Expression and functional studies of genes involved in transport and metabolism of glycerol in Pachysolen tannophilus
© Liu et al.; licensee BioMed Central Ltd. 2013
Received: 21 December 2012
Accepted: 13 March 2013
Published: 21 March 2013
Pachysolen tannophilus is a non-conventional yeast, which can metabolize many of the carbon sources found in low cost feedstocks including glycerol and xylose. The xylose utilisation pathways have been extensively studied in this organism. However, the mechanism behind glycerol metabolism is poorly understood. Using the recently published genome sequence of P. tannophilus CBS4044, we searched for genes with functions in glycerol transport and metabolism by performing a BLAST search using the sequences of the relevant genes from Saccharomyces cerevisiae as queries.
Quantitative real-time PCR was performed to unveil the expression patterns of these genes during growth of P. tannophilus on glycerol and glucose as sole carbon sources. The genes predicted to be involved in glycerol transport in P. tannophilus were expressed in S. cerevisiae to validate their function. The S. cerevisiae strains transformed with heterologous genes showed improved growth and glycerol consumption rates with glycerol as the sole carbon source.
P. tannophilus has characteristics relevant for a microbial cell factory to be applied in a biorefinery setting, i.e. its ability to utilise the carbon sources such as xylose and glycerol. However, the strain is not currently amenable to genetic modification and transformation. Heterologous expression of the glycerol transporters from P. tannophilus, which has a relatively high growth rate on glycerol, could be used as an approach for improving the efficiency of glycerol assimilation in other well characterized and applied cell factories such as S. cerevisiae.
KeywordsP. tannophilus Glycerol Transport S. cerevisiae
Pachysolen tannophilus is known for its ability to ferment D-xylose, one of the major components of hemicellulose plant residues, to ethanol . However, P. tannophilus has also been shown to be capable of converting crude glycerol to ethanol under microaerobic conditions . This ability is interesting since glycerol, a by-product of biodiesel production, has also been considered as a potential alternative carbon source for industrial bioprocesses due to the recent dramatic increase in production of biodiesel.
The whole genome of P. tannophilus CBS4044 has been sequenced , and with this, the possibility for understanding and exploiting glycerol transport in this yeast has arisen. A number of studies have investigated glycerol transport, consumption and production by different types of yeast [4–6], but the glycerol transport and metabolic pathways in P. tannophilus have not been studied so far. P. tannophilus has a relatively high growth rate on glycerol (0.3 h-1) , but improvement of the strain for industrial applications through engineering the metabolism is hampered due to the lack of tools for genetic modification. However, with glycerol becoming an increasingly abundant substrate, it is highly relevant to characterize strains which are capable of its utilization, and consider the possibility of expressing genes from these strains in industrial cell factories for which genetic engineering strategies are already well defined.
Glycerol transport is the first barrier to glycerol utilization in a microbial cell. In S. cerevisiae, glycerol enters the cell by two different mechanisms: a low affinity transport system (facilitated diffusion) and a high affinity proton symport system (active transport) . When glucose is present, glycerol diffuses into the cells through a glycerol channel by facilitated transport, a process dependent on the FPS1 gene [8, 9]. The physiological role of the facilitator Fps1p in S. cerevisiae has been described to be glycerol export rather than uptake during hypo-osmotic shock . However, when only non-fermentable carbon sources (glycerol, acetate, ethanol) are present, an active uptake system driven by a proton motive force ensures the uptake of glycerol [7, 10]. Two multi-membrane-spanning proteins encoded by GUP1 and GUP2 (Glycerol Uptake Protein), were first identified as being involved in active glycerol uptake in S. cerevisiae. However, in later studies Gup1p and 2p were proposed to have different roles than glycerol transport . A screen for genes encoding membrane proteins involved in glycerol assimilation in S. cerevisiae identified a gene, STL1, involved in active glycerol uptake. Stl1p is localized in the plasma membrane, is glucose repressed and inactivated by growth in glucose [7, 12]. Importantly, it was concluded that the protein is a member of the sugar transporter family and acts as a glycerol proton symporter . Stl1p has also been shown to have a function in glycerol uptake in several other yeasts. It has been shown that in Candida albicans glycerol was actively transported into the cells by a proton symporter encoded by the C. albicans STL1. It has also been reported that S. cerevisiae strains harboring the STL1 gene from D. hansenii slightly improved their growth and doubling times on glycerol .
After glycerol is transported into the cells, two different routes have been identified in yeasts for further assimilation: a phosphorylation route and an oxidation route. Within the first pathway, glycerol is dissimilated by glycerol kinase encoded by GUT1 and then by glycerol-3-phosphate dehydrogenase encoded by GUT2, which is located at the outer surface of the mitochondrial membrane . Another pathway is catalyzed by glycerol dehydrogenase encoded by GCY1 followed by dihydroxyacetone kinase encoded by DAK1 and DAK2. In Schizosaccharomyces pombe, it has been reported that glycerol is utilized solely by this pathway [15, 16]. In both pathways, glycerol is converted to dihydroxyacetone phosphate, which then enters glycolysis. The pathways involved in glycerol utilization differ in different yeasts. In some yeast strains, all four enzymes in both pathways are present, but only one pathway functions for glycerol dissimilation [4, 6]. In S. cerevisiae, it has been shown that glycerol is degraded by the phosphorylation pathway and that mutants lacking one of the two genes are incapable of utilizing glycerol . Although the fermentative pathway was also discovered to be present in S. cerevisiae, the function is unknown. The production of glycerol has two functions in S. cerevisiae: redox balance and protection against osmotic stress as a compatible solute (osmolyte) [6, 19]. Glycerol is commonly produced in the cytosol of yeasts from the glycolytic intermediate dihydroxyacetone phosphate. This compound is converted into glycerol in two steps that are catalyzed by glycerol-3- phosphate dehydrogenase (Gpd) and glycerol-3-phosphatase (Gpp) respectively. Each of these enzymatic activities has two isoenzymes, Gpd1p and Gpd2p, Gpp1p and Gpp2p .
Although there have been several studies on glycerol metabolism in S. cerevisiae, many industrially used baker’s yeast strains and laboratory strains grow poorly on glycerol. For example, the specific growth rate of the well-studied lab strain S. cerevisiae CBS 8066 under aerobic conditions was found to be very low, 0.010 ± 0.002 h-1, during shake flask cultivations in minimal medium containing glycerol as sole carbon source . Strategies based on adaptive evolution, and overexpression or knock out of the genes involved in glycerol metabolism have been performed with the aim of increasing the host strain’s growth on glycerol and production of different value-added products have been pursued [21, 22]. By adaptive evolution, strains with growth rates of up to 0.2 h-1 have been achieved . However, one possible rate-limiting step during glycerol metabolism, glycerol transport, was not addressed in those approaches .
In this study, a comparison of the genome sequences of S. cerevisiae and P. tannophilus has been performed to study the genes involved in glycerol transport and glycerol metabolism in these two organisms. Quantitative real-time PCR was performed to compare transcript levels during growth on glycerol. The genes involved in glycerol transport in P. tannophilus were heterologously expressed in a S. cerevisiae STL1 knockout strain to assess their function and to evaluate whether they could possibly contribute as transporter genes to improve the growth of S. cerevisiae on glycerol.
Identification of genes in glycerol metabolism and analysis of glycerol transporters in P. tannophilus
Genes potentially involved in glycerol metabolism in P. tannophilus
Gene name S. cerevisiae
Gene name P. tannophilus
For FPS1, the lengths of PtFps1p, PtFps2p, PpFps1p, YaFps1p and GlpF were relatively short compared to other Fps1p proteins. PtFps1p was shown to be 54% identical to PpFps1p, 45% to YlFps1p, 35% to ScFps1p and only 31% to GlpF. PtFps2p was shown to be 60% identical to PpFps1p, 49% to YlFps1p, 34% to GlpF and only 32% to ScFps1p. PtFps1p and PtFps2p were grouped together with YlFps1p and PpFps1p, while they were in a separate branch than ScFps1p. For STL1, the identity between PtStl1p and ScStl1p was low, 34%, compared to the 56% identity observed between PtStl1p and YlPtStl1p.
Quantitative real time PCR
Performance of S. cerevisiae stl1Δ harboring different glycerol transporter genes
The glycerol symporter gene ScSTL1, which is responsible for glycerol transport in the absence of glucose, was knocked out. In order to validate the function of the glycerol transporters from P. tannophilus, all the predicted transporter genes from P. tannophilus were heterologously expressed in S. cerevisiae stl1Δ. Meanwhile, the glycerol facilitator Fps1p and glycerol symporter Stl1p in S. cerevisiae were also expressed in S. cerevisiae stl1Δ. To analyze the performance of the recombinant strains, growth in defined medium with 1% (v/v) glycerol as the sole carbon source was analyzed. The stl1Δ strain expressing different transporter genes PtFPS1, PtFPS2, PtSTL1, PtSTL2, ScFPS1, ScSTL1 and the S. cerevisiae CENPK 113-5D strain were tested under aerobic conditions in shake flasks.
With regard to glycerol transport, two glycerol facilitator homologues PtFPS1, PtFPS2 and two glycerol symporter homologues PtSTL1, PtSTL2 were found in the genome of P. tannophilus. Based on qPCR results, all the gene homologues identified to be involved in glycerol transport and assimilation were induced on glycerol as carbon source relative to the levels observed on glucose. The exceptions were the predicted glycerol symporters PtSTL1 and PtSTL2, which were constitutively expressed on both glycerol and glucose as carbon sources. PtFPS2 and PtGUT1 (19.6 fold and 17.6 fold) were the two most strongly up-regulated genes, which might lead to the hypothesis that they are most relevant for glycerol assimilation in P. tannophilus. Moreover, the two genes are located closely together in the genome in agreement with a model where the two genes are co-regulated and contribute to the same overall process, i.e. glycerol assimilation. Next, we focused our efforts on understanding the function of the genes involved in glycerol transport as this function may be the rate limiting step for glycerol metabolism. In this paper the functions of the two types of glycerol transporters from P. tannophilus were addressed by transferring them individually to the well characterized yeast S. cerevisiae.
The high-affinity transporter Stl1p was identified as the glycerol proton symporter in S. cerevisiae, which is a member of the sugar permease family of the major facilitator superfamily (MFS) . It was demonstrated that the transcription of STL1 gene was significantly induced with glycerol as the sole carbon source, and the STL1 gene was subject to glucose repression based on microarray-based transcriptome analysis [25, 26]. However, glycerol uptake by STL1 from C. albicans was not affected by the carbon source and salt stress . In D. hansenii, it was shown that the active glycerol transport system was constitutively expressed and not subject to glucose repression . In agreement with these experiments, we showed that the glycerol symporter genes PtSTL1 and PtSTL2 in P. tannophilus are constitutively expressed on glycerol and glucose based on qPCR expression analysis. However, the presence of the PtSTL1 and PtSTL2 genes had no obvious effect on the physiology of S. cerevisiae, while the glycerol consumption and growth in strains that overexpressed ScSTL1 was improved compared to stl1Δ strains. The symporter PtStl1p showed a low degree of sequence identities to ScStl1p with 34%. However, the PtStl1p from P. tannophilus exhibited 52% identity to the Stl1p from D. hansenii (DEHA2A12364p) and 56% to the Stl1p from Yarrowia lipolytica (YALI0C16522p). It has been reported that Y. lipolytica can grow on glycerol with a μmax around 0.3 h-1 and Pichia pastoris can grow on glycerol with a μmax 0.26 h-1. P. tannophilus can grow on glycerol with μmax around 0.29 h-1, while S. cerevisiae grows relatively slowly on glycerol with μmax of 0.02 h-1 (CEN.PK 113-5D). Both the facilitator and symporter similarities among P. tannophilus, Y. lipolytica and P. pastoris were higher than that compared to S. cerevisiae. However, more protein sequence data with verified functions are needed to provide a definitive conclusion.
Since glycerol transport might be the rate-limiting step for glycerol utilization, heterologous expression of the glycerol transporters from yeasts which have relatively high growth rates on glycerol could be used as the approach for improving the efficiency of glycerol assimilation in S. cerevisiae. Improved glycerol transport has been demonstrated here with an increased glycerol consumption rate and growth rate under aerobic conditions with S. cerevisiae.
The current study demonstrates the function of the glycerol transporters from P. tannophilus. Our studies open new possibilities for further improvement of glycerol fermentation in industrial yeast strains with heterologous expression of glycerol transporters from the glycerol utilizing P. tannophilus. This study thus proposes a possible route to development of glycerol-based bioprocesses in S. cerevisiae.
Strains and plasmids
Strains and plasmids used in this study
Plasmid or strain
Source or reference
Plasmid with loxP-kanMX-loxP disruption cassette
2μ-based URA3 plasmid with TEF1/PGK1 promoter
Integrative USER vector, with reusable URA3 Marker
Centromeric plasmid, URA3, PGAL1-Cre- TCYC1
pXI-5 with TEF1/PGK1 promoter and PtFPS1 gene
pXI-5 with TEF1/PGK1 promoter and PtFPS2 gene
pXI-5 with TEF1/PGK1 promoter and PtSTL1 gene
pXI-5 with TEF1/PGK1 promoter and PtSTL2 gene
pXI-5 with TEF1/PGK1 promoter and ScFPS1 gene
pXI-5 with TEF1/PGK1 promoter and ScSTL1 gene
MAT a MAL2-8 c SUC2 ura3-52
CEN.PK 113-5D Stl1 Δ (490,1279 )::loxP
stl1Δ with plasmid pXI-5-PtFPS1
stl1Δ with plasmid pXI-5-PtFPS2
stl1Δ with plasmid pXI-5-PtSTL1
stl1Δ with plasmid pXI-5-PtSTL2
stl1Δ with plasmid pXI-5-ScFPS1
stl1Δ with plasmid pXI-5- ScSTL1
Medium and culture conditions
For quantitative real-time PCR (qPCR) experiments, P. tannophilus was grown at 30°C in defined medium, containing (per liter) 0.67 g yeast nitrogen base w/o amino acids, 10 g succinic acid and 6 g NaOH as buffer system (initial pH around 5.6), 2% (v/v) glycerol or 2% (w/v) glucose as the carbon source. Cells were harvested at mid exponential growth phase at the same optical density by centrifugation at 5000 g, 4°C for 5mins, resuspended in 1 ml ice cold ddH2O, and the cell pellet was stored at −80°C. S. cerevisiae stl1Δ harbouring different glycerol transporter genes were cultivated at 30°C for 96 hours in agitated flasks with defined medium, containing (per liter) 7.25 g synthetic complete (SC) powder, 10 g succinic acid and 6 g NaOH as buffer system, 1% (v/v) glycerol as the sole carbon source.
Sequence retrieval and analysis
The genome sequence from P. tannophilus CBS4044 was obtained from previous work and is available in the EMBL database . The sequences of genes involved in glycerol transport and metabolism in S. cerevisiae FPS1, STL1, GUT1, GUT2, GCY1, Dak1/2, GPD1/2, GPP1/2 were used as queries in a BLAST search against the genome sequence of P. tannophilus. The genes with high similarities and high identities were listed as potential orthologous genes. Gene sequences were registered in GenBank at NCBI. The multiple sequence alignments with the amino acid sequences of FPS1 and STL1 transporters from different yeast strains and E. coli were performed by using ClustalW2 free program at PDBe. Phylogenetic analyses were performed by using PAUP* 4.0b10 . Unweighted parsimony analysis was performed. Trees were inferred using the heuristic search option with tree bisection-reconnection branch swapping and 1000 random sequence additions. Maxtrees were unlimited, branches of zero length were collapsed and all multiple parsimonious trees were saved. Clade stability was assessed in a bootstrap analysis with 1000 replicates. Trees were visualized in Treeview .
Quantitative real-time PCR
Total RNA isolation and cDNA synthesis
Total RNA was isolated from frozen cells using an RNeasy Mini Kit (QIAGEN, USA) according to the manufacturer’s protocol. The quantity and quality of the isolated RNA were measured by NanoDrop ND-1000. The total RNA was treated with DNaseI (Fermentas) prior to cDNA synthesis. Five μg total RNA were used to synthesize cDNA employing the RevertAid™ First Strand cDNA Synthesis kit (Fermentas) following the manufacturer’s recommendations using oligo(dT)18 primer. The cDNA was used as the template for quantitative real-time PCR for determining the transcript levels of the target genes under different growth conditions.
qPCR was performed on a Stratagene Mx3005P using the SYBR Green technology. The qPCR reaction mixture was prepared with 5 μl of 5 times dilution of cDNA as template, 10 μl SYBR Green master mix, 2 μl of 1 μM forward primer and 2 μl of 1 μM reverse primer and ddH2O to 20 μl. The PCR program for qPCR was as follows: 10 min of incubation at 95°C, followed by 40 cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 30 s, and finally the temperature was increased from 55°C to 94°C to check for unspecific products. The number of fluorescence threshold cycles (Ct) was calculated with the set threshold value by using Mx3005P software. Results presented are mean values of three independent experiments. Suitable primer pairs for all the genes investigated were designed using the Primer Express software v3.0 (Applied Biosystems) software with the following parameters: product size 140 - 180 bp and melting temperature (Tm) 57-59°C. The primers used in this work are listed in Additional file 1. In addition, control reactions which include all components for qPCR, except for the reverse transcriptase, were performed to detect the genomic DNA contamination. The absence of products under these conditions meant the absence of enough genomic DNA for successful amplification.
The relative expression levels were calculated approximately based on 2-ΔΔCT, where ΔΔCT = (CT gene of interest - CT internal control) sample A - (CT gene of interest - CT internal control) sample B. CT represents the cycle number at which a sample reaches a predetermined threshold signal value for the specific target gene. Relative expression data were normalized to the relative expression value of the housekeeping gene TAF12 in each sample, thus giving normalized relative expression for a target gene.
S. cerevisiae STL1 knockout strain construction
The construction of the STL1 knockout strain was done through the loxP-kanMX-loxP /Cre recombinase system . The primers for amplification of loxP-kanMX-loxP fragment from pUG6 were: KanMX_fw_STL1 (sequence 5′- AAAGCTGAAAATAGAGGGTTGCTGGTCAATTTAGAAGGTTCCACAATTGCCTGAAGCTTCGTACGCTG -3′) and KanMX_re_STL1 (sequence 5′- AAGCGTTTGTTGATGCACGAACTTTCATTGATGCAATTTCTGGTGGGTATCTAGTGGATCTGATATCAC -3′). The loxP-kanMX-loxP fragment with 50 bp homologous sequences in both ends to the gene STL1 was transformed into CENPK-113-5D  for obtaining the knockout strain by selection colonies on YPD supplemented with 200 μg/ml Geneticin (G418). The resistant cells were transformed with pSH47 and selected on SC medium lacking uracil. Positive clones were purified and grown in liquid YPD overnight. The next day, the cells were washed once with distilled water and then incubated in YPG medium for 2 h in order to induce expression of the Cre recombinase. The culture medium was diluted and spread on YPD plates. The colonies were picked and restreaked on YPD plates supplemented with and without G418. Clones that were not able to grow on the G418 containing medium were the knockout strains. The knockout strains were streaked on 5-FOA (740 mg/L) plates to eliminate the pSH47 plasmid. Yeast extract-peptone-dextrose (YPD) medium, synthetic complete (SC) medium, and SC lacking specific amino acids were prepared as described previously .
Constructs harboring different glycerol transporters
In this study, six expression plasmids carrying the glycerol transporter genes from P. tannophilus and S.cerevisiae were constructed. The USER vector pXI-5 was digested with AsiSI and then with the nicking endonuclease Nb.BsmI for making the AsiSI/Nb.BsmI USER cassettes . The proofreading PfuX7  was used for amplification of fragments PtFPS1, PtFPS2, PtSTL1, PtSTL2, ScFPS1, ScSTL1 and bidirectional promoter TEF1/PGK1 with appropriate USER tails for insertion into the designated USER cassette AsiSI/Nb.BsmI. Primers used are listed in Additional file 1. PCR was performed with PfuX7 DNA polymerase according to manufacturer’s instructions. DNA mixtures were prepared from purified digested vector, glycerol transporter gene, and bidirectional promoter TEF1/PGK1, 5× Phusion HF buffer (Fermentas), and 1 U of USER enzyme (Fermentas), adjusted to 10 μl by adding ddH2O. The mixture was incubated at 37°C for 20 min, followed by 25°C for 20 min. The 10 μl reaction mix was used directly to transform chemically competent E. coli DH5α cells. All the glycerol transporter genes were cloned under control of the TEF1 promoter. Constructs were named as: pXI-5-PtFPS1, pXI-5-PtFPS2, pXI-5-PtSTL1, pXI-5-PtSTL2, pXI-5-ScFPS1, pXI-5-ScSTL1 (Table 2). The plasmids were verified by sequencing and digested with NotI (Fermentas) for 1 h at 37°C and then subjected to gel purification. Each linearized construct was homologously integrated into the S. cerevisiae stl1Δ genome by transformation; six yeast strain lines were constructed as summarized in Table 2. Transformants were plated onto SC-Ura for selection. The S. cerevisiae transformants were restreaked on SC-Ura plates for single colonies, and the desired homologous integration was checked by colony PCR. Sequences and descriptions of primers are presented in Additional file 1.
Growth was monitored by measuring OD450nm with a Shimadzu UV mini-1240 spectrophotometer (Shimadzu, Japan). Samples were taken periodically from the flasks and filtered through a 0.22 μM syringe filter, and supernatants were preserved at −20°C for later HPLC analysis. Concentrations of the substrate glycerol in supernatants were measured by HPLC refractive index detector RID-10A using an Aminex87H column (Bio-Rad, USA). Separations were performed at 60°C, flow rate of 0.6 ml/min and 5 mM H2SO4 as mobile phase.
This work was funded by the European Community’s 7th Framework Research Programme under Grant Agreement Number 213506 (Project GLYFINERY) and the EU European Regional Development Fund InterReg IVA project Øresund Biorefinery, providing financial support to XL and MW.
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