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

Overview

Journal J Bacteriol

Publisher American Society for Microbiology

Specialty Microbiology

Date 1992 Sep 1

PMID 1522074

Citations 16

Authors

Y Sakai

Y Tani

Affiliations

Soon will be listed here.

Abstract

A model system for one-step gene disruption for an asporogenous methylotrophic yeast, Candida boidinii, is described. In this system, the 3-isopropylmalate dehydrogenase gene (C. boidinii LEU2) was selected as the target gene for disruption to derive new host strains for transformation. First, the C. boidinii LEU2 gene was cloned, and its complete nucleotide sequence was determined. Next, the LEU2 disruption vectors, which had the C. boidinii URA3 gene as the selectable marker, were constructed. Of the Ura+ transformants obtained with these plasmids, more than half showed a Leu- phenotype. Finally, the double-marker strains of C. boidinii were derived. When vectors with repeated flanking sequences of the C. boidinii URA3 gene were used for gene disruption, Leu- Ura+ transformants changed spontaneously to a Leu- Ura- phenotype ca. 100 times more frequently than they did when plasmids without the repeated sequences were used. Southern analysis showed that these events included a one-step gene disruption and a subsequent popping out of the C. boidinii URA3 sequence from the transformant chromosome.

Citing Articles

The GMC superfamily of oxidoreductases revisited: analysis and evolution of fungal GMC oxidoreductases.

Sutzl L, Foley G, Gillam E, Boden M, Haltrich D Biotechnol Biofuels. 2019; 12:118.

PMID: 31168323 PMC: 6509819. DOI: 10.1186/s13068-019-1457-0.

Yeast nitrogen utilization in the phyllosphere during plant lifespan under regulation of autophagy.

Shiraishi K, Oku M, Kawaguchi K, Uchida D, Yurimoto H, Sakai Y Sci Rep. 2015; 5:9719.

PMID: 25900611 PMC: 5386246. DOI: 10.1038/srep09719.

Molecular characterization of hap complex components responsible for methanol-inducible gene expression in the methylotrophic yeast Candida boidinii.

Oda S, Yurimoto H, Nitta N, Sasano Y, Sakai Y Eukaryot Cell. 2015; 14(3):278-85.

PMID: 25595445 PMC: 4346567. DOI: 10.1128/EC.00285-14.

Trm1p, a Zn(II)2Cys6-type transcription factor, is a master regulator of methanol-specific gene activation in the methylotrophic yeast Candida boidinii.

Sasano Y, Yurimoto H, Yanaka M, Sakai Y Eukaryot Cell. 2008; 7(3):527-36.

PMID: 18203863 PMC: 2268522. DOI: 10.1128/EC.00403-07.

Cis-acting elements sufficient for induction of FDH1 expression by formate in the methylotrophic yeast Candida boidinii.

Komeda T, Yurimoto H, Kato N, Sakai Y, Kondo K Mol Genet Genomics. 2003; 270(3):273-80.

PMID: 14534783 DOI: 10.1007/s00438-003-0917-0.

References

Hinnen A, Hicks J, Fink G . Transformation of yeast. Proc Natl Acad Sci U S A. 1978; 75(4):1929-33. PMC: 392455. DOI: 10.1073/pnas.75.4.1929. View

Ratzkin B, Carbon J . Functional expression of cloned yeast DNA in Escherichia coli. Proc Natl Acad Sci U S A. 1977; 74(2):487-91. PMC: 392314. DOI: 10.1073/pnas.74.2.487. View

Cryer D, Eccleshall R, MARMUR J . Isolation of yeast DNA. Methods Cell Biol. 1975; 12:39-44. DOI: 10.1016/s0091-679x(08)60950-4. View

Gellissen G, Janowicz Z, Merckelbach A, Piontek M, Keup P, Weydemann U . Heterologous gene expression in Hansenula polymorpha: efficient secretion of glucoamylase. Biotechnology (N Y). 1991; 9(3):291-5. DOI: 10.1038/nbt0391-291. View

Picataggio S, Deanda K, Mielenz J . Determination of Candida tropicalis acyl coenzyme A oxidase isozyme function by sequential gene disruption. Mol Cell Biol. 1991; 11(9):4333-9. PMC: 361294. DOI: 10.1128/mcb.11.9.4333-4339.1991. View

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; 173(23):7458-63. PMC: 212510. DOI: 10.1128/jb.173.23.7458-7463.1991. View

Garrard L, Goodman J . Two genes encode the major membrane-associated protein of methanol-induced peroxisomes from Candida boidinii. J Biol Chem. 1989; 264(23):13929-37. View

Vieira J, Messing J . Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985; 33(1):103-19. DOI: 10.1016/0378-1119(85)90120-9. View

Kelly R, Miller S, KURTZ M, Kirsch D . Directed mutagenesis in Candida albicans: one-step gene disruption to isolate ura3 mutants. Mol Cell Biol. 1987; 7(1):199-208. PMC: 365057. DOI: 10.1128/mcb.7.1.199-208.1987. View

10.

Grimm C, Kohli J . Observations on integrative transformation in Schizosaccharomyces pombe. Mol Gen Genet. 1988; 215(1):87-93. DOI: 10.1007/BF00331308. View

11.

Hamasawa K, Kobayashi Y, Harada S, Yoda K, Yamasaki M, Tamura G . Molecular cloning and nucleotide sequence of the 3-isopropylmalate dehydrogenase gene of Candida utilis. J Gen Microbiol. 1987; 133(4):1089-97. DOI: 10.1099/00221287-133-4-1089. View

12.

Vieira J, Messing J . Production of single-stranded plasmid DNA. Methods Enzymol. 1987; 153:3-11. DOI: 10.1016/0076-6879(87)53044-0. View

13.

Zaret K, Sherman F . DNA sequence required for efficient transcription termination in yeast. Cell. 1982; 28(3):563-73. DOI: 10.1016/0092-8674(82)90211-2. View

14.

Feinberg A, Vogelstein B . A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983; 132(1):6-13. DOI: 10.1016/0003-2697(83)90418-9. View

15.

Ito H, Fukuda Y, Murata K, Kimura A . Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983; 153(1):163-8. PMC: 217353. DOI: 10.1128/jb.153.1.163-168.1983. View

16.

Boeke J, Lacroute F, Fink G . A positive selection for mutants lacking orotidine-5'-phosphate decarboxylase activity in yeast: 5-fluoro-orotic acid resistance. Mol Gen Genet. 1984; 197(2):345-6. DOI: 10.1007/BF00330984. View

17.

SANGER F, Nicklen S, Coulson A . DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977; 74(12):5463-7. PMC: 431765. DOI: 10.1073/pnas.74.12.5463. View

18.

Pribnow D . Bacteriophage T7 early promoters: nucleotide sequences of two RNA polymerase binding sites. J Mol Biol. 1975; 99(3):419-43. DOI: 10.1016/s0022-2836(75)80136-7. View

19.

Southern E . Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975; 98(3):503-17. DOI: 10.1016/s0022-2836(75)80083-0. View

20.

Tani Y . Production of useful chemicals by methylotrophs. Biotechnology. 1991; 18:253-70. DOI: 10.1016/b978-0-7506-9188-8.50018-8. View