Cloning, sequencing, and expression of the cellulase genes of Humicola grisea var. thermoidea

We have cloned an endoglucanase (EGI) gene and a cellobiohydrolase (CBHI) gene of Humicola grisea var. thermoidea using a portion of the Trichoderma reesei endoglucanase I gene as a probe, and determined their nucleotide sequences. The deduced amino acid sequence of EGI was 435 amino acids in length and the coding region was interrupted by an intron. The EGI lacks a hinge region and a cellulose-binding domain. The deduced amino acid sequence of CBHI was identical to the H. grisea CBHI previously reported, with the exception of three amino acids. The H. grisea EGI and CBHI show 39.8% and 37.7% identity with the T. Reesei EGI, respectively. In addition to TATA box and CAAT motifs, putative CREA binding sites were observed in the 5′ upstream regions of both genes. The cloned cellulase genes were expressed in Aspergillus oryzae and the gene products were purified. The optimal temperatures of CBHI and EGI were 60 °C and 55–60 °C, respectively. The optimal pHs of these enzymes were 5.0. CBHI and EGI had distinct substrate specificities: CBHI showed high activity toward Avicel, whereas EGI showed high activity toward carboxymethyl cellulose (CMC).     

Identification and expression of cellobiohydrolase (CBHI) gene from an endophytic fungus, Fusicoccum sp. (BCC4124) in Pichia pastoris

A gene encoding a cellobiohydrolase (CBHI) was isolated from Fusicoccum sp. (BCC4124), an endophytic fungus belongs in phylum Ascomycota, using 5' and 3' rapid amplification of cDNA end (RACE) technique. This CBHI gene contains 1395 nucleotides and encodes a 465-amino acid protein with a molecular weight of approximately 50 kDa. The deduced amino acid sequence showed significant similarity to those of other fungal CBHI belonging to family 7 of glycosyl hydrolase. Interestingly, the result from the amino acid alignment revealed that this CBHI does not contain the cellulose binding domain nor the linker region. The CBHI gene was successfully expressed in Pichia pastoris KM71. The purified recombinant CBHI has ability to hydrolyze Avicel, filter paper and 4-methylumbelliferyl beta-d-cellobioside (MUC) but not carboxymethylcellulose (CMC). It showed an optimal working condition at 40 degrees C, pH 5 with K(m) and V(max) toward MUC of 0.57 mM and 3.086 nmol/min/mg protein, respectively. The purified enzyme was stable at pH range of 3-11. The enzyme retained approximately 50% of its maximal activity after incubating at 70-90 degrees C for 30 min. Due to its stability through wide range of pH, and moderately stable at high temperature, this enzyme has potential in various biotechnology applications.     

PcMtr, an aromatic and neutral aliphatic amino acid permease of Penicillium chrysogenum

The gene encoding an aromatic and neutral aliphatic amino acid permease of Penicillium chrysogenum was cloned, functionally expressed and characterized in Saccharomyces cerevisiae M4276. The permease, designated PcMtr, is structurally and functionally homologous to Mtr of Neurospora crassa, and unrelated to the Amino Acid Permease (AAP) family which includes most amino acid permeases in fungi. Database searches of completed fungal genome sequences reveal that Mtr type permeases are not widely distributed among fungi, suggesting a specialized function.     

Asexual cephalosporin C producer Acremonium chrysogenum carries a functional mating type locus

Acremonium chrysogenum, the fungal producer of the pharmaceutically relevant beta-lactam antibiotic cephalosporin C, is classified as asexual because no direct observation of mating or meiosis has yet been reported. To assess the potential of A. chrysogenum for sexual reproduction, we screened an expressed sequence tag library from A. chrysogenum for the expression of mating type (MAT) genes, which are the key regulators of sexual reproduction. We identified two putative mating type genes that are homologues of the alpha-box domain gene, MAT1-1-1 and MAT1-1-2, encoding an HPG domain protein defined by the presence of the three invariant amino acids histidine, proline, and glycine. In addition, cDNAs encoding a putative pheromone receptor and pheromone-processing enzymes, as well as components of a pheromone response pathway, were found. Moreover, the entire A. chrysogenum MAT1-1 (AcMAT1-1) gene and regions flanking the MAT region were obtained from a genomic cosmid library, and sequence analysis revealed that in addition to AcMAT1-1-1 and AcMAT1-1-2, the AcMAT1-1 locus comprises a third mating type gene, AcMAT1-1-3, encoding a high-mobility-group domain protein. The alpha-box domain sequence of AcMAT1-1-1 was used to determine the phylogenetic relationships of A. chrysogenum to other ascomycetes. To determine the functionality of the AcMAT1-1 locus, the entire MAT locus was transferred into a MAT deletion strain of the heterothallic ascomycete Podospora anserina (the PaDeltaMAT strain). After fertilization with a P. anserina MAT1-2 (MAT(+)) strain, the corresponding transformants developed fruiting bodies with mature ascospores. Thus, the results of our functional analysis of the AcMAT1-1 locus provide strong evidence to hypothesize a sexual cycle in A. chrysogenum.     

Molecular cloning and expression of a laccase from Ganoderma lucidum, and its antioxidative properties

Laccases are multicopper-containing oxidases that catalyze the oxidation of many aromatic compounds with concomitant reduction of oxygen to water. Interest in this enzyme has arisen in many fields of industry, including detoxification, wine stabilization, paper processing, and enzymatic conversion of chemical intermediates. In this study, we cloned a laccase gene (GLlac1) from the white-rot fungus Ganoderma lucidum. The cloned gene consists of 4,357 bp, with its coding region interrupted by nine introns, and the upstream region has putative CAAT and TATA boxes as well as several metal responsive elements (MREs). We also cloned a full-length cDNA of GLlac1, which contains an uninterrupted open reading frame (ORF) of 1,560 bp coding for 520 amino acids with a putative 21-residue signal sequence. The DNA and deduced amino acid sequences of GLlac1 were similar but not identical to those of other fungal laccases. GLlac1 was released from the cells when expressed in P. pastoris, and had high laccase activity. In addition, GLlac1 conferred antioxidative protection from protein degradation, and thus may be useful in bio-medical applications.     

脱乙酰氧基头孢菌素C合成酶／羟化酶基因cefEF的基因克隆及序列分析

利用氯化苄分别从真菌顶头孢（Cephalosporium acremonium)和产黄头孢（Acremonium chrysogenum)中提取总DNA,通过PCR方法扩增脱乙酰氧基头孢菌素C合成酶／羟化酶基因cefEF,结果只能从黄头孢DNA趸扩增出cefEF基因。测序结果表明,其与已报道的基因序列只有3个碱基的差异,推断的氨基酸序列只有2个氨基酸有差异,并未涉及活性中心。同时表明,国外指所指的与该酶有关的顶头孢（Cephalosporium acremonium或Acremonium chryso-geum)对应的是国内的产黄头孢（Acremonium chrysogenum)。     

Molecular characterization of the acyl-coenzyme A:isopenicillin N acyltransferase gene (penDE) from Penicillium chrysogenum and Aspergillus nidulans and activity of recombinant enzyme in Escherichia coli.

The final step in the biosynthesis of beta-lactam antibiotics in Penicillium chrysogenum and Aspergillus nidulans involves removal of the L-alpha-aminoadipyl side chain from isopenicillin N (IPN) and exchange with a nonpolar side chain. The enzyme catalyzing this reaction, acyl-coenzyme A:isopenicillin N acyltransferase (acyltransferase), was purified from P. chrysogenum and A. nidulans. Based on NH2-terminal amino acid sequence information, the acyltransferase gene (penDE) from P. chrysogenum and A. nidulans were cloned. In both organisms, penDE was located immediately downstream from the isopenicillin N synthetase gene (pcbC) and consisted of four exons encoding an enzyme of 357 amino acids (approximately 40 kilodaltons [kDa]). The DNA coding sequences showed approximately 73% identity, while the amino acid sequences were approximately 76% identical. Noncoding DNA regions (including the region between pcbC and penDE) were not conserved. Acyltransferase activity from Escherichia coli producing the 40-kDa protein accepted either 6-aminopenicillanic acid or IPN as the substrate and made a penicillinase-sensitive antibiotic in the presence of phenylacetyl coenzyme A. Therefore, a single gene is responsible for converting IPN to penicillin G. The active form of the enzyme may result from processing of the 40-kDa monomeric precursor to a heterodimer containing subunits of 11 and 29 kDa.     

Inactivation of the lys7 gene, encoding saccharopine reductase in Penicillium chrysogenum, leads to accumulation of the secondary metabolite precursors piperideine-6-carboxylic acid and pipecolic acid from alpha-aminoadipic acid

Pipecolic acid serves as a precursor of the biosynthesis of the alkaloids slaframine and swainsonine (an antitumor agent) in some fungi. It is not known whether other fungi are able to synthesize pipecolic acid. Penicillium chrysogenum has a very active alpha-aminoadipic acid pathway that is used for the synthesis of this precursor of penicillin. The lys7 gene, encoding saccharopine reductase in P. chrysogenum, was target inactivated by the double-recombination method. Analysis of a disrupted strain (named P. chrysogenum SR1-) showed the presence of a mutant lys7 gene lacking about 1,000 bp in the 3'-end region. P. chrysogenum SR1- lacked saccharopine reductase activity, which was recovered after transformation of this mutant with the intact lys7 gene in an autonomously replicating plasmid. P. chrysogenum SR1- was a lysine auxotroph and accumulated piperideine-6-carboxylic acid. When mutant P. chrysogenum SR1- was grown with L-lysine as the sole nitrogen source and supplemented with DL-alpha-aminoadipic acid, a high level of pipecolic acid accumulated intracellularly. A comparison of strain SR1- with a lys2-defective mutant provided evidence showing that P. chrysogenum synthesizes pipecolic acid from alpha-aminoadipic acid and not from L-lysine catabolism.     

Uptake of the beta-lactam precursor alpha-aminoadipic acid in Penicillium chrysogenum is mediated by the acidic and the general amino acid permease

External addition of the beta-lactam precursor alpha-aminoadipic acid to the filamentous fungus Penicillium chrysogenum leads to an increased intracellular alpha-aminoadipic acid concentration and an increase in penicillin production. The exact route for alpha-aminoadipic acid uptake is not known, although the general amino acid and acidic amino acid permeases have been implicated in this process. Their corresponding genes, PcGAP1 and PcDIP5, of P. chrysogenum were cloned and functionally expressed in a mutant of Saccharomyces cerevisiae (M4276) in which the acidic amino acid and general amino acid permease genes (DIP5 and GAP1, respectively) are disrupted. Transport assays show that both PcGap1 and PcDip5 mediated the uptake of alpha-aminoadipic acid, although PcGap1 showed a higher affinity for alpha-aminoadipic acid than PcDip5 (K(m) values, 230 and 800 microM, respectively). Leucine strongly inhibits alpha-aminoadipic acid transport via PcGap1 but not via PcDip5. This difference was exploited to estimate the relative contribution of each transport system to the alpha-aminoadipic acid flux in beta-lactam-producing P. chrysogenum. The transport measurements demonstrate that both PcGap1 and PcDip5 contribute to the alpha-aminoadipic acid flux.     

The cephamycin biosynthetic genes pcbAB, encoding a large multidomain peptide synthetase, and pcbC of Nocardia lactamdurans are clustered together in an organization different from the same genes in Acremonium chrysogenum and Penicillium chrysogenum

A 34 kb fragment of the Nocardia lactamdurans DNA carrying the cluster of early cephamycin biosynthetic genes was cloned in lambda EMBL3 by hybridization with probes internal to the pcbAB and pcbC genes of Penicillium chrysogenum and Streptomyces griseus. The pcbAB and pcbC genes were found to be closely linked together in the genome of N. lactamdurans. The pcbAB gene of N. lactamdurans showed the same orientation as the pcbC gene, in contrast to the divergent expression of the genes in the pcbAB-pcbC cluster of P. chrysogenum and Acremonium chrysogenum. The pcbAB gene encodes a large (3649 amino acids) multidomain delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase with a deduced Mr of 404,134. This enzyme contains three repeated domains and a consensus thioesterase active-site sequence. The pcbC gene encodes a protein of 328 amino acids with a deduced Mr of 37,469, which is similar to other isopenicillin N synthases except that it lacks one of two cysteine residues conserved in all other isopenicillin N synthases. The different organization of the pcbAB-pcbC gene cluster in N. lactamadurans and Streptomyces clavuligerus relative to P. chrysogenum and A. chrysogenum is intriguing in relation to the hypothesis of horizontal transference of these genes from actinomycetes to filamentous fungi by a single transfer event.     

Conversion of Pipecolic Acid into Lysine in Penicillium chrysogenum Requires Pipecolate Oxidase and Saccharopine Reductase: Characterization of the lys7 Gene Encoding Saccharopine Reductase

Pipecolic acid is a component of several secondary metabolites in plants and fungi. This compound is useful as a precursor of nonribosomal peptides with novel pharmacological activities. In Penicillium chrysogenum pipecolic acid is converted into lysine and complements the lysine requirement of three different lysine auxotrophs with mutations in the lys1, lys2, or lys3 genes allowing a slow growth of these auxotrophs. We have isolated two P. chrysogenum mutants, named 7.2 and 10.25, that are unable to convert pipecolic acid into lysine. These mutants lacked, respectively, the pipecolate oxidase that converts pipecolic acid into piperideine-6-carboxylic acid and the saccharopine reductase that catalyzes the transformation of piperideine-6-carboxylic acid into saccharopine. The 10.25 mutant was unable to grow in Czapek medium supplemented with alpha-aminoadipic acid. A DNA fragment complementing the 10.25 mutation has been cloned; sequence analysis of the cloned gene (named lys7) revealed that it encoded a protein with high similarity to the saccharopine reductase from Neurospora crassa, Magnaporthe grisea, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. Complementation of the 10.25 mutant with the cloned gene restored saccharopine reductase activity, confirming that lys7 encodes a functional saccharopine reductase. Our data suggest that in P. chrysogenum the conversion of pipecolic acid into lysine proceeds through the transformation of pipecolic acid into piperideine-6-carboxylic acid, saccharopine, and lysine by the consecutive action of pipecolate oxidase, saccharopine reductase, and saccharopine dehydrogenase.     

Cloning, nucleotide sequence, and overexpression of the gene coding for delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B.

The structural gene coding for the delta 5-3-ketosteroid isomerase (KSI) of Pseudomonas putida biotype B has been cloned, and its entire nucleotide sequence has been determined by a dideoxynucleotide chain termination method. A 2.1-kb DNA fragment containing the ksi gene was cloned from a P. putida biotype B genomic library in lambda gt11. The open reading frame of ksi encodes 393 nucleotides, and the amino acid sequence deduced from the nucleotide sequence agrees with the directly determined amino acid sequence (K. Linden and W. F. Benisek, J. Biol. Chem. 261:6454-6460, 1986). A putative purine-rich ribosome binding site was found 8 bp upstream of the ATG start codon. Escherichia coli BL21(DE3) transformed with the pKK-KSI plasmid containing the ksi gene expressed a high level of isomerase activity when induced by isopropyl-beta-D-thiogalactopyranoside. KSI was purified to homogeneity by a simple and rapid procedure utilizing fractional precipitation and an affinity column of deoxycholate-ethylenediamine-agarose as a major chromatographic step. The molecular weight of KSI was 14,535 (calculated, 14,536) as determined by electrospray mass spectrometry. The purified KSI showed a specific activity (39,807 mumol min-1 mg-1) and a Km (60 microM) which are close to those of KSI originally obtained from P. putida biotype B.