chemical causing callus formation, suggesting that it may be A basic chitinase was secreted into culture medium of pumpkin cell suspension cultures. The chitinase was purified from independent of the presence of 2,4-D. Perhaps, induction is caused by osmotic or wounding stress. Levels of chitinase the culture medium. A cDNA encoding the pumpkin chitinase mRNA markedly increased at 4 h after transfer of pumpkin was cloned by reverse transcription (RT)-PCR and rapid amplification of cDNA ends (RACE) methods. The chitinase callus cells into fresh culture liquid medium. They were also high at later stages of cell suspension culture. In transgenic gene was strongly expressed in pumpkin callus cells, but little or not at all in mature leaf, young leaf, cotyledon, stem, tobacco BY-2 cells, into which the pumpkin chitinase cDNA hypocotyl and root of pumpkin. No chitinase mRNA was was introduced, the recombinant pumpkin chitinase was expressed and secreted into the culture medium, suggesting that detected in intact pumpkin fruit tissues. However, chitinase was induced during callus formation from sliced pumpkin fruit the signal peptide of pumpkin chitinase also functions for secretion from tobacco BY-2 cells. tissues. Induction also occurred in the absence of 2,4-D, a perhaps by releasing endogenous factors that are related to nodulation factors (De Jong et al. 1992, Vijn et al. 1993.In plants, four classes of chitinases have so far been proposed on the basis of their primary structures (Collinge et al. 1993). Class I chitinases contain an amino-terminal cysteine-rich domain of about 40 amino acids, a chitinbinding domain that is homologous to hevein and wheat germ agglutinin, and a highly conserved main structure, separated by a variable hinge region. Most class I chitinases have a basic isoelectric point and are localized in vacuoles. Indeed, many prochitinases have vacuolar targeting signals, i.e seven carboxy-terminal extra amino acids (Neuhaus et al. 1991). Class II chitinases are similar to class I chitinases but lack an amino-terminal cysteine-rich domain. They appear to be acidic proteins and are located in the apoplast. Class III chitinases have sequences very different from most of class I and class II chitinases. They can be acidic or basic proteins, and may be localized in the apoplast. Class IV chitinases contain a cysteine-rich domain and a conserved main structure, resembling class I chitinases, but they are significantly smaller as a consequence of four major deletions.
The synthesis of dicarboxylic acids (DCAs) in Candida tropicalis is thought to be induced by a decrease in fatty acyl-CoA-oxidase activity. However, in the present study we demonstrate that repression of the POX4 gene, encoding fatty acyl-CoA oxidase, does not directly lead to high-level production of DCAs. No fatty acyl-CoA-oxidase activity was detected if the POX4 gene of C. tropicalis strain 1098 (wild-type strain) was disrupted. Furthermore, introduction of the POX4 gene from C. tropicalis strain M1210A3, which is a mutant derived from strain 1098 and is used as an industrial DCA-producing strain, still exhibited low-level fatty acyl-CoA-oxidase activity. Nevertheless, production of DCA was not observed in either case. Furthermore, the increase in acyl-CoA-oxidase activity by expression of the POX4 gene in strain M1210A3 did not reduce high-level production of DCA. These results suggest that alterations in acyl-CoA-oxidase activity are not necessarily related to production of DCA in industrial DCA-producing C. tropicalis M1210A3.
In the long-chain dicarboxylic acids (DCA)-hyperproducing mutant Candida maltosa strains, methyl-ends of n-alkanes and fatty acids are hydroxylated by n-alkane inducible cytochromes P450 (P450alk), presumably as an essential step in DCA production. A significantly higher production of P450alks was observed in response to n-alkane in the DCA-hyperproducing mutant strain M2030 than in the wild-type strain 1098. Northern analysis demonstrated that n-tetradecane induction levels of mRNAs of all four ALK genes encoding major P450alk isoforms involved in n-alkane assimilation were significantly higher in the DCA-hyperproducing mutant than in the wild-type strain. Among these four ALK genes, enhancement of the transcriptional induction level of ALK5, which prefers fatty acids as substrates, was prominent in the mutant. In agreement with Northern analysis, promoters of ALK genes, especially that of ALK5, more strongly responded to n-alkanes in the DCA-hyperproducing mutant than in the wild-type strain. These results suggest that the transcriptional control of ALK genes in the DCA-hyperproducing mutant strains was altered preferably to accelerate DCA production.
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.
Phylogenetic relationships of several species within the n-alkane assimilating Candida yeasts were investigated by using characters from the nucleotide sequence of the variable D1/D2 region at the 5' end of a large-subunit (26S) ribosomal DNA (rDNA) gene. First the nucleotide sequences of D1/D2 domain of Candida sp. 1098 (formerly identified as C. tropicalis 1098) and its dicarboxylic acid-producing-mutant strain M1210 were investigated. These two nucleotide sequences were identical and lacked only one base pair compared with that of C. maltosa CBS 5611 (type strain), and they were identified as C. maltosa. We then showed that C. maltosa IFO 1978 (formerly identified as C. cloacae) and C. maltosa IFO 1975 (formerly identified as C. subtropicalis) had the same nucleotide sequence and had only one base pair substitution compared with C. maltosa CBS 5611 (type strain), which is consistent with conventional classification. We also found that another widely studied n-alkane assimilating Candida yeast, C. tropicalis pk233, to be C. viswanathii.
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