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Enhanced butanol production by eukaryotic Saccharomyces cerevisiae engineered to contain an improved pathway a
ab
a
ab
Hiroshi Sakuragi , Hironobu Morisaka , Kouichi Kuroda & Mitsuyoshi Ueda a
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan b
Kyoto Industrial Science and Technology Innovation Center, Kyoto, Japan Published online: 28 Oct 2014.
Click for updates To cite this article: Hiroshi Sakuragi, Hironobu Morisaka, Kouichi Kuroda & Mitsuyoshi Ueda (2015) Enhanced butanol production by eukaryotic Saccharomyces cerevisiae engineered to contain an improved pathway, Bioscience, Biotechnology, and Biochemistry, 79:2, 314-320, DOI: 10.1080/09168451.2014.972330 To link to this article: http://dx.doi.org/10.1080/09168451.2014.972330
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Bioscience, Biotechnology, and Biochemistry, 2015 Vol. 79, No. 2, 314–320
Enhanced butanol production by eukaryotic Saccharomyces cerevisiae engineered to contain an improved pathway Hiroshi Sakuragi1, Hironobu Morisaka1,2, Kouichi Kuroda1 and Mitsuyoshi Ueda1,2,* 1
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan; 2Kyoto Industrial Science and Technology Innovation Center, Kyoto, Japan
Received August 1, 2014; accepted September 14, 2014
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http://dx.doi.org/10.1080/09168451.2014.972330
Compared with ethanol, butanol has more advantageous physical properties as a fuel, and biobutanol is thus considered a promising biofuel material. Biobutanol has often been produced by Clostridium species; however, because they are strictly anaerobic microorganisms, these species are challenging to work with. We attempted to introduce the butanol production pathway into yeast Saccharomyces cerevisiae, which is a well-known microorganism that is tolerant to organic solvents. 1-Butanol was found to be produced at very low levels when the butanol production pathway of Clostridium acetobutylicum was simply introduced into S. cerevisiae. The elimination of glycerol production pathway in the yeast contributed to the enhancement of 1-butanol production. In addition, by the use of trans-enoylCoA reductase in the engineered pathway, 1-butanol production was markedly enhanced to yield 14.1 mg/L after 48 h of cultivation. Key words:
1-butanol production; Saccharomyces cerevisiae; Clostridium acetobutylicum; glycerol pathway; trans-enoyl-CoA reductase
The production of biofuels from renewable biomass such as grain and wood is facilitated by microbes. Bioethanol is regarded as one of the carbon neutral products, and thus, to date, it has been one of the most promising biofuels. Bioethanol is normally produced by Saccharomyces cerevisiae, an organism that has been used in the production of alcohols and breads for a long time. Recently, butanol has been attracting attention as a fuel more preferable to ethanol because of various significant advantages associated with the use of butanol as a fuel. Butanol can be used either in its pure form or as a mixture with gasoline at any concentration and, because of its lower vapor pressure, butanol is safer to use than ethanol.1) It is well known that 1-butanol can be produced by clostridia fermentation. Clostridia comprise a diverse group of anaerobic, spore-forming, Gram-positive bacteria that includes notable pathogens and industrially *Corresponding author. Email: [email protected] © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
significant microorganisms.2,3) Clostridium acetobutylicum has been found to produce acetone/butanol/ethanol at a ratio of 3:6:1 in a process called ABE fermentation.4,5) Bacterial production of butanol and acetone using the ABE fermentation process has been shown to be valuable in the production of the lacquer solvent butylacetate as well as in synthetic rubber industry. Due to the possibility of oil depletion, biofuel production is currently practiced worldwide. Thus, research and development into microbial butanol production is again being actively pursued.6) Using the several genes of C. acetobutylicum involved in the biosynthesis of 1-butanol, engineered Escherichia coli under aerobic conditions has been constructed.7) E. coli has also been shown to exhibit a tolerance to up to 1.5% butanol,8) which is comparable with the tolerance exhibited by clostridia. On the other hand, among eukaryotic cells, yeast S. cerevisiae has been described as an ideal host for butanol production. S. cerevisiae can be grown under both aerobic and anaerobic conditions, and has an inherent tolerance to organic solvents due to its extensive use in the industrial production of ethanol. S. cerevisiae is known to tolerate up to 2% butanol.9) Several approaches to the construction of a butanol-producing S. cerevisiae strain have been examined.10,11) In this study, we attempted to produce 1-butanol in S. cerevisiae by introducing the 1-butanol production pathway from C. acetobutylicum into the yeast. Eight genes are required for the ABE fermentation pathway,5) such as acetoacetyl-CoA thiolase (thl), β-hydroxybutyryl-CoA dehydrogenase (hbd), 3-hydroxybutyryl-CoA dehydratase (crotonase, crt), butyrylCoA dehydrogenase (bcd), electron transfer flavoprotein α subunit (etfA), electron transfer flavoprotein β subunit (etfB), aldehyde dehydrogenase (ad), and aldehyde-alcohol dehydrogenase (aad) (Fig. 1). In this study, the acetoacetyl-CoA thiolase-encoding gene from Candida tropicalis was introduced,12) and glycerol production pathway was eliminated from S. cerevisiae. The trans-enoyl-CoA reductase (ter) gene from Treponema denticola11) was furthermore introduced into the yeast instead of the genes of bcd, etfA, and etfB. As a result, the engineered yeast exhibited enhanced 1-butanol production.
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Butanol production using Saccharomyces cerevisiae
Fig. 1. Metabolic pathway for butanol production. Notes: The 1-butanol production pathway introduced into Saccharomyces cerevisiae. Enzymes required in the 1-butanol production pathway are represented as words enclosed in boxes. THL, acetoacetyl-CoA thiolase; HBD, β-hydroxybutyryl-CoA dehydrogenase; CRT, 3-hydroxybutyryl-CoA dehydratase (crotonase); BCD, butyryl-CoA dehydrogenase; ETFA, electron transfer flavoprotein α subunit; ETFB, electron transfer flavoprotein β subunit; AD, aldehyde dehydrogenase; AAD, aldehyde-alcohol dehydrogenase; TER, trans-enoyl-CoA reductase.
Materials and methods Strains and media. The E. coli strain DH5α (F−, endA1, hsdR17 [rK− mK+], supE44, thi-l, λ−, recA1, gyrA96, deoR, relA1, Δ[lacZYA-argF]U169, ϕ80dlacZΔM15) (Toyobo, Osaka, Japan) was used as a host for DNA manipulation. S. cerevisiae W303-1A (MATa; ade2-1; his3-11,15; leu2-3,112; trp1-1; ura3-1; can1-100)13) was used as the host for the 1-butanol pathway. E. coli was grown in Luria-Bertani medium [1% (w/v) tryptone, 0.5% (w/v) yeast extract, and 1% (w/v) sodium chloride] containing 100 μg/mL ampicillin. Yeast host cells were grown in yeast peptone dextrose (YPD) medium [1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) glucose] for transformation. For 1-butanol production, cells were cultivated in synthetic dextrose (SD) medium [0.67% (w/v) yeast nitrogen base without amino acids (Difco, New Jersey, USA), 2% (w/v) glucose, 0.5% (w/v) casamino acids, 0.002% (w/v) L-adenine, 0.002% (w/v) L-histidine, 0.003% (w/v) L-leucine, 0.002% (w/v) L-uracil, and 0.002% (w/v) L-tryptophan]. This medium was named SDC + AHLUW medium. Construction of 1-butanol-producing yeast. All primers used in plasmid construction are listed in Table 1. The thl gene derived from C. tropicalis was amplified from pWTIA,12) and the other seven genes (hbd, crt, bcd, etfA, etfB, ad, and aad) were amplified from C. acetobutylicum (NBRC 13948) genomic DNA by PCR. The ter gene14) was amplified from T. denticola (ATCC 35405D-5) genomic DNA by PCR. Following PCR, the amplified DNA fragments were inserted into the EcoRISalI (for thl, hbd, crt, etfA, etfB, ad, aad, and ter) or BamHI-SalI (for bcd) of the pULI1 vector,15) a multicopy vector with a constitutive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter (PGAP) and a
315
GAPDH terminator (TGAP). The sequences of the inserted DNA fragments were confirmed by DNA sequencing, and the resulting plasmids were named pULI1-thl, pULI1-hbd, pULI1-crt, pULI1-bcd, pULI1etfA, pULI1-etfB, pULI1-ad, pULI1-aad, and pULI1-ter, respectively. The nine genes were then amplified from these plasmids together with PGAP and TGAP, and were cloned into the BamHI-XhoI (for bcd), the NotI-BamHI (for etfA), or the SacI-SacII (for etfB) site of the pRS403 plasmid; into the BamHI-XhoI site (for ter) of the pRS403 plasmid; into the PstI-XhoI (for ad) or XbaI-NotI (for aad) site of the pRS405 plasmid; or into the SacI-SacII (for thl), the SpeI-XbaI (for hbd), or BamHI-XhoI (for crt) site of the pRS406 plasmid.16) The three constructed plasmids were digested with BstXI (for pRS403), BanIII (for pRS405), and PstI (for pRs406), and were introduced into the laboratory haploid W3031A S. cerevisiae strain using the lithium acetate method.17) The overexpression of genes for 1-butanol production in yeast strains were confirmed by transcription level of its gene using real-time reverse transcription (RT)-PCR. Total RNAs were isolated using an RNeasy mini kit (QIAGEN, Hilden, Germany) from cells grown to the mid-log phase. Synthesis of cDNA was performed using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems by Life Technologies, CA, USA) with 2 μg of the total RNA as a template. The reaction was carried out according to the manufacturer’s protocol. For the quantitative PCR, the ACT1 gene was used as an endogenous control to normalize the expression data for each gene. The primers, which are listed in Table 1, were designed using Primer Express® software (Applied Biosystems). Amplification was carried out using Power SYBR® Green PCR Master Mix (Applied Biosystems) in the 7500 Real-Time PCR System (Applied Biosystems). The reporter signals were analyzed using the 7500 Real-Time PCR System.18) For the deletion of the glycerol-3-phosphate dehydrogenase (gpd1 and gpd2) genes, the promoter sequences of these genes were amplified using the gpd1-Pro-F and gpd1-Pro-R primers (Table 1) for the gpd1 gene, and the gpd2-Pro-F and gpd2-Pro-R primers (Table 1) for the gpd2 gene. In the same way, the terminator sequences of these genes were amplified using the gpd1-Term-F and gpd1-Term-R primers (Table 1) for the gpd1 gene, and the gpd2-Term-F and gpd2-Term-R primers (Table 1) for the gpd2 gene. The promoter and terminator sequences were combined by overlappingPCR. The resulting sequences were inserted into the pAUR135 vector (Takara Bio, Kyoto, Japan) at the KpnI-EcoRI (for gpd1) and KpnI-SacI (for gpd2) sites. Each plasmid was digested (SacI for pAUR135-gpd1, and HindIII for pAUR135-gpd2) and introduced into S. cerevisiae W303-1A as described above, using Aureobasidin A (AbA)-resistance. Cultivation conditions. SDC + AHLUW medium buffered with citric acid (pH 5.0) was used for all yeast cultivation. The engineered yeasts were pre-cultivated in 10 mL medium at 30 °C on a rotary shaker (250 rpm) for 24 h under aerobic conditions. The yeasts were collected by centrifugation for 5 min at 3000 × g and 4 °C, and then transferred into 100 mL fresh medium at an OD600 of 0.1. After further cultivation at
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Table 1.
H. Sakuragi et al. Primers used in this study.
Primers
Sequence
thl-F thl-R hbd-F hbd-R crt-F crt-R bcd-F bcd-R etfA-F etfA-R etfB-F etfB-R ad-F ad-R aad-F aad-R ter-F ter-R GAPDH-Pro-F GAPDH-Term-R gpd1-Pro-F gpd1-Pro-R gpd1-Term-F gpd1-Term-R gpd2-Pro-F gpd2-Pro-R gpd2-Term-F gpd2-Term-R
5′-CAGTGAATTCATGGCTCTCCCACCAGTCTA-3′ 5′-CAGTCTCGAGTCTAGAGGATCCTTACAACTTGGC-3′ 5′-CAGTGAATTCATGAAAAAGGTATGTGTTATAGGTGCAGGT-3′ 5′-CAGTCTCGAGTTATTTTGAATAATCGTAGAAACCTTTTCC-3′ 5′-CAGTGAATTCATGGAACTAAACAATGTCATCCTTGAAAAG-3′ 5′-CAGTCTCGAGCTATCTATTTTTGAAGCCTTCAATTTTTCT-3′ 5′-CAGTAGATCTATGGATTTTAATTTAACAAGAGAACAAGAATTAGT-3′ 5′-CAGTCTCGAGTTATCTAAAAATTTTTCCTGAAATAACTAATTTCTGAA-3′ 5′-CAGTGAATTCATGAATAAAGCAGATTACAAGGGCGTAT-3′ 5′-CAGTCTCGAGTTAATTATTAGCAGCTTTAACTTGAGCTATTAATTCTG-3′ 5′-CAGTGAATTCATGAATATAGTTGTTTGTTTAAAACAAGTTCCAGATACA-3′ 5′-CAGTCTCGAGTTAATTTTGAGACAACATATCAGCTGCTTCC-3′ 5′-CAGTGAATTCATGAAAGTCACAACAGTAAAGGAATTAG-3′ 5′-CAGTCTCGAGTTAAGGTTGTTTTTTAAAACAATTTATATA-3′ 5′-CAGTGAGCTCATGAAAGTTACAAATCAAAAAGAACT-3′ 5′-CAGTCTCGAGTTAAAATGATTTTATATAGATATCCTTAAG-3′ 5′-CAGTGAATTCATGATTGTAAAACCAATGGTTAGGAACAATATT-3′ 5′-CAGTCTCGAGTTAAATCCTGTCGAACCTTTCTACC-3′ 5′-CAGTXXXXXXAAGCTTACCAGTTCTCACACGG-3′ 5′-CAGTXXXXXXGGTACCTCAATCAATGAATCG-3′ 5′-CAGTGGTACCGAAGAAAACAGAAGGCCAAGACAGG-3′ 5′-AGTCAGTCAGTCAGTCTTTATATTATCAATATTTGTGTTTGTGGAGGGGG-3′ 5′-ACTGACTGACTGACTATTTATTGGAGAAAGATAACATATCATACTTTCCCCC-3′ 5′-CAGTGAATTCCCTTAACAAGAACAATGTCATGACATTGGATGG-3′ 5′-CAGTGGTACCGGGCGAAAAATAAAAACTGGAGCAAGGAATTA-3′ 5′-AGTCAGTCAGTCAGTTGATAAGGAAGGGGAGCGAAGGA-3′ 5′-ACTGACTGACTGACTACACTCTCCCCCCCC-3′ 5′-CAGTGAGCTCCGTTGACCTGTACAGCTGG-3′
Primers for real-time PCR analysis act1-Frt act1-Rrt thl-Frt thl-Rrt hbd-Frt hbd-Rrt crt-Frt crt-Rrt bcd-Frt bcd-Rrt etfA-Frt etfA-Rrt etfB-Frt etfB-Rrt ad-Frt ad-Rrt aad-Frt aad-Rrt ter-Frt ter-Rrt
5′-TCGTTCCAATTTACGCTGGTT-3′ 5′-ACCGGCCAAATCGATTCTC-3′ 5′-TGGGTGTCGCTGCTGAAA-3′ 5′-CCTGGTCTTCTCTGCTGAATCC-3′ 5′-CCGGAACAGTTGACCTTAATATGG-3′ 5′-CCATTCTTTCAACAGCTGCTTCT-3′ 5′-GGTCTCGGAATAACACCTGGTT-3′ 5′-GCCATGCCCATTCCAACTAA-3′ 5′-GAAGGAGAGAAAACAATTTGGAAGA-3′ 5′-TGCCATCATCCATGCAAGAC-3′ 5′-GAGGAGTTGGAAGCAAAGAAAACT-3′ 5′-GCTGCTCTTGAAGCGGCTAT-3′ 5′-TGCTGGAAGGCAGGCTATAGA-3′ 5′-GGTATTCCAAGATGCTCAGCTATTT-3′ 5′-GATGAAATCTTTAGAAATGCAGCAAT-3′ 5′-GAAACCGGTATGGGCTTAGTTG-3′ 5′-AGGGAGCAAGCGGAGATTTAT-3′ 5′-GCCGCATCCAAGAGTAAATGA-3′ 5′-GCCGAATGCTGCCGTTAT-3′ 5′-GGTTCTCGGCTGCTCGAA-3′
Notes: Underlined sequences indicate the restriction sites. X indicates A or T or G or C. F indicates the forward primer, and R indicates the reverse primer.
30 °C on a rotary shaker (250 rpm) for 24 h under aerobic conditions, the yeasts were collected by centrifugation for 5 min at 3000 × g and 4 °C, and then transferred into 50 mL fresh medium (2% glucose or 1% glucose) at an OD600 of 10. Fermentation was carried out under anaerobic conditions at 30 °C with a magnetic stirrer (130 rpm). For fermentation, CO2 was bubbled into the medium for 1 min before autoclave treatment. After 24 h of cultivation, an additional 1% glucose was added to the cultures grown with an initial glucose concentration of 1%. This experiment was performed in triplicate.
Measurement of the production of 1-butanol and glycerol. For the quantification of 1-butanol production, 500 μL samples of each yeast culture were subjected to centrifugation (5 min, 3000 × g, 4 °C) and 380 μL of each of the resulting supernatants was collected. 2-Butanol (20 μL 0.1% (v/v) (Nacalai Tesque, Inc., Kyoto, Japan)) was added to the supernatants as an internal standard. Ethyl acetate (400 μL) (Nacalai Tesque) was added to each of the sample, which was subsequently vortexed for 1 min and left to stand for 10 min. The ethyl acetate phase was recovered and analyzed with a GCMS-QP2010 Ultra gas chromatograph
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Butanol production using Saccharomyces cerevisiae
mass spectrometer (Shimadzu, Kyoto, Japan) using a DB-WAX capillary column (30 m, 0.32 mm i.d., 0.50 mm film thickness) (Agilent Technologies, Santa Clara, CA).10) Aliquots (1 μL) were injected in the split mode (1/1) at 225 °C using helium as a carrier gas at a flow rate of 7.7 mL/min. The column temperature was maintained isothermally at 40 °C for 5 min, raised to 120 °C (15 °C/min), and then to 230 °C (25 °C/min). The temperature was then maintained isothermally at 230 °C for 4 min. The interface and MS source temperatures were 230 °C and 240 °C, respectively, and the ion voltage used was −0.1 kV. Data were collected and calibrated using GCMS solution software (Shimadzu). This experiment was performed in triplicate. For the quantification of glycerol production, 500 μL samples of each yeast culture were filtered using an Ultrafree-MC 0.45 mm centrifugal filter device (Millipore). Glycerol concentration was determined by highperformance liquid chromatography (Shimadzu) together with an RID-10A refractive index detector (Shimadzu) using a YMC-Pack Polyamine II column (YMC Co., LTD, Japan). This experiment was performed in triplicate.
Results Construction of yeasts producing enzymes of the 1-butanol production pathway During 1-butanol production, acetyl-CoA is converted into 1-butanol via the pathway from clostridia (Fig. 1). Yeasts normally produce the enzyme acetylCoA acetyltransferase (thiolase), which converts acetylCoA into acetoacetyl-CoA.19) However, in order to enhance this conversion into acetoacetyl-CoA, thl (thiolase) derived from C. tropicalis was utilized based on a report of the successful overproduction and high enzyme activity of thiolase from C. tropicalis in S. cerevisiae.12) Five S. cerevisiae strains were constructed to contain different combinations of introduced genes (Table 2). Each plasmid was integrated into the yeast genome, and each gene was confirmed to be successfully expressed in the yeast strains using real-time PCR (data not shown). After cultivation of the constructed strains in medium (2% initial glucose concentration) under anaerobic conditions, the supernatants of these cultures were collected by centrifugation. The supernatants containing 1-butanol were analyzed by GC/MS. A control strain S. cerevisiae harboring three empty
Table 2.
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vectors produced little 1-butanol; however, regardless of whether the thiolase gene from C. tropicalis was present or not, the engineered strains produced 1-butanol. Especially, the engineered strain which was not introduced the thiolase gene into produced 1.2 mg/L 1-butanol after 60 h of cultivation (Fig. 2). Effect of glycerol on butanol production by engineered yeasts S. cerevisiae produces glycerol under anaerobic conditions (Fig. 3(a)),20) and elimination of the glycerol pathway leads to higher ethanol production.21,22) A deletion mutant lacking elements of the glycerol production pathway (gpd1Δ and gpd2Δ) was thus created (Fig. 3(b) and (c)). The deletion mutant was found to have no glycerol-3-phosphate dehydrogenase (GPD) activity, and may produce dihydroxyacetone phosphate (DHAP) from glucose. DHAP may furthermore be rapidly converted to pyruvate by glyceraldehyde-3phosphate (GAP). A double-deletion mutant lacking the gpd1 and gpd2 genes was engineered from the strain containing the genes for 1-butanol production. In the double-deletion strain, glycerol production was found to be substantially decreased compared with the corresponding strain containing the gpd1 and gpd2 genes (Fig. 3(c)) and the deletion mutant was also shown to produce higher levels of 1-butanol (Figs. 2 and 3(b)). When grown in the presence of 2% glucose, the double-deletion mutant produced 1.5 mg/L 1-butanol after 48 h of cultivation (Fig. 3(b)). When an initial glucose concentration of 5% was used, less 1-butanol was produced by the deletion mutant strain (data not shown). The cultivation time was furthermore found to have little effect on butanol production (Fig. 2). The addition of glucose during cultivation as described in Materials and Methods had a pronounced effect on the production of butanol: 1.8 mg/ L 1-butanol was produced after 48 h cultivation (Fig. 3(b)). The amount of 1-butanol production after 60 h of cultivation was as much as after 48 h of cultivation (data not shown). Effect of the rate-limiting step on butanol production in engineered yeasts According to previous reports, the conversion of crotonyl-CoA to butyryl-CoA is the rate-limiting step of the butanol production pathway,5) and the gene of
Engineered strains.
Strains \genes
thl
hbd
crt
bcd
etfA&B
ter
ad
aad
gpd1, gpd2
#1 #2 #3 #4 #5 #6 (N.C.)
+ − − + − −
+ + + + + −
+ + + + + −
+ + + − − −
+ + + − − −
− − − + + −
+ + + + + −
+ + + + + −
+ + − + + +
Notes: #1: W303-1A harboring pRS406-thl, hbd, crt, pRS403-bcd, etfA&B, and pRS405-ad, aad, #2: W303-1A harboring pRS406-hbd, crt, pRS403-bcd, etfA&B, and pRS405-ad, aad, #3: W303-1A harboring pRS406-hbd, crt, pRS403-bcd, etfA&B, and pRS405-ad, aad, gpd1Δ and gpd2Δ, #4: W303-1A harboring pRS406-thl, hbd, crt, pRS403-ter, and pRS405-ad, aad, #5: W303-1A harboring pRS406-hbd, crt, pRS403-ter, and pRS405-ad, aad, #6: W303-1A harboring pRS406, pRS403, and pRS405 (N.C.; negative control).
H. Sakuragi et al.
1-Butanol production (mg/L)
318
1.4
(a)
1.2
36 h
1
60 h
0.8 0.6 0.4 0.2 0 #6
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Fig. 2. 1-Butanol production by various engineered yeast strains. Notes: #1: W303-1A harboring pRS406-thl, hbd, crt, pRS403-bcd, etfA&B, and pRS405-ad, aad, #2: W303-1A harboring pRS406-hbd, crt, pRS403-bcd, etfA&B, and pRS405-ad, aad, #6 W303-1A harboring pRS406, pRS403, and pRS405 (negative control). White bars represent strains cultivated for 36 h and black bars represent strains cultivated for 60 h. Error bars represent standard deviations (n = 3).
ter derived from T. denticola reportedly catalyzes the reaction more efficiently in E. coli.23) The bcd, etfA, and etfB genes which function to convert crotonyl-CoA into butyryl-CoA were thus replaced by ter. As shown in Fig. 4, the yeast strain engineered to contain the ter gene instead of the bcd, etfA, and etfB genes produced more 1-butanol compared with the strain containing the bcd, etfA, and etfB genes. At the initial glucose concentration (2%), the ter-containing strain produced 13.1 mg/L 1-butanol after 48 h of cultivation, an amount 11 times higher than that produced by the strain containing the bcd, etfA, and etfB genes (Fig. 2). After 48 h of cultivation, the ter-containing strain produced 14.1 mg/L 1-butanol when it was cultivated with an initial glucose concentration of 1% for the first 24 h and with an additional 1% glucose for the second 24 h of cultivation (Fig. 4). The amount of 1-butanol production after 60 h of cultivation was as much as after 48 h of cultivation (data not shown).
Discussion The Clostridium species, C. acetobutylicum, produces acetate, 1-butanol, and ethanol via ABE fermentation. Clostridium spp. are anaerobic and have a lower tolerance for solvents than the yeast S. cerevisiae does. We thus engineered S. cerevisiae to serve as a 1-butanol-producing host. Unlike clostridia, S. cerevisiae is able to grow in aerobic conditions. Compared with prokaryotes, S. cerevisiae, a eukaryotic organism, is also thought to produce more CoA and NADH, which are required for the Clostridium metabolic pathway of butanol production (acetoacetyl-CoA to β-hydroxybutyrylCoA, crotonyl-CoA to butyryl-CoA, butyryl-CoA to butyraldehyde, and butyraldehyde to 1-butanol). The engineered strains, however, were found to produce very little 1-butanol (Fig. 2). When comparing 1-butanol production in yeast strains with or without the thl gene, which allows for the conversion of acetyl-CoA to acetoacetyl-CoA, the effect of the thl gene on the 1-butanol production was unexpectedly small (Fig. 2).
(b) 1-Butanol production (mg/L)
#2
2 2% glucose
1.5
1%+1% glucose 1
0.5 0 24 h
(c) Glycerol production (g/L)
#1
48 h
0.2 #2 (2% glucose)
0.15
#3 (2% glucose)
0.1
#2 (1%+1% glucose)
0.05
#3 (1%+1% glucose)
0
48 h Fig. 3. The effect of eliminating the glycerol production pathway. Notes: (a) The glycerol production pathway in yeast. Fructose1,6-P, fructose 1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde-3-phosphate; Glycerol-3-P, glycerol-3-phosphate. (b) 1-Butanol production by the double-deletion mutant S. cerevisiae strain lacking genes of the glycerol pathway. White bars represent strain #3, grown with 2% glucose, and black bars represent strain #3 grown with 1% glucose to which an additional 1% glucose was added after 24 h of cultivation. Error bars represent standard deviations (n = 3). (c) Glycerol production in engineered S. cerevisiae strains after 48 h of cultivation. White bar represents strain #2 grown with 2% glucose, light gray bar represents strain #3 grown with 2% glucose, dark gray bar represents strain #2 grown with 1% glucose to which an additional 1% glucose was added after 24 h of cultivation, black bar represents strain #3 grown with 1% glucose to which an additional 1% glucose was added after 24 h of cultivation.
Low levels of 1-butanol production by S. cerevisiae may be a result of producing large amounts of glycerol under anaerobic conditions, and of lacking the NADH coenzyme factor involved in redox balance. Besides, several aroma compounds were increased by a glyceroldefective mutant.24) The double-deletion mutant yeast engineered to lack the gpd1 and gpd2 genes was found to produce less glycerol, leading to the reduced consumption of NADH in the conversion of DHAP into glycerol-3-P (Fig. 3(a) and (c)). Other enzymes such as glycerol dehydrogenase were still in yeasts, and thus glycerol production would be not zero.25) The deletion
1-Butanol production (mg/L)
Butanol production using Saccharomyces cerevisiae 16 14 12 10 8 6 4 2 0
#4 (2% glucose) #4 (1%+1% glucose) #5 (2% glucose) #5 (1%+1% glucose)
24 h
48 h
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Fig. 4. 1-Butanol production in yeast strains engineered to contain the ter gene. Notes: White bars represent strain #4 grown with 2% glucose and black bars represent strain #4 grown with 1% glucose to which an additional 1% glucose was added after 24 h of cultivation. Light gray bars represent strain #5 grown with 2% glucose and dark gray bars represent strain #5 grown with 1% glucose to which an additional 1% glucose was added after 24 h of cultivation. Error bars represent standard deviations (n = 3).
strain might exhibit increased 1-butanol production compared with the strains containing the gpd genes. The ter gene from T. denticola was introduced into S. cerevisiae to replace the bcd, etfA, and etfB genes. Previous research showed the conversion of crotonylCoA to butyryl-CoA to be the rate-limiting step in the butanol production pathway.5) Indeed, ter functioned more effectively than the bcd, etfA, and etfB genes, yielding increased 1-butanol production in S. cerevisiae. This can be explained by the fact that butyryl-CoA dehydrogenase (BCD) is required for the formation of the BCD-ETFAB complex, whereas TER functions without flavoproteins.23) The amount of 1-butanol produced by the ter-containing yeast (14.1 mg/L) was 5.6 times higher than that reported in the previous study.10) The cultivation time for maximum production (48 h) is furthermore shorter than previously reported.10) The elimination of the glycerol pathway in yeast expressing the ter gene was thus shown to further enhance 1-butanol production. The ethanol production pathway in S. cerevisiae comprises two reactions,26) and the pdc1, pdc5, and pdc6 genes encode the enzymes that convert pyruvate to acetaldehyde.27) The conversion of acetaldehyde to ethanol also requires NADH, and thus, the disruption of genes involved in ethanol production (pdc1, pdc5, and pdc6) may also lead to increased 1-butanol production in S. cerevisiae. Although thl gene was shown to have a negative effect on 1-butanol production in the yeast strains containing the bcd, etfA, and etfB genes for the conversion of crotonyl-CoA to butyryl-CoA in this study, the thl gene may facilitate 1-butanol production in ter-containing yeasts. This may be a result of more butyryl-CoA being produced by TER than by BCD-ETFAB causing a shortage of crotonyl-CoA, β-hydroxybutyryl-CoA, and acetoacetyl-CoA. In the case of the thl-containing strain, the equilibrium reaction between acetyl-CoA and acetoacetyl-CoA, thus, led to an increase in acetoacetyl-CoA production compared with the strain lacking the thl gene. In conclusion, we demonstrated that the engineered 1-butanol-producing yeast using the Clostridium pathway for butanol production coupled with the elimination of glycerol production pathway genes and using the ter gene derived from T. denticola was shown
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to strongly enhance 1-butanol production. The findings of processes to enhance the production of 1-butanol in yeast presented here provide important information for further application and enhancement of 1-butanol production by S. cerevisiae.
Author contribution HS, HM, KK, and MU conceived and designed the study. HS and HM analyzed the data. HS, KK, and MU drafted the manuscript. All authors read and approved the final manuscript.
Supplemental material The supplemental material for this paper is available at http://dx.doi.org/10.1080/09168451.2014.972330.
Acknowledgments This research was supported by CREST and Science and technology research promotion program for agriculture, forestry, fisheries, and food industry.
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[13] Thomas BJ, Rothstein R. Elevated recombination rates in transcriptionally active DNA. Cell. 1989;56:619–630. [14] Tucci S, Martin W. A novel prokaryotic trans-2-enoyl-CoA reductase from the spirochete Treponema denticola. FEBS Lett. 2007;581:1561–1566. [15] Miura N, Kirino A, Endo S, Morisaka H, Kuroda K, Takagi M, Ueda M. Tracing putative trafficking of the glycolytic enzyme enolase via SNARE-driven unconventional secretion. Eukaryotic Cell. 2012;11:1075–1082. [16] Sikorski RS, Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989;122:19–27. [17] Ito H, Fukuda Y, Murata K, Kimura A. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 1983;153:163–168. [18] Nishida N, Ozato N, Matsui K, Kuroda K, Ueda M. ABC transporters and cell wall proteins involved in organic solvent tolerance in Saccharomyces cerevisiae. J. Biotechnol. 2013;165:145–152. [19] Hiser L, Basson ME, Rine J. ERG10 from Saccharomyces cerevisiae encodes acetoacetyl-CoA thiolase. J. Biol. Chem. 1994; 269:31383–31389. [20] Costenoble R, Valadi H, Gustafsson L, Niklasson C, Franzen CJ. Microaerobic glycerol formation in Saccharomyces cerevisiae. Yeast. 2000;16:1483–1495.
[21] Yu KO, Kim SW, Han SO. Reduction of glycerol production to improve ethanol yield in an engineered Saccharomyces cerevisiae using glycerol as a substrate. J. Biotechnol. 2010;150:209–214. [22] Jain VK, Divol B, Prior BA, Bauer FF. Elimination of glycerol and replacement with alternative products in ethanol fermentation by Saccharomyces cerevisiae. J. Ind. Microbiol. Biotechnol. 2011;38:1427–1435. [23] Shen CR, Lan EI, Dekishima Y, Baez A, Cho KM, Liao JC. Driving forces enable high-titer anaerobic 1-butanol synthesis in Escherichia coli. Appl. Environ. Microbiol. 2011;77:2905–2915. [24] Jain VK, Divol B, Prior BA, Bauer FF. Effect of alternative NAD+-regenerating pathways on the formation of primary and secondary aroma compounds in a Saccharomyces cerevisiae glycerol-defective mutant. Appl. Microbiol. Biotechnol. 2012;93:131–141. [25] Costenoble R, Valadi H, Gustafsson L, Niklasson C, Franzen CJ. Microaerobic glycerol formation in Saccharomyces cerevisiae. Yeast. 2000;16:1483–1495. [26] Buijs NA, Siewers V, Nielsen J. Advanced biofuel production by the yeast Saccharomyces cerevisiae. Curr. Opin. Chem. Biol. 2013;17:480–488. [27] Chen X, Nielsen KF, Borodina I, Kielland-Brandt MC, Karhumaa K. Increased isobutanol production in Saccharomyces cerevisiae by overexpression of genes in valine metabolism. Biotechnol. Biofuels. 2011;4:21.
Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
Enhanced butanol production by eukaryotic Saccharomyces cerevisiae engineered to contain an improved pathway a
ab
a
ab
Hiroshi Sakuragi , Hironobu Morisaka , Kouichi Kuroda & Mitsuyoshi Ueda a
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan b
Kyoto Industrial Science and Technology Innovation Center, Kyoto, Japan Published online: 28 Oct 2014.
Click for updates To cite this article: Hiroshi Sakuragi, Hironobu Morisaka, Kouichi Kuroda & Mitsuyoshi Ueda (2015) Enhanced butanol production by eukaryotic Saccharomyces cerevisiae engineered to contain an improved pathway, Bioscience, Biotechnology, and Biochemistry, 79:2, 314-320, DOI: 10.1080/09168451.2014.972330 To link to this article: http://dx.doi.org/10.1080/09168451.2014.972330
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Bioscience, Biotechnology, and Biochemistry, 2015 Vol. 79, No. 2, 314–320
Enhanced butanol production by eukaryotic Saccharomyces cerevisiae engineered to contain an improved pathway Hiroshi Sakuragi1, Hironobu Morisaka1,2, Kouichi Kuroda1 and Mitsuyoshi Ueda1,2,* 1
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan; 2Kyoto Industrial Science and Technology Innovation Center, Kyoto, Japan
Received August 1, 2014; accepted September 14, 2014
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http://dx.doi.org/10.1080/09168451.2014.972330
Compared with ethanol, butanol has more advantageous physical properties as a fuel, and biobutanol is thus considered a promising biofuel material. Biobutanol has often been produced by Clostridium species; however, because they are strictly anaerobic microorganisms, these species are challenging to work with. We attempted to introduce the butanol production pathway into yeast Saccharomyces cerevisiae, which is a well-known microorganism that is tolerant to organic solvents. 1-Butanol was found to be produced at very low levels when the butanol production pathway of Clostridium acetobutylicum was simply introduced into S. cerevisiae. The elimination of glycerol production pathway in the yeast contributed to the enhancement of 1-butanol production. In addition, by the use of trans-enoylCoA reductase in the engineered pathway, 1-butanol production was markedly enhanced to yield 14.1 mg/L after 48 h of cultivation. Key words:
1-butanol production; Saccharomyces cerevisiae; Clostridium acetobutylicum; glycerol pathway; trans-enoyl-CoA reductase
The production of biofuels from renewable biomass such as grain and wood is facilitated by microbes. Bioethanol is regarded as one of the carbon neutral products, and thus, to date, it has been one of the most promising biofuels. Bioethanol is normally produced by Saccharomyces cerevisiae, an organism that has been used in the production of alcohols and breads for a long time. Recently, butanol has been attracting attention as a fuel more preferable to ethanol because of various significant advantages associated with the use of butanol as a fuel. Butanol can be used either in its pure form or as a mixture with gasoline at any concentration and, because of its lower vapor pressure, butanol is safer to use than ethanol.1) It is well known that 1-butanol can be produced by clostridia fermentation. Clostridia comprise a diverse group of anaerobic, spore-forming, Gram-positive bacteria that includes notable pathogens and industrially *Corresponding author. Email: [email protected] © 2014 Japan Society for Bioscience, Biotechnology, and Agrochemistry
significant microorganisms.2,3) Clostridium acetobutylicum has been found to produce acetone/butanol/ethanol at a ratio of 3:6:1 in a process called ABE fermentation.4,5) Bacterial production of butanol and acetone using the ABE fermentation process has been shown to be valuable in the production of the lacquer solvent butylacetate as well as in synthetic rubber industry. Due to the possibility of oil depletion, biofuel production is currently practiced worldwide. Thus, research and development into microbial butanol production is again being actively pursued.6) Using the several genes of C. acetobutylicum involved in the biosynthesis of 1-butanol, engineered Escherichia coli under aerobic conditions has been constructed.7) E. coli has also been shown to exhibit a tolerance to up to 1.5% butanol,8) which is comparable with the tolerance exhibited by clostridia. On the other hand, among eukaryotic cells, yeast S. cerevisiae has been described as an ideal host for butanol production. S. cerevisiae can be grown under both aerobic and anaerobic conditions, and has an inherent tolerance to organic solvents due to its extensive use in the industrial production of ethanol. S. cerevisiae is known to tolerate up to 2% butanol.9) Several approaches to the construction of a butanol-producing S. cerevisiae strain have been examined.10,11) In this study, we attempted to produce 1-butanol in S. cerevisiae by introducing the 1-butanol production pathway from C. acetobutylicum into the yeast. Eight genes are required for the ABE fermentation pathway,5) such as acetoacetyl-CoA thiolase (thl), β-hydroxybutyryl-CoA dehydrogenase (hbd), 3-hydroxybutyryl-CoA dehydratase (crotonase, crt), butyrylCoA dehydrogenase (bcd), electron transfer flavoprotein α subunit (etfA), electron transfer flavoprotein β subunit (etfB), aldehyde dehydrogenase (ad), and aldehyde-alcohol dehydrogenase (aad) (Fig. 1). In this study, the acetoacetyl-CoA thiolase-encoding gene from Candida tropicalis was introduced,12) and glycerol production pathway was eliminated from S. cerevisiae. The trans-enoyl-CoA reductase (ter) gene from Treponema denticola11) was furthermore introduced into the yeast instead of the genes of bcd, etfA, and etfB. As a result, the engineered yeast exhibited enhanced 1-butanol production.
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Butanol production using Saccharomyces cerevisiae
Fig. 1. Metabolic pathway for butanol production. Notes: The 1-butanol production pathway introduced into Saccharomyces cerevisiae. Enzymes required in the 1-butanol production pathway are represented as words enclosed in boxes. THL, acetoacetyl-CoA thiolase; HBD, β-hydroxybutyryl-CoA dehydrogenase; CRT, 3-hydroxybutyryl-CoA dehydratase (crotonase); BCD, butyryl-CoA dehydrogenase; ETFA, electron transfer flavoprotein α subunit; ETFB, electron transfer flavoprotein β subunit; AD, aldehyde dehydrogenase; AAD, aldehyde-alcohol dehydrogenase; TER, trans-enoyl-CoA reductase.
Materials and methods Strains and media. The E. coli strain DH5α (F−, endA1, hsdR17 [rK− mK+], supE44, thi-l, λ−, recA1, gyrA96, deoR, relA1, Δ[lacZYA-argF]U169, ϕ80dlacZΔM15) (Toyobo, Osaka, Japan) was used as a host for DNA manipulation. S. cerevisiae W303-1A (MATa; ade2-1; his3-11,15; leu2-3,112; trp1-1; ura3-1; can1-100)13) was used as the host for the 1-butanol pathway. E. coli was grown in Luria-Bertani medium [1% (w/v) tryptone, 0.5% (w/v) yeast extract, and 1% (w/v) sodium chloride] containing 100 μg/mL ampicillin. Yeast host cells were grown in yeast peptone dextrose (YPD) medium [1% (w/v) yeast extract, 2% (w/v) peptone, and 2% (w/v) glucose] for transformation. For 1-butanol production, cells were cultivated in synthetic dextrose (SD) medium [0.67% (w/v) yeast nitrogen base without amino acids (Difco, New Jersey, USA), 2% (w/v) glucose, 0.5% (w/v) casamino acids, 0.002% (w/v) L-adenine, 0.002% (w/v) L-histidine, 0.003% (w/v) L-leucine, 0.002% (w/v) L-uracil, and 0.002% (w/v) L-tryptophan]. This medium was named SDC + AHLUW medium. Construction of 1-butanol-producing yeast. All primers used in plasmid construction are listed in Table 1. The thl gene derived from C. tropicalis was amplified from pWTIA,12) and the other seven genes (hbd, crt, bcd, etfA, etfB, ad, and aad) were amplified from C. acetobutylicum (NBRC 13948) genomic DNA by PCR. The ter gene14) was amplified from T. denticola (ATCC 35405D-5) genomic DNA by PCR. Following PCR, the amplified DNA fragments were inserted into the EcoRISalI (for thl, hbd, crt, etfA, etfB, ad, aad, and ter) or BamHI-SalI (for bcd) of the pULI1 vector,15) a multicopy vector with a constitutive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter (PGAP) and a
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GAPDH terminator (TGAP). The sequences of the inserted DNA fragments were confirmed by DNA sequencing, and the resulting plasmids were named pULI1-thl, pULI1-hbd, pULI1-crt, pULI1-bcd, pULI1etfA, pULI1-etfB, pULI1-ad, pULI1-aad, and pULI1-ter, respectively. The nine genes were then amplified from these plasmids together with PGAP and TGAP, and were cloned into the BamHI-XhoI (for bcd), the NotI-BamHI (for etfA), or the SacI-SacII (for etfB) site of the pRS403 plasmid; into the BamHI-XhoI site (for ter) of the pRS403 plasmid; into the PstI-XhoI (for ad) or XbaI-NotI (for aad) site of the pRS405 plasmid; or into the SacI-SacII (for thl), the SpeI-XbaI (for hbd), or BamHI-XhoI (for crt) site of the pRS406 plasmid.16) The three constructed plasmids were digested with BstXI (for pRS403), BanIII (for pRS405), and PstI (for pRs406), and were introduced into the laboratory haploid W3031A S. cerevisiae strain using the lithium acetate method.17) The overexpression of genes for 1-butanol production in yeast strains were confirmed by transcription level of its gene using real-time reverse transcription (RT)-PCR. Total RNAs were isolated using an RNeasy mini kit (QIAGEN, Hilden, Germany) from cells grown to the mid-log phase. Synthesis of cDNA was performed using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems by Life Technologies, CA, USA) with 2 μg of the total RNA as a template. The reaction was carried out according to the manufacturer’s protocol. For the quantitative PCR, the ACT1 gene was used as an endogenous control to normalize the expression data for each gene. The primers, which are listed in Table 1, were designed using Primer Express® software (Applied Biosystems). Amplification was carried out using Power SYBR® Green PCR Master Mix (Applied Biosystems) in the 7500 Real-Time PCR System (Applied Biosystems). The reporter signals were analyzed using the 7500 Real-Time PCR System.18) For the deletion of the glycerol-3-phosphate dehydrogenase (gpd1 and gpd2) genes, the promoter sequences of these genes were amplified using the gpd1-Pro-F and gpd1-Pro-R primers (Table 1) for the gpd1 gene, and the gpd2-Pro-F and gpd2-Pro-R primers (Table 1) for the gpd2 gene. In the same way, the terminator sequences of these genes were amplified using the gpd1-Term-F and gpd1-Term-R primers (Table 1) for the gpd1 gene, and the gpd2-Term-F and gpd2-Term-R primers (Table 1) for the gpd2 gene. The promoter and terminator sequences were combined by overlappingPCR. The resulting sequences were inserted into the pAUR135 vector (Takara Bio, Kyoto, Japan) at the KpnI-EcoRI (for gpd1) and KpnI-SacI (for gpd2) sites. Each plasmid was digested (SacI for pAUR135-gpd1, and HindIII for pAUR135-gpd2) and introduced into S. cerevisiae W303-1A as described above, using Aureobasidin A (AbA)-resistance. Cultivation conditions. SDC + AHLUW medium buffered with citric acid (pH 5.0) was used for all yeast cultivation. The engineered yeasts were pre-cultivated in 10 mL medium at 30 °C on a rotary shaker (250 rpm) for 24 h under aerobic conditions. The yeasts were collected by centrifugation for 5 min at 3000 × g and 4 °C, and then transferred into 100 mL fresh medium at an OD600 of 0.1. After further cultivation at
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Table 1.
H. Sakuragi et al. Primers used in this study.
Primers
Sequence
thl-F thl-R hbd-F hbd-R crt-F crt-R bcd-F bcd-R etfA-F etfA-R etfB-F etfB-R ad-F ad-R aad-F aad-R ter-F ter-R GAPDH-Pro-F GAPDH-Term-R gpd1-Pro-F gpd1-Pro-R gpd1-Term-F gpd1-Term-R gpd2-Pro-F gpd2-Pro-R gpd2-Term-F gpd2-Term-R
5′-CAGTGAATTCATGGCTCTCCCACCAGTCTA-3′ 5′-CAGTCTCGAGTCTAGAGGATCCTTACAACTTGGC-3′ 5′-CAGTGAATTCATGAAAAAGGTATGTGTTATAGGTGCAGGT-3′ 5′-CAGTCTCGAGTTATTTTGAATAATCGTAGAAACCTTTTCC-3′ 5′-CAGTGAATTCATGGAACTAAACAATGTCATCCTTGAAAAG-3′ 5′-CAGTCTCGAGCTATCTATTTTTGAAGCCTTCAATTTTTCT-3′ 5′-CAGTAGATCTATGGATTTTAATTTAACAAGAGAACAAGAATTAGT-3′ 5′-CAGTCTCGAGTTATCTAAAAATTTTTCCTGAAATAACTAATTTCTGAA-3′ 5′-CAGTGAATTCATGAATAAAGCAGATTACAAGGGCGTAT-3′ 5′-CAGTCTCGAGTTAATTATTAGCAGCTTTAACTTGAGCTATTAATTCTG-3′ 5′-CAGTGAATTCATGAATATAGTTGTTTGTTTAAAACAAGTTCCAGATACA-3′ 5′-CAGTCTCGAGTTAATTTTGAGACAACATATCAGCTGCTTCC-3′ 5′-CAGTGAATTCATGAAAGTCACAACAGTAAAGGAATTAG-3′ 5′-CAGTCTCGAGTTAAGGTTGTTTTTTAAAACAATTTATATA-3′ 5′-CAGTGAGCTCATGAAAGTTACAAATCAAAAAGAACT-3′ 5′-CAGTCTCGAGTTAAAATGATTTTATATAGATATCCTTAAG-3′ 5′-CAGTGAATTCATGATTGTAAAACCAATGGTTAGGAACAATATT-3′ 5′-CAGTCTCGAGTTAAATCCTGTCGAACCTTTCTACC-3′ 5′-CAGTXXXXXXAAGCTTACCAGTTCTCACACGG-3′ 5′-CAGTXXXXXXGGTACCTCAATCAATGAATCG-3′ 5′-CAGTGGTACCGAAGAAAACAGAAGGCCAAGACAGG-3′ 5′-AGTCAGTCAGTCAGTCTTTATATTATCAATATTTGTGTTTGTGGAGGGGG-3′ 5′-ACTGACTGACTGACTATTTATTGGAGAAAGATAACATATCATACTTTCCCCC-3′ 5′-CAGTGAATTCCCTTAACAAGAACAATGTCATGACATTGGATGG-3′ 5′-CAGTGGTACCGGGCGAAAAATAAAAACTGGAGCAAGGAATTA-3′ 5′-AGTCAGTCAGTCAGTTGATAAGGAAGGGGAGCGAAGGA-3′ 5′-ACTGACTGACTGACTACACTCTCCCCCCCC-3′ 5′-CAGTGAGCTCCGTTGACCTGTACAGCTGG-3′
Primers for real-time PCR analysis act1-Frt act1-Rrt thl-Frt thl-Rrt hbd-Frt hbd-Rrt crt-Frt crt-Rrt bcd-Frt bcd-Rrt etfA-Frt etfA-Rrt etfB-Frt etfB-Rrt ad-Frt ad-Rrt aad-Frt aad-Rrt ter-Frt ter-Rrt
5′-TCGTTCCAATTTACGCTGGTT-3′ 5′-ACCGGCCAAATCGATTCTC-3′ 5′-TGGGTGTCGCTGCTGAAA-3′ 5′-CCTGGTCTTCTCTGCTGAATCC-3′ 5′-CCGGAACAGTTGACCTTAATATGG-3′ 5′-CCATTCTTTCAACAGCTGCTTCT-3′ 5′-GGTCTCGGAATAACACCTGGTT-3′ 5′-GCCATGCCCATTCCAACTAA-3′ 5′-GAAGGAGAGAAAACAATTTGGAAGA-3′ 5′-TGCCATCATCCATGCAAGAC-3′ 5′-GAGGAGTTGGAAGCAAAGAAAACT-3′ 5′-GCTGCTCTTGAAGCGGCTAT-3′ 5′-TGCTGGAAGGCAGGCTATAGA-3′ 5′-GGTATTCCAAGATGCTCAGCTATTT-3′ 5′-GATGAAATCTTTAGAAATGCAGCAAT-3′ 5′-GAAACCGGTATGGGCTTAGTTG-3′ 5′-AGGGAGCAAGCGGAGATTTAT-3′ 5′-GCCGCATCCAAGAGTAAATGA-3′ 5′-GCCGAATGCTGCCGTTAT-3′ 5′-GGTTCTCGGCTGCTCGAA-3′
Notes: Underlined sequences indicate the restriction sites. X indicates A or T or G or C. F indicates the forward primer, and R indicates the reverse primer.
30 °C on a rotary shaker (250 rpm) for 24 h under aerobic conditions, the yeasts were collected by centrifugation for 5 min at 3000 × g and 4 °C, and then transferred into 50 mL fresh medium (2% glucose or 1% glucose) at an OD600 of 10. Fermentation was carried out under anaerobic conditions at 30 °C with a magnetic stirrer (130 rpm). For fermentation, CO2 was bubbled into the medium for 1 min before autoclave treatment. After 24 h of cultivation, an additional 1% glucose was added to the cultures grown with an initial glucose concentration of 1%. This experiment was performed in triplicate.
Measurement of the production of 1-butanol and glycerol. For the quantification of 1-butanol production, 500 μL samples of each yeast culture were subjected to centrifugation (5 min, 3000 × g, 4 °C) and 380 μL of each of the resulting supernatants was collected. 2-Butanol (20 μL 0.1% (v/v) (Nacalai Tesque, Inc., Kyoto, Japan)) was added to the supernatants as an internal standard. Ethyl acetate (400 μL) (Nacalai Tesque) was added to each of the sample, which was subsequently vortexed for 1 min and left to stand for 10 min. The ethyl acetate phase was recovered and analyzed with a GCMS-QP2010 Ultra gas chromatograph
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Butanol production using Saccharomyces cerevisiae
mass spectrometer (Shimadzu, Kyoto, Japan) using a DB-WAX capillary column (30 m, 0.32 mm i.d., 0.50 mm film thickness) (Agilent Technologies, Santa Clara, CA).10) Aliquots (1 μL) were injected in the split mode (1/1) at 225 °C using helium as a carrier gas at a flow rate of 7.7 mL/min. The column temperature was maintained isothermally at 40 °C for 5 min, raised to 120 °C (15 °C/min), and then to 230 °C (25 °C/min). The temperature was then maintained isothermally at 230 °C for 4 min. The interface and MS source temperatures were 230 °C and 240 °C, respectively, and the ion voltage used was −0.1 kV. Data were collected and calibrated using GCMS solution software (Shimadzu). This experiment was performed in triplicate. For the quantification of glycerol production, 500 μL samples of each yeast culture were filtered using an Ultrafree-MC 0.45 mm centrifugal filter device (Millipore). Glycerol concentration was determined by highperformance liquid chromatography (Shimadzu) together with an RID-10A refractive index detector (Shimadzu) using a YMC-Pack Polyamine II column (YMC Co., LTD, Japan). This experiment was performed in triplicate.
Results Construction of yeasts producing enzymes of the 1-butanol production pathway During 1-butanol production, acetyl-CoA is converted into 1-butanol via the pathway from clostridia (Fig. 1). Yeasts normally produce the enzyme acetylCoA acetyltransferase (thiolase), which converts acetylCoA into acetoacetyl-CoA.19) However, in order to enhance this conversion into acetoacetyl-CoA, thl (thiolase) derived from C. tropicalis was utilized based on a report of the successful overproduction and high enzyme activity of thiolase from C. tropicalis in S. cerevisiae.12) Five S. cerevisiae strains were constructed to contain different combinations of introduced genes (Table 2). Each plasmid was integrated into the yeast genome, and each gene was confirmed to be successfully expressed in the yeast strains using real-time PCR (data not shown). After cultivation of the constructed strains in medium (2% initial glucose concentration) under anaerobic conditions, the supernatants of these cultures were collected by centrifugation. The supernatants containing 1-butanol were analyzed by GC/MS. A control strain S. cerevisiae harboring three empty
Table 2.
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vectors produced little 1-butanol; however, regardless of whether the thiolase gene from C. tropicalis was present or not, the engineered strains produced 1-butanol. Especially, the engineered strain which was not introduced the thiolase gene into produced 1.2 mg/L 1-butanol after 60 h of cultivation (Fig. 2). Effect of glycerol on butanol production by engineered yeasts S. cerevisiae produces glycerol under anaerobic conditions (Fig. 3(a)),20) and elimination of the glycerol pathway leads to higher ethanol production.21,22) A deletion mutant lacking elements of the glycerol production pathway (gpd1Δ and gpd2Δ) was thus created (Fig. 3(b) and (c)). The deletion mutant was found to have no glycerol-3-phosphate dehydrogenase (GPD) activity, and may produce dihydroxyacetone phosphate (DHAP) from glucose. DHAP may furthermore be rapidly converted to pyruvate by glyceraldehyde-3phosphate (GAP). A double-deletion mutant lacking the gpd1 and gpd2 genes was engineered from the strain containing the genes for 1-butanol production. In the double-deletion strain, glycerol production was found to be substantially decreased compared with the corresponding strain containing the gpd1 and gpd2 genes (Fig. 3(c)) and the deletion mutant was also shown to produce higher levels of 1-butanol (Figs. 2 and 3(b)). When grown in the presence of 2% glucose, the double-deletion mutant produced 1.5 mg/L 1-butanol after 48 h of cultivation (Fig. 3(b)). When an initial glucose concentration of 5% was used, less 1-butanol was produced by the deletion mutant strain (data not shown). The cultivation time was furthermore found to have little effect on butanol production (Fig. 2). The addition of glucose during cultivation as described in Materials and Methods had a pronounced effect on the production of butanol: 1.8 mg/ L 1-butanol was produced after 48 h cultivation (Fig. 3(b)). The amount of 1-butanol production after 60 h of cultivation was as much as after 48 h of cultivation (data not shown). Effect of the rate-limiting step on butanol production in engineered yeasts According to previous reports, the conversion of crotonyl-CoA to butyryl-CoA is the rate-limiting step of the butanol production pathway,5) and the gene of
Engineered strains.
Strains \genes
thl
hbd
crt
bcd
etfA&B
ter
ad
aad
gpd1, gpd2
#1 #2 #3 #4 #5 #6 (N.C.)
+ − − + − −
+ + + + + −
+ + + + + −
+ + + − − −
+ + + − − −
− − − + + −
+ + + + + −
+ + + + + −
+ + − + + +
Notes: #1: W303-1A harboring pRS406-thl, hbd, crt, pRS403-bcd, etfA&B, and pRS405-ad, aad, #2: W303-1A harboring pRS406-hbd, crt, pRS403-bcd, etfA&B, and pRS405-ad, aad, #3: W303-1A harboring pRS406-hbd, crt, pRS403-bcd, etfA&B, and pRS405-ad, aad, gpd1Δ and gpd2Δ, #4: W303-1A harboring pRS406-thl, hbd, crt, pRS403-ter, and pRS405-ad, aad, #5: W303-1A harboring pRS406-hbd, crt, pRS403-ter, and pRS405-ad, aad, #6: W303-1A harboring pRS406, pRS403, and pRS405 (N.C.; negative control).
H. Sakuragi et al.
1-Butanol production (mg/L)
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1.4
(a)
1.2
36 h
1
60 h
0.8 0.6 0.4 0.2 0 #6
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Fig. 2. 1-Butanol production by various engineered yeast strains. Notes: #1: W303-1A harboring pRS406-thl, hbd, crt, pRS403-bcd, etfA&B, and pRS405-ad, aad, #2: W303-1A harboring pRS406-hbd, crt, pRS403-bcd, etfA&B, and pRS405-ad, aad, #6 W303-1A harboring pRS406, pRS403, and pRS405 (negative control). White bars represent strains cultivated for 36 h and black bars represent strains cultivated for 60 h. Error bars represent standard deviations (n = 3).
ter derived from T. denticola reportedly catalyzes the reaction more efficiently in E. coli.23) The bcd, etfA, and etfB genes which function to convert crotonyl-CoA into butyryl-CoA were thus replaced by ter. As shown in Fig. 4, the yeast strain engineered to contain the ter gene instead of the bcd, etfA, and etfB genes produced more 1-butanol compared with the strain containing the bcd, etfA, and etfB genes. At the initial glucose concentration (2%), the ter-containing strain produced 13.1 mg/L 1-butanol after 48 h of cultivation, an amount 11 times higher than that produced by the strain containing the bcd, etfA, and etfB genes (Fig. 2). After 48 h of cultivation, the ter-containing strain produced 14.1 mg/L 1-butanol when it was cultivated with an initial glucose concentration of 1% for the first 24 h and with an additional 1% glucose for the second 24 h of cultivation (Fig. 4). The amount of 1-butanol production after 60 h of cultivation was as much as after 48 h of cultivation (data not shown).
Discussion The Clostridium species, C. acetobutylicum, produces acetate, 1-butanol, and ethanol via ABE fermentation. Clostridium spp. are anaerobic and have a lower tolerance for solvents than the yeast S. cerevisiae does. We thus engineered S. cerevisiae to serve as a 1-butanol-producing host. Unlike clostridia, S. cerevisiae is able to grow in aerobic conditions. Compared with prokaryotes, S. cerevisiae, a eukaryotic organism, is also thought to produce more CoA and NADH, which are required for the Clostridium metabolic pathway of butanol production (acetoacetyl-CoA to β-hydroxybutyrylCoA, crotonyl-CoA to butyryl-CoA, butyryl-CoA to butyraldehyde, and butyraldehyde to 1-butanol). The engineered strains, however, were found to produce very little 1-butanol (Fig. 2). When comparing 1-butanol production in yeast strains with or without the thl gene, which allows for the conversion of acetyl-CoA to acetoacetyl-CoA, the effect of the thl gene on the 1-butanol production was unexpectedly small (Fig. 2).
(b) 1-Butanol production (mg/L)
#2
2 2% glucose
1.5
1%+1% glucose 1
0.5 0 24 h
(c) Glycerol production (g/L)
#1
48 h
0.2 #2 (2% glucose)
0.15
#3 (2% glucose)
0.1
#2 (1%+1% glucose)
0.05
#3 (1%+1% glucose)
0
48 h Fig. 3. The effect of eliminating the glycerol production pathway. Notes: (a) The glycerol production pathway in yeast. Fructose1,6-P, fructose 1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde-3-phosphate; Glycerol-3-P, glycerol-3-phosphate. (b) 1-Butanol production by the double-deletion mutant S. cerevisiae strain lacking genes of the glycerol pathway. White bars represent strain #3, grown with 2% glucose, and black bars represent strain #3 grown with 1% glucose to which an additional 1% glucose was added after 24 h of cultivation. Error bars represent standard deviations (n = 3). (c) Glycerol production in engineered S. cerevisiae strains after 48 h of cultivation. White bar represents strain #2 grown with 2% glucose, light gray bar represents strain #3 grown with 2% glucose, dark gray bar represents strain #2 grown with 1% glucose to which an additional 1% glucose was added after 24 h of cultivation, black bar represents strain #3 grown with 1% glucose to which an additional 1% glucose was added after 24 h of cultivation.
Low levels of 1-butanol production by S. cerevisiae may be a result of producing large amounts of glycerol under anaerobic conditions, and of lacking the NADH coenzyme factor involved in redox balance. Besides, several aroma compounds were increased by a glyceroldefective mutant.24) The double-deletion mutant yeast engineered to lack the gpd1 and gpd2 genes was found to produce less glycerol, leading to the reduced consumption of NADH in the conversion of DHAP into glycerol-3-P (Fig. 3(a) and (c)). Other enzymes such as glycerol dehydrogenase were still in yeasts, and thus glycerol production would be not zero.25) The deletion
1-Butanol production (mg/L)
Butanol production using Saccharomyces cerevisiae 16 14 12 10 8 6 4 2 0
#4 (2% glucose) #4 (1%+1% glucose) #5 (2% glucose) #5 (1%+1% glucose)
24 h
48 h
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Fig. 4. 1-Butanol production in yeast strains engineered to contain the ter gene. Notes: White bars represent strain #4 grown with 2% glucose and black bars represent strain #4 grown with 1% glucose to which an additional 1% glucose was added after 24 h of cultivation. Light gray bars represent strain #5 grown with 2% glucose and dark gray bars represent strain #5 grown with 1% glucose to which an additional 1% glucose was added after 24 h of cultivation. Error bars represent standard deviations (n = 3).
strain might exhibit increased 1-butanol production compared with the strains containing the gpd genes. The ter gene from T. denticola was introduced into S. cerevisiae to replace the bcd, etfA, and etfB genes. Previous research showed the conversion of crotonylCoA to butyryl-CoA to be the rate-limiting step in the butanol production pathway.5) Indeed, ter functioned more effectively than the bcd, etfA, and etfB genes, yielding increased 1-butanol production in S. cerevisiae. This can be explained by the fact that butyryl-CoA dehydrogenase (BCD) is required for the formation of the BCD-ETFAB complex, whereas TER functions without flavoproteins.23) The amount of 1-butanol produced by the ter-containing yeast (14.1 mg/L) was 5.6 times higher than that reported in the previous study.10) The cultivation time for maximum production (48 h) is furthermore shorter than previously reported.10) The elimination of the glycerol pathway in yeast expressing the ter gene was thus shown to further enhance 1-butanol production. The ethanol production pathway in S. cerevisiae comprises two reactions,26) and the pdc1, pdc5, and pdc6 genes encode the enzymes that convert pyruvate to acetaldehyde.27) The conversion of acetaldehyde to ethanol also requires NADH, and thus, the disruption of genes involved in ethanol production (pdc1, pdc5, and pdc6) may also lead to increased 1-butanol production in S. cerevisiae. Although thl gene was shown to have a negative effect on 1-butanol production in the yeast strains containing the bcd, etfA, and etfB genes for the conversion of crotonyl-CoA to butyryl-CoA in this study, the thl gene may facilitate 1-butanol production in ter-containing yeasts. This may be a result of more butyryl-CoA being produced by TER than by BCD-ETFAB causing a shortage of crotonyl-CoA, β-hydroxybutyryl-CoA, and acetoacetyl-CoA. In the case of the thl-containing strain, the equilibrium reaction between acetyl-CoA and acetoacetyl-CoA, thus, led to an increase in acetoacetyl-CoA production compared with the strain lacking the thl gene. In conclusion, we demonstrated that the engineered 1-butanol-producing yeast using the Clostridium pathway for butanol production coupled with the elimination of glycerol production pathway genes and using the ter gene derived from T. denticola was shown
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to strongly enhance 1-butanol production. The findings of processes to enhance the production of 1-butanol in yeast presented here provide important information for further application and enhancement of 1-butanol production by S. cerevisiae.
Author contribution HS, HM, KK, and MU conceived and designed the study. HS and HM analyzed the data. HS, KK, and MU drafted the manuscript. All authors read and approved the final manuscript.
Supplemental material The supplemental material for this paper is available at http://dx.doi.org/10.1080/09168451.2014.972330.
Acknowledgments This research was supported by CREST and Science and technology research promotion program for agriculture, forestry, fisheries, and food industry.
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