Peroxisomal amine oxidase of Hansenula polymorpha does not require its SRL-containing C-terminal sequence for targeting

Amine oxidase (AMO) is a peroxisomal matrix protein of Hansenula polymorpha, which is induced during growth of the yeast in media containing primary amines as a sole nitrogen source. The deduced amino acid sequence of the protein contains an SRL sequence at nine amino acids from the C-terminus. In this study, we have examined the possible role of the SRL motif in sorting of AMO to peroxisomes by mutating the corresponding gene sequence. For this purpose, we have developed a DNA construct that is specifically integrated into the AMO locus of the H. polymorpha genome, placing the mutant gene under the control of the endogenous AMO promoter and eliminating expression of the wild-type gene. Analysis of a stable transformant, containing the desired gene configuration, showed that mutation of the C-terminal sequence neither interfered with correct targeting of the protein into the peroxisome nor displayed significant effects on its activity. From this, it was concluded that the SRL-containing C-terminus is not essential for peroxisomal targeting of AMO in H. polymorpha.     

Cloning and characterization of the Hansenula polymorpha PEP4 gene encoding proteinase A

The Hansenula polymorpha PEP4 gene encoding proteinase A was cloned by Southern blot hybridization using the Saccharomyces cerevisiae PEP4 gene as probe and characterized by gene disruption and overexpression. Nucleotide sequence analysis revealed an open reading frame (ORF) of 1239 nucleotides corresponding to a polypeptide of 413 amino acids, sharing about 67.2% sequence similarity with that of S. cerevisiae proteinase A. That the cloned H. polymorpha PEP4 gene encodes proteinase A was supported by a gene disruption experiment, which showed that the H. polymorpha pep4 mutant strain showed significantly reduced level of carboxypeptidase Y activity when assayed with an artificial substrate. When the PEP4 gene is overproduced in pep4 mutant strain, mature proteinase A could be found in the growth medium. N-terminal amino acid sequencing of extracellular proteinase A revealed the presence of a putative propeptide of 55 amino acids ending with a dibasic peptide (Lys-Arg), probably processed by Kex2p-like endopeptidase of H. polymorpha. The nucleotide sequence of the H. polymorpha PEP4 gene has been submitted to GenBank under Accession No. U67173.     

Characterization of the Hansenula polymorpha CPY gene encoding carboxypeptidase Y

We have isolated the Hansenula polymorpha CPY gene encoding carboxypeptidase Y (Hp-CPY). The deduced amino acid sequence revealed that Hp-CPY consists of 541 amino acids and has a calculated Mr of 60,793. The protein is highly similar to Saccharomyces cerevisiae CPY (61.8% identity). At the N-terminus of Hp-CPY signals for the entry into the secretory pathway and subsequent sorting to the vacuole were identified. Immunocytochemically, using monospecific antibodies raised against Hp-CPY, the protein was localized to the vacuole. On Western blots, a diffuse protein band was observed in extracts of H. polymorpha cells, suggesting that the protein is glycosylated. This was confirmed by endoglycosidase H treatment, which resulted in a strong reduction of the apparent Mr of the protein. We have investigated the effect of CPY deletion on the degradation of peroxisomes, an autophagous process that occurs when the organelles become redundant for growth. In deltacpy cells peroxisomal proteins were degraded in the vacuole as efficiently as in wild-type H. polymorpha cells, indicating that CPY is not a major proteinase in this pathway.     

Cloning and characterization of the Hansenula polymorpha homologue of the Saccharomyces cerevisiae MNN9 gene

A gene homologous to Saccharomyces cerevisiae MNN9 has been cloned and characterized in the methylotrophic yeast Hansenula polymorpha. This gene was cloned from a H. polymorpha genomic DNA library using the S. cerevisiae MNN9 gene as a probe. The H. polymorpha MNN9 homologue (HpMNN9) contained a 1062 bp open reading frame encoding a predicted protein of 354 amino acids. The deduced amino acid sequence showed 58% and 51% identity, respectively, with the S. cerevisiae and Candida albicans Mnn9 proteins. Disruption of HpMNN9 leads to phenotypic effects suggestive of cell wall defects, including detergent sensitivity and hygromycin B sensitivity. The hygromycin B sensitivity of S. cerevisiae mnn9 null mutant was complemented in the presence of the HpMNN9 gene. The DNA sequence of the H. polymorpha homologue has been submitted to GenBank with the Accession No. AF264786.     

Expression of the alpha-galactosidase from Cyamopsis tetragonoloba (guar) by Hansenula polymorpha.

The methylotrophic yeast Hansenula polymorpha, a host organism for the production of heterologous proteins, has been applied to produce the alpha-galactosidase from the plant Cyamopsis tetragonoloba (guar). The yeast/Escherichia coli shuttle expression vector used is based on the origin of replication of the endogenous 2 microns plasmid of Saccharomyces cerevisiae and the LEU2 gene of S. cerevisiae for selection in H. polymorpha. In the expression vector, the alpha-galactosidase is controlled by the methanol-regulated promoter from the methanol oxidase gene, MOX, of H. polymorpha. The signal sequence of SUC2 (invertase) from the yeast S. cerevisiae, was used to ensure secretion of the alpha-galactosidase enzyme. After transformation and stabilization, the expression vector was stably integrated in the genome. The active alpha-galactosidase enzyme was efficiently secreted (greater than 85%) and after methanol induction, the expression level was 42 mg/l. Amino-terminal sequencing of the purified alpha-galactosidase enzyme synthesized by H. polymorpha showed that the S. cerevisiae invertase signal sequence was correctly processed by H. polymorpha. The secreted alpha-galactosidase was glycosylated and had a sugar content of 9.5%. The specific activity of the alpha-galactosidase produced by H. polymorpha was 38 U mg-1 compared to 100 U mg-1 for the guar alpha-galactosidase. Deglycosylation of the H. polymorpha alpha-galactosidase restored the specific activity completely.     

Molecular characterization of the PEX5 gene encoding peroxisomal targeting signal 1 receptor from the methylotrophic yeast Pichia methanolica

In this study, we describe the molecular characterization of the PEX5 gene encoding the peroxisomal targeting signal 1 (PTS1) receptor from the methylotrophic yeast Pichia methanolica. The P. methanolica PEX5 (PmPEX5) gene contains a open reading frame corresponding to a gene product of 646 amino acid residues, and its deduced amino acid sequence shows a high similarity to those of Pex5ps from other methylotrophic yeasts. Like other Pex5ps, the PmPex5p possesses seven repeats of the TPR motif in the C-terminal region and three WXXXF/Y motifs. A strain with the disrupted PEX5 gene (pex5Delta) lost its ability to grow on peroxisome-inducible carbon sources, methanol and oleate, but grew normally on glucose and glycerol. Disruption of PmPEX5 caused a drastic decrease in peroxisomal enzyme activities and mislocalization of GFP-PTS1 and some peroxisomal methanol-metabolizing enzymes in the cytosol. Expression of the PmPEX5 gene was regulated by carbon sources, and it was strongly expressed by peroxisome-inducible carbon sources, especially methanol. Taken together, these findings show that PmPex5p has an essential physiological role in peroxisomal metabolism of P. methanolica, including methanol metabolism, and in peroxisomal localization and activation of methanol-metabolizing enzymes, e.g. AOD isozymes, DHAS and CTA.     

Cloning and sequencing of the chromosomal DNA and cDNA encoding the mitochondrial citrate synthase of Aspergillus niger WU-2223L

The complementary DNA (cDNA) and chromosomal DNA encoding the citrate synthase (EC 4.1.3.7) gene (cit1) of Aspergillus niger WU-2223L, a citric acid-producing strain, were cloned. Synthetic oligonucleotide primers were designed according to the amino acid sequences of already known eukaryotic citrate synthases and the codon bias of A. niger genes. The 920-bp DNA fragment was amplified by polymerase chain reaction with these primers using chromosomal DNA of WU-2223L as a template, and was employed to screen a cDNA library of A. niger. One full-length cDNA clone was isolated and sequenced, within which an ORF of 1425 by encoding a protein of 475 as with a molecular weight of 52,153 Da was found. Its N-terminal region contains a typical mitochondrial-targeting motif. The predicted as sequence was 82, 68, and 65% homologous with the mitochondrial citrate synthases of Neurospora crassa, Saccharomyces cerevisiae, and pig, respectively, but it showed lower homology to bacterial citrate synthases. The full-length cDNA clone was used to screen a chromosomal library of A. niger WU-2223L, and a 7.5 kb-SalI fragment containing the corresponding chromosomal gene was isolated. Comparison of the chromosomal and cDNA sequences revealed that the cit1 gene is interrupted by six introns. In the chromosomal DNA, upstream of the coding region, a CT-rich region, but not the TATAAA or CAAT motifs, was found. Escherichia coli MOB150, a citrate synthase-deficient mutant showing a glutamate-requiring phenotype, was transformed with the plasmid pKAC-35S, which is the expression vector pKK223-3 containing the cDNA fragment encoding a putative mature protein of A. niger citrate synthase. The transformant harboring pKAC-35S showed citrate synthase activity and a glutamate-nonrequiring phenotype.     

Analysis of the THR4 region on chromosome III of the yeast Saccharomyces cerevisiae

The gene encoding threonine synthase (THR4) from the yeast Saccharomyces cerevisiae was cloned by complementation of a thr4 mutant. This gene was also found on a lambda clone (5239) consisting of a fragment of chromosome III inserted in the vector lambdaMG3. The THR4 gene encodes a protein of 514 amino acids (M.W. 58 kDa), which has extensive homologies with E. coli threonine synthase (thrC) and B subtilis threonine synthase. The 5' flanking region of the gene contains three regulatory sequences [TGACT(C)] for the general amino acid control (GCN). About 130 bp downstream of the THR4 gene another large open reading frame (563 amino acids) is found in the opposite orientation. This may imply that this open reading frame, called CTR86, shares a terminator region with THR4. The function of the protein encoded by CTR86 is not yet clear, but the fact that the upstream region contains a GCN4 responsive site suggests that the gene product may also be involved in amino acid biosynthesis.     

Structural comparison of the Pichia pastoris alcohol oxidase genes

In methylotrophic yeasts, alcohol oxidase is the first enzyme in the methanol-utilization pathway. The genome of one such yeast, Pichia pastoris, contains two alcohol oxidase genes, AOX1 and AOX2. Sequence analysis indicated that each gene encodes a similar protein of 663 amino acids. The protein-coding regions of the genes were 92% and 97% homologous at the nucleotide and predicted amino acid sequence levels, respectively. In contrast to homology observed within the protein-coding portions of the AOX genes, no homology was found in either the 5′ or 3′ non-coding regions. Although alcohol oxidase is found in peroxisomes of P. pastoris, the AOX amino acid sequences did not contain a peptide sequence similar to the peroxisomal transport sequence found at the C-terminus of some peroxisomally located proteins in higher eukaryotes.     

Molecular characterization of the Hansenula polymorpha FLD1 gene encoding formaldehyde dehydrogenase.

Glutathione-dependent formaldehyde dehydrogenase (FLD) is a key enzyme required forthe catabolism of methanol as a carbon source and certain primary amines, such as methylamine as nitrogen sources in methylotrophic yeasts. Here we describe the molecular characterization of the FLD1 gene from the yeast Hansenula polymorpha. Unlike the recently described Pichia pastoris homologue, the H. polymorpha gene does not contain an intron. The predicted FLD1 product (Fld1p) is a protein of 380 amino acids (ca. 41 kDa) with 82% identity to P. pastoris Fld1p, 76% identity to the FLD protein sequence from n-alkane-assimilating yeast Candida maltosa and 63-64% identity to dehydrogenase class III enzymes from humans and other higher eukaryotes. The expression of FLD1 is strictly regulated and can be controlled at two expression levels by manipulation of the growth conditions. The gene is strongly induced under methylotrophic growth conditions; moderate expression is obtained under conditions in which a primary amine, e.g. methylamine, is used as nitrogen source. These properties render the FLD1 promoter of high interest for heterologous gene expression. The availability of the H. polymorpha FLD1 promoter provides an attractive alternative for expression of foreign genes besides the commonly used alcohol oxidase promoter.     

Subcellular localization of fructosyl amino acid oxidases in peroxisomes of Aspergillus terreus and Penicillium janthinellum

Fructosyl amino acid oxidase (FAOD) is the enzyme catalyzing the oxidative deglycation of Amadori compounds, such as fructosyl amino acids, yielding the corresponding amino acids, glucosone, and H(2)O(2). In a previous report, we determined the primary structures of cDNAs coding for FAODs from two fungal strains Aspergillus terreus AP1 and Penicillium janthinellum and we found that both fungal FAODs included the putative peroxisome targeting signal 1 (PTS1) at the carboxyl terminal (Yoshida, N. et al., Eur. J. Biochem., 242, 499-505, 1996). In this study, we determined the intracellular localization of FAODs in these two fungi. Subcellular fractionation experiments and immuno-electronmicroscopic observations, together with the previous findings indicated that the FAODs were localized in peroxisomes of A. terreus AP1 and P. janthinellum. These FAODs were also found to belong to a new member of "peroxisomal sarcosine oxidase family protein" in eucaryotic cells.     

Short-chain prenyl diphosphate synthase that condenses isopentenyl diphosphate with dimethylallyl diphosphate in ispA null Escherichia coli strain lacking farnesyl diphosphate synthase

A short-chain prenyl diphosphate synthase in an Escherichia coli mutant that lacked the gene coding for farnesyl diphosphate synthase, ispA, was separated from other prenyl diphosphate synthases by DEAE-Toyopearl column chromatography. The purified enzyme catalyzed the condensation of isopentenyl diphosphate with dimethylallyl diphosphate to form farnesyl diphosphate and geranylgeranyl diphosphate.     

A 12·8 kb segment,on the right arm of chromosome II from Saccharomyces cerevisiae including part of the DUR1, 2 gene,contains five putative new genes

A 12 820 bp fragment from the right arm of chromosome II of Saccharomyces cerevisiae was sequenced and analysed. This fragment contains six non-overlapping long open reading frames (ORFs) designated from the centromere- to the telomere-proximal ends as: YBR1441, 1443, 1444, 1445, 1446 and 1448. YBR1441 encodes a polypeptide of 845 amino acids which shares a long consensus domain with products of S. cerevisiae MCM2, MCM3, CDC46 and Schizosaccharomyces pombe cdc21⁺ genes. These genes are involved in DNA replication. YBR1445 encodes a polypeptide of 404 amino acids which has strong similarity with the S. cerevisiae KRE2/MNT1, YUR1, KTR1 gene products. The KRE2/MNT1 protein is an α-1,2-mannosyltransferase. The product of YBR1444, which encodes a protein of 375 amino acids, presents a lipase signature sequence and a peroxisomal targeting signal. YBR1448, whose sequence extends further on the telomere-proximal end of the fragment, is identical to the 3′ end of the DUR1,2 gene encoding urea amidolyase. The two ORFs, YBR1443 and YBR1446, exhibit no significant similarity with any known gene.     

昆明假单胞菌HL22-2海藻糖合酶基因的克隆、表达及酶学性质研究

采用热不对称交错聚合酶链式反应（Tail-PCR）克隆云南磷矿来源昆明假单胞菌（Pseudomonas kunmingensis）HL22-2的海藻糖合酶（TreS）基因HL22-2TreS,将该基因与表达载体pETM3C连接后在大肠杆菌（Escherichia coli）BL21（DE3）pLysS中进行异源表达,通过Ni-NTA柱纯化重组酶HL22-2TreS,并对其酶学特性进行分析。结果表明,HL22-2TreS基因全长3 336 bp,编码1 111个氨基酸,氨基酸序列与Genbank数据库中相关的海藻糖合酶具有极高的相似性。重组酶HL22-2TreS的分子质量约126 kDa,该酶的最适反应温度和pH值分别为40 ℃和7.0,在温度20～50 ℃及pH值6.0～9.0条件下比较稳定,Cu2+、Hg2+、Ba2+及Al3+对海藻糖合酶的活力有强烈的抑制效果。HL22-2TreS基因对麦芽糖和海藻糖的米氏常数（Km）分别为20.6 mmol/L和87.5 mmol/L,对麦芽糖具有更高的亲和性,更容易将麦芽糖转化成海藻糖。     

Catalytic triad of intracellular poly(3-hydroxybutyrate) depolymerase (PhaZ1) in Ralstonia eutropha H16

The amino acid sequence of an intracellular poly[D(-)-3-hydroxybutyrate] (PHB) depolymerase (PhaZ1) from Ralstonia eutropha H16 was compared with the sequences of various proteins using the BLAST search. It showed a number of matches including with intracellular PHB depolymerases, conserved hypothetical proteins, and PHB synthases. From an alignment of these proteins, we constructed nine mutants: C87A, S118A, H120Q, C183A, C183S, D355A, D356A, C370A, and H388Q. The C183A, D355A, and H388Q mutants lost their activities, but C183S and the other mutants did not. C183, D355, and H388 in PhaZ1 were positioned similarly to the amino acids of the catalytic triad of PHB synthase. These results indicated that C183, D355, and H388 make up the catalytic triad of PhaZ1.     

褐藻胶裂解酶的基因克隆及表达条件优化

采用大肠杆菌（Escherichia coli）表达海洋弧菌（Vibrio sp.）X511的褐藻胶裂解酶基因,以实现对该酶的大量制备及酶学性质研究。通过序列分析,预测该酶的前20个氨基酸为信号肽,去除信号肽后的蛋白质分子质量约为30 254.28 Da,等电点8.75,分子式为C1368H2100N364O401S6 ,对应的基因序列命名为alg1987。按如下方案构建重组菌：设计特异性核酸引物,PCR扩增得到alg1987;将重组质粒pET-30(a)-alg1987导入大肠杆菌Trans5α进行测序;提取阳性pET-30(a)-alg1987质粒导入大肠杆菌BL21,利用异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达。十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）检测诱导后的重组菌胞液上清,发现异源表达的褐藻胶裂解酶分子质量大小与预测值相近。对重组菌中的褐藻胶裂解酶进行表达条件优化,结果显示,最适参数为诱导温度16 ℃,诱导时间16 h,诱导剂IPTG浓度0.8 mmol/L。     

Cloning and sequence analysis of the estA gene encoding enzyme for producing (R)-beta-acetylmercaptoisobutyric acid from Pseudomonas aeruginosa 1001

The estA gene encoding the enzyme that catalyzes the production of (R)-beta-acetylmercaptoisobutyric acid from (R,S)-ester from Pseudomonas aeruginosa 1001, was cloned in Escherichia coli and its nucleotide sequence was determined, revealing the presumed open reading frame encoding a polypeptide of 316 amino acid residues (948 nucleotides). The overall A + T and C + G compositions were 32.59% and 67.41%, respectively. The amino acid sequence of the estA gene product showed a significant similarity with that of the triacylglycerol lipase from Psychrobacter immobilis (38% identity), triacylglycerol lipase from Moraxella sp. (36% identity), and two forms of carboxyl esterases from Acinetobacter calcoaceticus (17% and 17% identities). The deduced amino acid sequences have a pentapeptide consensus sequence, G-X-S-X-G, having an active serine residue, and another active site, dipeptides H-G, located at 70-100 amino acids upstream of the G-X-S-X-G consensus sequence.