Moriya Ohkuma

Proliferation of intracellular membrane structures upon homologous overproduction of cytochrome P-450 in Candida maltosa

In an alkane-assimilating yeast, Candida maltosa, a cultivation on alkane causes both induction of endoplasmic reticulum (ER)-resident membrane proteins, such as cytochrome P-450, and proliferation of ER. In this study, individual genes for alkane-inducible forms of cytochrome P-450 (P-450alk) were homologously overexpressed in C. maltosa using a galactose-inducible expression system developed in this yeast. Immunoelectron microscopy revealed that, upon the overexpression, a dramatic proliferation of ER occurred, in which overproduced P-450alk protein accumulated. The proliferated membranes were mainly tubular forms and stacks of paired membranes were also observed after prolonged expression. The tubular forms were morphologically very similar to the proliferated ER in alkane-induced C. maltosa cells. The observed proliferation of ER membranes by homologous overproduction of P-450alk, here depicted, will provide a unique opportunity for investigating the mechanisms by which cells regulate ER biogenesis, in comparison with the intrinsic form of ER proliferation.

An improved host-vector system for Candida maltosa using a gene isolated from its genome that complements the hiss mutation of Saccharomvces cerevisiae

SummaryThe host-vector system of an n-lkaneassimilating-yeast, Candida maltosa, which we previously constructed using an autonomously replicating sequence (ARS) region isolated from the genome of this yeast, utilizes C. maltosa J288 (leu2−) as a host. As this host had a serious growth defect on n-alkane, we developed an improved host-vector system using C. maltosa CHI (his) as host. The vectors were constructed with the Candida ARS region and a DNA fragment isolated from the genome of C. maltosa. Since this DNA fragment could complement histidine auxotrophy of both C. maltosa CH1 and S. cerevisiae (hiss−), we termed the gene contained in this DNA fragment C-HIS5. The vectors were characterized in terms of transformation frequency and stability, and the nucleotide sequence of C-HISS was determined. The deduced amino acid sequence (389 residues) shared 51% homology with that of HISS of S. cerevisiae (384 residues; Nishiwaki et al. 1987).

Cloning of the C-URA3 gene and construction of a triple auxotroph (his5, ade1, ura3) as a useful host for the genetic engineering of Candida maltosa.

The C-URA3 gene of the n-alkane assimilating-yeast Candida maltosa was cloned by complementation of the ura3 mutation of Saccharomyces cerevisiae. The nucleotide sequence of C-URA3 and its deduced amino-acid sequence showed significant homology to those of the orotidine 5′-phosphate decarboxylases of other fungal species. To construct a useful host for genetic engineering of C. maltosa using C-URA3 as a marker, one allele of C-URA3 in a double auxotroph (his5, ade1) was disrupted by C-ADE1, and subsequently two kinds of ura3 mutants were isolated by selecting for spontaneous 5-fluoro-orotic acid (5FOA) resistance. One of the mutants was homozygous for the disruption (ura3::C-ADE1/ura3::C-ADE1); the other was heterozygous (ura3::C-ADE1/ura3). The ura3::C-ADE1 allele in the latter strain was re-substituted by C-URA3 to rescue the adenine auxotroph (his5, ade1, C-URA3/ura3). Finally, by selecting a 5FOA-resistant mutant, a triple auxotroph (his5, ade1, ura3/ura3) was isolated.

Identification of a centromeric activity in the autonomously replicating TRA region allows improvement of the host-vector system for Candida maltosa

A centromeric activity was identified in the previously isolated 3.8 kb DNA fragment that carries an autonomously replicating sequence (ARS) from the yeast Candida maltosa. Plasmids bearing duplicated copies of the centromeric DNA (dicentric plasmids) were physically unstable and structural rearrangements of the dicentric plasmids occurred frequently in the transformed cells. The centromeric DNA activity was dissociated from the ARS, which is 0.2 kb in size, and was delimited to a fragment at least 325 by in length. The centromeric DNA region included the consensus sequences of CDEI (centromeric DNA element I) and an AT-rich CDEII-like region of Saccharomyces cerevisiae but had no homology to the functionally critical CDEIII consensus. A plasmid bearing the whole 3.8 kb fragment was present in 1–2 copies per cell and was maintained stably even under non-selective culture conditions, while a plasmid having only the 0.2 kb ARS was unstable and accumulated to high copy numbers. The high-copy-number plasmid allowed us to overexpress a gene to a high level, which had never been attained before, under the control of both constitutive and inducible promoters in C. maltosa.

Expression of an endogenous and a heterologous gene in Candida maltosa by using a promoter of a newly-isolated phosphoglycerate kinase (PGK) gene.

A gene encoding phosphoglycerate kinase (PGK) was isolated from the genomic library of C. maltosa to construct an expression vector for this yeast. The PGK gene had an open reading frame of 1251 base pairs encoding approximately 47-kDA polypeptide of 417 amino-acid residues. Expression of this gene assayed by Northern-blot analysis was significantly induced in cells grown on glucose but not in cells grown on n-tetradecane, n-tetradecanol, or oleic acid. By using the promoter region of this gene, an expression vector (termed pMEA1) for C. maltosa was constructed and expression of an endogenous gene (P450alkl encoding one of cytochrome P450s for n-alkane hydroxylation in C. maltosa) and a heterologous gene (LAC4 encoding Kluyveromyces lactis β-galactosidase) was tested. Expression of P450alkl gene was confirmed at both mRNA and protein levels. LAC4 gene expression was confirmed by determining β-galactosidase activity. The activity in cells grown on various carbon sources correlated very well with the expression levels of PGK mRNA in these cells.

Galactose-inducible expression systems in Candida maltosa using promoters of newly-isolated GAL1 and GAL10 genes.

The GAL1 and GAL10 gene cluster encoding the enzymes of galactose utilization was isolated from an asporogenic yeast, Candida maltosa. The structure of the gene cluster in which both genes were divergently transcribed from the central promoter region resembled those of some other yeasts. The expression of both genes was strongly induced by galactose and repressed by glucose in the medium. Galactose‐inducible expression vectors in C. maltosa were constructed on low‐ and high‐copy number plasmids using the promoter regions of both genes. With these vectors and the β‐galactosidase gene from Kluyveromyces lactis as a reporter, galactose‐inducible expression was confirmed. Homologous overexpression of members of the cytochrome P‐450 gene family in C. maltosa was also successful by using a high‐copy‐number vector under the control of these promoters.

Draft Genome Sequence of Paenibacillus pini JCM 16418T, Isolated from the Rhizosphere of Pine Tree

ABSTRACT Paenibacillus pini strain JCM 16418T is a cellulolytic bacterium isolated from the rhizosphere of pine trees. Here, we report the draft genome sequence of this strain. This genome information will be useful for studies of rhizosphere bacteria.