Sep 1, 1987 - xylulose (2, 10), P. tannophilus utilizes D-xylose reductase. (D-xylose .... L brothb. Glycerol. Glycerol + xylose. GM8. 0.2. 0. 5.2. SR14c. 0.2. 0.2.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1987, p. 2975-2977
Vol. 53, No. 12
0099-2240/87/122975-03$02.00/0
Copyright © 1987, American Society for Microbiology
Cloning of the Pachysolen tannophilus Xylulokinase Gene by Complementation in Escherichia coli PANAYIOTIS E. STEVIS, JAMES J. HUANG,t
AND
NANCY W. Y. HO*
Laboratory of Renewable Resources Engineering, Purdue University, West Lafayette, Indiana 47907 Received 20 April 1987/Accepted 1 September 1987
The gene coding for xylulokinase has been isolated from the yeast Pachysolen tannophilus by complementation of Escherichia coli xylulokinase (xylB) mutants. Through subcloning, the gene has been localized at one end of a 3.2-kilobase EcoRI-PstI fragment. Expression of the cloned gene was insensitive to glucose inhibition. Furthermore, the cloned gene did not cross-hybridize with E. coli and Saccharomyces cerevisiae xylulokinase genes.
Pachysolen tannophilus is a D-xylose-utilizing yeast isolated from wood sulfite liquor by Wickerham (14). Although this organism has been studied extensively relative to its ability to ferment Xylose to ethanol 3, 4, 9, 11), very little is known about its genetic organization and gene expression. Unlike procaryotes, such as Escherichia coli and Salmonella typhimurium, which isomerize xylose directly to xylulose (2, 10), P. tannophilus utilizes D-xylose reductase (D-xylose
-*
xylitol) and xylitol dehydrogenase (xylitol
resulted in the isolation of pLPK3 (Fig. 1). The latter plasmid conferred a Xyl+, Tcr, Aps phenotype on SR14. This analysis localized the gene to a 3.2-kb PstI-PstI-EcoRI fragment distal to the gene for Apr in pLPK2 (Fig. 1). Deletion of the 1.4-kb HindlIl fragment from pLPK3 abolished the ability of pLPK3 to complement strain SR14. The 1.4-kb HindIlI fragment from pLPK3 subcloned into pUC9 also failed to complement SR14. Furthermore, the 2.3-kb PstI fragment cloned into pUC9 also did not complement SR14. In addition, removal of the HindIII or PstI sites, spaced 450 base pairs apart on pLPK2 and pLPK3, disrupted the ability of the cloned fragment to complement the xylB mutants. The EcoRI fragment derived from pLPK2 was also subcloned into pUC9 in both orientations. The orientation which placed the large (approximately 1.3-kb) AvaI fragment proximal to the lac promoter yielded transformants on minimal medium with 0.4% xylose after a 12-h incubation. Transformants containing the plasmid in the opposite orientation yielded smaller colonies after 48 h. In addition to XYK-Pa of P. tannophilus, the xylulokinase genes of E. coli (7, 8) and Saccharomyces cerevisiae (S. F. Chang and N. W. Y. Ho, Appl. Biochem. Biotechnol., in press) have also been isolated by complementation of E. coli xylulokinase mutants. The cloned xylulokinase genes from these three organisms have different restriction enzyme patterns and do not cross-hybridize with each other (data not shown). The identity of the XYK-Pa gene was further established by the fact that the cloned gene hybridized to P. tannophilus cellular DNA and not to other yeast DNA (Fig. 2). The xylulokinase assay results (Table 1) demonstrate that the expression of the cloned XYK-Pa gene in E. coli is insensitive to glucose inhibition, while the expression of the E. coli chromosomal xylB gene is repressed in the presence of glucose. This further indicates that the cloned gene is not the E. coli xylB gene and that it does not code for a P. tannophilus suppressor tRNA. A preliminary analysis showed that yeast (S. cerevisiae) transformants harboring pLPK1 or pLPK4 (pLPK3 with a fragment containing TRPJ-ARS [12] cloned into the EcoRI site) did not exhibit increased rates of xylulose utilization. Furthermore, xylulokinase assays of the yeast transformants harboring pLPK4 did not reveal the presence of any plasmidborne xylulokinase activity. The absence of plasmid-borne xylulokinase activity could be due to differences in the transcription or translation mechanism or both in the two
>
D-xylulose) to achieve this conversion. Xylulokinase then converts xylulose to xylulose-5-phosphate, an intermediate of the pentose phosphate pathway. Lachke and Jeffries (6) found that xylulokinase is critical for the aerobic and anaerobic utilization of xylose. In this report we describe the isolation of the P. tannophilus xylulokinase gene (XYK-Pa) by complementation of E. coli xylB mutants. A genomic bank was constructed for P. tannophilus by the insertion of Sau3A-digested P. tannophilus DNA onto the BamHI site of the yeast-E. coli shuttle vector YEp13 (1). Transformation of the E. coli xylulokinase mutant strain SR14 (8) with the genomic bank resulted in the isolation of five Xyl+ colonies. These positive transformants were detected as red colonies on MacConkey-xylose fermentation indicator plates against a background of nonpigmented (white) colonies. Digestion by SalI of plasmid minipreparations isolated from the Xyl+ colonies indicated the presence of additional Sall fragments in these plasmids other than those in YEp13. Retransformation of strain SR14 with plasmid DNA from Xyl+ transformants resulted in all colonies being Xyl+. One of these plasmids, pLPK1 (structure not shown), was chosen for further analysis. The initial plasmid, pLPK1, consisted of a 9-kilobase (kb) Sau3A fragment inserted into the BamHI site of YEp13. To facilitate restriction site mapping, a 4.5-kb EcoRI fragment from pLPK1 was subcloned into pBR322, yielding pLPK2 (Fig. 1). In addition to complementing SR14, pLPK2 also complemented four transposon-induced E. coli xylulokinase mutants (XK104, XK106, XK201, and XK202) which were generated with the TnJO derivatives described by Way et al. (13). This indicates that the cloned gene is not a Pachysolen suppressor tRNA gene. Partial digestion of pLPK2 by PstI followed by religation and transformation of strain SR14 * Corresponding author. t Present address: E. I. du Pont de Nemours & Co., Inc., Glenolden, PA 19036.
2975
2976
NOTES
0\~i_1.5
APPL. ENVIRON. MICROBIOL.
Ava I
EcoRI
TABLE 1. Xylulokinase activities of E. coli strains and transformants Strain or transformant GM8
AvalI^
H
M
pLPK-3
(7 Kb)
5 '\IP
Pvu
EcoR I
/HindEX
/
Sal I
I
Ava I
FIG. 1. Restriction maps of pLPK2 and pLPK3. nophilus DNA; , pBR322 DNA sequences.
OGO,
P. tan-
organisms or to the absence in the cloned gene of the intact control elements for gene expression in yeasts. The XYK-Pa gene is, to our knowledge, the first gene isolated from P. tannophilus. Analysis of the nucleotide A
B
C
kb
4.5'
#
2. Southern hybridization to determine the origin of the xylulokinase gene. Yeast DNA and plasmid were digested by HindIII. Lanes: A, P. tannophilus DNA; B, Candida utilis DNA; and C, pLPK1. pLPK1 was used as the 32P_labeled probe. C. utilis
FIG.
cloned
DNA
was
selected
as
the control for
isolation of C. utilis xylulokinase gene when the isolation of XYK-Pa
already
was
hybridization because the was also being attempted
in progress.
5.2
gene. d pLPK1 is a low-copy-number plasmid (-10 copies per cell). e pLPK3 is a high-copy-number plasmid (-50 copies per cell).
Hind
9
0
0.2 0.2 1.0 0.6 1.1 2.6 3.1 1.6 2.1 aNanomoles of product formed per minute per milligram of protein. bGlucose-containing medium. c Xylulokinase mutant which has residual xylulokinase activity and exhibits a fivefold induction of its xylulokinase activity in the presence of xylose. The slight effect of xylose on the xylulokinase activities of SR14(pLPK1) and SR14(pLPK3) is due most likely to its effect on the expression of the mutated xylulokinase gene of SR14 rather than that of the plasmid-borne XYK-Pa
I k>-,vAva -J~~~yk AvasI
PstIg
0.2
Xylulokinase activitya in: Glycerol Glycerol + xylose
SR14c SR14(pLPK1)c,d SR14(pLPK3)c,e
EcoRI
(8.8 Kb)
L brothb
Furthermore,
had sufficient evidence to prove that the XYK-Pa gene not from E. coli or S. cerevisiae (see the text).
we
was
sequence of the cloned gene will provide the necessary data for comparing the mechanisms of gene expression in P. tannophilus and S. cerevisiae. Such analysis is important because the former organism is one of the few yeasts capable of directly fermenting xylose, while the latter organism, a major industrial microorganism for ethanol production, is not able to utilize xylose. This is especially relevant to the utilization of renewable biomass, of which xylose is a major component. Furthermore, there is no cloning system available for P. tannophilus at present. The cloned XYK-Pa gene can also serve as a selection marker in establishing such a system. This work was supported in part by U.S. Department of Agriculture grant 85-FSTY-9-0109 and National Science Foundation grant DBM-8305043. LITERATURE CITED 1. Broach, J. R., J. N. Strathern, and J. B. Hicks. 1979. Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN] gene. Gene 8:121-133. 2. David, J. D., and H. Weismeyer. 1970. Control of xylose metabolism in Escherichia coli. Biochim. Biophys. Acta 201: 497-499. 3. Debus, D., H. Methner, D. Schulz, D. Dellweg, and H. Dellweg. 1983. Fermentation of xylose with the yeast Pachysolen tannophilus. Eur. J. Appl. Microbiol. Biotechnol. 17:287-291. 4. Dekker, R. F. H. 1982. Ethanol production from D-xylose and other sugars by the yeast Pachysolen tannophilus. Biotechnol. Lett. 4:411-416. 5. Hicks, J., and G. R. Fink. 1977. Identification of chromosomal location of yeast DNA from hybrid plasmid pYeleu,1O. Nature (London) 269:265-267. 6. Lachke, A. H., and T. W. Jeffries. 1986. Levels of enzymes of the pentose phosphate pathway in Pachysolen tannophilus Y-2460 and selected mutants. Enzyme Microb. Technol. 8:353-359. 7. Lawlis, V. B., M. S. Dennis, E. Y. Chen, D. H. Smith, and J. Henner. 1984. Cloning and sequencing of the xylose isomerase and xylulose kinase genes of Escherichia coli. Appl. Environ. Microbiol. 47:15-21. 8. Rosenfeld, S. A., P. E. Stevis, and N. W. Y. Ho. 1984. Cloning and characterization of the xyl genes from Escherichia coli. Mol. Gen. Genet. 194:410-415. 9. Schneider, H., P. Y. Wang, Y. K. Chan, and R. Maleszka. 1981. Conversion of D-xylose into ethanol by the yeast Pachysolen tannophilus. Biotechnol. Lett. 3:89-92. 10. Shamanna, D. K., and K. E. Sanderson. 1979. Uptake and catabolism of D-xylose in Salmonella typhimurium LT2. J. Bacteriol. 139:64-70. 11. Slininger, P. J., R. J. Bothast, J. E. Van Cawenberge, and C. P.
VOL. 53, 1987 Kurtzman. 1982. Conversion of D-xylose to ethanol by the yeast Pachysolen tannophilus. Biotechnol. Bioeng. 24:371-384. 12. Tschumper, G., and J. Carbon. 1980. Sequence of a yeast DNA fragment containing a chromosomal replicator and the TRPI gene. Gene 10:157-166. 13. Way, J. C., M. A. Davis, D. Morisato, D. E. Roberts, and N.
NOTES
2977
Kleckner. 1984. New TnWO derivatives for transposon mutagenesis and for construction of lacZ operon fusion by transposition. Gene 32:369-379. 14. Wickerham, L. J. 1970. Genus 14. Pachysolen Boidin et Adzet, p. 448-454. In J. Lodder (ed.), The yeasts. A taxonomic study, 2nd ed. North-Holland Publishing Co., Amsterdam.
