Xun Chen, School of Chemical Engineering and Technology, Tianjin University, Weijing Road 92, Tianjin, China. E-mail: email@example.com
Aims: To investigate the effect of FPS1 deletion on the fermentation properties of Saccharomyces cerevisiae and to evaluate whether FPS1 deletion would result in higher ethanol yield.
Methods and Results: FPS1 of S. cerevisiae was knocked out using the one-step gene replacement method. The fermentation properties of the fps1Δ mutant under microaerobic conditions were investigated and compared with that of the wild type. Consumption of glucose, yield of ethanol, yield of glycerol, acetic acid and pyruvic acid were monitored. Compared with the wild type, the ethanol yield of the fps1Δ mutant was improved by 10 ± 2% and glycerol yield decreased by 18·8 ± 2%. Meanwhile, acetic acid yield decreased by 5·4 ± 1%, and pyruvic acid yield decreased by 58·6 ± 1%.
Conclusions: FPS1 deletion of S. cerevisiae resulted in reduced glycerol yield and higher ethanol yield.
Significance and Impact of the Study: The cost of carbon source in ethanol fermentation is an important factor in determining ethanol production. Approximately 5% carbon source is converted into glycerol in ethanol fermentation. Eliminating formation of glycerol through FPS1 deletion can be used to increase ethanol yield of S. cerevisiae without increasing the overall cost of carbon source.
With the inevitable depletion of the world's energy supply, there has been an increasing worldwide interest in renewable sources of energy, such as fuel ethanol. Nearly all fuel ethanol is produced by fermentation of corn glucose in the US or sucrose in Brazil (Rosillo-Calle and Cortez 1998; MacDonald et al. 2001). The substrate cost is a primary factor in determining the overall economy of ethanol production. From the practical view it is necessary to ensure an efficient utilization of the carbon source for the overall economy of ethanol production. In anaerobic fermentation of Saccharomyces cerevisiae (S. cerevisiae), in addition to biomass and carbon dioxide, a number of byproducts are produced, such as glycerol and organic acids (e.g. acetic acid and pyruvic acid, succinic acid). Eliminating formation of glycerol is expected to increase ethanol yield of S. cerevisiae.
Glycerol formation has two roles in the fermentation of S. cerevisiae (Nevoigt and Stahl 1997). Under anaerobic fermentations when the respiratory chain is not functioning, net formation of NADH produced during synthesis of biomass and organic acids, i.e. acetic acid, pyruvic acid, and succinic acid, must be reoxidized to NAD+ by formation of glycerol in order to avoid a serious imbalance in the NAD+/NADH ratio. Synthesis of 1 mol glycerol from glucose leads to reoxidation of 1 mol NADH. Furthermore, during growth under osmotic stress conditions glycerol is formed and accumulated inside the cell where it works as an efficient osmolyte that protects the cell against lysis.
The amount of intracellular glycerol is controlled by its biosynthetic pathway as well as by a regulated transmembrane transport system. Glycerol is produced from the glycolytic intermediate dihydroxyacetone phosphate in two steps catalysed by NAD+-dependent glycerol-3-phosphate dehydrogenase and glycerol-3-phosphate phosphatase. Both enzymes are encoded by two similar isogenes, GPD1 plus GPD2 and GPP1 plus GPP2, respectively. Expression of GPD1 and GPP2 is induced by high osmolarity, whereas that of GPD2 and GPP1 is stimulated under anaerobic conditions (Albertyn et al. 1994; Eriksson et al. 1995; Ohmiya et al. 1995; Ansell et al. 1997; Martijn et al. 1999). Glycerol-3-phosphate dehydrogenase but not glycerol phosphatase is rate limiting for glycerol production in S. cerevisiae (Remize et al. 2001). Glycerol-3-phosphate dehydrogenase mutants result in a higher ethanol production compared with the wild type (Valadi et al. 1998; Nissen et al. 2000a,b; Bakker et al. 2001).
Glycerol can cross the plasma membrane by passive diffusion or by facilitated diffusion through the MIP protein channel Fps1p (Oliveira et al. 2003). Fps1p can transport glycerol in both directions; however, the main role of this glycerol facilitator is to regulate glycerol export rather than its uptake (Luyten et al. 1995, Tamás et al.1999). The opening and closing of the Fps1p channel is responsible for changes in glycerol permeability during osmotic adaptation.
Recent advances on the regulation of glycerol synthesis and transport in S. cerevisiae led to the development of metabolic engineering approaches to reduce glycerol yield during industrial ethanol fermentation processes. The purpose of this study was to investigate the effects of FPS1 deletion on the fermentation properties of S. cerevisiae, and to evaluate whether FPS1 deletion would result in reduced glycerol yield and higher ethanol yield.
Materials and methods
Yeast strains and media
The S. cerevisiae strains used in this study were all isogonics to DC124 as described in Table 1.
Table 1. Strains used in this study. The genetic background of all the strains is DC124
Matαleu2 ura3 trp1 his3 ade8 can1
M. Wigler (Cold Spring Harbor, NY, USA)
Matαleu2 ura3 trp1 his3 ade8 can1 fps1Δ::LEU2
The strains of S. cerevisiae cells were routinely grown in medium containing 2% peptone and 1% yeast extract supplemented with 2% glucose as carbon source (YPD). Selective media SC (Burke et al. 2000) minus leucine were used for selection of transformants containing LEU2-selective marker.
Growth conditions and experimental procedures
Incubation conditions were standardized at 30°C and 200 rev min−1 orbital shaking. Standard techniques were applied as described in Sambrook et al. (1989) for all gene cloning experiments. DNA fragments were purified using DNA recycle kits (Tiangen, Beijing, China) and PCR products were purified using phenol deproteinization and ethanol precipitation. Restriction and modification enzymes were used according to the manufacturers’ instructions. Yeast transformation was performed by the lithium acetate method. Escherichia coli Top10′ was used for subcloning. All yeast strains were maintained at 4°C on YPD plates, prepared monthly from a glycerol stock kept at −75°C.
Microanaerobic cultivations were performed at 30°C in the unbaffled shake flasks kept at constant stirring speed of 100 rev min−1 with 0·1 l medium (containing 2% peptone and 1% yeast extract supplemented with 8% glucose as carbon source). Initial biomass concentrations were set at OD660 nm 1·0 after inoculation. Fermentation experiments were performed in triplicate and one representative experiment was shown.
Plasmids and strains constructions
The plasmids and primers used in this study were described in Tables 2 and 3, respectively.
Primer FPS1C-5′ containing restriction enzyme site for EcoRI and SmalI in front of nucleotides 460 to 441 upstream of the ATG start codon of FPS1, and primer FPS1C-3′ containing nucleotides 2243 to 2224 of the complementary strand downstream of the ATG start codon of FPS1, were used to clone a 2703 bp fragment containing the open reading frame of FPS1 by PCR with the Taq DNA polymerase (TAKARA, Dalian, China). The PCR products were digested with EcoRIII and HindIII, then ligated into EcoRI and HindIII digestion sites of the plasmid pUC18, resulting in the plasmid pUC18-FPS1C.
Primer oligo1 containing the restriction enzyme site for PstI in front of nucleotides of 2008 to 2030 downstream of the ATG start codon of FPS1, and oligo2 containing the restriction enzyme site for XbaIII in front of nucleotides 27 to 9 of the complementary strand upstream of the ATG start codon of FPS1, were used to clone a 3206-bp fragment (named as pUC18-FPS1C-1) of the pUC18-FPS1C containing 450 and 120 bp of FPS1 on the two ends. The DNA fragment of was digested with XbaIII and PstIII.
pUC18-FPS1C-1-LEU2 plasmid construction
Primer LEU2-U containing the restriction enzyme site for XbaI in front of 20 nucleotides of plasmid YEplac181, and LEU2-D containing restriction enzyme site for PstI in front of 20 nucleotides of plasmid YEplac181, were used to clone a LEU2 selectable marker gene of plasmid YEplac181.The PCR product was 1560 bp, and digested with PstI and XbaI, then ligated with a DNA fragment of pUC18-FPS1C-1 digested with XbaI and PstI, resulting in plasmid pUC18-FPS1C-1-LEU2.
Yeast strain constructions
Plasmid pUC18-FPS1C-1-LEU2 was digested with EcoRI and HindIII. DNA fragments of FPS1C-1-LEU2 were recycled using DNA recycle kits. DNA fragments of FPS1C-1-LEU2 were transformed into wild-type strains, and the transformation mixture was incubated on SC minus leucine plates for 2–3 days to obtain one-step gene replacement. Correct deletion of FPS1 was verified by PCR analysis with primers FPS1C-5′ and FPS1C-3′.
Growth was followed by measuring the absorbance of the cultures at 660 nm in a Bioquest CE2502 spectrophotometer (Progen Scientific, UK).
Measurement of glucose, ethanol, glycerol, acetic acid and pyruvic acid
The samples (0·0015 l each) were centrifuged for 5 min at 18 000 g and the resulting supernatants were frozen (−20°C) until analysis. The content of glycerol and glucose in the fermentation broth was determined by HPLC using differential refractive index detector and Agilent ZORBAX carbohydrate column (Agilent, Beijing, China) eluted by 75% acetonitrile with 0·001 l min−1. The content of acetic acid and ethanol was determined using a gas chromatograph (Shimadzu GC-2010; Shimadzu, Kyoto, Japan) with a DB-WAX capillary column and a flame ionization detector (FID). The temperatures of inlet, oven and detector were respectively kept at 200, 150 and 200°C. The content of pyruvic acid was determined using an RP18 column (Waters, Milford, MA, USA) eluted with 0·1 mol l−1 KH2PO4 (pH 3·0) at a flow rate of 0·0006 l min−1 at 30°C and a photodiode array (PDA) detector.
Determination of dry weight
Samples (0·05 l) were centrifuged at 5000 g for 10 min and washed twice with water, and subsequently the pellets were kept at 110°C for 24 h before temperature equilibration and weighing.
The fermentation properties of DC124 (wild type) and fps1Δ mutant were studied under microaerobic conditions. As described in Fig. 1a, the growth profile of the fps1Δ mutant was similar to the wild type under microaerobic conditions. But, at the end point of the fermentations (when glucose in the medium was consumed completely, i.e. 16 h), dry weight (biomass concentration) of the fps1Δ mutant had a slight decrease as described in Fig. 2e.
As given in Fig. 1a,b, yield of ethanol and yield of glycerol of DC124 and the fps1Δ mutant increased slowly during the first 4 h of the fermentations, but increased sharply during the log phase. At the end point of the fermentations (16 h), the fps1Δ mutant decreased by 18·8 ± 2% in glycerol yield compared with the wild type as described in Fig. 2d, while the ethanol yield was improved by 10 ± 2%, shown in Fig. 2c, and the acetic acid and pyruvic acid yield of the fps1Δ mutant decreased 5·4 ± 1% and 58·6 ± 1%, respectively, as shown in Fig. 2a,b. However, glucose consumption profiles of the fps1Δ mutant were similar when compared with the wild type during the growth phases according to Fig. 1d.
The main role of Fps1p is to regulate glycerol export. Because of the inactivity of glycerol export channel protein Fps1p, intracellular glycerol of fps1Δ mutant cannot efflux quickly, so extracellular glycerol increased more slowly in the first period of fermentations. The accumulated intracellular glycerol may induce other regulation systems of S. cerevisiae to reduce glycerol biosynthesis. On the other hand, other glycerol transport systems may regulate the efflux of the accumulated intracellular glycerol when the content of intracellular glycerol reaches a flash point, so the extracellular glycerol increased quickly during the last period of fermentations. But the glycerol yield of the fps1Δ mutant decreased significantly compared with that of the wild type.
The glycerol produced under anaerobic conditions would lead to an increase in turgor if not effectively exported. Tamás et al. (1999) reported that Fps1p is required for glycerol export under anaerobic conditions, and mutants lacking Fps1p grew poorly under anaerobiosis. But in our research, under microaerobic conditions, the growth profile of fps1Δ mutant was similar to that of the wild type. This can be explained by, on the one hand, diffusion through the membrane or other less efficient export pathways may be involved in the adaptation of microaerobic conditions, or on the other hand, there were some differences between microaerobic conditions and anaerobic conditions.
Although the production of ethanol from glucose is redox neutral, the formation of certain metabolites, such as organic acids, i.e. acetic acid and pyruvic acid, leads to surplus production of NADH (Ohmiya et al. 1995; Valadi et al. 1998; Nissen et al. 2000a,b; Bakker et al. 2001). Glycerol metabolism is essential for anaerobic growth of yeast when a surplus of NADH cannot be reoxidized by respiration. When glycerol synthesis is hampered, the yeast cells may reduce organic acids biosynthesis to solve the redox problem. Our data have shown that acetic acid and pyruvic acid yield of fps1Δ mutant decreased 5·4 ± 1% and 58·6 ± 1%, respectively, compared with that of the wild type. The decrease in acetic acid and pyruvic acid formation is an example of a metabolic adjustment by the cells to minimize the NADH surplus when glycerol synthesis capacity is hampered. According to metabolic flux analysis of S. cerevisiae (Nissen et al. 1997), when glycerol yield, acetic acid yield and pyruvic acid yield decreased, the metabolism must shift towards ethanol. In our research, glycerol yield, acetic acid yield and pyruvic acid yield of fps1Δ mutant decreased 18·8 ± 2%, 5·4 ± 1% and 58·6 ± 1% respectively, while ethanol yield of fps1Δ mutant increased 10 ± 2%, which verified the metabolic flux analysis of Nissen et al. (1997).
fps1Δ mutant may solve the occurring redox balance problems partly by reducing acetic acid and pyruvic acid formation, but the redox balance problem may still exist during ethanol fermentation. Further modifications of the redox metabolism are necessary to further increase ethanol yield. A suitable way to solve the redox balance problem could be simultaneous over-expression of GLN1 and GLT1 which encode glutamine synthetase and glutamate synthase, respectively (Nissen et al. 2000a,b). In S. cerevisiae, ammonium is assimilated into glutamate by reaction with 2-oxoglutarate. In wild-type cells this reaction is catalysed by an NADPH-dependent glutamate dehydrogenase encoded by GDH1 (Equation 1):
Another system that synthesizes glutamate exists in S. cerevisiae. This consists of two coupled reactions, catalysed by glutamate synthase (Equation 2), encoded by GLT1, and glutamine synthetase (Equation 3), encoded by GLN1:
So simultaneous over-expression of GLN1 and GLT1, which encode glutamine synthetase and glutamate synthase respectively, to consume surplus NADH, can resolve the redox balance problem.
In conclusion, in our research, the glycerol yield of fps1Δ mutant decreased 18·8 ± 2%, while the ethanol yield increased about 10 ± 2%. The present work demonstrated, for the first time, that the FPS1 deletion mutant of S. cerevisiae can be used to improve ethanol yield.
This work was supported by National ‘863 Program’ project of China (2002AA647040).