Correspondence to: B. Zhuge, Key Laboratory of Industrial Biotechnology of Ministry of Education, Research Centre of Industrial Microorganisms, School of Biotechnology, Jiangnan University, Wuxi 214122, People's Republic of China.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme of glycolysis, catalyses the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate using NAD+ as the co-enzyme (Harris and Waters, 1976). GAPDH is considered to be a classical glycolytic protein, which is utilized as a model for protein and also as an internal control factor for relative quantitation of gene expression (Mazzola and Sirover, 2003; Sirover, 2005). One minor isoform in Saccharomyces cerevisiae, TDH1, was regulated by reductive stress caused by an excess of cytoplasmic NADH (Valadi et al., 2004). GAPDH was determined to fuse with triose phosphate isomerase (TPI) and form a single transcriptional unit (tigA) in Phytophthora species (Unkles et al., 1997). This TPI–GAPDH fusion protein was demonstrated to indicate a mitochondrial origin of the eukaryotic glycolytic pathway, all of these suggesting a positive role of GAPDH in an important structure and enlightening our study on GAPDH in C. glycerinogenes.
Candida glycerinogenes has been commercially exploited to produce glycerol (Zhuge et al., 2001). Although the physiological and fermentation properties of C. glycerinogenes have been investigated (Chen et al., 2008; Wang et al., 2001; Jin et al., 2003), fewer molecular studies have been conducted compared to other yeasts. Unlike other osmotolerant yeasts, some other pathways might be involved in glycerol biosynthesis in addition to glycolysis (Zhang et al., 2007). Therefore, research on glycerol metabolism and the interaction of metabolic pathways would be valuable and practical. Based on the mechanisms of the hexose monophosphate pathway (HMP), which plays an important role in polyol biosynthesis in osmotolerant yeast exposed to high osmotic stress (Spencer and Shu, 1957; Spencer et al., 1956) [11,12], GAP formed from HMP is catalysed by the triose phosphate isomerase activity. In this study, the CgGAP gene encoding glyceraldehyde-3-phosphate dehydrogenase of C. glycerinogenes was cloned and its sequence was analysed.
Materials and methods
Yeast strains, plasmids, growth and stress conditions
Candida glycerinogenes WL2002-5 (Zhuge et al., 2001) was grown in YPD medium (yeast extract 1% w/v, bactopeptone 2% w/v, glucose 2% w/v and agarose 1.5% w/v for solid medium). Saccharomyces cerevisiae W303-1A (MATa, leu2-3, ura3-1, trp1-1, his3-11, ade2-1, can1-100) was used for the construction of S. cerevisiae mutant. As necessary, 150 µg zeocin/ml were added to the solid medium. Escherichia coli DH5a was cultured in Luria–Bertani (LB) medium supplemented with 100 µg/ml ampicillin or 25 µg/ml Zeocin and used for plasmid propagation.
Liquid growth assays were performed by pregrowing the strains in YNBG medium (0.67% w/v YNB, 2% w/v glucose). Shake-flask cultivations were performed following Zhuge et al. (2001)) and Chen et al. (2008)).
Cloning of a putative CgGAP gene
Genomic DNA of C. glycerinogenes was extracted as described previously (Burke et al., 2000). The degenerate primers CgG-F (TBRTBGGITCHGGYAAYTGGG), encoding IKVGINGFG (amino acid residues 3–11), and CgG-R (RGCVAYVAYRTTYTTYARIGC), encoding YDNEYGYSTR (amino acid residues 315–324), were designed corresponding to conserved regions of amino acid sequences of different GAP protein sequences. The degenerate PCR was performed for 30 cycles consisting of 94°C for 50 s, 56°C for 1 min and 72°C for 2 min, with a final 72°C for 10 min using Hot Start Taq (Takara) polymerase, and then sequenced. Based on the sequence obtained, two inverse PCR primers, CgI-F (CCTTATTTCCGTGTTCGTGTTATTAGTG) and CgI-R (CGCCTTCAATCAATTCTTCATAGAC) were designed to complete the putative CgGAP full-length gene. C. glycerinogenes genomic DNA was digested with NheI and SacI (Takara). After extraction, the precipitate was circularized with T4 DNA ligase (Takara). Inverse PCR was performed for 35 cycles consisting of 94°C for 1 min, 58°C for 1 min and 72°C for 3 min, using LA Taq (Takara) polymerase. Both 2.3 and 2.2 kb PCR products were purified, cloned into pMD18-T vector, sequenced and analysed.
Sequence alignment and phylogenetic analysis
The nucleotide sequence, deduced amino acid sequence and open reading frame (ORF) were analysed using SeqMan (DNAStar) and DNAMAN sequence analysis software (Lynnon Biosoft), and the sequence comparison was conducted using the BLAST program (NCBI; http://www.ncbi. nlm.nih.gov). The phylogenetic analysis of CgGAP was aligned with Clustal W (v. 2.0), using default parameters. A phylogenetic tree was constructed using MEGA version 4.0 (Tamura et al., 2007). The neighbour-joining method (Saitou and Nei, 1987) was used to construct the tree.
Functional complementation of CgGAP in Saccharomyces cerevisiae
A 3.9 kb DNA fragment containing CgGAP and its flanking sequence was amplified by PCR from C. glycerinogenes genomic DNA, using LA Taq (Takara) polymerase and primers CgGA-F (ACGGAATTCATGGCTTTCACAGTTG) and CgGA-R (ACGCTCGAGTCAGTTAGAGAGAG CTG). PCR products (1.1 kb long) were inserted into plasmid PYX212, previously digested with the same restriction enzymes. The plasmid was cloned in Escherichia coli DH5a and the positive clone was selected and designated as PYX212–CgGAP. Yeast transformation was performed according to standard protocols (Adams et al., 1997).
Enzyme assay and determination of NADH, glucose and glycerol
Cell extracts were prepared as described previously (van Hoek et al., 2000). The protein concentration of cell-free extract was determined by the method of Bradford (1976). The specific activity of the CgGAP was determined on cell extracts according to Serrano et al. (1993).
Determination of intracellular NADH was performed as described previously (Jennifer et al., 2008). Glucose was determined by using a glucose analyser. Glycerol was monitored as described previously (Harris and Waters, 1976).
Construction of the reporter gene vector PYX212–zeocin–PCgGAP–gfp
In this study, green fluorescent protein (gfp) was used as a reporter. The vector (PYX212–zeocin–PCgGAP–gfp), which carries a gfp gene under the control of the C. glycerinogenes CgGAP promoter, was used to establish the transformation protocol and as a proof of principle to demonstrate the functionality of the promoter approach in Saccharomyces cerevisiae. For the construction of this vector, a 1068 bp CgGAP promoter fragment (position 1–1068, 3′ from the AUG of the CgGAP gene) was amplified from the genomic DNA of C. glycerinogenes by PCR as well as the 756 bp gfp gene fragment, which was amplified out of the commercial available plasmid pCAMBIA1302 and ligated together via a HindIII restriction site. The resulting fragment (PCgGAP–gfp) was ligated into the PYX212–zeocin vector using T4 DNA ligase (Takara).
Photomicrography and fluorescence microscopy
Transformant exposed to 4°C for 4 h was collected and suspended in buffer PBS (20 mm Tris–HCl, pH 8.0, 1.0 mm EDTA) and the microorganism observed by fluorescence microscopy (Olympus, Tokyo) at × 1000 magnification. The excitation and emission filters used were ca. 485 and 520 nm, respectively.
Results and discussion
Cloning and analysis of CgGAP from C. glycerinogenes
A 0.93 kb PCR fragment was obtained from C. glycerinogenes genomic DNA by PCR amplification using two degenerate primers. A 2300 bp fragment containing the entire putative CgGAP was obtained by inverse PCR (Figure 1). Sequence analysis revealed that the open reading frame (ORF) of the putative CgGAP gene consisted of 1164 nucleotides encoding a polypeptide of 387 amino acids. One putative translational start codon (ATG) was found, whereas the nucleotides were AAAAATGG, having A, A and G at positions −3, –1 and +4, respectively. This position corresponds very well with the consensus initiation signal in yeast (Kozak, 1991). This observation indicates that the putative start codon in the CgGAP gene may be the ATG at position 1. In the 5′-flanking non-coding region of the CgGAP gene, six stress-responsive elements (STREs) characterized by the core sequence motif AGGGG or CCCCT (Schmitt and McEntee, 1996) were found at positions −1290, –1027, –542, –477, –283 and −147. Several genes containing STREs, including GPD1 and CTT1 in S. cerevisiae, are induced by environmental stresses such as heat shock, oxidative stress and, especially, osmotic stress (Schuller et al., 1994; Marchler et al., 1993). This result suggests that CgGAP may be a stress-response gene. Some general cis-elements, including the TATA box at positions −97 and −87 and GAAT at position −128 were revealed in the upstream sequences, and a putative polyadenylation signal sequence, AAAATA, was found 356 nt downstream of the stop codon. Since the genome is AT-rich, it will be interesting to compare the sequence elements with those of CG-rich promoters.
Comparative analysis of the deduced amino acid sequence and phylogenetic relationships
Comparison of the CgGAP with amino acid sequences was conducted and the results revealed that CgGAP shows the highest identity to other GAPs. The amino acid sequences of GAPs were aligned according to the Clustal W algorithm and showed that these GAPs had high similarity throughout the entire coding region. On the basis of amino acid sequence homologies with known eukaryotic GAPs, the gene was therefore designated CgGAP.
CgGAP was the first GAP gene cloned from C. glycerinogenes; therefore, it would be interesting to investigate the evolutionary relationship of various GAPs. The GXGXXG motif predicted as the binding site of the NAD+ co-enzyme (Otto et al., 1980) was identified in the deduced protein (Figure 1). Using MEGA version 4.0 from CLUSTAL W alignments, a phylogenetic tree of GAPs was constructed from different organisms (Figure 2).
Expression of the CgGAP gene in S. cerevisiae
In order to test the function of CgGAP in S. cerevisiae, complementation analysis was performed in the constructed S. cerevisiae. When the constructed plasmid pYX212–CgGAP was transformed in S. cerevisiae, the cells harbouring CgGAP were observed to grow on minimal YNB medium with glucose as the sole carbon resource, which indicated that the CgGAP was functional in S. cerevisiae (Table 1).
Table 1. Fermentation parameter analysis of strains modified
In S. cerevisiae, glycerol is chiefly produced to re-establish the balance in cytosolic redox equivalents by oxidizing the additional NADH generated during yeast biomass accumulation, like those found during table wine fermentation. Glycerol synthesis also permits yeast to fulfil the demand for oxidized co-factor, NAD+, in response to a build-up of NADH generated in glycolysis to cope with the initial stages of sugar assimilation upon inoculation (Bisson, 1993).
In conclusion, this study has shown that C. glycerinogenes has a CgGAP similar to GAP genes in other yeasts, which points to the possibility that C. glycerinogenes follows similar molecular mechanisms in the synthesis of glycerol, and other factors, including the HOG pathway and the glycerol export mechanism, are involved in the ability of this yeast to produce high amounts of extracellular glycerol. An investigation of GAP regulation and glycerol export may yield fruitful information.
Expression of the gfp gene in S. cerevisiae under the control of the CgGap promoter
The 1068 bp promoter fragment of the CgGAP gene was placed directly in front of the AUG sequence of the gfp gene (Figure 3). The resulting vector, PYX212–zeocin–PCgGAP–gfp, was transformed into S. cerevisiae. The fluorescence of the strains was analysed as described above after microscopic examination of cells grown on YEPD medium. It is obvious that the transformed S. cerevisiae strain shows a bright green fluorescence after light activation (Figure 4B). Compared to the transformant carrying the gfp construct, this indicates that the CgGAP promoter is strong enough to exert a fluorescent signal after expression of the gfp gene if it is fully induced due to growth on YEPD medium.
In conclusion, the CgGAP gene promoter, PCgGAP, constitutively expresses genes in S. cerevisiae cells grown on glucose. In addition, green fluorescent protein (gfp) expressed under transcriptional control of PCgGAP can be used as a suitable reporter protein to study protein import in S. cerevisiae.
Here we show that the CgGAP gene encoding a glyceraldehyde-3-phosphate dehydrogenase was cloned and characterized with inverse PCR. Sequence analysis revealed a 1164 bp open reading frame encoding a putative peptide of 387 deduced amino acids with a molecular mass of 36 kDa. The CgGAP gene consisted of an N-terminal NAD+-binding domain and a central catalytic domain, whereas six stress-response elements were found in the upstream region. Functional analysis revealed that C. glycerinogenes has a CgGAP similar to GAP genes in other yeasts, based on their deduced amino acid sequence, which points to the possibility that C. glycerinogenes follows similar molecular mechanisms in the synthesis of glycerol, and that other factors, including the HOG pathway and the glycerol export mechanism, are involved in the ability of this yeast to produce high amounts of extracellular glycerol. An investigation of GAP regulation and glycerol export may yield fruitful information. Promoter studies in S. cerevisiae using green fluorescent protein (gfp) as a reporter showed that the GAP promoter (PCgGAP) is constitutively expressed in S. cerevisiae cells grown on glucose.
This work was funded by China National “863” High-Technology Research and Development Program (No. 2012AA021201, No. 2011AA02A207) and supported by the National Natural Science Foundation of China (No. 31270080).