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Keywords:

  • fatty acid ethyl esters;
  • glycerol;
  • acyltransferase;
  • Saccharomyces cerevisiae;
  • endogenously produced ethanol

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. REFERENCES

The high price of petroleum-based diesel fuel has led to the development of alternative fuels, such as ethanol. Saccharomyces cerevisiae was metabolically engineered to utilize glycerol as a substrate for ethanol production. For the synthesis of fatty acid ethyl esters (FAEEs) by engineered S. cerevisiae that utilize glycerol as substrate, heterologous expression of an unspecific acyltransferase from Acinetobacter baylyi with glycerol utilizing genes was established. As a result, the engineered YPH499 (pGcyaDak, pGupWs-DgaTCas) strain produced 0.24 g/L FAEEs using endogenous ethanol produced from glycerol. And this study also demonstrated the possibility of increasing FAEE production by enhancing ethanol production by minimizing the synthesis of glycerol. The overall FAEE production in strain YPH499 fps1Δ gpd2Δ (pGcyaDak, pGupWs-DgaTCas) was 2.1-fold more than in YPH499 (pGcyaDak, pGupWs-DgaTCas), with approximately 0.52 g/L FAEEs produced, while nearly 17 g/L of glycerol was consumed. These results clearly indicated that FAEEs were synthesized in engineered S. cerevisiae by esterifying exogenous fatty acids with endogenously produced ethanol from glycerol. This microbial system acts as a platform in applying metabolic engineering that allows the production of FAEEs from cheap and abundant substrates specifically glycerol through the use of endogenous bioethanol. Biotechnol. Bioeng. 2012;109: 110–115. © 2011 Wiley Periodicals, Inc.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. REFERENCES

Biodiesel is an interesting alternative energy source and is a potential substitute for petroleum-based fuel. However, a broader use of biodiesel and a more significant substitution for petroleum-based fuels in the future will only be possible if production processes are developed that are not solely based on oilseed crops but on less expensive sources (Azocar et al., 2010; Robles-Medina et al., 2009). For this reason, major efforts have been focused on microbial production of high-energy fuels by metabolic engineering (Canakci and Sanli, 2008).

Biodiesel is made from renewable biomass mainly by alkali-catalyzed transesterification of triacylglycerol (TAGs) from plant oils (Kaieda et al., 1999). It consists of monoalkyl esters of long-chain fatty acids with short-chain alcohols, primarily methanol and ethanol, resulting in fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs). Currently, FAMEs are produced primarily due to the lower price of methanol compared to ethanol. However, methanol has some limitations, namely, the fact that it is highly toxic and hazardous compare to ethanol. Thus, the use of ethanol for the production of FAEE-based biodiesel would result in a fully sustainable fuel, albeit at the expense of much higher production costs (Kalscheuer et al., 2006).

For synthesis of FAEEs, particular factors are needed, such as the generation of ethanol from carbohydrates and catalysis of the transesterification reaction yielding ethyl esters (Elbahloul and Steinbuchel, 2010; Kalscheuer et al., 2006). Esterification is defined as the formation of esters from alcohols and carboxylic acids and is catalyzed by acyltransferase (Du et al., 2008; Li et al., 2008). The acyltransferase reaction is mediated by wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT; the atfA gene product), the key enzyme for wax ester and TAG biosynthesis, utilizing long-chain fatty alcohols or diacylglycerols and fatty acid coenzyme A thioesters (acyl-CoA) as substrates. It has been reported that Acinetobacter bifunctional WS/DGAT can utilize acyl-CoAs of variable lengths, and its utility in producing fatty acid esters has been exploited by its overexpression in different hosts (Kalscheuer et al., 2004; Waltermann et al., 2007).

Microbial fermentation of glycerol has been extensively studied due to several advantages that include highly reduced substrate, glycerol, and the cost advantage of anerobic processes with less energy requirements. Glycerol can be used to replace traditional carbohydrates, such as sucrose, glucose, and starch, in some industrial fermentation processes due to its generation as an inevitable byproduct of biodiesel fuel production that has resulted in a dramatic decrease in crude glycerol prices (Choi, 2008). Previously, the conversion of glycerol to glycolytic intermediate dihydroxyacetone phosphate was demonstrated by overexpression of glycerol utilizing genes with a glycerol-uptake gene in S. cerevisiae (Yu et al., 2010a, b).

Microbial metabolic engineering presents a unique opportunity to lower the costs associated with the raw materials used in biodiesel production. The purpose of this study was to develop biological processes for production of biodiesel fuels, such as FAEEs, from low-priced glycerol a byproduct of the chemical transesterification process. Bioethanol is of great importance in that it is itself a biofuel and it is also required for the transesterification process in microdiesel biosynthesis. To this end, the use of glycerol to produce ethanol was accomplished and the glycerol production pathway was effectively disabled to increase ethanol production by engineered S. cerevisiae (Yu et al., 2010a, b). In this study, the biosynthesis of FAEEs using glycerol as a substrate was established by heterologous expression of the unspecific acyltransferase from A. baylyi strain in engineered S. cerevisiae. The engineered S. cerevisiae strain successfully produced FAEEs using endogenously produced ethanol. Furthermore, this study also demonstrated the possibility of increasing ethanol production by minimizing glycerol production, resulting in enhanced FAEEs synthesis. The aims of this study are to achieve a more competitive production cost and develop bioprocess for the production of fuels from glycerol.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. REFERENCES

Strains and Cultivation Conditions

The strains and plasmids used in this study are listed in Table I. The condition of cultivation was determined as described previously (Yu et al., 2010a, b). Strains of S. cerevisiae were cultivated at 30°C in synthetic minimal dropout medium lacking tryptophan and containing 0.6% yeast nitrogen basal medium (Difco, Detroit, MI), 0.13% yeast synthetic dropout supplement without tryptophan (Sigma, Deisenhofen, Germany), 0.0075% adenine hemisulfate, and 2% glucose or galactose.

Table I. Strains, plasmids, and primers used in this study.
Strain/plasmids/primerDescription/genotype/sequenceRefs.
Strains
 YPH499MATa ura3-52 lys2-801_amber ade2-101_ochre trp1-Δ63 his3-Δ200 leu2-Δ1 
 YPH499 fps1Δ gpd2ΔMATa ura3-52 lys2-801_amber ade2-101_ochre trp1-Δ63 his3-Δ200 leu2-Δ1 fps1Δ::KM gpd2Δ::ZEOYu et al. (2010b)
Plasmids
 pGcyaDakS. cerevisiae Gcy and Dak gene under control of Gal1Yu et al. (2010a)
 pGupCasS. cerevisiae Gup1 cassette gene under control of Gal10Yu et al. (2010a)
 pGupWs-DgaTCasS. cerevisiae Gup1 with atfA from A. baylyi cassette gene under control of Gal10This study
Primers
 WS-DgaT-FactagtcccgccgccaccaaggagatgcgcccttacatcThis study
 WS-DgaT-RactagtttaattggctgttttaatatcttcThis study

Heterologous Expression of WS/DGAT in S. cerevisiae

The atfA gene encoding WS/DGAT was amplified from A. baylyi genomic DNA by PCR with the oligonucleotides 5′-ACTAGTCCCGCCGCCACCAAGGAGATGCGCCCTTACATC-3′ containing a SpeI restriction site and a Kozak translation initiation sequence and 5′-ACTAGTTTAATTGGCTGTTTTAATATCTTC-3′ containing a SpeI restriction site. The PCR product was cloned into the SpeI-restricted vector pGup1 (Yu et al., 2010a, b) colinear to the GAL1 promoter inducible by galactose. The Gal1p-Gup1-atfA-CYC1t cassette was inserted into the BamHI site of YIP-5. The resulting integrating vector, pGupWs-DgaTCas (10.04 kb), contains a target sequence for homologous recombination and the URA3 blaster cassette for selection. The pGupWs-DgaTCas plasmid was linearized by SalI digestion and transformed into strains. Transformation of the integration plasmid pGupWs-DgaTCas into S. cerevisiae was carried out by the lithium acetate method with a YEASTMAKER yeast transformation system (Clontech Laboratories, Mountain View, CA). Yeast transformants were selected on SD agar plates containing G418 after 2–3 days (Yu et al., 2010a, b).

Determination of Enzyme Activities

Enzyme activities from engineered S. cerevisiae strains were determined as described previously (Kalscheuer and Steinbuchel, 2003) using 100 µg of protein in each assay. The sample assay was incubated at 35°C for 30 min, and the reaction was stopped by extraction with 500 µL of chloroform–methanol solution (1:1 v/v).

Fermentation Condition and Analytical Methods

Cultivations were performed at 30°C in 50 mL closed bottles with a 20 mL culture volume containing 0.02% galactose, 0.17% yeast YNB, and 0.13% TRP dropout amino acids supplemented with 2% glycerol, and kept at a constant stirring speed of 130 rpm. Initial concentrations were set at OD600 = 1 after inoculation. Fermentation experiments were performed in triplicate. The ethanol concentration was measured by gas chromatography (GC). GC conditions were determined as described previously (Yu et al., 2010a). For quantification of FAEEs, 5 mL culture broth was extracted with 5 mL chloroform/methanol (1:1 v/v) by vigorous vortexing for 5 min. After phase separation, the organic phase was withdrawn, evaporated to dryness, and redissolved in 1 mL chloroform/methanol (1:1 v/v). TLC was done as described previously using the petroleum ether/diethyl ether/acetic acid solvent system (75:5:1 v/v). Lipids were visualized by spraying with 40% sulfuric acid and charring. Ethyl oleate was purchased from Sigma–Aldrich Chemie (Steinheim, Germany) and used as a reference substance for FAEEs. The conditions for GC and GC/MS were determined as previously described (Kalscheuer et al., 2006).

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. REFERENCES

Establishment of FAEE Biosynthesis in Recombinant S. cerevisiae

The main purpose of this study is the development of processes to convert low-priced glycerol into higher-value production. In our previous study, we successfully increased ethanol production from glycerol through expression of glycerol utilization genes (Yu et al., 2010a, b). Using this system, efficient ethanol production from glycerol was achieved. To further develop the strain for the synthesis of FAEEs from glycerol, an artificial system was devised in S. cerevisiae through overexpression of WS/DGAT from A. baylyi in tandem with glycerol utilizing genes (Fig. 1, Table II). The plasmid map of the pGupWS-DgaTCas vector that contains the Gup1 (Yu et al., 2010a, b), plays a role in the uptake of glycerol with the atfA gene under a GAL1 promoter is shown in Figure 2. pGupWS-DgaTCas plasmid vector was integrated into S. cerevisiae genomic DNA for increased stability and reduced use of antibiotics (Fig. 2).

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Figure 1. Pathway involved in the metabolic engineering strategy. Relevant genes and corresponding enzymes are included. The names of the genes are shown beside the arrows. The abbreviations correspond to glycerol uptake protein (Gup1), glycerol dehydrogenase (Gcy1), dihydroxyacetone (DHA), dihydroxyacetone kinase (Dak), dihydroxyacetone phosphate (DHAP), glycerol 3-phosphate (G3p), glycerol 3-phosphate dehydrogenase (GPD2), glycerol facilitator (FPS1), wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT), coenzyme A (CoA), and pyrophosphate (PPi).

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Table II. Reaction scheme of the strategy for synthesis of FAEEs using glycerol.
StrategyInserted/overexpressed reaction
Strategy 1
Overexpression of Gcy1, Dak1, and Gup1Glycerol (ext) [RIGHTWARDS ARROW] Glycerol
Glycerol + NADP [RIGHTWARDS ARROW] NADPH + DHA
DHA + ATP [RIGHTWARDS ARROW] DHAP + ADP
DHAP + 2NAD + 2ADP [RIGHTWARDS ARROW] PYR + 2NADH + 2ATP
PYR [RIGHTWARDS ARROW] Acetaldehyde + CO2
Acetaldehyde + NADH [RIGHTWARDS ARROW] Ethanol + NAD
Strategy 2
Deletion of GPD2 and FPS1DHAP + NADH [RIGHTWARDS ARROW] G3P + NAD
Glycerol [RIGHTWARDS ARROW] Glycerol (ext)
Strategy 3
Overexpression of WS/DGATOleic acid + ATP [RIGHTWARDS ARROW] Oleoyl-coA + AMP + Pyrophosphate
Oleoyl-coA + Ethanol [RIGHTWARDS ARROW] Ethyl-oleate + Coenzyme A
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Figure 2. A diagram illustrating the constructions of plasmid pGupWs-DgaTCas used in this study.

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To confirm the enzyme activities, the reaction was done with crude extracts of recombinant S. cerevisiae strains. WS/DGAT from A. baylyi was successfully expressed as a functionally active enzyme in S. cerevisiae YPH499. The engineered YPH499 (pGcyaDak, pGupWs-DgaTCas) strain showed high levels of WS and DGAT activity, whereas YPH499 (pESC-TRP) and YPH499 (pGcyaDak, pGupCas) showed low levels of enzymatic activity (Table III). WS/DGAT, the key enzyme for fatty acid esters biosynthesis from A. baylyi was successfully expressed in engineered YPH499 (pGcyaDak, pGupCas) using glycerol as a substrate. As a result, the accumulation of TAGs and FAEEs in engineered S. cerevisiae expressing WS/DGAT was reflected by an increased of total fatty acid content, whereas recombinant storage lipid synthesis had no significant influence on fatty acid composition (data not shown).

Table III. WS and DGAT activities in crude extracts of engineered S. cerevisiae.*
StrainEnzyme specific activity (pmol [mg/min])
WSDGAT
  • *

    Yeast cells were cultivated for 24 h at 30°C in synthetic minimal dropout medium without tryptophan and containing 2% galactose. Data are the mean values of at least three independent experiments ± SD.

YPH499 (pESC-TRP)1.01 ± 0.033.32 ± 0.07
YPH499 (pGcyaDak, pGupWs-DgaTCas)32.10 ± 2.1225.17 ± 1.18

Ethanol Production and FAEE Biosynthesis by Engineered S. cerevisiae Using Glycerol

To confirm the synthesis of FAEEs deriving from endogenously produced ethanol from glycerol by expression of WS/DGAT, the engineered S. cerevisiae was examined from heterologous expression of the WS/DGAT gene with or without sodium oleate in the medium.

Ethanol production was improved when pGcyaDak and pGupWs-DgaTCas were overexpressed without sodium oleate in the medium (Fig. 3A). YPH499 (pGcyaDak, pGupWs-DgaTCas) produced 5.2 g/L ethanol, whereas YPH499 (pGcyaDak, pGupCas) produced 3.2 g/L after 72 h of cultivation. However, the synthesis of FAEEs was not increased. As shown in Figure 3B, FAEE synthesis was achieved when pGcyaDak and pGupWs-DgaTCas were overexpressed with sodium oleate in the medium. YPH499 (pGcyaDak, pGupWs-DgaTCas) produced 1.1 g/L ethanol, whereas YPH499 (pGcyaDak, pGupCas) produced 2.2 g/L after 72 h of cultivation (Fig. 3). Although ethanol yields were reduced, YPH499 (pGcyaDak, pGupWs-DgaTCas) was found to produce 0.24 g/L FAEEs (Fig. 3B). These results suggested that YPH499 (pGcyaDak, pGupWs-DgaTCas) successfully produced FAEEs using endogenous ethanol upon additional of exogenous fatty acids. Thus, these results clearly indicated that FAEEs were synthesized by overexpression of WS/DGAT using endogenously produced ethanol from glycerol.

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Figure 3. Ethanol production and TLC analysis of intracellular lipid accumulated by engineered strains. Cells were cultivated for 72 h at 30°C in synthetic minimal dropout medium without tryptophan supplemented with 0.02% galactose (A) or 0.02% galactose plus 0.1% sodium oleate (B). Symbols: triangles, YPH499 (pGcyaDak, pGupWs-DgaTCas); squares, YPH499 (pGcyaDak, pGupCas); and diamonds, YPH499 (pESC-TRP). Lane 1: YPH499 (pGcyaDak, pGupCas). Lane 2: YPH499 (pGcyaDak, pGupWs-DgaTCas).

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Escherichia coli was engineered to produce FAEEs directly from glucose and ethanol (Steen et al., 2010). E. coli does not produce FAEEs via its natural metabolism and also has some limitations regarding ethanol production, as it only synthesizes ethanol anerobically. E. coli was engineered for FAEE synthesis, by including the expression of pyruvate decarboxylase and alcohol dehydrogenase from Zymomonase mobilis and the unspecific acyltransferase from A. baylyi strain ADP1 (Kalscheuer et al., 2006).

In this study, S. cerevisiae strain were chosen for the synthesis of FAEEs as it is the classic ethanologenic yeast with good ethanol tolerant characteristics as compared to other yeasts and engineered bacteria (Alves-Rodrigues et al., 2006; Sharma et al., 1996). By combining the pathway for ethanol and FAEE biosynthesis, FAEE was successfully produced from glycerol as the carbon source using endogenously produced ethanol by engineered S. cerevisiae.

Enhanced FAEEs Biosynthesis in Engineered S. cerevisiae Using Glycerol

Ethanol is produced in amounts that are sufficient for the transesterification process to yield substantial amounts of FAEEs. It has been reported that additional ethanol results in increased FAEE yields in E. coli (Steen et al., 2010). To show the possibility of enhanced FAEEs synthesis, ethanol production in S. cerevisiae was successfully increased by metabolic engineering in overexpression or knockout strains. Recently, the performance of an integrated biocatalyst to improve the rate of ethanol production by deleting glycerol production pathway was evaluated. FPS1, a gene in the glycerol transport pathway, was first knocked out to prevent Fps1p from constitutively releasing glycerol and deletion of GPD2 in the glycerol synthesis pathway resulted in a significant reduction of glycerol, which increased ethanol yield as a consequence (Yu et al., 2010b). Hypothetically, this study assumed that as the ethanol production in engineered strains increases, the FAEE yields would also consequently increase. To further increase FAEE biosynthesis, the YPH499 fps1Δ gpd2Δ strain that minimized glycerol production to directly enhance ethanol production (Yu et al., 2010b).

FAEE yields were increased in YPH499 fps1Δ gpd2Δ (pGcyaDak, pGupWs-DgaTCas) compared to YPH499 (pGcyaDak, pGupWs-DgaTCas). YPH499 fps1Δ gpd2Δ (pGcyaDak, pGupWs-DgaTCas) produced 0.52 g/L FAEE, whereas YPH499 (pGcyaDak, pGupWs-DgaTCas) produced 0.24 g/L after 72 h of cultivation. Synthesis of FAEEs increased 2.1-fold in comparison to YPH499 (pGcyaDak, pGupWs-DgaTCas) after 72 h of cultivation (Fig. 4). Strain YPH499 fps1Δ gpd2Δ (pGcyaDak, pGupWs-DgaTCas) consumed almost 17 g/L of glycerol, while strain YPH499 (pGcyaDak, pGupWs-DgaTCas) consumed about 16.4 g/L glycerol over a period of 72 h. After 72 h of cultivation, YPH499 fps1Δ gpd2Δ (pGcyaDak, pGupWs-DgaTCas) produced 0.52 g/L FAEEs and produced 1.4 g/L ethanol (Fig. 5). From the results obtained, this study successfully increased the FAEEs synthesis in genetically enhanced ethanol producing strains using metabolic engineering. Thus, the microdiesel production from a cheap carbon source like glycerol using S. cerevisiae, as a production platform was accomplished. The main purpose of this article is that we demonstrated the possibility to recycle the byproduct of FAEEs for produce FAEEs. To this end we used endogenously produced ethanol for production of FAEEs using ethanol tolerance strain S. cerevisiae and also show the possibility of improved production through simply modified genetic engineering. To our knowledge, FAEEs production in S. cerevisiae from glycerol has not been reported yet.

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Figure 4. FAEE production and glycerol utilization by YPH499 (pGcyaDak, pGupWs-DgaTCas) and YPH499 fps1Δ gpd2Δ (pGcyaDak, pGupWs-DgaTCas). Symbols: squares, YPH499 fps1Δ gpd2Δ (pGcyaDak, pGupWs-DgaTCas); and triangles, YPH499 (pGcyaDak, pGupWs-DgaTCas). Lane 1: YPH499 (pGcyaDak, pGupWs-DgaTCas). Lane 2: YPH499 fps1Δ gpd2Δ (pGcyaDak, pGupWs-DgaTCas).

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Figure 5. FAEE (squares) and ethanol (triangles) production by YPH499 fps1Δ gpd2Δ (pGcyaDak, pGupWs-DgaTCas) strains using glycerol with sodium oleate.

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The development of cost-effective process to convert glycerol into fuels is hampered by significant roadblocks such as optimizing metabolic pathways and balancing the redox state in the engineered microbes. Furthermore, this study provides the basis to achieve more competitive production costs, and, therefore, a more substantial substitution of petroleum-derived fuels with biofuels in the future.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. REFERENCES

Biodiesel is gaining more attention due to the energy needs and environmental awareness. A substantial increase in biodiesel production and a more significant substitution of petroleum-based diesel fuel in the future will probably only be feasible when processes are developed that enable biodiesel synthesis from cheap and renewable carbon sources such as glycerol. Modification of metabolic pathways could be a powerful approach for production of biodiesel from renewable sources. Microdiesel production by engineered microorganisms could finally offer some major advantages over established conventional production processes.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. REFERENCES

This work was supported by the Advenced Biomass R&D Center (ABC) of Korea Grant funded by the Ministry of Education, Science and Technology (ABC-2010-0029799.

REFERENCES

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgements
  8. REFERENCES
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