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. 1997 Jul;179(14):4480–4485. doi: 10.1128/jb.179.14.4480-4485.1997

Regulation of the formate dehydrogenase gene, FDH1, in the methylotrophic yeast Candida boidinii and growth characteristics of an FDH1-disrupted strain on methanol, methylamine, and choline.

Y Sakai 1, A P Murdanoto 1, T Konishi 1, A Iwamatsu 1, N Kato 1
PMCID: PMC179282  PMID: 9226256

Abstract

The structural gene (FDH1) coding for NAD(+)-dependent formate dehydrogenase (FDH) was cloned from a genomic library of Candida boidinii, and the FDH1 gene was disrupted in the C. boidinii genome (fdh1 delta) by one-step gene disruption. In a batch culture experiment, although the fdh1 delta strain was still able to grow on methanol, its growth was greatly inhibited and a toxic level of formate was detected in the medium. In a methanol-limited chemostat culture at a low dilution rate (0.03 to 0.05 h[-1]), formate was not detected in the culture medium of the fdh1 delta strain; however, the fdh1 delta strain showed only one-fourth of the growth yield of the wild-type strain. Expression of FDH1 was found to be induced by choline or methylamine (used as a nitrogen source), as well as by methanol (used as a carbon source). Induction of FDH1 was not repressed in the presence of glucose when cells were grown on methylamine, choline, or formate, and expression of FDH1 was shown to be regulated at the mRNA level. Growth on methylamine or choline as a nitrogen source in a batch culture was compared between the wild type and the fdh1 delta mutant. Although the growth of the fdh1 delta mutant was impaired and the level of formate was higher in the fdh1 delta mutant than in the wild-type strain, the growth defect caused by FDH1 gene disruption was small and less severe than that caused by growth on methanol. As judged from these results, the main physiological role of FDH with all of the FDH1-inducing growth substrates seems to be detoxification of formate, and during growth on methanol, FDH seems to contribute significantly to the energy yield.

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Selected References

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  1. Allen S. J., Holbrook J. J. Isolation, sequence and overexpression of the gene encoding NAD-dependent formate dehydrogenase from the methylotrophic yeast Candida methylica. Gene. 1995 Aug 30;162(1):99–104. doi: 10.1016/0378-1119(95)00347-9. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cregg J. M., Vedvick T. S., Raschke W. C. Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnology (N Y) 1993 Aug;11(8):905–910. doi: 10.1038/nbt0893-905. [DOI] [PubMed] [Google Scholar]
  5. Cryer D. R., Eccleshall R., Marmur J. Isolation of yeast DNA. Methods Cell Biol. 1975;12:39–44. doi: 10.1016/s0091-679x(08)60950-4. [DOI] [PubMed] [Google Scholar]
  6. Davis R. W., Thomas M., Cameron J., St John T. P., Scherer S., Padgett R. A. Rapid DNA isolations for enzymatic and hybridization analysis. Methods Enzymol. 1980;65(1):404–411. doi: 10.1016/s0076-6879(80)65051-4. [DOI] [PubMed] [Google Scholar]
  7. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  8. Gellissen G., Janowicz Z. A., Merckelbach A., Piontek M., Keup P., Weydemann U., Hollenberg C. P., Strasser A. W. Heterologous gene expression in Hansenula polymorpha: efficient secretion of glucoamylase. Biotechnology (N Y) 1991 Mar;9(3):291–295. doi: 10.1038/nbt0391-291. [DOI] [PubMed] [Google Scholar]
  9. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  10. Murdanoto A. P., Sakai Y., Konishi T., Yasuda F., Tani Y., Kato N. Purification and properties of methyl formate synthase, a mitochondrial alcohol dehydrogenase, participating in formaldehyde oxidation in methylotrophic yeasts. Appl Environ Microbiol. 1997 May;63(5):1715–1720. doi: 10.1128/aem.63.5.1715-1720.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. NASH T. The colorimetric estimation of formaldehyde by means of the Hantzsch reaction. Biochem J. 1953 Oct;55(3):416–421. doi: 10.1042/bj0550416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Sakai Y., Akiyama M., Kondoh H., Shibano Y., Kato N. High-level secretion of fungal glucoamylase using the Candida boidinii gene expression system. Biochim Biophys Acta. 1996 Jul 31;1308(1):81–87. doi: 10.1016/0167-4781(96)00075-9. [DOI] [PubMed] [Google Scholar]
  13. Sakai Y., Goh T. K., Tani Y. High-frequency transformation of a methylotrophic yeast, Candida boidinii, with autonomously replicating plasmids which are also functional in Saccharomyces cerevisiae. J Bacteriol. 1993 Jun;175(11):3556–3562. doi: 10.1128/jb.175.11.3556-3562.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Sakai Y., Kazarimoto T., Tani Y. Transformation system for an asporogenous methylotrophic yeast, Candida boidinii: cloning of the orotidine-5'-phosphate decarboxylase gene (URA3), isolation of uracil auxotrophic mutants, and use of the mutants for integrative transformation. J Bacteriol. 1991 Dec;173(23):7458–7463. doi: 10.1128/jb.173.23.7458-7463.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Sakai Y., Murdanoto A. P., Sembiring L., Tani Y., Kato N. A novel formaldehyde oxidation pathway in methylotrophic yeasts: methylformate as a possible intermediate. FEMS Microbiol Lett. 1995 Apr 1;127(3):229–234. doi: 10.1111/j.1574-6968.1995.tb07478.x. [DOI] [PubMed] [Google Scholar]
  16. Sakai Y., Rogi T., Takeuchi R., Kato N., Tani Y. Expression of Saccharomyces adenylate kinase gene in Candida boidinii under the regulation of its alcohol oxidase promoter. Appl Microbiol Biotechnol. 1995 Mar;42(6):860–864. doi: 10.1007/BF00191182. [DOI] [PubMed] [Google Scholar]
  17. Sakai Y., Rogi T., Yonehara T., Kato N., Tani Y. High-level ATP production by a genetically-engineered Candida yeast. Biotechnology (N Y) 1994 Mar;12(3):291–293. doi: 10.1038/nbt0394-291. [DOI] [PubMed] [Google Scholar]
  18. Sakai Y., Saiganji A., Yurimoto H., Takabe K., Saiki H., Kato N. The absence of Pmp47, a putative yeast peroxisomal transporter, causes a defect in transport and folding of a specific matrix enzyme. J Cell Biol. 1996 Jul;134(1):37–51. doi: 10.1083/jcb.134.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sakai Y., Tani Y. Directed mutagenesis in an asporogenous methylotrophic yeast: cloning, sequencing, and one-step gene disruption of the 3-isopropylmalate dehydrogenase gene (LEU2) of Candida boidinii to derive doubly auxotrophic marker strains. J Bacteriol. 1992 Sep;174(18):5988–5993. doi: 10.1128/jb.174.18.5988-5993.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Schüte H., Flossdorf J., Sahm H., Kula M. R. Purification and properties of formaldehyde dehydrogenase and formate dehydrogenase from Candida boidinii. Eur J Biochem. 1976 Feb 2;62(1):151–160. doi: 10.1111/j.1432-1033.1976.tb10108.x. [DOI] [PubMed] [Google Scholar]
  21. Subramani S. Protein import into peroxisomes and biogenesis of the organelle. Annu Rev Cell Biol. 1993;9:445–478. doi: 10.1146/annurev.cb.09.110193.002305. [DOI] [PubMed] [Google Scholar]

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