Cloning and characterization of the POX2 gene in Candida maltosa

Masuda, Yutaka; Park, Sun Mee; Ohta, Akio; Takagi, Masamichi

doi:10.1016/0378-1119(95)00655-9

Cited by 15 publications

(7 citation statements)

References 14 publications

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“…Some P450alk isoforms should play important roles for the production because considerable fatty acid -hydroxylation activity has been reported for them (5,(11)(12)(13). In combination with the functional block of the ␤-oxidation system by gene disruption as described in C. tropicalis (22,23) and C. maltosa (24) and gene disruption of the appropriate P450alk forms as described here, it may be possible to construct an efficient strain for production.…”

Section: Discussionmentioning

confidence: 99%

Isozyme Function of n-Alkane-inducible Cytochromes P450 in Candida maltosa Revealed by Sequential Gene Disruption

Ohkuma¹,

Zimmer²,

Iida³

et al. 1998

Journal of Biological Chemistry

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An n-alkane-assimilating yeast Candida maltosa contains multiple n-alkane-inducible forms of cytochromes P450 (P450alk), which can be assumed to catalyze terminal hydroxylation of n-alkanes in the assimilation pathway. Eight structurally related P450alk genes have been identified. In the present study, the function of four major isoforms of P450alk (encoded by ALK1, ALK2, ALK3, and ALK5 genes) was investigated by sequential gene disruption. Auxotrophic markers used for the selection of disrupted strains were regenerated repeatedly through either mitotic recombination between heterozygous alleles of the diploid genome or directed deletion of the marker gene, to allow sequential gene disruptions within a single strain. The strain depleted of all four isoforms could not utilize n-alkanes for growth, providing direct evidence that P450alk is essential for n-alkane assimilation. Growth properties of a series of intermediate disrupted strains, plasmid-based complementation, and enzyme assays after heterologous expression of single isoforms revealed (i) that each of the four individual isoforms is alone sufficient to allow growth on long chain n-alkane; (ii) that the ALK1-encoding isoform is the most versatile and efficient P450alk form, considering both its enzymatic activity and its ability to confer growth on n-alkanes of different chain length; and (iii) that the ALK5-encoding isoform exhibits a rather narrow substrate specificity and thus cannot support the utilization of short chain n-alkanes.

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Section: Discussionmentioning

confidence: 99%

Isozyme Function of n-Alkane-inducible Cytochromes P450 in Candida maltosa Revealed by Sequential Gene Disruption

Ohkuma¹,

Zimmer²,

Iida³

et al. 1998

Journal of Biological Chemistry

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“…The consensus sequence of 13-oxidation box, AACGGGGAT, concluded by the peroxisomal acyl-CoA oxidase and the peroxisomal trifunctiona113-oxidation enzyme of Saccharomyces cerevisiae (17) Kb with six introns that vary in length between 62 bp to 9.7 Kb. Such complexity can also be found in the aeyl-CoA oxidase gene of human and rat (3,18 (20). This is also the ease for POX2 and POX4 genes in Candida tropicalis (6).…”

Section: Isolation and Nucleotide Sequence Analysis Of Paco1 Gene Fromentioning

confidence: 94%

Gene structure of PACO1, a petal senescence‐related related gene from Phalaenopsis encoding peroxisomal acyl‐CoA oxidase homolog

Huang

1997

IUBMB Life

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SUMMARYThe putative peroxisomal acyl-CoA oxidase gene PACO1 from Phalaenopsis was isolated from a genomic library constructed in the )~EMBL3 vector by screening with PACO1 cDNA. This gene encompasses approximately 14 Kb and is divided into seven exons by six introns. The consensus GT-AG dinucleotides situated at all the boundaries between exons and introns. According to the genomic Southern analysis with different fragments of the first intron, PACO1 gene possesses a large intron about 10 Kb interrupting the first and secondary exon.

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“…Oligonucleotides H801 and H802 were used as PCR primers. These oligonucleotides were designed from C. maltosa POX4 (Masuda et al 1995). The amplified fragment was inserted into pT7Blue(R), sequenced, and identified as the POX4 gene.…”

Section: Cotransformation Methodsmentioning

confidence: 99%

Novel and convenient methods for Candida tropicalis gene disruption using a mutated hygromycin B resistance gene

Hara

Arie

Kanai

et al. 2001

Archives of Microbiology

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We established a novel and convenient method to construct a ura3 strain (ura3/ura3) of the asporogenous and diploid yeast, Candida tropicalis, that produces dicarboxylic acid. One copy of the URA3 gene was disrupted using a mutated hygromycin B resistance gene (HYG#). The obtained hygromycin-resistant strain was further transformed with a URA3 disruption cassette and selected on a plate containing 5-fluoroorotic acid. The obtained strains were analyzed and the disruption of the gene was confirmed by PCR and Southern blot analysis. The results showed that the strains were obtained in which allelic URA3 genes were simultaneously disrupted. Furthermore, we established a cotransformation method for this gene disruption, using HYG# in C. tropicalis. In order to disrupt the allelic POX4 genes (encoding acyl-CoA oxidase) of dicarboxylic acid-producing strains, the ARS plasmid (which contained HYG#) and a POX4 disruption cassette (which carried the LAC4 gene encoding beta-galactosidase of Kluyveromyces lactis) were simultaneously introduced by transformation. As a result, the allelic POX4 gene was successfully disrupted.

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Cloning and characterization of the POX2 gene in Candida maltosa

Cited by 15 publications

References 14 publications

Isozyme Function of n-Alkane-inducible Cytochromes P450 in Candida maltosa Revealed by Sequential Gene Disruption

Isozyme Function of n-Alkane-inducible Cytochromes P450 in Candida maltosa Revealed by Sequential Gene Disruption

Gene structure of PACO1, a petal senescence‐related related gene from Phalaenopsis encoding peroxisomal acyl‐CoA oxidase homolog

Novel and convenient methods for Candida tropicalis gene disruption using a mutated hygromycin B resistance gene

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