Skip to main content

The Kluyveromyces lactis CPY homologous genes — Cloning and characterization of the KlPCL1 gene

Abstract

A 3.85-kb genomic fragment containing the KlPCL1 gene, with an open reading frame (ORF) of 1359 bp, was isolated from Kluyveromyces lactis genomic library by heterologous colony hybridization using the Saccharomyces cerevisiae PRC1 (ScPRC1) gene as a probe. The KlPCL1 nucleotide sequence was identical to the KLLAOC17490g ORF of K. lactis and showed >55 % identity with S. cerevisiae YBR139w and PRC1 genes encoding carboxypeptidases. The deduced KlPcl1p amino acid sequence displayed strong similarities to yeast and higher eukaryotic carboxypeptidases. In silico analyses revealed that KlPcl1p contained several highly conserved regions characteristic of the serine-type carboxypeptidases, such as the catalytic triad in the active site and the LNGGPGCSS, FHIAGESYAGHYIP and ICNWLGN motifs involved in the substrate binding. All this suggests that the KlPCL1 gene product belongs to the serine carboxypeptidase family. Sporulation and ascus dissection of a diploid strain heterozygous for single-copy disruption of KlPCL1 revealed that this gene is not essential in K. lactis. Further analyses of haploid and diploid deletion mutants demonstrated that disruption of the KlPCL1 gene neither impaired sporulation nor affected growth abilities of K. lactis cells under a variety of physiological conditions, e.g., growth on different carbon sources, at various temperatures or pH of the medium, and under nitrogen depletion.

This is a preview of subscription content, access via your institution.

Abbreviations

CPY:

carboxypeptidase Y

PCR:

polymerase chain reaction

G418:

geneticin

PRC:

proteinase C

Kl :

Kluyveromyces lactis

SM:

synthetic medium

ME:

malt extract (medium)

Sc :

Saccharomces cerevisiae

MM:

minimal medium

YP:

yeast extract-peptone

ORF:

open reading frame

YPD:

yeast extract-peptone-dextrose

PCL:

proteinase C-like

References

  • Baxter S.M., Rosenblum J.S., Knutson S., Nelson M.R., Montimurro J.S., Di Gennaro J.A., Speir J.A., Burbaum J.J., Fet-Row J.S.: Synergistic computational and experimental proteomics approaches for more accurate detection of active serine hydrolases in yeast. Mol.Cell.Proteomics 3, 209–225 (2004).

    PubMed  Article  CAS  Google Scholar 

  • Bendtsen J.D., Nielsen H., von Heijne G., Brunak S.: Improved prediction of signal peptides: signal P 3.0. J.Mol.Biol. 340, 783–795 (2004).

    PubMed  Article  Google Scholar 

  • Bianchi M.M., Falcone C., Chen X.J., Weśolowski-Louvel M., Frontali L., Fukuhara H.: Transformation of the yeast Kluyveromyces lactis by new vectors derived from the 1.6 μm circular plasmid pKD1. Curr.Genet. 12, 185–192 (1987).

    Article  CAS  Google Scholar 

  • Billard P., Ménart S., Blaisonneau J., Bolotin-Fukuhara M., Fukuhara H., Weśolowski-Louvel M.: Glucose uptake in Kluyveromyces lactis. Role of the HGT1 gene in glucose transport. J.Bacteriol. 178, 5860–5866 (1996).

    PubMed  CAS  Google Scholar 

  • Brown A.J., Planta R.J., Restuhadi F., Bailey D., Butler P., Cadahia J., Cerdan M., De Jonge M., Gardner D., Gent M., Hayes A., Kolen C., Lombardia L., Murad A., Oliver R., Sefton M., Thevelein J., Tournu H., Van Delft Y., Verbart D., Winderickx J., Oliver S.: Transcript analysis of 1003 novel yeast genes using high-throughput Northern hybridizations. EMBO J. 20, 3177–3186 (2001).

    PubMed  Article  CAS  Google Scholar 

  • Byrne K.P., Wolfe K.H.: Visualizing syntenic relationships among the hemiascomycetes with the Yeast Gene Order Browser. Nucl. Acids Res. 34 (Database issue), D452–D455 (2006).

    PubMed  Article  CAS  Google Scholar 

  • Dujon B., Sherman D., Fisher G.: Genome evolution in yeasts. Nature 430, 35–44 (2004).

    PubMed  Article  Google Scholar 

  • Flores M.V., Cuellas A., Voget C.E.: The proteolytic system of the yeast Kluyveromyces lactis. Yeast 15, 1437–1448 (1999).

    PubMed  Article  CAS  Google Scholar 

  • Gasch A.P., Spellman P.T., Kao C.M., Carmel-Harel O., Eisen M.B., Storz G., Botstein D., Brown P.O.: Genomic expression programs in the response of yeast cells to environmental changes. Mol.Biol.Cell 11, 4241–4257 (2000).

    PubMed  CAS  Google Scholar 

  • Hayashi R., Moore S., Stein W.H.: Carboxypeptidase from yeast. Large scale preparation and the application to COOH-terminal analysis of peptides and proteins. J.Biol.Chem. 248, 2296–2302 (1973).

    PubMed  CAS  Google Scholar 

  • Van den Hazel H.B., Kielland-Brandt M.C., Winther J.R.: Review: biosynthesis and function of yeast vacuolar proteases. Yeast 12, 1–16 (1996).

    Article  Google Scholar 

  • Hoffman C.S., Winston F.: A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57, 267–272 (1987).

    PubMed  Article  CAS  Google Scholar 

  • Hussein L., Elasyed S., Foda S.: Reduction of lactose in milk by purified lactase produced by Kluyveromyces lactis. J.Food Prot. 52, 30–34 (1989).

    CAS  Google Scholar 

  • Inoue H., Nojima H., Okayama H.M.: High efficiency transformation of Escherichia coli with plasmids. Gene 96, 23–28 (1990).

    PubMed  Article  CAS  Google Scholar 

  • Jones E.W., Webb G.C., Hiller M.A.: Biogenesis and function of yeast vacuole, pp. 363–470 in J.R. Pringle, J.R. Broach, E.W. Jones (Eds): The Molecular and Cellular Biology of the Yeast Saccharomyces, Vol. 3. Cold Spring Harbor, New York 1997.

    Google Scholar 

  • Jung G., Ueno H., Hayashi R.: Carboxypeptidase Y: structural basis for protein sorting and catalytic triad. J.Biochem.(Tokyo) 126, 1–6 (1999).

    CAS  Google Scholar 

  • Liao D.I., Remington S.J.: Structure of wheat serine carboxypeptidase II at 3.5-Å resolution. A new class of serine proteinase. J.Biol. Chem. 265, 6528–6531 (1990).

    PubMed  CAS  Google Scholar 

  • Lundblad V., Hartzog G., Moqtaderi Z.: Manipulation of cloned yeast DNA, unit 13.10 in F.M. Ausubel, R. Brent, R. Kingston, D. Moore, J. Seidman, J. Smith, K. Struhl (Eds): Current Protocols in Molecular Biology. John Wiley & Sons, New York 1989; Supplement 1997.

    Google Scholar 

  • Nakase H., Murata S., Ueno H., Hayashi R.: Substrate recognition mechanism of carboxypeptidase Y. Biosci.Biotechnol.Biochem. 65, 2465–2471 (2001).

    PubMed  Article  CAS  Google Scholar 

  • Nasr F., Becam A.-M., Grzybowska E., Zagulski M., Slonimski P.P., Herbert C.J.: An analysis of the sequence of part of the right arm of chromosome II of S. cerevisiae reveals new genes encoding an amino-acid permease and a carboxypeptidase. Curr.Genet. 26, 1–7 (1994).

    PubMed  Article  CAS  Google Scholar 

  • Van Ooyen A., Dekker P., Huang M., Olsthoorn M., Jacobs D., Colussi P., Taron C.: Heterologous protein production in the yeast Kluyveromyces lactis. FEMS Yeast Res. 6, 381–392 (2006).

    PubMed  Article  Google Scholar 

  • Pearson B.M., Hernando Y., Schweizer M.: Construction of PCR-ligated long flanking homology cassettes for use in the functional analysis of six unknown open reading frames from the left and right arms of Saccharomyces cerevisiae chromosome XV. Yeast 14, 391–399 (1998).

    PubMed  Article  CAS  Google Scholar 

  • Sambrook J., Fritsch E.F., Maniatis T.: Molecular Cloning: a Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, New York 1989.

    Google Scholar 

  • Stennicke H.R., Mortensen U.H., Breddam K.: Studies on the hydrolytic properties of (serine) carboxypeptidase Y. Biochemistry 35, 7131–7141 (1996).

    PubMed  Article  CAS  Google Scholar 

  • Van den Berg J.A., Van der Laken K.J., Van Ooyen A., Renniers T., Rietveld K., Schaap A., Brake A., Bishop R., Schultz K., Moyer D., Richman M., Shuster J.R.: Kluyveromyces as a host for heterologous gene expression: expression and secretion of prochymosin. Bio/Technology 8, 135–139 (1990).

    PubMed  Article  Google Scholar 

  • Wach A., Brachat A., Pohlmann R., Philippsen P.: New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10, 1793–1808 (1994).

    PubMed  Article  CAS  Google Scholar 

  • Weśolowski-Louvel M., Tanguy-Rougeau C., Fukuhara H.: A nuclear gene required for the expression of the linear DNA-associated killer system in the yeast Kluyveromyces lactis. Yeast 4, 71–81 (1988).

    PubMed  Article  Google Scholar 

  • Weśolowski-Louvel M., Breunig K.D., Fukuhara H.: Kluyveromyces lactis, pp. 139–202 in K. Wolf (Ed.): Nonconventional Yeasts in Biotechnology: a Handbook. Springer-Verlag, Berlin 1996.

    Google Scholar 

  • Winston F., Dollard C., Ricupero-Hovasse S.L.: Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast 11, 53–55 (1995).

    PubMed  Article  CAS  Google Scholar 

  • Winzeler E., Shoemaker D., Astromoff A., Liang H., Anderson K., Andre B., Bangham R., Benito R., Boeke J., Bussey H., Chu A., Connelly C., Davis K., Dietrich F., Dow S., Bakkoury M., Foury F., Friend S., Gentalen E., Giaever G., Hegemann J., Jones T., Laub M., Liao H., Liebundguth N., Lockhart D., Lucau-Danila A., Lussier M., M’rabet N., Menard P., Mittmann M., Pai C., Rebischung C., Revuelta J., Riles L., Roberts C., Ross-Macdonald P., Scherens B., Snyder M., Sookhai-Mahadeo S., Storms R., Véronneau S., Voet M., Volckaert G., Ward T., Wy-Socki R., Yen G., Yu K., Zimmermann K., Philippsen P., Johnston M., Davis R.: Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901–906 (1999).

    PubMed  Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to D. Staneva.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Staneva, D., Uccelletti, D., Venkov, P. et al. The Kluyveromyces lactis CPY homologous genes — Cloning and characterization of the KlPCL1 gene. Folia Microbiol 53, 325–332 (2008). https://doi.org/10.1007/s12223-008-0051-9

Download citation

  • Received:

  • Revised:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12223-008-0051-9

Keywords

  • Lactis
  • Catalytic Triad
  • Nitrogen Depletion
  • Disruption Cassette
  • Serine Carboxypeptidase