Development of an autonomously replicating linear vector of the yeast Cryptococcus humicola by using telomere-like sequence repeats.

The yeast Cryptococcus humicola has several attractive properties for practical applications such as in bioremediation and as a source of industrially useful enzymes and compounds. We have developed an autonomously replicating vector of C. humicola to improve its properties. We initially tried to isolate an autonomously replicating sequence (ARS) from genomic DNA by transformation using a genomic DNA library. We obtained a candidate plasmid vector harboring an ARS that gave high transformation efficiency. Southern blot analysis of transformants revealed the autonomous replication of the introduced vector in some transformants. However, the vector was not only variously altered in length but also linearized. PCR analysis indicated that a telomere-like sequence repeat (TTAGGGGG)( n ) was added to the termini of linearized vector. Thus, we constructed an autonomously replicating linear vector having ten repeats of the telomere-like sequence at both ends. The vector transformed the yeast cells with high transformation efficiency (3230 CFU/µg of DNA), which was approximately 25-fold higher than that of a control vector lacking the repeats, and was autonomously replicated at a roughly constant size. The copy number was estimated to be less than one copy, and Ura(+) mitotic stability varied widely among the transformants and was related to plasmid segregation efficiency.

Isolation and identification of persistent chlorinated organophosphorus flame retardant-degrading bacteria.

Tris(2-chloroethyl) and tris(1,3-dichloro-2-propyl) phosphates are chlorinated persistent flame retardants that have recently emerged as environmental pollutants. Two bacterial strains that can degrade the compounds when they are the sole phosphorus sources have been isolated and identified as members of the sphingomonads. The strains can be useful for the bioremediation of environments contaminated with these compounds.

Cloning and expression of carp acetylcholinesterase gene in Pichia pastoris and characterization of the recombinant enzyme.

The gene encoding acetylcholinesterase (AChE) was cloned from common carp muscle tissue. The full-length cDNA was 2368 bp that contains a coding region of 1902 bp, corresponding to a protein of 634 amino acids. The deduced amino acid sequence showed a significant homology with those of ichthyic AChEs and several common features among them, including T peptide encoded by exon T in the C-terminus. Three yeast expression vectors were constructed and introduced into the yeast Pichia pastoris. The transformant harboring carp AChE gene lacking exon T most effectively produced AChE activity extracellularly. The replacement of the native signal sequence with the yeast alpha-factor prepro signal sequence rather decreased the production. A decrease in cultivation temperature from 30 to 15 degrees C increased the activity production 32.8-fold. The purified recombinant AChE lacking T peptide, eluted as a single peak with a molecular mass of about 230 kDa on the gel filtration chromatography, exhibited the specific activity of 4970 U/mg. On the SDS-PAGE, three proteins with molecular masses of 73, 54, and 22 kDa were observed. These proteins were N-glycosylated, and their N-terminal sequence showed that the latter two were produced from the former probably by proteolytic cleavage at the C-terminal region. Thus, the recombinant AChE is homotrimer of three identical subunits with 73 kDa. The optimal temperature and pH of the recombinant were comparable to those of the native enzyme purified previously, but the values of kinetic parameters and the sensitivities to substrate inhibition and inhibitors were considerably different between them.

Enrichment and characterization of chlorinated organophosphate ester-degrading mixed bacterial cultures.

Chlorinated organophosphate ester (OPE)-degrading enrichment cultures were obtained using tris(2-chloroethyl) phosphate (TCEP) or tris(1,3-dichloro-2-propyl) phosphate (TDCPP) as the sole phosphorus source. In cultures with 46 environmental samples, significant TCEP and TDCPP degradation was observed in 10 and 3 cultures, respectively, and successive subcultivation markedly increased their degradation rates. 67E and 45D stable enrichment cultures obtained with TCEP and TDCPP, respectively, completely degraded 20 muM of the respective compounds within 6 h and also the other, although the degradation rate of TCEP by 45D was relatively slow. We confirmed chloride ion generation on degradation in both cases and the generation of 2-chloroethanol (2-CE) and 1,3-dichloro-2-propanol (1,3-DCP) as metabolites of TCEP and TDCPP, respectively. 67E and 45D also showed dehalogenation ability toward 2-CE and 1,3-DCP, respectively. Addition of inorganic phosphate did not significantly influence their ability to degrade the chlorinated OPEs but markedly increased their dehalogenation ability, which was maximum at 0.2 mM of inorganic phosphate and decreased at a higher concentration. Denaturing gradient gel electrophoresis analysis showed that dominant bacteria in 67E are related to Acidovorax spp. and Sphingomonas spp. and those in 45D are Acidovorax spp., Aquabacterium spp., and Sphingomonas spp. This analysis indicated the relationship of the Sphingomonas- and Acidovorax-related bacteria with the cleavage of the phosphoester bond and dehalogenation, respectively, in both cultures. This is the first report on bacterial enrichment cultures capable of degrading both TCEP and TDCPP.

Occurrence and functions of free D-aspartate and its metabolizing enzymes.

D-Aspartate is one of a few D-amino acids that attracted attention at an early date, since it was detected in various tissues of mammals as a protein component. The occurrence of free D-aspartate in nature was recognized a little later, and raised questions about its physiological functions and metabolism. This amino acid has been gradually accepted, based on various experimental observations, to be a physiological substrate of D-aspartate oxidase, whose role had been considered enigmatic since its early discovery in the 1940s. Mammalian enzymes that serve to liberate D-aspartyl residue in proteins have been identified. One enzyme hydrolyzes peptide bond at the amino side of D-aspartyl residue in a dipeptide and another enzyme hydrolyzes that at the carbonyl side of the residue in proteins. The first pyridoxal 5'-phosphate-dependent aspartate racemase has been purified and cloned from a bivalve species. The enzyme supports the high contents of D-aspartate comparable to those of L-aspartate in the bivalve, and the enantiomers are consumed when hypoxia is imposed on the bivalve. In some yeast species, assimilation of D-aspartate has been found to depend on inducible D-aspartate oxidase, which also serves to detoxify acidic D-amino acids.

Cloning and expression of the pyridoxal 5'-phosphate-dependent aspartate racemase gene from the bivalve mollusk Scapharca broughtonii and characterization of the recombinant enzyme.

D-aspartate is present at high concentrations in the tissues of Scapharca broughtonii, and its production depends on aspartate racemase. This enzyme is the first aspartate racemase purified from animal tissues and unique in its pyridoxal 5'-phosphate (PLP)-dependence in contrast to microbial aspartate racemases thus far characterized. The enzyme activity is markedly increased in the presence of AMP and decreased in the presence of ATP. To analyze the structure-function relationship of the enzyme further, we cloned the cDNA of aspartate racemase, and then purified and characterized the recombinant enzyme expressed in Escherichia coli. The cDNA included an open reading frame of 1,017 bp encoding a protein of 338 amino acids, and the deduced amino acid sequence contained a PLP-binding motif. The sequence exhibits the highest identity (43-44%) to mammalian serine racemase, followed mainly by threonine dehydratase. These relationships are fully supported by phylogenetic analyses of the enzymes. The active recombinant aspartate racemase found in the Escherichia coli extract represented about 10% of total bacterial protein and was purified to display essentially identical physicochemical and catalytic properties with those of the native enzyme. In addition, the enzyme showed a dehydratase activity toward L-threo-3-hydroxyaspartate, similar to the mammalian serine racemase that produces pyruvate from D- and L-serine.

Physiological role of D-aspartate oxidase in the assimilation and detoxification of D-aspartate in the yeast Cryptococcus humicola.

The physiological role of D-aspartate oxidase (ChDASPO) in the yeast Cryptococcus humicola was analysed through the growth characteristics of a ChDASPO gene-disrupted strain (daspoDelta) and the expression profile of ChDASPO on various combinations of carbon and nitrogen sources. The daspoDelta strain, constructed by homologous integration of the yeast URA3 marker, grew as well as the wild-type strain on ammonium chloride, L-aspartate or D-alanine as the sole nitrogen source. In contrast, the daspoDelta strain did not grow at all on D-aspartate, not only as the sole nitrogen source but also as the sole carbon source or as the sole nitrogen and carbon source, and grew more slowly than the wild-type strain on D-glutamate as the sole nitrogen source. In the wild-type strain, the induction of ChDASPO activity strictly depended on the presence of D-aspartate and was little affected by the co-presence of ammonium chloride, but it was significantly reduced by the co-presence of both glucose and ammonium chloride, which, however, did not abolish the induction, allowing considerable expression of ChDASPO. This expression pattern was consistent with that shown by Northern blot analysis. The daspoDelta strain was more sensitive than the wild-type to the growth retardation by acidic D-amino acids, but not to that by the corresponding L-isomers or D-alanine. These results clearly show that in the yeast, DASPO plays an essential role in the assimilation of D-aspartate and acts as a detoxifying agent for D-aspartate.

Cloning and expression in Escherichia coli of the D-aspartate oxidase gene from the yeast Cryptococcus humicola and characterization of the recombinant enzyme.

The D-aspartate oxidase (DDO) from the yeast Cryptococcus humicola UJ1 (ChDDO) is highly specific to D-aspartate. The gene encoding ChDDO was cloned and expressed in Escherichia coli. Sequence analysis of the ChDDO gene showed that an open reading frame of 1,110 bp interrupted by two introns encodes a protein of 370 amino acids. The deduced amino acid sequence showed an FAD-binding motif and a peroxisomal targeting signal 1 in the N-terminal region and at the C-terminus, respectively, and also the presence of certain catalytically important amino acid residues corresponding to those catalytically important in D-amino acid oxidase (DAO). The sequence exhibited only a moderate identity to human (27.4%) and bovine (28.0%) DDOs, and a rather higher identity to yeast and fungal DAOs (30.4-33.2%). Similarly, phylogenetic analysis showed that ChDDO is more closely related to yeast and fungal DAOs than to mammalian DDOs. The gene expression was regulated at the transcriptional level and specifically induced by the presence of D-aspartate as the sole nitrogen source. ChDDO was expressed in an active form in E. coli to an approximately 5-fold greater extent than in yeast. The purified recombinant enzyme was identical to the native enzyme in physicochemical and catalytic properties.

Nucleotides modulate the activity of aspartate racemase of Scapharca broughtonii.

The activity of D-aspartate racemase purified from Scapharca broughtonii has been found to depend markedly on some nucleotides. Purine nucleoside monophosphates enhanced the enzyme activity, which was, on the contrary, lowered by purine nucleoside triphosphates and not affected by pyrimidine nucleotides. AMP produced the highest increase of seven-fold in the enzyme activity at 6 mM and a half-maximum increase at approximately 3.8 mM. ATP caused a half-maximum decrease in the activity at approximately 1.4 mM and the remaining activity was lower than 7% at saturating ATP concentrations. AMP and ATP both brought about changes in V(max) and not in K(m). Analysis of the effect of AMP and ATP suggests that each of them has its own primary binding site, which is different from the substrate-binding site. In view of these effects of the nucleotides, the roles of the racemase and D-aspartate in energy metabolism under anoxic conditions are discussed.

Purification and characterization of aspartate racemase from the bivalve mollusk Scapharca broughtonii.

High concentrations of D-aspartate occur in blood shell Scapharca broughtonii (Mollusca) tissues. We purified aspartate racemase from the foot muscle of the bivalve to electrophoretic homogeneity. The molecular mass shown by sodium dodecyl sulfate polyacrylamide gel was 39 kDa, while that shown by gel filtration ranged from 51 to 63 kDa. Pyridoxal 5'-phosphate-dependency of the enzyme was demonstrated by its absorption spectrum as well as the effects of amino-oxyacetate and other reagents on the activity and spectrum. The enzyme is highly specific to aspartate and does not racemize L-alanine, L-serine and L-glutamate. It showed the highest activity at pH 8 both in the conversion of L- to D- and D- to L-aspartate, and the optimal temperature was 25 degrees C. V(max) and K(m) values for L-aspartate were 7.39 micromolmin(-1)mg(-1) and 60.4 mM and those for D-aspartate were 22.6 micromolmin(-1)mg(-1) and 159 mM, respectively.

N-methyl-D-glutamate and N-methyl-L-glutamate in Scapharca broughtonii (Mollusca) and other invertebrates.

The presence of N-methyl-D-glutamate (NMDG) and N-methyl-L-glutamate (NMLG) has been demonstrated in the tissues of Scapharca broughtonii, which are known to contain N-methyl-D-aspartate (NMDA). To our knowledge, this is the first report on the natural occurrence of NMDG and the occurrence of NMLG in eukaryotes. These compounds were identified according to the following findings; (a) their derivatives with (+)- and (-)-l-(9-fluorenyl)ethyl chloroformate (FLEC) showed identical behaviors with those of authentic NMDG and NMLA, respectively, on high-performance liquid chromatography (HPLC), (b) the HPLC peak of NMDG disappeared when the extract, as well as the authentic compound, was treated with D-aspartate oxidase before derivatization, (c) they behaved identically with authentic compounds on thin-layer chromatography and differently from NMDA. Both or either of NMDG and NMLG were also detected in several mollusks and other animals. Concentrations of the enantiomers were comparable in the tissues of S. broughtonii and a few other species.

Isolation of the Cryptococcus humicolus URA3 gene encoding orotidine-5'-phosphate decarboxylase and its use as a selective marker for transformation.

A transformation system for a yeast, Cryptococcus humicolus, was constructed. As a selectable marker, the URA3 gene encoding orotidine-5'-phosphate decarboxylase (OMPdecase) was isolated from a C. humicolus genomic DNA library, and the equivalent cDNA was cloned. The coding region encompasses a polypeptide of 269 amino acids interrupted by two introns, which were located at the same positions as observed in the equivalent genes of some filamentous fungi. The deduced amino acid sequence showed significant homology to those of OMPdecases from other fungal species. Although no canonical TATA and CHAT sequences and polyadenylation sequence are in the flanking regions, two C + T-rich sequences are observed in the 5'-flanking region. The cDNA of the URA3 gene of C. humicolus was able to complement functionally the ura3 mutation of Saccharomyces cerevisiae. As a host, five uracil auxotrophic mutants were isolated by the selection of ethyl methanesulfonate mutagenized cells on 5-fluoroorotic acid. Three of them could be transformed to Ura+ phenotype with a linearized URA3-harboring vector using electroporation, and the best transformation frequency was 14 transformants per microg of DNA. Southern blot analysis of five independent transformants showed the integration of the vector into the host chromosomal DNA at the URA3 locus in one transformant, and also the integration at ectopic sites and the modified extrachromosomal forms in others.