Expression of a synthetic protein-based polymer (elastomer) gene in Aspergillus nidulans

A gene for a synthetic protein-based polymer, G-(VPGVG)₁₁₉-VPGV, coding for the EG-120mer (elastomer), was cloned into a fungal expression vector to allow constitutive expression of the polymer controlled by the gpdA (glyceraldehyde-3-phosphate dehydrogenase) promoter sequence of Aspergillus nidulans. Stable transformants of A. nidulans showed plasmid integration with varying copy number when analyzed by Southern-blot hybridization. Expression of the synthetic gene was demonstrated by Northern-blot hybridization. However, the translational efficiency for production of the polymer polypeptide was low, presumably because of certain codons in the polymer gene (CCG and GUA) that are rarely used by A. nidulans. Partial purification by reversible phase transition followed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of polymer protein in a transformant that contained multiple copies of the polymer gene. This study represents the first attempt to express a synthetic gene (with no natural analog) in a fungus. Received: 23 July 1996 / Received revision: 19 November 1996 / Accepted: 23 November 1996     

Overexpression of the Chitosanase Gene in <Emphasis Type="Italic">Fusarium solani</Emphasis> via <Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-Mediated Transformation

To overexpress the chitosanase gene (csn) in F. solani, a vector based on pCAMBIA 1300 was constructed. The csn gene, which is under control of the Aspergillus nidulans gpdA promoter and A. nidulans trpC terminator, was introduced back into the F. solani genome by Agrobacterium tumefaciens-mediated transformation, and the herbicide-resistance gene bar from Streptomyces hygroscopicus was used as the selection marker. Transformants which showed a significant increase in chitosanase production (~2.1-fold than control) were obtained. Southern blot analysis indicated that most transformants had a single-copy T-DNA integration.     

Effect of pH on the stability of Pleurotus eryngii versatile peroxidase during heterologous production in Emericella nidulans

Complementary DNA (cDNA) encoding the new versatile peroxidase from the ligninolytic basidiomycete Pleurotus eryngii has been expressed in the ascomycete Emericella nidulans. In recombinant E. nidulans cultures, the pH reached values as high as 8.3, correlating with a sharp decrease in peroxidase activity. Peroxidase was rapidly inactivated at alkaline pH, but was comparatively stable at acidic pH. The peroxidase inactivation in alkaline buffer could be reversed by adding Ca²⁺ and lowering the pH. However, reactivation did not result after incubating the enzyme in non-buffered E. nidulans cultures that reached pH 7.5. To optimize recombinant peroxidase production, the effect of controlling the pH in E. nidulans bioreactor cultures was studied. An extended growth period, and a significant increase in the recombinant peroxidase level (5.3-fold higher activity than in the bioreactor without pH control) was obtained when the pH was maintained at 6.8, showing that culture pH is an important parameter for recombinant peroxidase production.     

产甘油假丝酵母与酿酒酵母胞浆3-磷酸甘油脱氢酶基因的功能比较

胞浆3-磷酸甘油脱氢酶(GPD)是酿酒酵母细胞甘油合成过程中的关键限速酶．尽管高产甘油菌株产甘油假丝酵母基因组中编码该酶的基因CgGPD已经被克隆出来,但是具体的功能,特别是与酿酒酵母GPD1和GPD2基因的功能比较值得进一步研究．以酿酒酵母渗透压敏感型的gpd1/gpd2和gpd1突变株为宿主,分别导入CgGPD、GPD1和GPD2基因,比较分析了CgGPD、GPD1和GPD2基因在高渗透压胁迫条件下和厌氧环境中的表达调控,及其对细胞甘油合成能力的影响．研究发现,GPD1基因受到渗透压诱导表达,GPD2基因在细胞厌氧条件下起着氧化还原平衡调节作用,而CgGPD基因不仅能够在渗透压胁迫条件下通过过量快速合成甘油调节渗透压平衡,而且能够在厌氧培养环境中互补GPD2基因的缺失,使gpd1/gpd2缺失突变株能够正常生长,同时提高了突变株的甘油合成能力．结果表明,CgGPD基因在gpd1/gpd2缺失突变株中既具有GPD1基因的功能,又能发挥GPD2基因的功能．     

Isolation of the facA (acetyl-CoA synthetase) gene of Phycomyces blakesleeanus

A 5.6 kb DNA fragment from the fungus Phycomyces blakesleeanus has been cloned and sequenced. The fragment contains a gene that probably codes for the enzyme acetyl-coenzyme A synthetase (facA). The amino acid sequence deduced for the P. blakesleeanns protein is highly homologous to those of acetyl-coA-synthetases from other organisms. When placed under the control of a constitutive promoter from Aspergillus nidulans, the cloned gene complemented a facA mutation of this organism. In P. blakesleeanns, the expression of facA is induced by acetate.     

Gene cluster for biosynthesis of thermophilin 1277--a lantibiotic produced by Streptococcus thermophilus SBT1277, and heterologous expression of TepI, a novel immunity peptide

Aims: To identify genes cluster for thermophilin 1277 produced by Streptococcus thermophilus SBT1277. Methods and Results: To identify genes for thermophilin 1277 production, the chromosomal DNA region surrounding the structural gene, tepA, was sequenced using a primer‐walking method. The thermophilin 1277 biosynthesis gene locus (tep) is a 9·9‐kb region, which consists of at least ten open reading frames (ORFs) in the following order: tepAMTFEGKRI and ORF4. Homology analysis showed high similarity to genes involved in bovicin HJ50 production by Streptococcus bovis HJ50. tepI encodes a novel, small, positively charged hydrophobic peptide of 52 amino acids, which contains a putative transmembrane segment. By heterologous expression in Lactococcus lactis ssp. cremoris MG1363, the TepI‐expressing strain exhibited at least 1·3 times higher resistance to thermophilin 1277. Conclusions: Thermophilin 1277 biosynthesis genes were encoded by a 9·9‐kbp region containing at least ten ORFs. TepI is a novel immunity peptide, which protected Strep. thermophilus SBT1277 against thermophilin 1277 in addition to TepFEG, a putative ABC transporter. Significance and Impact of the Study: This is the first report regarding a lantibiotic gene cluster produced by Strep. thermophilus strain.     

Overexpressing <Emphasis Type="Italic">GLT1</Emphasis> in <Emphasis Type="Italic">gpd1</Emphasis>Δ mutant to improve the production of ethanol of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

To improve ethanol production in Saccharomyces cerevisiae, two yeast strains were constructed. In the mutant, KAM-4, the GPD1 gene, which encodes a glycerol 3-phosphate dehydrogenase of S. cerevisiae to synthesize glycerol, was deleted. The mutant KAM-12 had the GLT1 gene (encodes glutamate synthase) placed under the PGK1 promoter while harboring the GPD1 deletion. Notably, overexpression of GLT1 by the PGK1 promoter along with GPD1 deletion resulted in a 10.8% higher ethanol production and a 25.0% lower glycerol formation compared to the wild type in anaerobic fermentations. The growth rate of KAM-4 was slightly lower than that of the wild type under the exponential phase whereas KAM-12 and the wild type were indistinguishable in the biomass concentration at the end of growth period. Meanwhile, dramatic reduction of formation of acetate and pyruvic acid was observed in all the mutants compared to the wild type.     

ThePenicillium chrysogenum andAspergillus nidulans wetA developmental regulatory genes are functionally equivalent

Aspergillus nidulans andPenicillium chrysogenum are related fungi that reproduce asexually by forming multicellular conidiophores and uninucleate conidia. InA. nidulans, spore maturation is controlled by thewetA (AwetA) regulatory gene. We cloned a homologous gene (PwetA) fromP. chrysogenum to determine if spore maturation is regulated by a similar mechanism in this species. ThePwetA andAwetA genes are similar in structure and functional organization. The inferred polypeptides share 77% overall amino acid sequence similarity, with several regions having > 85% similarity. The genes also had significant, local sequence similarities in their 5 flanking regions, including conserved binding sites for the product of the regulatory geneabaA.PwetA fully complemented anA. nidulans wetA deletion mutation, demonstrating thatPwetA and its 5 regulatory sequences function normally inA. nidulans. These results indicate that the mechanisms controlling sporulation inA. nidulans andP. chrysogenum are evolutionarily conserved.     

紫色红曲霉Mp-21 MpMcrA基因的克隆与生物信息学分析

McrA为最近在构巢曲霉(Aspergillus nidulans)中发现的全局调控因子，具有调控丝状真菌生长发育和次级代谢的作用，利用生物信息学分析方法找到并克隆紫色红曲霉（Monascus purpureus）中mcrA基因，将其命名为MpMcrA。分析MpMcrA蛋白质理化性质、亲疏水性、亚细胞定位、信号肽、跨膜区域及磷酸化位点、转录因子结合位点以及蛋白质二级结构。利用ProtParam、ProtScale、PSORTII、SignalP4.1等生物信息学软件对MpMcrA进行系统分析。 结果表明，MpMcrA基因长1 356 bp，其中含有3个外显子，2个内含子，编码410个氨基酸，与构巢曲霉序列比对蛋白相似性高达64％。预测结果显示，MpMcrA属于亲水蛋白，位于细胞核可能性大，不存在跨膜区域，不属于膜蛋白；不存在剪切位点，不属于分泌蛋白；基因含有54个潜在的磷酸化位点；可能存在5个转录因子结合位点；蛋白结构大部分为无规则卷曲，整体结构较松散。对MpMcrA基因进行了生物信息学分析，得到了基因特征和分析结果。初步确定MpMcrA基因为构巢曲霉同源mcrA基因，在红曲霉中未见有报道。     

Genetic analysis of Aspergillus nidulans AmdS+ transformants

Summary To correlate the genetic background of the Aspergillus nidulans amdS deletion strain MH1277 with the integrational behaviour of transforming vectors, classical genetic methods were used to construct AmdS^- strains in which whole chromosomes had been exchanged with those of a master strain. Progeny strains were transformed to the AmdS⁺ phenotype with vector p3SR2. From Southern analysis it was concluded that transformants from all constructions contained tandemly repeated, multiple copy inserts of vector DNA as found for MH1277-derived AmdS⁺ transformants.AmdS⁺ transformants of MH1277 were analysed genetically to prove that the transformant phenotype is genome linked and that transformation by integration can take place on various chromosomes. In one case the AmdS⁺ property showed linkage to both chromosomes II and IV, due to a chromosomal translocation. Sexual analysis of two transformants with AmdS⁺ insertions on the same chromosome revealed a considerable instability of the AmdS⁺ phenotype in one of the strains upon selfing. Due to this instability no decisive answer could be given for the degree of linkage between the AmdS⁺ insertions in these transformants.     

Development of a new transformant selection system for Penicillium chrysogenum: isolation and characterization of the P. chrysogenum acetyl-coenzyme A synthetase gene (facA) and its use as a homologous selection marker

A new transformation system for the filamentous fungus Penicillium chrysogenum is described, based on the use of the homologous acetyl-coenzyme A synthetase (facA) gene as a selection marker. Acetate-non-utilizing (Fac^–) strains of P. chrysogenum were obtained by positive selection for spontaneous resistance to fluoroacetate. Among these fac mutants putative facA strains were selected for a loss of acetyl-coenzyme A (CoA) synthetase activity. The facA gene, coding for the enzyme acetyl-CoA synthetase, was isolated from a P. chrysogenum genomic library using synthetic oligonucleotides derived from conserved regions from the corresponding genes of Aspergillus nidulans and Neurospora crassa. Vector pPC2-3, comprising a genomic 6.5 kb PstI fragment, was able to complement P. chrysogenum facA strains with frequencies up to 27 transformants·g^–1 DNA. Direct selection of transformants was accomplished using acetate and low amounts (0.001%) of glucose as carbon sources. About 50% of the transformants arose by integration of pPC2-3 DNA at the homologous facA locus and 50% by integration elsewhere in the genome. Determination of the nucleotide sequence of part of the cloned fragment showed the presence of an open reading frame of 2007 nucleotides, interrupted by five putative introns. Comparison of the nucleotide and the amino acid sequence of the facA gene of P. chrysogenum with the facA gene of A. nidulans reveals similarities of 80% and 89%, respectively. The putative introns present in the P. chrysogenum facA gene appear at identical positions as those in the A. nidulans facA gene, but show no significant sequence similarity. Correspondence to: R. F. M.van Gorcom     

Highly efficient production of laccase by the basidiomycete Pycnoporus cinnabarinus

An efficient transformation and expression system was developed for the industrially relevant basidiomycete Pycnoporus cinnabarinus. This was used to transform a laccase-deficient monokaryotic strain with the homologous lac1 laccase gene placed under the regulation of its own promoter or that of the SC3 hydrophobin gene or the glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of Schizophyllum commune. SC3-driven expression resulted in a maximal laccase activity of 107 nkat ml(-1) in liquid shaken cultures. This value was about 1.4 and 1.6 times higher in the cases of the GPD and lac1 promoters, respectively. lac1-driven expression strongly increased when 25 g of ethanol liter(-1) was added to the medium. Accordingly, laccase activity increased to 1,223 nkat ml(-1). These findings agree with the fact that ethanol induces laccase gene expression in some fungi. Remarkably, lac1 mRNA accumulation and laccase activity also strongly increased in the presence of 25 g of ethanol liter(-1) when lac1 was expressed behind the SC3 or GPD promoter. In the latter case, a maximal laccase activity of 1,393 nkat ml(-1) (i.e., 360 mg liter(-1)) was obtained. Laccase production was further increased in transformants expressing lac1 behind its own promoter or that of GPD by growth in the presence of 40 g of ethanol liter(-1). In this case, maximal activities were 3,900 and 4,660 nkat ml(-1), respectively, corresponding to 1 and 1.2 g of laccase per liter and thus representing the highest laccase activities reported for recombinant fungal strains. These results suggest that P. cinnabarinus may be a host of choice for the production of other proteins as well.     

Cloning and characterisation of the cytochrome c gene of Aspergillus nidulans

The cytochrome c gene (cycA) of the filamentous fungus Aspergillus nidulans has been isolated and sequenced. The gene is present in a single copy per haploid genome and encodes a polypeptide of 112 amino acid residues. The nucleotide sequence of the A. nidulans cycA gene shows 87% identity to the DNA sequence of the Neurospora crassa cytochrome c gene, and approximately 72% identity to the sequence of the Saccharomyces cerevisiae iso-1-cytochrome c gene (CYC1). The S. cerevisiae CYC1 gene was used as a heterologous probe to isolate the homologous gene in A. nidulans. The A. nidulans cytochrome c sequence contains two small introns. One of these is highly conserved in terms of position, but the other has not been reported in any of the cytochrome c genes so far sequenced. Expression of the cycA gene is not affected by glucose repression, but has been shown to be induced approximatly tenfold in the presence of oxygen and three- to fourfold under heatshock conditions.     

Cloning, characterization of the acyl-CoA : 6-amino penicillanic acid acyltransferase gene of Aspergillus nidulans and linkage to the isopenicillin N synthase gene

Summary The penDE gene encoding acyl-CoA:6-amino penicillanic acid acyltransferase (AAT), the last enzyme of the penicillin biosynthetic pathway, has been cloned from the DNA of Aspergillus nidulans. The gene contains three introns which are located in the 5 region of the open reading frame. It encodes a protein of 357 amino acids with a molecular weight of 39 240 Da. The penDE gene of A. nidulans shows 73% similarity at the nucleotide level with the penDE gene of Penicillium chrysogenum. The A. nidulans gene was expressed in P. chrysogenum and complemented the AAT deficiency of the non-producer mutants of P. chrysogenum, npe6 and npe8. The penDE gene of A. nidulans is linked to the pcbC gene, which encodes the isopenicillin N synthase, as also occurs in P. chrysogenum. Both genes show the same orientation and are separated by an intergenic region of 822 nucleotides.     

Molecular characteristics and expression profiles of glycerol-3-phosphate dehydrogenase 1 (GPD1) gene in pig

The cytosolic activity of glycerol-3-phosphate dehydrogenase 1 (GPD1, EC 1.1.1.8) plays an important role in the synthesis of triacylglycerol and in the transport of reducing equivalents from the cytosol to mitochondria. Here we report the full-length genomic sequence of porcine GPD1 gene including promoter region. Porcine GPD1 gene contains eight exons and seven introns. Using the ImpRH, the GPD1 gene was mapped on chromosome 5. Sub-cellular localization of the pig GPD1 was localized in cytoplasm by GFP reporter gene. The full-length CDS of porcine GPD1 gene comprises 1050 nucleotides and it encodes 349 amino acids. Using the CDS sequences of 17 species, we built the phylogeny tree of GPD1 gene. We investigated the expression level of the gene in 13 different tissues and time course from birth to postnatal day 28 in longissinus doris muscle (LD) and in cerebrum. The result shows that porcine GPD1 gene is expressed in almost all tissues we tested but its levels of expression varies widely over 2 orders of magnitude. LD and the cerebrum have similar expression pattern that is at a low level at birth and increasing with aging to the highest level at postnatal day 8 in LD and postnatal day 14 in cerebrum. But weaning decreased the expression level of the GPD1 gene. This may partially explains the effects of weaning on energy metabolism.     

Gene inactivation in the plant pathogen Glomerella cingulata: three strategies for the disruption of the pectin lyase gene pnIA

The feasibility of performing routine transformation-mediated mutagenesis in Glomerella cingulata was analysed by adopting three one-step gene disruption strategies targeted at the pectin lyase gene pnIA. The efficiencies of disruption following transformation with gene replacement- or gene truncation-disruption vectors were compared. To effect replacement-disruption, G. cingulata was transformed with a vector carrying DNA from the pnlA locus in which the majority of the coding sequence had been replaced by the gene for hygromycin B resistance. Two of the five transformants investigated contained an inactivated pnlA gene (pnlA ^– );both also contained ectopically integrated vector sequences. The efficacy of gene disruption by transformation with two gene truncation-disruption vectors was also assessed. Both vectors carried a 5and 3truncated copy of the pnlA coding sequence, adjacent to the gene for hygromycin B resistance. The promoter sequences controlling the selectable marker differed in the two vectors. In one vector the homologous G. cingulata gpdA promoter controlled hygromycin B phosphotransferase expression (homologous truncation vector), whereas in the second vector promoter elements were from the Aspergillus nidulans gpdA gene (heterologous truncation vector). Following transformation with the homologous truncation vector, nine transformants were analysed by Southern hybridisation; no transformants contained a disrupted pnlA gene. Of nineteen heterologous truncation vector transformants, three contained a disrupted pnlA gene; Southern analysis revealed single integrations of vector sequence at pnlA in two of these transformants. pnlA mRNA was not detected by Northern hybridisation in pnlA-transformants. pnlA-transformants failed to produce a PNLA protein with a pI identical to one normally detected in wild-type isolates by silver and activity staining of isoelectric focussing gels. Pathogenesis on Capsicum and apple was unaffected by disruption of the pnlA gene, indicating that the corresponding gene product, PNLA, is not essential for pathogenicity. Gene disruption is a feasible method for selectively mutating defined loci in G. cingulata for functional analysis of the corresponding gene products.