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Cloning and sequencing of two d -xylose reductase genes (xyrA and xyrB) from Candida tropicalis
JOURNALOF FERMBNTA~ION ANDBIOENOINEERING Vol. 80, No. 6, 603-605. 1995
NOTES Cloning and Sequencing of Two D-Xylose Reductase Genes (xyrA and xyrB) from Candida tropicalis SHIN-ICHIRO
YOKOYAMA,’ YOSHIKO KINOSHITA,z TOHRU SUZUKI,2 KEIICHI KAWAI,2y3* HIROYUKI HORITSU,g AND KAZUHIRO TAKAMIZAWA2
The United Graduate School of Agricultural Sciences,’ Department of Biotechnology, Faculty of Agriculture,2 and Gene Research Center,3 Gifu University, I-I Yanagido, Gifu 501-11, Japan Received 10 July 1995/Accepted 29 August 1995
Two D-xyloae reductase (XR) genes, xyrA and xyrB, of Candida tropicalis IF0 0618 were cloned and sequenced. Each XR gene had an open reading frame consisting of 324 amino acids (972 bp). The deduced amino acid sequences of each XR gene were very similar to each other, except for three amino acids. The molecular weights of the deduced gene products were 36,551 (xyrA) and 36,596 (xvre). These genes were 73.0% identical to the Pichia stipitis XR gene. [Key words:
Candida tropicalis, xylose reductase,
nucleotide
sequence]
peptidase showed regions which were highly homologous (85%) to Pichia stipitis XR (2, 3). In this note, we describe the cloning and sequencing of XR genes from C. tropicalis. In order to construct a hybridization probe for C. tropicalis XR genes (1). Polymerase chain reaction (PCR)-amplification of a DNA fragment coding two adjacent peptide fragments (32 amino acids) from XRl was attempted. As shown in Fig. 1, P-l was used as the forward primer and P-2 the reverse primer. The PCR
n-Xylose reductase (XR) reduces o-xylose to xylitol in the presence of NADPH as a cofactor. In this study, we investigated NADPH-dependent XRs from Candida tropicalis IF0 0618, which produces three XR isomers (XRl, XR2 and XR3). In a previous paper, we reported the purification and characterization of two major XRs, XRl and XR2 (1). The molecular masses of XRl and XR2 determined by ion spray-mass spectrometry were 36,497.91 and 36,539.68, respectively. The amino acid sequence of the fragments of XRl digested by lysylendoP-l GAA ACA GCA CTA AAC AAA AC G C C C T G G G G T T T AC GAA ACA GCA TTA AAC AM G C C G T G G
G
T
T
FIG. 1. PCR strategy based on amino acid sequences of peptide fragments from C. tropicalis XR. The primers (P-l and P-2) used were designed from the amino acid sequence. The PCR method was as follows; denaturation at 93’C for 30 s, annealing at 45OC for 30, and elongation at 73’C for 30. The closed box represents the determined nucleotide sequence of the 77 bp insert of pCTXR77.
* Corresponding author. 5 Present address: Faculty of Home Economics, Department of Food and Nutrition, Chukyo Women’s University, 55 Natakayama, Yokone-cho, Ohbu, Aichi-ken, Japan.
603
604
YOKOYAMA ET AL.
J.
xyrA
kb
1.0
1.7
0.6
1.7
0.6
4.2
1.6
0.4
4.4
2.5
3.3
BIOENO.,
method involved denaturation at 93°C for 3Os, annealing at 45°C for 30, and elongation at 73°C for 30. The PCR products were cloned into the HincII site of pUC19 and sequenced by the dideoxy-mediated chaintermination method (4). The results showed that a plasmid containing 77 bp of the target sequence was obtained (Fig. 1). This plasmid clone was designated pCTXR77 and employed to generate a probe by a PCR labeling method using [a32P]-dCTP (5). Southern hybridization of genomic DNA digested with restriction enzymes revealed that there were two reactive fragments (Fig. 2). This finding suggests that there are two similar genes which are related to XR in the C. tropicalis genome. Cloning of the XR genes was carried out as follows. R-DASH II genomic libraries that carried the partially Sau3AI-digested genomic DNA of C. tropicalis IF0 0618
XY_
kbp
FERMENT.
1.0
FIG. 2. Restriction map of C. tropicufis genomic DNA near the XR coding regions. Southern analysis was performed as follows; Genomic DNA was digested with restriction enzymes, electrophoresed on 0.7% agarose gel and then transferred to Zeta-Probe@ GT Membrane (Bio-Rad Laboratories, Inc.) using a capillary system. Hybridization was performed with PCR labeled pCTXR77 as a probe. Panel A, Myra; panel B, xyrB. E, EcoRI; H, HindID; X, XbaI; Xo, XhoI; SC, SacI. The thick arrows indicate XR ORFs. -227
161 61
GvTGAAGvYmAxmAoccA!rYAAAGAAGGATmmTAAAxAGAAGAAmAllcAlc G I G I NRA I I[ I G L V K
R
II
1
L
F
I
361 121
mAAAml3rrCCAATrGMGMAAAT&zCCACCToarTlcTAClwrwrGmmrGAT F K F V P I K E A Y P P G F
Y
C
G
D
G
D
I
K
L
V
E
P
Y
L
421MCTfCCK:aToM~orrccATaTTAOATACT~AMOCT~DMAM~01T 111 N F B Y K D V P L L D T
W
K
541 181
TTG~~CQTDCTlLCTAn:AMCCADCT~TTACM~OMCATCIY:CCATAC'ITG L I R G A T I K P A V L Q I
721
TYATlTGAACATGhAACT~AAA K
~ATYGCTGATAAA~~AAAY~T~~A~TCM D
A
E
G
K
6
P
A
Q
A
V
I
P
K
8
N
N
P
641OMKIATTAOCTCMAACTP131PCT~~~mc3u:m:~TIUQ~OATTR3GIY: 281 E R L A Q R L 6 V V D F D
L
T
K
D
D
L
D
961 321
E
ii
A
781 261
L
L
S
~~~~TOD~TACTCM~AATATTQCT(XTATP~AM~~AATCCA V L L R W A T Q RN I
241
F
AL
T
I
K
L 6
I
CCAAlCllTQPl'TAA~~~~~AT P I F V end *****
FIG. 3. Nucleotide and deduced amino acid sequences of xyrA and xyB. The deduced TATAA promoter sites are underlined (thin lines). Amino acid sequences which are identical to a portion of the amino acid sequence of XRl are underlined (thick line). The 5 asterisks (*) represent a putative polyadenylation signal. The double-underlined region shows the proposed cofactor binding site. For overlapping sequences, the lower sequences belong to xyrB.
NOTES
VOL. 80, 1995
were constructed and screened with the probe described above. Fifty-six positive clones were obtained from 6 x lo6 plaques. In these clones, 1 clone contained a 0.6 kb HindIII-EcoRI fragment and 5 clones contained a 0.8 kb HindIII-Hind111 fragment (Fig. 2). These fragments and their neighbors were subcloned to pUC19 and sequenced. The results showed that each clone had the same length (972 bp) as one open reading frame (ORF) which encoded a 324 amino acid polypeptide (Fig. 3). These ORF amino acid sequences were 73.0% homologous to that of P. stipitis XR (2, 3). The deduced amino acid sequences of these genes were identical to each other except that the 29th Thr was changed to Asn, the 31st Ala to Glu and the 249th Leu to Ser (Fig. 3). Four bases in the ORFs, which did not have altered amino acids, were different; 99 C (in xyrA)+T (in xyrB), 120 C+T, 123 T+C and 351 C-*T, respectively. The amino acid compositions of these ORFs corresponded to the results of amino acid analysis of XRl and XR2 (1). The molecular weights of these polypeptides deduced from the nucleotide sequences were about 36 kDa, a value which corresponds to the experimental data (1). TATA boxes were present in the 5’ non-coding regions of both genes (Fig. 3). A typical polyadenylation signal (AATAA) also existed in each 3’ non-coding region. These results suggest that the two genes could be expressed as isomers, and that the two ORFs were XR structural genes which had no introns. Thus, these genes were named xyrA and xyrB, respectively. The molecular masses of XRs deduced from the xyrA and xyrB genes were calculated to be 36,551 and 36,596, respectively. These values are slightly higher than previously described experimental values. These inconsistencies may be explained by post-translational modifications because Edman degradation of XRl and XR2 did not proceed (1). On the basis of these results, we believe that the first Met residues of the N-termini of XRs may be removed in accordance with the N-end rule (6, 7) and that the second Ser was then modified with a compound. The suggested cofactor binding site (-Ile-Pro-Lys-Ser-) for aldose reductases was located between amino acids 274 to 277 in both genes (Fig. 3). Although P. stipitis XR utilizes both NADPH and NADH, C. tropicalis XRs require NADPH and not NADH (1). In spite of this difference in cofactor dependency, the two genes are quite similar to each other. The downstream region of this cofactor binding site, 280 to 301
605
(as amino acid number), showed relatively lower homology compared to that of P. stipitis XR (2, 3). Therefore, we believe that this region may be responsible for cofactor binding specificity. In this study, it was demonstrated that there are two XR genes on the C. tropicalis genome when a PCR labeled DNA fragment from pCTXR77 was used as a probe. It is clear that one gene codes for XRl, although we did not determine exactly which gene codes XRl. The other codes for either XR2 or XR3. Since the third XR gene may not hybridize with the DNA probe from pCTXR77, the amino acid components of the XR product of this gene may be different from those of XRl. Expression experiments are needed to math the XR genes to either XRl, XR2 or XR3. These XR genes can be expected to be expressed in Escherichia coli or Saccharomyces cerevisiae. We are currently studying the expression of the genes in these host cells. REFERENCES 1. Yokoyama, S., Suzuki, T., Kawai, K., Horitsu, H., and Takamizawa, K.: Purification, characterization and structure analysis of NADPH-dependent p-xylose reductases from Cundidu tropicufis. J. Ferment. Bioeng., 79, 217-223 (1995). 2. Amore, R., Kiitter, P., Ktister, C., Ciriacy, M., and HollenCloning and expression in Saccharomyces berg, C.P.: cerevisiae of the xylose-assimilating yeast Pichia stipitis. Gene, 109, 89-97 (1991). 3. Hallborn, J., Walfrfdsson, M., Airaksinen, U., Ojamo, H., Hahn-Hiigerdal, B., Penttilii, M., and Keritnen, S.: Xylitol production by recombinant Saccharomyces cerevisiae. Bio/ Technol., 9, 1090-1095 (1991). 4. Sanger, F., Nickfen, S., and Coulson, A. R.: DNA sequencing with chain-terminating inhibitors. Proc. Nat. Acad. Sci. USA, 74, 5463-5467 (1977). 5. Schowalter, D.B. and Sommer, S. S.: The generation of radiolabeled DNA and RNA probes with polymerase chain reaction. Anal. Biochem., 177, 90-94 (1989). 6. Sherman, F., Stewart, J. W., and Tsunasawa, S.: Methionine or not methionine at the beginning of a protein? BioEssays, 3, 27-31 (1985). 7. Affin, S. M. and Bradshaw, R. A.: Cotranslational processing and protein turnover in eukaryotic cells. Biochemistry, 27, 7979-7984 (1988). 8. Verduyn, C., van-Kfeef, R., Frank, J., Schreuder, H., van-Dijken, J. P., and Scheffers, W. A.: Properties of the NAD(P)Hdependent xylose reductase from the xylose-fermenting yeast Pichiu stipitis. Biochem. J., 226, 669-677 (1985).
NOTES Cloning and Sequencing of Two D-Xylose Reductase Genes (xyrA and xyrB) from Candida tropicalis SHIN-ICHIRO
YOKOYAMA,’ YOSHIKO KINOSHITA,z TOHRU SUZUKI,2 KEIICHI KAWAI,2y3* HIROYUKI HORITSU,g AND KAZUHIRO TAKAMIZAWA2
The United Graduate School of Agricultural Sciences,’ Department of Biotechnology, Faculty of Agriculture,2 and Gene Research Center,3 Gifu University, I-I Yanagido, Gifu 501-11, Japan Received 10 July 1995/Accepted 29 August 1995
Two D-xyloae reductase (XR) genes, xyrA and xyrB, of Candida tropicalis IF0 0618 were cloned and sequenced. Each XR gene had an open reading frame consisting of 324 amino acids (972 bp). The deduced amino acid sequences of each XR gene were very similar to each other, except for three amino acids. The molecular weights of the deduced gene products were 36,551 (xyrA) and 36,596 (xvre). These genes were 73.0% identical to the Pichia stipitis XR gene. [Key words:
Candida tropicalis, xylose reductase,
nucleotide
sequence]
peptidase showed regions which were highly homologous (85%) to Pichia stipitis XR (2, 3). In this note, we describe the cloning and sequencing of XR genes from C. tropicalis. In order to construct a hybridization probe for C. tropicalis XR genes (1). Polymerase chain reaction (PCR)-amplification of a DNA fragment coding two adjacent peptide fragments (32 amino acids) from XRl was attempted. As shown in Fig. 1, P-l was used as the forward primer and P-2 the reverse primer. The PCR
n-Xylose reductase (XR) reduces o-xylose to xylitol in the presence of NADPH as a cofactor. In this study, we investigated NADPH-dependent XRs from Candida tropicalis IF0 0618, which produces three XR isomers (XRl, XR2 and XR3). In a previous paper, we reported the purification and characterization of two major XRs, XRl and XR2 (1). The molecular masses of XRl and XR2 determined by ion spray-mass spectrometry were 36,497.91 and 36,539.68, respectively. The amino acid sequence of the fragments of XRl digested by lysylendoP-l GAA ACA GCA CTA AAC AAA AC G C C C T G G G G T T T AC GAA ACA GCA TTA AAC AM G C C G T G G
G
T
T
FIG. 1. PCR strategy based on amino acid sequences of peptide fragments from C. tropicalis XR. The primers (P-l and P-2) used were designed from the amino acid sequence. The PCR method was as follows; denaturation at 93’C for 30 s, annealing at 45OC for 30, and elongation at 73’C for 30. The closed box represents the determined nucleotide sequence of the 77 bp insert of pCTXR77.
* Corresponding author. 5 Present address: Faculty of Home Economics, Department of Food and Nutrition, Chukyo Women’s University, 55 Natakayama, Yokone-cho, Ohbu, Aichi-ken, Japan.
603
604
YOKOYAMA ET AL.
J.
xyrA
kb
1.0
1.7
0.6
1.7
0.6
4.2
1.6
0.4
4.4
2.5
3.3
BIOENO.,
method involved denaturation at 93°C for 3Os, annealing at 45°C for 30, and elongation at 73°C for 30. The PCR products were cloned into the HincII site of pUC19 and sequenced by the dideoxy-mediated chaintermination method (4). The results showed that a plasmid containing 77 bp of the target sequence was obtained (Fig. 1). This plasmid clone was designated pCTXR77 and employed to generate a probe by a PCR labeling method using [a32P]-dCTP (5). Southern hybridization of genomic DNA digested with restriction enzymes revealed that there were two reactive fragments (Fig. 2). This finding suggests that there are two similar genes which are related to XR in the C. tropicalis genome. Cloning of the XR genes was carried out as follows. R-DASH II genomic libraries that carried the partially Sau3AI-digested genomic DNA of C. tropicalis IF0 0618
XY_
kbp
FERMENT.
1.0
FIG. 2. Restriction map of C. tropicufis genomic DNA near the XR coding regions. Southern analysis was performed as follows; Genomic DNA was digested with restriction enzymes, electrophoresed on 0.7% agarose gel and then transferred to Zeta-Probe@ GT Membrane (Bio-Rad Laboratories, Inc.) using a capillary system. Hybridization was performed with PCR labeled pCTXR77 as a probe. Panel A, Myra; panel B, xyrB. E, EcoRI; H, HindID; X, XbaI; Xo, XhoI; SC, SacI. The thick arrows indicate XR ORFs. -227
161 61
GvTGAAGvYmAxmAoccA!rYAAAGAAGGATmmTAAAxAGAAGAAmAllcAlc G I G I NRA I I[ I G L V K
R
II
1
L
F
I
361 121
mAAAml3rrCCAATrGMGMAAAT&zCCACCToarTlcTAClwrwrGmmrGAT F K F V P I K E A Y P P G F
Y
C
G
D
G
D
I
K
L
V
E
P
Y
L
421MCTfCCK:aToM~orrccATaTTAOATACT~AMOCT~DMAM~01T 111 N F B Y K D V P L L D T
W
K
541 181
TTG~~CQTDCTlLCTAn:AMCCADCT~TTACM~OMCATCIY:CCATAC'ITG L I R G A T I K P A V L Q I
721
TYATlTGAACATGhAACT~AAA K
~ATYGCTGATAAA~~AAAY~T~~A~TCM D
A
E
G
K
6
P
A
Q
A
V
I
P
K
8
N
N
P
641OMKIATTAOCTCMAACTP131PCT~~~mc3u:m:~TIUQ~OATTR3GIY: 281 E R L A Q R L 6 V V D F D
L
T
K
D
D
L
D
961 321
E
ii
A
781 261
L
L
S
~~~~TOD~TACTCM~AATATTQCT(XTATP~AM~~AATCCA V L L R W A T Q RN I
241
F
AL
T
I
K
L 6
I
CCAAlCllTQPl'TAA~~~~~AT P I F V end *****
FIG. 3. Nucleotide and deduced amino acid sequences of xyrA and xyB. The deduced TATAA promoter sites are underlined (thin lines). Amino acid sequences which are identical to a portion of the amino acid sequence of XRl are underlined (thick line). The 5 asterisks (*) represent a putative polyadenylation signal. The double-underlined region shows the proposed cofactor binding site. For overlapping sequences, the lower sequences belong to xyrB.
NOTES
VOL. 80, 1995
were constructed and screened with the probe described above. Fifty-six positive clones were obtained from 6 x lo6 plaques. In these clones, 1 clone contained a 0.6 kb HindIII-EcoRI fragment and 5 clones contained a 0.8 kb HindIII-Hind111 fragment (Fig. 2). These fragments and their neighbors were subcloned to pUC19 and sequenced. The results showed that each clone had the same length (972 bp) as one open reading frame (ORF) which encoded a 324 amino acid polypeptide (Fig. 3). These ORF amino acid sequences were 73.0% homologous to that of P. stipitis XR (2, 3). The deduced amino acid sequences of these genes were identical to each other except that the 29th Thr was changed to Asn, the 31st Ala to Glu and the 249th Leu to Ser (Fig. 3). Four bases in the ORFs, which did not have altered amino acids, were different; 99 C (in xyrA)+T (in xyrB), 120 C+T, 123 T+C and 351 C-*T, respectively. The amino acid compositions of these ORFs corresponded to the results of amino acid analysis of XRl and XR2 (1). The molecular weights of these polypeptides deduced from the nucleotide sequences were about 36 kDa, a value which corresponds to the experimental data (1). TATA boxes were present in the 5’ non-coding regions of both genes (Fig. 3). A typical polyadenylation signal (AATAA) also existed in each 3’ non-coding region. These results suggest that the two genes could be expressed as isomers, and that the two ORFs were XR structural genes which had no introns. Thus, these genes were named xyrA and xyrB, respectively. The molecular masses of XRs deduced from the xyrA and xyrB genes were calculated to be 36,551 and 36,596, respectively. These values are slightly higher than previously described experimental values. These inconsistencies may be explained by post-translational modifications because Edman degradation of XRl and XR2 did not proceed (1). On the basis of these results, we believe that the first Met residues of the N-termini of XRs may be removed in accordance with the N-end rule (6, 7) and that the second Ser was then modified with a compound. The suggested cofactor binding site (-Ile-Pro-Lys-Ser-) for aldose reductases was located between amino acids 274 to 277 in both genes (Fig. 3). Although P. stipitis XR utilizes both NADPH and NADH, C. tropicalis XRs require NADPH and not NADH (1). In spite of this difference in cofactor dependency, the two genes are quite similar to each other. The downstream region of this cofactor binding site, 280 to 301
605
(as amino acid number), showed relatively lower homology compared to that of P. stipitis XR (2, 3). Therefore, we believe that this region may be responsible for cofactor binding specificity. In this study, it was demonstrated that there are two XR genes on the C. tropicalis genome when a PCR labeled DNA fragment from pCTXR77 was used as a probe. It is clear that one gene codes for XRl, although we did not determine exactly which gene codes XRl. The other codes for either XR2 or XR3. Since the third XR gene may not hybridize with the DNA probe from pCTXR77, the amino acid components of the XR product of this gene may be different from those of XRl. Expression experiments are needed to math the XR genes to either XRl, XR2 or XR3. These XR genes can be expected to be expressed in Escherichia coli or Saccharomyces cerevisiae. We are currently studying the expression of the genes in these host cells. REFERENCES 1. Yokoyama, S., Suzuki, T., Kawai, K., Horitsu, H., and Takamizawa, K.: Purification, characterization and structure analysis of NADPH-dependent p-xylose reductases from Cundidu tropicufis. J. Ferment. Bioeng., 79, 217-223 (1995). 2. Amore, R., Kiitter, P., Ktister, C., Ciriacy, M., and HollenCloning and expression in Saccharomyces berg, C.P.: cerevisiae of the xylose-assimilating yeast Pichia stipitis. Gene, 109, 89-97 (1991). 3. Hallborn, J., Walfrfdsson, M., Airaksinen, U., Ojamo, H., Hahn-Hiigerdal, B., Penttilii, M., and Keritnen, S.: Xylitol production by recombinant Saccharomyces cerevisiae. Bio/ Technol., 9, 1090-1095 (1991). 4. Sanger, F., Nickfen, S., and Coulson, A. R.: DNA sequencing with chain-terminating inhibitors. Proc. Nat. Acad. Sci. USA, 74, 5463-5467 (1977). 5. Schowalter, D.B. and Sommer, S. S.: The generation of radiolabeled DNA and RNA probes with polymerase chain reaction. Anal. Biochem., 177, 90-94 (1989). 6. Sherman, F., Stewart, J. W., and Tsunasawa, S.: Methionine or not methionine at the beginning of a protein? BioEssays, 3, 27-31 (1985). 7. Affin, S. M. and Bradshaw, R. A.: Cotranslational processing and protein turnover in eukaryotic cells. Biochemistry, 27, 7979-7984 (1988). 8. Verduyn, C., van-Kfeef, R., Frank, J., Schreuder, H., van-Dijken, J. P., and Scheffers, W. A.: Properties of the NAD(P)Hdependent xylose reductase from the xylose-fermenting yeast Pichiu stipitis. Biochem. J., 226, 669-677 (1985).