminal fusion with mitochondria localization signal from subunit IV of the yeast cytochrome c oxidase (encoded by COX4) [16, 17]. Compartmentalization of this pathway into the mitochondria enabled high-level production of branched-chain alcohols. Finally, by combining these engineering strategies with high-cell-density fermentation, over 230 mg/L FASBEs were produced, which represents the highest titer reported in yeast to date.Fig. 1

Bottom Line: However, while fatty acid methyl or ethyl esters are useful biodiesel produced commercially, fatty acid esters with branched-chain alcohol moieties have superior fuel properties.Crucially, this includes improved cold flow characteristics, as one of the major problems associated with biodiesel use is poor low-temperature flow properties.Both enzymes were found to catalyze the formation of fatty acid esters, with different alcohol preferences.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, Singapore, 117597 Singapore ; NUS Synthetic Biology for Clinical and Technological Innovation (SynCTI), Life Sciences Institute, National University of Singapore, 28 Medical Drive, Singapore, 117456 Singapore.

ABSTRACT

Background: Biodiesel is a mixture of fatty acid short-chain alkyl esters of different fatty acid carbon chain lengths. However, while fatty acid methyl or ethyl esters are useful biodiesel produced commercially, fatty acid esters with branched-chain alcohol moieties have superior fuel properties. Crucially, this includes improved cold flow characteristics, as one of the major problems associated with biodiesel use is poor low-temperature flow properties. Hence, microbial production as a renewable, nontoxic and scalable method to produce fatty acid esters with branched-chain alcohol moieties from biomass is critical.

Results: We engineered Saccharomyces cerevisiae to produce fatty acid short- and branched-chain alkyl esters, including ethyl, isobutyl, isoamyl and active amyl esters using endogenously synthesized fatty acids and alcohols. Two wax ester synthase genes (ws2 and Maqu_0168 from Marinobacter sp.) were cloned and expressed. Both enzymes were found to catalyze the formation of fatty acid esters, with different alcohol preferences. To boost the ability of S. cerevisiae to produce the aforementioned esters, negative regulators of the INO1 gene in phospholipid metabolism, Rpd3 and Opi1, were deleted to increase flux towards fatty acyl-CoAs. In addition, five isobutanol pathway enzymes (Ilv2, Ilv5, Ilv3, Aro10, and Adh7) targeted into the mitochondria were overexpressed to enhance production of alcohol precursors. By combining these engineering strategies with high-cell-density fermentation, over 230 mg/L fatty acid short- and branched-chain alkyl esters were produced, which is the highest titer reported in yeast to date.

Conclusions: In this work, we engineered the metabolism of S. cerevisiae to produce biodiesels in the form of fatty acid short- and branched-chain alkyl esters, including ethyl, isobutyl, isoamyl and active amyl esters. To our knowledge, this is the first report of the production of fatty acid isobutyl and active amyl esters in S. cerevisiae. Our findings will be useful for engineering S. cerevisiae strains toward high-level and sustainable biodiesel production.

No MeSH data available.