Curr Microbiol (2014) 69:909–914 DOI 10.1007/s00284-014-0670-0
Identification of a Novel HOG1 Homologue from an Industrial Glycerol Producer Candida glycerinogenes Hao Ji • Xinyao Lu • Chengyin Wang • Hong Zong • Huiying Fang • Jin Sun • Jian Zhuge • Bin Zhuge
Received: 10 October 2013 / Accepted: 10 July 2014 / Published online: 14 August 2014 Ó Springer Science+Business Media New York 2014
Abstract Candida glycerinogenes, a glycerol production industrial strain with hyperosmo-adaptation can grow well in 15 % (w/v) NaCl or 55 % (w/v) glucose. To understand the osmo-adaptation mechanism in C. glycerinogenes, the mitogen-activated protein kinase HOG1 gene (CgHOG1), which plays an essential role in the yeast hyperosmotic response, was isolated by degenerate PCR and SEFAFormed Adaptor PCR. The CgHOG1 gene was then transformed in Saccharomyces cerevisiae hog1D null mutant, which restored the recombination S. cerevisiae to the wild-type phenotype with osmo-adaptation. To further clarify the function of CgHOG1, the phosphorylation of CgHOG1 and transcription of the glycerol-3-phosphate dehydrogenase gene (GPD1) of the CgHOG1-harbouring S. cerevisiae mutant was detected, and found to be similar to that of wild-type S. cerevisiae. In addition, the recombination S. cerevisiae with CgHOG1 gene significantly accumulated intracellular glycerol when stressed with NaCl.
Hao Ji and Xinyao Lu contributed equally to this study and share first authorship. H. Ji (&) X. Lu C. Wang H. Zong H. Fang J. Zhuge B. Zhuge The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China e-mail: [email protected] B. Zhuge e-mail: [email protected] J. Sun Zhejiang Condiments Industry Research Center, Zhejiang Zhengwei Food Co., Ltd, Yiwu, China
Introduction Candida glycerinogenes, which was isolated from glazed fruit in Southern China, is a novel osmo-tolerant yeast with an extremely high productivity of glycerol [23]. Because of its several special properties, such as a tolerance to 15 % (w/v) NaCl or 55 % (w/v) glucose, rapid growth, overproducing (137 g/L) and high yield (64.5 %) of extra-cellular glycerol, C. glycerinogenes has been used for industrial production of glycerol over the last ten years in China. When C. glycerinogenes is exposed to high osmolarity, glycerol is produced and accumulated as compatible solute to maintain the proper cytosolic osmolarity. In S. cerevisiae, the production of compatible solutes is regulated by a stress activated protein kinase cascade, the evolutionarily conserved high-osmolarity glycerol (HOG) pathway [5, 8, 9]. The most important component of HOG pathway is Hog1p, a mitogenactivated protein kinase, which is activated by the signaling cascade. When cells suffering stress stimuli, Hog1p is transiently phosphorylated and rapidly translocated into the nucleus to launch a series of transcription of specific genes, including some crucial enzymes involved in the synthesis of compatible solutes [11, 14]. In recent years, many attentions have been paid to HOG pathway of other important fungi, such as Candida lusitaniae [18], Candida albicans [1], and Aspergillus fumigatus [16]. Some HOG1 homologous genes such as DHOG1 from Debaryomyces hansenii, THOG1 from Torulopsis versatilis, WiHog1A and WiHog1B from Wallemia ichthyophaga, HwHog1 from Hortea werneckii and Kmhog1 from Kluyveromyces marxianus, have also been reported and proved to be functional genes similar with the HOG1 from S. cerevisiae by genetic manipulation [2, 4, 13, 15, 19]. These researches confirmed evolutionarily conservation of the HOG pathway. Thus, we proposed a hypothesis that a similar HOG pathway also existed in C. glycerinogenes.
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H. Ji et al.: Identification of a Novel HOG1 Homologue
Table 1 Primers used in this study Primers
Primer sequence (50 –30 )
DHOG1U
AAGATATCARATHTTYGGNAC
DHOG1R
CAGGATYTCNSWRTACATCAT
CgHOG1-r
CGCGAATTCATGTCTACCGACCAA
CgHOG1-f
CGGAAGCTTTTATTGCTGTTGTTG
CgHOG1-his-tagf
CTCAAGCTTTTAATGGTGATGGTGATGATGTTGCTGTTGTTGTTGGTGC
ScHOG1-r
CCTAAGCTTATGACCACTAACGAGGAATT
ScHOG1-f
CTTCTCGAGTTACTGTTGGAACTCATTAGC
ScHOG1-his-tagf
CTTCTCGAGTTAATGGTGATGGTGATGATGCTGTTGGAACTCATTAGCG
5SP1
ACATAGCCTGTCATTTGAGGGTCCTG
5SP2
GGTCAGCAATCTATGTAAATCC
5SP3 3SP1
TTTTTTAATTNNNNNNNNNTGATTTGTTAG GGGATGGGCGCATTTGGACTAGTTTGTTCTGC
3SP2
GGATTTACATAGATTGCTGACC
3SP3
AGATTATGCTAANNNNNNNNNAATACGATA
PDAr
GCCTTGTTCCACAGACCTTACT
PDAf
GGTTCTTCATTGGGTAGTTGTTG
GPD1r
TGCTGACATCCTTGTTTTCAAC
GPD1f
GTTACCCCATCCCATACCTTCT
Identification of such pathway is imperative for a clear understanding of the mechanism of the hyperosmo-adaptation in C. glycerinogenes. The cytol NAD?-glycerol 3-phospate dehydrogenase of C. glycerinogenes (CgGPD) which may be regulated by the HOG pathway was cloned and supposed to be the key gene involved in the efficient glycerol biosynthesis under hyperosmotic stress in our previous study [6]. However, other information of the HOG pathway in C. glycerinogenes was reported rarely. In this study, the HOG1 homologous gene from C. glycerinogenes (CgHOG1) was coloned and its function was analyzed by complementation in S. cerevisiae hog1 D null mutant strain. The phosphorylation of CgHOG1 and transcription levels of the glycerol synthesis gene GPD1 regulated by HOG1 homologous genes and the glycerol production upon hyperosmotic stress stimuli were detected to further confirm the function of CgHOG1.
glucose; w/v) supplemented with the appropriate concentrations of essential nutrients. Cloning CgHOG1 and Structure Analysis Two primers DHOG1U and DHOG1R (Table 1) were defined according to the consensus sequences of the HOG1 genes from Genbank and used to amplify a 570 bp CgHOG1 gene fragment from C. glycerinogenes genomic template. Then four gene specific primers (5SP1, 5SP2, 3SP1, and 3SP2) and two non-specific primers (5SP3 and 3SP3) were designed to amplify the upstream and downstream flanking sequences by Self-Formed Adaptor PCR [20]. All sequence data were analyzed using the BLAST programs (http://blast. ncbi.nlm.nih.gov/). Multiple sequence alignment was conducted with ClustalW and displayed by GeneDoc program. Complementation of S. cerevisiae hog1D Null Mutant by CgHOG1
Materials and Methods Strains and Culture Conditions Wild C. glycerinogenes WL2002-5, S. cerevisiae W3031A (MATa leu2-3/112 ura3-1 trp1-1 his3-11/15 ade2-1 can1-100 GAL SUC2 mal0) and hog1D null mutant of S. cerevisiae W303-1A were cultured in yeast peptone dextrose (YPD) medium or synthetic complete (SC) medium (0.67 % yeast nitrogen base without amino acid, 2 %
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CgHOG1 was cloned with primers CgHOG1-r and CgHOG1-f listed in Table 1 and then cloned into the plasmid pYX212 generating the plasmid pYX212-CgHOG1. The resulted plasmid was then transformed into S. cerevisiae hog1D null mutant by Electrotransformation, forming the strain W303 hog1D-pYX212-CgHOG1. The plasmid pYX212 was also transformed into S. cerevisiae W303 and the hog1D null mutant to obtain the control strain S. cerevisiae W303-pYX212 and S. cerevisiae hog1D-pYX212.
H. Ji et al.: Identification of a Novel HOG1 Homologue
Western Blotting Assay To determine whether the CgHOG1 kinase expressed in the S. cerevisiae hog1D mutant phosphorylated under hyperosmotic as a ScHOG1 kinase, the transformants S. cerevisiae hog1D-pYX212-ScHOG1 and S. cerevisiae hog1DpYX212-CgHOG1 introduced with a His-tag in the C-terminal, were harvested before and after NaCl shocked. The protein extractions were prepared as described in [13]. The anti-phpspho-p38 antibody and anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP) were purchase from Beyotime Institute of Biotechonology. An anti-His[HPR]mouse monoclonal antibody from CWbiotech was used to detect the His-tag. QRT-PCR After cultivated in YEPD at 30 °C for 16 h, S. cerevisiae cells were collected and later exposed to NaCl for 30 min. The treated cells were recollected and frozen in liquid N2 for total RNA extracted using Trizol, and the RNA was then used as template for reverse transcription following the instruction of Fermentas RevertAidTM First Strand cDNA Synthesis Kit after treated with DNaseI (Sigma). QRT-PCR was performed by using the cDNA samples as template with the primers GPD1r and GPD1f. PDA1 was used as internal reference with primers PDAr and PDAf. Determination of Intracellular Glycerol
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The 1164 bp CgHOG1 gene encodes a putative protein of 387 amino acids with a predicted molecular weight of 44.6 kDa and a theoretical isoelectric point (pI) of 5.31 (Fig. 1). Homology studies on the deduced amino acid sequence showed a high identity to MAP kinases of Ogataea parapolymorpha (86 %), Wickerhamomyces ciferrii (85 %), Komagataella pastoris GS115 (85 %), Eremothecium cymbalariae (84 %) and S. cerevisiae (82 %). Structure Analysis As Fig. 1 shown, a catalytic protein kinase domain was found from Tyr-24 to Leu-302, which was similar to other MAP kinases [3]. Inside the catalytic domain, there were an active site with Asp-144 and a TGY motif at amino acids 174-177, which is specific for MAPK activated by hyperosmolarity [3]. The conserved ATP-binding signature of protein kinases was identified at amino acids 30–54. A common docking (CD) motif from Asp-299 to Glu-316 contains acidic and hydrophobic residues [7]. Asp-307 and Asp-310 could establish critical electrostatic interactions with the positively charged residues of docking domains of upstream and downstream effectors together with the hydrophobic residues Tyr-305 and His-306. Complementation of CgHOG1 in S. cerevisiae hog1D Null Mutant
To determine intracellular glycerol concentrations, samples were withdrawn from cultures after being cultured in SC media for 16 h. Cells were collected by centrifugation and suspended in different hypertonic solution s for 30 min, and then cell extracts were prepared and the glycerol content was determined by HPLC [6].
S. cerevisiae hog1D mutant is osmosensitive and its growth is defective under high osmotic pressure. Figure 2 revealed that the recombinants with pYX212-CgHOG1 were obviously more resistant to osmotic stress than the control. In SC with 1.0 M NaCl, growth of the hog1D null mutant completely ceased, while the growth pattern of the mutant harboring pYX212-CgHOG1 was very similar to that of the wild-type S. cerevisiae.
Results
Function of CgHOG1 Expressed in S. cerevisiae hog1D Null Mutant Cells
Cloning of CgHOG1 from C. glycerinogenes A 0.57 kb PCR fragment was obtained from C. glycerinogenes genomic DNA by PCR amplification using two degenerate primers. BLAST search revealed that the sequence was homologous to known HOG1 encoding genes. Specific primers (5SP1, 2, 3 and 3SP1, 2, 3) were used to amplify its upstream and downstream sequences by SEFA PCR. Two DNA fragments, with a length of 2.4 and 1.5 kb, were respectively obtained after amplification. After purification, the PCR products were cloned into pMD18-T vector, and sequenced to finally assemble the full-length C. glycerinogenes HOG1 gene (Accession number KC480066 in GenBank).
To further clarify whether CgHOG1 is functionally equivalent to ScHOG1 when expressed in the S. cerevisiae hog1D null mutant cells, the phosphorylations of CgHOG1 upon NaCl shock were detected by a anti-P-p38 antibody. As shown in Fig. 3, the phosphorylation level of CgHOG1raised after 0.5 M NaCl shocked. QRT-PCR on GPD1 was carried out in S. cerevisiae W303 hog1D-pYX212CgHOG. After NaCl shock, the transcription of GPD1 in CgHOG1-harbouring strains was induced markedly to the same level as the wild-type S. cerevisiae W303-1A, but the S. cerevisiae hog1D null mutant failed to enhance the transcription of GPD1 under the same conditions (Fig. 4). Moreover, we detected that the intracellular glycerol yield
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912 Fig. 1 Nucleotide sequences and predicted amino acid sequence of the CgHOG1 gene. Deduced amino acid sequence is given above the nucleotide sequence of the coding region. Conserved TGY motif is underlined. D denotes active site. Boxed amino acids represent the C-terminal common docking (CD) motif, in which the conserved hydrophobic amino acids tyrosine (Y) and histidine (H) are underlined, and conserved acidic aspartic acids (D) are depicted by apostrophes
H. Ji et al.: Identification of a Novel HOG1 Homologue
1 1 61 21
ATGTCTACCGACCAAGATTTTGTGCGTTCGAATATTTTTGGTACTGTCTTTGAGACAACA M S
T
D
Q
D
F V
R
S
N
I
F
G
T
V
F
E
T
T
ACTCGATACTCTGATTTAAATCCAATTGGGATGGGCGCATTTGGACTAGTTTGTTCTGCT T
R
Y
S
D
L
N
P
I
G
M
G
A
F
G
L
V
C
S
A
Protein kinase ATP binding region 121 41 181 61 241 81
AAGGATAAACTAACAAATCAAAATGTTGCAATTAAAAAAGTGATGAAACCTTTTTCCACT K
D
K
L
T
N
Q
N
V
A
I
K
K
V
M
K
P
F
S
T
CCGGTGTTGGCGAAAAGGACTTATAGAGAGTTAAAATTGTTAAATCATTTAAGACACGAA P
V
L
A
K
R
T
Y
R
E
L
K
L
L
N
H
L
R
H
E
AACCTTATATCATTGGAGGATATCTTTTTATCACCATTAGAAGATATTTATTTTGTTACA N
L
I
S
L
E
D
I
F
L
S
P
L
E
D
I
Y
F
V
T
MAP-Kinase signature 301 101 361 121 421 141
GATTTACAAGGTACGGATTTACATAGATTGCTGACCTCCAGACCATTGGAAAAACAGTTT D
L
Q
G
T
D
L
H
R
L
L
T
S
R
P
L
E
K
Q
F
GTTCAATATTTTCTCTACCAGATTTTACGTGGTTTAAAGTATGTTCATTCAGCTGGTGTT V
Q
Y
F
L
Y
Q
I
L
R
G
L
K
Y
V
H
S
A
G V
ATTCATAGAGATTTAAAGCCTTCAAACATTTTGGTTAACGAAAACTGTGATTTGAAGATT I
H
R
D
L
K
P
S
N
I
L
V
N
E
N
C
D
L
K I
Serine/threonine Protein kinase site 481 161 541 181 601 201 661 221 721 241 781 261 841 281 901 301 961 321 1021 341 1081 361 1141 381
TGCGACTTTGGTTTAGCTCGTGTTCAGGACCCTCAAATGACAGGCTATGTCTCAACAAGA C
D
F
G
A
R
V
Q
D
P
Q
M
T
G
Y
V
S
T R
Y
Y
R
A
P
E
I
M
L
T
W
Q
K
Y
D
T
E
V
D
I
TGGTCTGTTGGCTGTATCTTTGCTGAAATGATAGAGGGCAAGCCATTGTTCCCAGGTAAG W
S
V
G
C
I
F
A
E
M
I
E
G
K
P
L
F
P
G
K
GATCATGTTCATCAGTTTTCTATTATTACTGAATTATTGGGATCTCCACCACCAGATGTT D
H
V
H
Q
F
S
I
I
T
E
L
L
G S
P
P
P
D
V
ATCGATACAATTTGTTCTGAGAATACGTTGAAGTTTGTCCAGTCATTACCGCACAAGGAA I
D
T
I
C
S
E
N
T
L
K
F
V
Q
S
L
P
H
K
E
GCAGTGCCTTTTACCGAACGATTCAAAGGTGTCGACCCTGATGCGATTGACTTGTTATCT A
V
P
F
T
E
R
F
K
G
V
D
P
D
A
I
D
L
L
S
AAAATGTTGGTTTTTGATCCAAGAAAAAGGATCACGGCGGCGGAAGCATTGGCACATCCT K
M
L
V
F
D
P
R
K
R
I
T
A
A
E
A
L
A
H
P
TATTTAGCTCCATATCATGATCCAAGTGATGAGCCGGTCAGTGAGGAGAAATTCGATTGG Y
L
A
P
Y
H
P
S
E
P
V
S
E
E
K
F
D
W
TCTTTCAACAATGCTGACTTGCCAATCGAAAACTGGAAAATTATGATGTATTCTGAAATT S
F
N
N
A
D
L
P
I
E
N
W
K
I
M
M
Y
S
E
I
CTAGACTTCCATGAAATTGAAGGTGCTGGTACTTTTGACAATGCCAACCTACAGCAATAT L
D
F
H
E
I
E
G
A
G
T
F
D
N
A
N
L
Q
Q
Y
GAAAACCACATATTAGAGCAACAACAAATACAGGGTCAAAATAATATCAATCAGCACCAC E
N
H
I
L
E
Q
Q
Q
I
Q
G
Q
N
N
I
N
Q
H
H
GAGCACCAACAACAACAGCAATAA E
H
Q
Fig. 2 Serial dilutions (10-1–10-5) of the exponentially growing cells (from a culture at an A600 nm of 0.6) of the strains S. cerevisiae W303-pYX212 (a), S. cerevisiae hog1D-pYX212-CgHOG1 (b), S.
123
L
TATTACAGAGCTCCCGAGATTATGCTAACTTGGCAAAAATACGATACCGAGGTTGATATT
Q
Q
Q
Q *
cerevisiae hog1D-pYX212 (c) and S. cerevisiae hog1D-pYX212ScHOG1 (d) were spotted (4 lL) onto SC plates containing NaCl at the concentrations indicated and incubated at 30 °C for 3 days
H. Ji et al.: Identification of a Novel HOG1 Homologue 0M NaCl
913 70
0.5 M NaCl
anti-P-p38 anti-His-tag 1
2
3
3
1
2
Fig. 3 The western blotting analysis of S. cerevisiae hog1D-pYX212ScHOG1-His-tag(1), S. cerevisiae hog1D-pYX212 -CgHOG1-His-tag (2), and S. cerevisiae hog1D-pYX212 (3) under 0.5 and 0 M NaCl
0M NaCl
transcription levels/(IOD)
50
0M NaCl 0.5M NaCl
40 30 20 10
1.2 1
µg glycerol / g dry cells
60
0 1
0.5M NaCl
2
3
4
Fig. 5 Intracellular glycerol content of strains S. cerevisiae hog1D null mutant (1), S. cerevisiae hog1D-pYX212-CgHOG1 (2), S. cerevisiae W303-pYX212 (3) and S. cerevisiae hog1D-pYX212 -ScHOG1 (4) in the presence of 0 M (black) and 0.5 M (white) NaCl, respectively
0.8 0.6 0.4 0.2 0 1
2
3
4
Fig. 4 The transcription levels of GPD1 gene in S. cerevisiae hog1D null mutant (1), S. cerevisiae hog1D-pYX212-CgHOG1 (2), S. cerevisiae W303-pYX212 (3) and S. cerevisiae hog1D-pYX212 ScHOG1 (4) under 0 and 0.5 M NaCl
of mutant strain with pYX212-CgHOG1 was obviously higher than that of control (Fig. 5).These results further confirmed that CgHogl have the similar function as Hogl in regulating the transcription of GPD1 in S. cerevisiae.
Discussion Candida glycerinogenes is industrial hyperosmo-tolerant yeast which can adjust the intracellular osmotic state by overproducing and accumulating glycerol as a compatible osmolyte when exposed to high external osmolarity [22]. This adaptive response to osmotic stress has been elucidated in S. cerevisiae, which is regulated by an osmosensing and signaling system called the HOG pathway. In S. cerevisiae, the key components of HOG pathway, MAP kinase Hoglp has been reported to regulate the transcription of genes encoding stress-induced proteins to control glycerol accumulation. Those include the genes encoding the two key enzymes in glycerol biosynthesis, GPD1 and
glycerol 3-phosphatase gene (GPP) [12]. We assumed that a pathway similar to the HOG pathway controlling the glycerol production might be present in C. glycerinogenes. In this paper, we cloned a gene homologous to S. cerevisiae HOG1 from C. glycerinogenes by degenerate PCR and Self-Formed Adaptor PCR. The deduced amino acid sequence encoded by CgHOG1 was highly homologous to the conserved consensus sequences of HOG1 from other fungi. Moreover, the TGY motif was found according to a structure prediction of the sequence of CgHog1p (Fig. 1). This motif can be phosphorylated rapidly by MAP kinase Pbs2p, an upstream kinase of Hog1p in S. cerevisiae, upon an osmotic shock. Such cells are apparently fully resistant to high osmolarity [10]. Complementation of S. cerevisiae hog1D null mutant was carried out by expression of CgHOG1 gene, and it made the recombinant recovered osmotolerant phenotype. The western blotting assay released that CgHOG1 kinase from C. glycerinogenes phosphorylated upon the NaCl shocked similar as the HOG1 kinase from S. cerevisiae. We infer that the CgHOG1 also plays an important role on osmo-tolerant and glycerol production of C. glycerinogenes. What puzzled us is why the glycerol yield of C. glycerinogenes was extraordinarily high comparing with S. cerevisiae and other yeasts (data no shown). S. cerevisiae possess two isogenes coding for GPD (GPD1 and GPD2) with separate functions in osmo-regulation and redox balancing [17], while GPD gene may exist as a single copy in C. glycerinogenes with both the functions [6]. Our previous study also suggested that GPD maintains
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at high activity around the whole cells cycle and the activity of another crucial enzyme GPP is much higher than that of GPD, which are different from that in S. cerevisiae [21]. These differences may imply a special mechanism present in C. glycerinogenes for glycerol over-producing and hyperosmo-tolerant. Above all, we obtained a mitogenactivated protein kinase HOG1 gene which is functional for regulating the transcription of GPD, and more work is needed, to clarify how CgHOG1 regulated glycerol overproducing in response to the hyperosmotic stress in C. glycerinogenes. Acknowledgments This work was supported by the National High Technology Research and Development Program of China (863 Program, NO.2011AA02A207, NO.2012AA021201), Natural Science Foundation of China (NO.31270080), Natural Science Foundation of Jiangsu Province (No. BK20140138), Jiangnan University Independent Scientific Research Program (No. JUSRP1008, No. JUSRP11431) and the 111 Project (No. 111-2-06).
References 1. Alonso-Monge R, Roman E, Nombela C, Pla J (2006) The MAP kinase signal transduction network in Candida albicans. Microbiology 152:905–912 2. Bansal PK, Mondal AK (2000) Isolation and sequence of the HOG1 homologue from Debaryomyces hansenii by complementation of the hog1 delta strain of Saccharomyces cerevisiae. Yeast 16:81–88 3. Cano E, Mahadevan LC (1995) Parallel signal processing among mammalian MAPKs. Trends Biochem Sci 20:117–122 4. Cao XX, Meng M, Wang YY, Wang CL, Hou LH (2011) Identification of salt-tolerant gene HOG1 in Torulopsis versatilis. Biotechnol Lett 33:1449–1456 5. Chen RE, Thorner J (2007) Function and regulation in MAPK signaling pathways. Biochim Biophys Acta 1773(8):1311–1340 6. Chen XZ, Fang HY, Rao ZM, Shen W, Zhuge B, Wang ZX, Zhuge J (2008) Cloning and characterization of a NAD?dependent glycerol-3-phosphate dehydrogenase gene from Candida glycerinogenes, an industrial glycerol producer. FEMS Yeast Res 8:725–734 7. Enslen H, Davis RJ (2001) Regulation of MAP kinases by docking domains. Biol Cell 93:5–14
123
H. Ji et al.: Identification of a Novel HOG1 Homologue 8. de Eula`lia N, Paula MA, Francesc P (2002) Dealing with osostress through MAP kinase actvation. EMBO Rep 3:735–740 9. Gustin MC, Albertyn J, Alexander M, Davenport K (1998) MAP kinase pathways in the yeast Saccharomyces cerevisiae. Microbiol Mol Biol Rev 62:1264–1300 10. Hohmann S (2002) Osmotic stress signaling and osmoadaptation in yeasts. Microbiol Mol Biol Rev 66:300–372 11. Hohmann S, Krantz M, Nordlander B (2007) Yeast osmoregulation. Methods Enzymol 428:29–45 12. Hohmann S (2009) Control of high osmolarity signalling in the yeast Saccharomyces cerevisiae. FEBS Lett 583:4025–4029 13. Konte T, Plemenitas A (2013) The HOG signal transduction pathway in the halophilic fugus Wallemia ichthyophaga : identification and characterization of MAP kinases WiHog1A and WiHog1B. Extremophiles 17(4):623–636 14. Pelet S, Rudolf F, Nadal-Ribelles M, de Nadal E, Posas F, Peter M (2011) Transient activation of the HOG MAPK pathway regulates bimodal gene expression. Science 332:732–735 15. Qian JC, Qin XL, Y Q, Chu J, Wang YH (2011) Cloning and characterization of Kluyveromyces marxianus Hog1 gene. Biotechnol Lett 33:571–575 16. Reyes G, Romans A, Nguyen CK, May GS (2006) Novel mitogenactivated protein kinase MpkC of Aspergillus fumigatus is required for utilization of polyalcohol sugars. Eukaryot Cell 5:1934–1940 17. Ricky A, Katarina G, Stefan H, Johan MT, Lennart A (1997) The two isoenzymes for yeast NAD?-dependent glycerol 3-phosphate dehydrogenase encoded by GPD1 and GPD2 have distinct roles in osmoadaptation and redox regulation. EMBO J 16(9):2179–2187 18. Ste´phanie B, Gwenael RR, Martine F, Bruno DS, Florence B, Nicolas P (2008) Insight into the role of HOG pathway components Ssk2p, Pbs2p, and Hog1p in the opportunistic yeast Candida lusitaniae. Eukaryot Cell 7(12):2179–2183 19. Turk M, Plemenitas A (2002) The HOG pathway in the halophilic black yeast Hortea werneckii: isolation of the HOG1 homolog gene and activation of HwHog1p. FEMS Microbiol Lett 216:193–199 20. Wang SM, He J, Cui ZL, Li SP (2007) Self-Formed Adaptor PCR: a Simple and Efficient Method for Chromosome Walking. Appl Environ Microbiol 73(15):5048–5051 21. Wang ZX, Zhuge J, Cao Y, Chen J, Fang HY (2000) The key enzymes of metabolisms of glycerol in Candida glycerolgenesis. Acta Microbiol Sin 40:180–187 22. Wang ZX, Zhuge J, Fang HY, Prior BA (2001) Glycerol production by microbial fermentation: a review. Biotechnol Adv 19:201–223 23. Zhuge J, Fang HY, Wang ZX, Chen DZ, Jin HR, Gu HL (2001) Glycerol production by a novel osmotolerant yeast Candida glycerinogenes. Appl Microbiol Biotechnol 55:686–692
Identification of a Novel HOG1 Homologue from an Industrial Glycerol Producer Candida glycerinogenes Hao Ji • Xinyao Lu • Chengyin Wang • Hong Zong • Huiying Fang • Jin Sun • Jian Zhuge • Bin Zhuge
Received: 10 October 2013 / Accepted: 10 July 2014 / Published online: 14 August 2014 Ó Springer Science+Business Media New York 2014
Abstract Candida glycerinogenes, a glycerol production industrial strain with hyperosmo-adaptation can grow well in 15 % (w/v) NaCl or 55 % (w/v) glucose. To understand the osmo-adaptation mechanism in C. glycerinogenes, the mitogen-activated protein kinase HOG1 gene (CgHOG1), which plays an essential role in the yeast hyperosmotic response, was isolated by degenerate PCR and SEFAFormed Adaptor PCR. The CgHOG1 gene was then transformed in Saccharomyces cerevisiae hog1D null mutant, which restored the recombination S. cerevisiae to the wild-type phenotype with osmo-adaptation. To further clarify the function of CgHOG1, the phosphorylation of CgHOG1 and transcription of the glycerol-3-phosphate dehydrogenase gene (GPD1) of the CgHOG1-harbouring S. cerevisiae mutant was detected, and found to be similar to that of wild-type S. cerevisiae. In addition, the recombination S. cerevisiae with CgHOG1 gene significantly accumulated intracellular glycerol when stressed with NaCl.
Hao Ji and Xinyao Lu contributed equally to this study and share first authorship. H. Ji (&) X. Lu C. Wang H. Zong H. Fang J. Zhuge B. Zhuge The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China e-mail: [email protected] B. Zhuge e-mail: [email protected] J. Sun Zhejiang Condiments Industry Research Center, Zhejiang Zhengwei Food Co., Ltd, Yiwu, China
Introduction Candida glycerinogenes, which was isolated from glazed fruit in Southern China, is a novel osmo-tolerant yeast with an extremely high productivity of glycerol [23]. Because of its several special properties, such as a tolerance to 15 % (w/v) NaCl or 55 % (w/v) glucose, rapid growth, overproducing (137 g/L) and high yield (64.5 %) of extra-cellular glycerol, C. glycerinogenes has been used for industrial production of glycerol over the last ten years in China. When C. glycerinogenes is exposed to high osmolarity, glycerol is produced and accumulated as compatible solute to maintain the proper cytosolic osmolarity. In S. cerevisiae, the production of compatible solutes is regulated by a stress activated protein kinase cascade, the evolutionarily conserved high-osmolarity glycerol (HOG) pathway [5, 8, 9]. The most important component of HOG pathway is Hog1p, a mitogenactivated protein kinase, which is activated by the signaling cascade. When cells suffering stress stimuli, Hog1p is transiently phosphorylated and rapidly translocated into the nucleus to launch a series of transcription of specific genes, including some crucial enzymes involved in the synthesis of compatible solutes [11, 14]. In recent years, many attentions have been paid to HOG pathway of other important fungi, such as Candida lusitaniae [18], Candida albicans [1], and Aspergillus fumigatus [16]. Some HOG1 homologous genes such as DHOG1 from Debaryomyces hansenii, THOG1 from Torulopsis versatilis, WiHog1A and WiHog1B from Wallemia ichthyophaga, HwHog1 from Hortea werneckii and Kmhog1 from Kluyveromyces marxianus, have also been reported and proved to be functional genes similar with the HOG1 from S. cerevisiae by genetic manipulation [2, 4, 13, 15, 19]. These researches confirmed evolutionarily conservation of the HOG pathway. Thus, we proposed a hypothesis that a similar HOG pathway also existed in C. glycerinogenes.
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H. Ji et al.: Identification of a Novel HOG1 Homologue
Table 1 Primers used in this study Primers
Primer sequence (50 –30 )
DHOG1U
AAGATATCARATHTTYGGNAC
DHOG1R
CAGGATYTCNSWRTACATCAT
CgHOG1-r
CGCGAATTCATGTCTACCGACCAA
CgHOG1-f
CGGAAGCTTTTATTGCTGTTGTTG
CgHOG1-his-tagf
CTCAAGCTTTTAATGGTGATGGTGATGATGTTGCTGTTGTTGTTGGTGC
ScHOG1-r
CCTAAGCTTATGACCACTAACGAGGAATT
ScHOG1-f
CTTCTCGAGTTACTGTTGGAACTCATTAGC
ScHOG1-his-tagf
CTTCTCGAGTTAATGGTGATGGTGATGATGCTGTTGGAACTCATTAGCG
5SP1
ACATAGCCTGTCATTTGAGGGTCCTG
5SP2
GGTCAGCAATCTATGTAAATCC
5SP3 3SP1
TTTTTTAATTNNNNNNNNNTGATTTGTTAG GGGATGGGCGCATTTGGACTAGTTTGTTCTGC
3SP2
GGATTTACATAGATTGCTGACC
3SP3
AGATTATGCTAANNNNNNNNNAATACGATA
PDAr
GCCTTGTTCCACAGACCTTACT
PDAf
GGTTCTTCATTGGGTAGTTGTTG
GPD1r
TGCTGACATCCTTGTTTTCAAC
GPD1f
GTTACCCCATCCCATACCTTCT
Identification of such pathway is imperative for a clear understanding of the mechanism of the hyperosmo-adaptation in C. glycerinogenes. The cytol NAD?-glycerol 3-phospate dehydrogenase of C. glycerinogenes (CgGPD) which may be regulated by the HOG pathway was cloned and supposed to be the key gene involved in the efficient glycerol biosynthesis under hyperosmotic stress in our previous study [6]. However, other information of the HOG pathway in C. glycerinogenes was reported rarely. In this study, the HOG1 homologous gene from C. glycerinogenes (CgHOG1) was coloned and its function was analyzed by complementation in S. cerevisiae hog1 D null mutant strain. The phosphorylation of CgHOG1 and transcription levels of the glycerol synthesis gene GPD1 regulated by HOG1 homologous genes and the glycerol production upon hyperosmotic stress stimuli were detected to further confirm the function of CgHOG1.
glucose; w/v) supplemented with the appropriate concentrations of essential nutrients. Cloning CgHOG1 and Structure Analysis Two primers DHOG1U and DHOG1R (Table 1) were defined according to the consensus sequences of the HOG1 genes from Genbank and used to amplify a 570 bp CgHOG1 gene fragment from C. glycerinogenes genomic template. Then four gene specific primers (5SP1, 5SP2, 3SP1, and 3SP2) and two non-specific primers (5SP3 and 3SP3) were designed to amplify the upstream and downstream flanking sequences by Self-Formed Adaptor PCR [20]. All sequence data were analyzed using the BLAST programs (http://blast. ncbi.nlm.nih.gov/). Multiple sequence alignment was conducted with ClustalW and displayed by GeneDoc program. Complementation of S. cerevisiae hog1D Null Mutant by CgHOG1
Materials and Methods Strains and Culture Conditions Wild C. glycerinogenes WL2002-5, S. cerevisiae W3031A (MATa leu2-3/112 ura3-1 trp1-1 his3-11/15 ade2-1 can1-100 GAL SUC2 mal0) and hog1D null mutant of S. cerevisiae W303-1A were cultured in yeast peptone dextrose (YPD) medium or synthetic complete (SC) medium (0.67 % yeast nitrogen base without amino acid, 2 %
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CgHOG1 was cloned with primers CgHOG1-r and CgHOG1-f listed in Table 1 and then cloned into the plasmid pYX212 generating the plasmid pYX212-CgHOG1. The resulted plasmid was then transformed into S. cerevisiae hog1D null mutant by Electrotransformation, forming the strain W303 hog1D-pYX212-CgHOG1. The plasmid pYX212 was also transformed into S. cerevisiae W303 and the hog1D null mutant to obtain the control strain S. cerevisiae W303-pYX212 and S. cerevisiae hog1D-pYX212.
H. Ji et al.: Identification of a Novel HOG1 Homologue
Western Blotting Assay To determine whether the CgHOG1 kinase expressed in the S. cerevisiae hog1D mutant phosphorylated under hyperosmotic as a ScHOG1 kinase, the transformants S. cerevisiae hog1D-pYX212-ScHOG1 and S. cerevisiae hog1DpYX212-CgHOG1 introduced with a His-tag in the C-terminal, were harvested before and after NaCl shocked. The protein extractions were prepared as described in [13]. The anti-phpspho-p38 antibody and anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP) were purchase from Beyotime Institute of Biotechonology. An anti-His[HPR]mouse monoclonal antibody from CWbiotech was used to detect the His-tag. QRT-PCR After cultivated in YEPD at 30 °C for 16 h, S. cerevisiae cells were collected and later exposed to NaCl for 30 min. The treated cells were recollected and frozen in liquid N2 for total RNA extracted using Trizol, and the RNA was then used as template for reverse transcription following the instruction of Fermentas RevertAidTM First Strand cDNA Synthesis Kit after treated with DNaseI (Sigma). QRT-PCR was performed by using the cDNA samples as template with the primers GPD1r and GPD1f. PDA1 was used as internal reference with primers PDAr and PDAf. Determination of Intracellular Glycerol
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The 1164 bp CgHOG1 gene encodes a putative protein of 387 amino acids with a predicted molecular weight of 44.6 kDa and a theoretical isoelectric point (pI) of 5.31 (Fig. 1). Homology studies on the deduced amino acid sequence showed a high identity to MAP kinases of Ogataea parapolymorpha (86 %), Wickerhamomyces ciferrii (85 %), Komagataella pastoris GS115 (85 %), Eremothecium cymbalariae (84 %) and S. cerevisiae (82 %). Structure Analysis As Fig. 1 shown, a catalytic protein kinase domain was found from Tyr-24 to Leu-302, which was similar to other MAP kinases [3]. Inside the catalytic domain, there were an active site with Asp-144 and a TGY motif at amino acids 174-177, which is specific for MAPK activated by hyperosmolarity [3]. The conserved ATP-binding signature of protein kinases was identified at amino acids 30–54. A common docking (CD) motif from Asp-299 to Glu-316 contains acidic and hydrophobic residues [7]. Asp-307 and Asp-310 could establish critical electrostatic interactions with the positively charged residues of docking domains of upstream and downstream effectors together with the hydrophobic residues Tyr-305 and His-306. Complementation of CgHOG1 in S. cerevisiae hog1D Null Mutant
To determine intracellular glycerol concentrations, samples were withdrawn from cultures after being cultured in SC media for 16 h. Cells were collected by centrifugation and suspended in different hypertonic solution s for 30 min, and then cell extracts were prepared and the glycerol content was determined by HPLC [6].
S. cerevisiae hog1D mutant is osmosensitive and its growth is defective under high osmotic pressure. Figure 2 revealed that the recombinants with pYX212-CgHOG1 were obviously more resistant to osmotic stress than the control. In SC with 1.0 M NaCl, growth of the hog1D null mutant completely ceased, while the growth pattern of the mutant harboring pYX212-CgHOG1 was very similar to that of the wild-type S. cerevisiae.
Results
Function of CgHOG1 Expressed in S. cerevisiae hog1D Null Mutant Cells
Cloning of CgHOG1 from C. glycerinogenes A 0.57 kb PCR fragment was obtained from C. glycerinogenes genomic DNA by PCR amplification using two degenerate primers. BLAST search revealed that the sequence was homologous to known HOG1 encoding genes. Specific primers (5SP1, 2, 3 and 3SP1, 2, 3) were used to amplify its upstream and downstream sequences by SEFA PCR. Two DNA fragments, with a length of 2.4 and 1.5 kb, were respectively obtained after amplification. After purification, the PCR products were cloned into pMD18-T vector, and sequenced to finally assemble the full-length C. glycerinogenes HOG1 gene (Accession number KC480066 in GenBank).
To further clarify whether CgHOG1 is functionally equivalent to ScHOG1 when expressed in the S. cerevisiae hog1D null mutant cells, the phosphorylations of CgHOG1 upon NaCl shock were detected by a anti-P-p38 antibody. As shown in Fig. 3, the phosphorylation level of CgHOG1raised after 0.5 M NaCl shocked. QRT-PCR on GPD1 was carried out in S. cerevisiae W303 hog1D-pYX212CgHOG. After NaCl shock, the transcription of GPD1 in CgHOG1-harbouring strains was induced markedly to the same level as the wild-type S. cerevisiae W303-1A, but the S. cerevisiae hog1D null mutant failed to enhance the transcription of GPD1 under the same conditions (Fig. 4). Moreover, we detected that the intracellular glycerol yield
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912 Fig. 1 Nucleotide sequences and predicted amino acid sequence of the CgHOG1 gene. Deduced amino acid sequence is given above the nucleotide sequence of the coding region. Conserved TGY motif is underlined. D denotes active site. Boxed amino acids represent the C-terminal common docking (CD) motif, in which the conserved hydrophobic amino acids tyrosine (Y) and histidine (H) are underlined, and conserved acidic aspartic acids (D) are depicted by apostrophes
H. Ji et al.: Identification of a Novel HOG1 Homologue
1 1 61 21
ATGTCTACCGACCAAGATTTTGTGCGTTCGAATATTTTTGGTACTGTCTTTGAGACAACA M S
T
D
Q
D
F V
R
S
N
I
F
G
T
V
F
E
T
T
ACTCGATACTCTGATTTAAATCCAATTGGGATGGGCGCATTTGGACTAGTTTGTTCTGCT T
R
Y
S
D
L
N
P
I
G
M
G
A
F
G
L
V
C
S
A
Protein kinase ATP binding region 121 41 181 61 241 81
AAGGATAAACTAACAAATCAAAATGTTGCAATTAAAAAAGTGATGAAACCTTTTTCCACT K
D
K
L
T
N
Q
N
V
A
I
K
K
V
M
K
P
F
S
T
CCGGTGTTGGCGAAAAGGACTTATAGAGAGTTAAAATTGTTAAATCATTTAAGACACGAA P
V
L
A
K
R
T
Y
R
E
L
K
L
L
N
H
L
R
H
E
AACCTTATATCATTGGAGGATATCTTTTTATCACCATTAGAAGATATTTATTTTGTTACA N
L
I
S
L
E
D
I
F
L
S
P
L
E
D
I
Y
F
V
T
MAP-Kinase signature 301 101 361 121 421 141
GATTTACAAGGTACGGATTTACATAGATTGCTGACCTCCAGACCATTGGAAAAACAGTTT D
L
Q
G
T
D
L
H
R
L
L
T
S
R
P
L
E
K
Q
F
GTTCAATATTTTCTCTACCAGATTTTACGTGGTTTAAAGTATGTTCATTCAGCTGGTGTT V
Q
Y
F
L
Y
Q
I
L
R
G
L
K
Y
V
H
S
A
G V
ATTCATAGAGATTTAAAGCCTTCAAACATTTTGGTTAACGAAAACTGTGATTTGAAGATT I
H
R
D
L
K
P
S
N
I
L
V
N
E
N
C
D
L
K I
Serine/threonine Protein kinase site 481 161 541 181 601 201 661 221 721 241 781 261 841 281 901 301 961 321 1021 341 1081 361 1141 381
TGCGACTTTGGTTTAGCTCGTGTTCAGGACCCTCAAATGACAGGCTATGTCTCAACAAGA C
D
F
G
A
R
V
Q
D
P
Q
M
T
G
Y
V
S
T R
Y
Y
R
A
P
E
I
M
L
T
W
Q
K
Y
D
T
E
V
D
I
TGGTCTGTTGGCTGTATCTTTGCTGAAATGATAGAGGGCAAGCCATTGTTCCCAGGTAAG W
S
V
G
C
I
F
A
E
M
I
E
G
K
P
L
F
P
G
K
GATCATGTTCATCAGTTTTCTATTATTACTGAATTATTGGGATCTCCACCACCAGATGTT D
H
V
H
Q
F
S
I
I
T
E
L
L
G S
P
P
P
D
V
ATCGATACAATTTGTTCTGAGAATACGTTGAAGTTTGTCCAGTCATTACCGCACAAGGAA I
D
T
I
C
S
E
N
T
L
K
F
V
Q
S
L
P
H
K
E
GCAGTGCCTTTTACCGAACGATTCAAAGGTGTCGACCCTGATGCGATTGACTTGTTATCT A
V
P
F
T
E
R
F
K
G
V
D
P
D
A
I
D
L
L
S
AAAATGTTGGTTTTTGATCCAAGAAAAAGGATCACGGCGGCGGAAGCATTGGCACATCCT K
M
L
V
F
D
P
R
K
R
I
T
A
A
E
A
L
A
H
P
TATTTAGCTCCATATCATGATCCAAGTGATGAGCCGGTCAGTGAGGAGAAATTCGATTGG Y
L
A
P
Y
H
P
S
E
P
V
S
E
E
K
F
D
W
TCTTTCAACAATGCTGACTTGCCAATCGAAAACTGGAAAATTATGATGTATTCTGAAATT S
F
N
N
A
D
L
P
I
E
N
W
K
I
M
M
Y
S
E
I
CTAGACTTCCATGAAATTGAAGGTGCTGGTACTTTTGACAATGCCAACCTACAGCAATAT L
D
F
H
E
I
E
G
A
G
T
F
D
N
A
N
L
Q
Q
Y
GAAAACCACATATTAGAGCAACAACAAATACAGGGTCAAAATAATATCAATCAGCACCAC E
N
H
I
L
E
Q
Q
Q
I
Q
G
Q
N
N
I
N
Q
H
H
GAGCACCAACAACAACAGCAATAA E
H
Q
Fig. 2 Serial dilutions (10-1–10-5) of the exponentially growing cells (from a culture at an A600 nm of 0.6) of the strains S. cerevisiae W303-pYX212 (a), S. cerevisiae hog1D-pYX212-CgHOG1 (b), S.
123
L
TATTACAGAGCTCCCGAGATTATGCTAACTTGGCAAAAATACGATACCGAGGTTGATATT
Q
Q
Q
Q *
cerevisiae hog1D-pYX212 (c) and S. cerevisiae hog1D-pYX212ScHOG1 (d) were spotted (4 lL) onto SC plates containing NaCl at the concentrations indicated and incubated at 30 °C for 3 days
H. Ji et al.: Identification of a Novel HOG1 Homologue 0M NaCl
913 70
0.5 M NaCl
anti-P-p38 anti-His-tag 1
2
3
3
1
2
Fig. 3 The western blotting analysis of S. cerevisiae hog1D-pYX212ScHOG1-His-tag(1), S. cerevisiae hog1D-pYX212 -CgHOG1-His-tag (2), and S. cerevisiae hog1D-pYX212 (3) under 0.5 and 0 M NaCl
0M NaCl
transcription levels/(IOD)
50
0M NaCl 0.5M NaCl
40 30 20 10
1.2 1
µg glycerol / g dry cells
60
0 1
0.5M NaCl
2
3
4
Fig. 5 Intracellular glycerol content of strains S. cerevisiae hog1D null mutant (1), S. cerevisiae hog1D-pYX212-CgHOG1 (2), S. cerevisiae W303-pYX212 (3) and S. cerevisiae hog1D-pYX212 -ScHOG1 (4) in the presence of 0 M (black) and 0.5 M (white) NaCl, respectively
0.8 0.6 0.4 0.2 0 1
2
3
4
Fig. 4 The transcription levels of GPD1 gene in S. cerevisiae hog1D null mutant (1), S. cerevisiae hog1D-pYX212-CgHOG1 (2), S. cerevisiae W303-pYX212 (3) and S. cerevisiae hog1D-pYX212 ScHOG1 (4) under 0 and 0.5 M NaCl
of mutant strain with pYX212-CgHOG1 was obviously higher than that of control (Fig. 5).These results further confirmed that CgHogl have the similar function as Hogl in regulating the transcription of GPD1 in S. cerevisiae.
Discussion Candida glycerinogenes is industrial hyperosmo-tolerant yeast which can adjust the intracellular osmotic state by overproducing and accumulating glycerol as a compatible osmolyte when exposed to high external osmolarity [22]. This adaptive response to osmotic stress has been elucidated in S. cerevisiae, which is regulated by an osmosensing and signaling system called the HOG pathway. In S. cerevisiae, the key components of HOG pathway, MAP kinase Hoglp has been reported to regulate the transcription of genes encoding stress-induced proteins to control glycerol accumulation. Those include the genes encoding the two key enzymes in glycerol biosynthesis, GPD1 and
glycerol 3-phosphatase gene (GPP) [12]. We assumed that a pathway similar to the HOG pathway controlling the glycerol production might be present in C. glycerinogenes. In this paper, we cloned a gene homologous to S. cerevisiae HOG1 from C. glycerinogenes by degenerate PCR and Self-Formed Adaptor PCR. The deduced amino acid sequence encoded by CgHOG1 was highly homologous to the conserved consensus sequences of HOG1 from other fungi. Moreover, the TGY motif was found according to a structure prediction of the sequence of CgHog1p (Fig. 1). This motif can be phosphorylated rapidly by MAP kinase Pbs2p, an upstream kinase of Hog1p in S. cerevisiae, upon an osmotic shock. Such cells are apparently fully resistant to high osmolarity [10]. Complementation of S. cerevisiae hog1D null mutant was carried out by expression of CgHOG1 gene, and it made the recombinant recovered osmotolerant phenotype. The western blotting assay released that CgHOG1 kinase from C. glycerinogenes phosphorylated upon the NaCl shocked similar as the HOG1 kinase from S. cerevisiae. We infer that the CgHOG1 also plays an important role on osmo-tolerant and glycerol production of C. glycerinogenes. What puzzled us is why the glycerol yield of C. glycerinogenes was extraordinarily high comparing with S. cerevisiae and other yeasts (data no shown). S. cerevisiae possess two isogenes coding for GPD (GPD1 and GPD2) with separate functions in osmo-regulation and redox balancing [17], while GPD gene may exist as a single copy in C. glycerinogenes with both the functions [6]. Our previous study also suggested that GPD maintains
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at high activity around the whole cells cycle and the activity of another crucial enzyme GPP is much higher than that of GPD, which are different from that in S. cerevisiae [21]. These differences may imply a special mechanism present in C. glycerinogenes for glycerol over-producing and hyperosmo-tolerant. Above all, we obtained a mitogenactivated protein kinase HOG1 gene which is functional for regulating the transcription of GPD, and more work is needed, to clarify how CgHOG1 regulated glycerol overproducing in response to the hyperosmotic stress in C. glycerinogenes. Acknowledgments This work was supported by the National High Technology Research and Development Program of China (863 Program, NO.2011AA02A207, NO.2012AA021201), Natural Science Foundation of China (NO.31270080), Natural Science Foundation of Jiangsu Province (No. BK20140138), Jiangnan University Independent Scientific Research Program (No. JUSRP1008, No. JUSRP11431) and the 111 Project (No. 111-2-06).
References 1. Alonso-Monge R, Roman E, Nombela C, Pla J (2006) The MAP kinase signal transduction network in Candida albicans. Microbiology 152:905–912 2. Bansal PK, Mondal AK (2000) Isolation and sequence of the HOG1 homologue from Debaryomyces hansenii by complementation of the hog1 delta strain of Saccharomyces cerevisiae. Yeast 16:81–88 3. Cano E, Mahadevan LC (1995) Parallel signal processing among mammalian MAPKs. Trends Biochem Sci 20:117–122 4. Cao XX, Meng M, Wang YY, Wang CL, Hou LH (2011) Identification of salt-tolerant gene HOG1 in Torulopsis versatilis. Biotechnol Lett 33:1449–1456 5. Chen RE, Thorner J (2007) Function and regulation in MAPK signaling pathways. Biochim Biophys Acta 1773(8):1311–1340 6. Chen XZ, Fang HY, Rao ZM, Shen W, Zhuge B, Wang ZX, Zhuge J (2008) Cloning and characterization of a NAD?dependent glycerol-3-phosphate dehydrogenase gene from Candida glycerinogenes, an industrial glycerol producer. FEMS Yeast Res 8:725–734 7. Enslen H, Davis RJ (2001) Regulation of MAP kinases by docking domains. Biol Cell 93:5–14
123
H. Ji et al.: Identification of a Novel HOG1 Homologue 8. de Eula`lia N, Paula MA, Francesc P (2002) Dealing with osostress through MAP kinase actvation. EMBO Rep 3:735–740 9. Gustin MC, Albertyn J, Alexander M, Davenport K (1998) MAP kinase pathways in the yeast Saccharomyces cerevisiae. Microbiol Mol Biol Rev 62:1264–1300 10. Hohmann S (2002) Osmotic stress signaling and osmoadaptation in yeasts. Microbiol Mol Biol Rev 66:300–372 11. Hohmann S, Krantz M, Nordlander B (2007) Yeast osmoregulation. Methods Enzymol 428:29–45 12. Hohmann S (2009) Control of high osmolarity signalling in the yeast Saccharomyces cerevisiae. FEBS Lett 583:4025–4029 13. Konte T, Plemenitas A (2013) The HOG signal transduction pathway in the halophilic fugus Wallemia ichthyophaga : identification and characterization of MAP kinases WiHog1A and WiHog1B. Extremophiles 17(4):623–636 14. Pelet S, Rudolf F, Nadal-Ribelles M, de Nadal E, Posas F, Peter M (2011) Transient activation of the HOG MAPK pathway regulates bimodal gene expression. Science 332:732–735 15. Qian JC, Qin XL, Y Q, Chu J, Wang YH (2011) Cloning and characterization of Kluyveromyces marxianus Hog1 gene. Biotechnol Lett 33:571–575 16. Reyes G, Romans A, Nguyen CK, May GS (2006) Novel mitogenactivated protein kinase MpkC of Aspergillus fumigatus is required for utilization of polyalcohol sugars. Eukaryot Cell 5:1934–1940 17. Ricky A, Katarina G, Stefan H, Johan MT, Lennart A (1997) The two isoenzymes for yeast NAD?-dependent glycerol 3-phosphate dehydrogenase encoded by GPD1 and GPD2 have distinct roles in osmoadaptation and redox regulation. EMBO J 16(9):2179–2187 18. Ste´phanie B, Gwenael RR, Martine F, Bruno DS, Florence B, Nicolas P (2008) Insight into the role of HOG pathway components Ssk2p, Pbs2p, and Hog1p in the opportunistic yeast Candida lusitaniae. Eukaryot Cell 7(12):2179–2183 19. Turk M, Plemenitas A (2002) The HOG pathway in the halophilic black yeast Hortea werneckii: isolation of the HOG1 homolog gene and activation of HwHog1p. FEMS Microbiol Lett 216:193–199 20. Wang SM, He J, Cui ZL, Li SP (2007) Self-Formed Adaptor PCR: a Simple and Efficient Method for Chromosome Walking. Appl Environ Microbiol 73(15):5048–5051 21. Wang ZX, Zhuge J, Cao Y, Chen J, Fang HY (2000) The key enzymes of metabolisms of glycerol in Candida glycerolgenesis. Acta Microbiol Sin 40:180–187 22. Wang ZX, Zhuge J, Fang HY, Prior BA (2001) Glycerol production by microbial fermentation: a review. Biotechnol Adv 19:201–223 23. Zhuge J, Fang HY, Wang ZX, Chen DZ, Jin HR, Gu HL (2001) Glycerol production by a novel osmotolerant yeast Candida glycerinogenes. Appl Microbiol Biotechnol 55:686–692
