High frequency one-step gene replacement in Trichoderma reesei. II. Effects of deletions of individual cellulase genes

Four cellulase genes of Trichoderma reesei, cbh1, cbh2, egl1 and egl2, have been replaced by the amdS marker gene. When linear DNA fragments and flanking regions of the corresponding cellulase locus of more than 1 kb were used, the replacement frequencies were high, ranging from 32 to 52%. Deletion of the major cellobiohydrolase 1 gene led to a 2-fold increase in the production of cellobiohydrolase II; however, replacement of the cbh2 gene did not affect the final cellulase levels and deletion of egl1 or egl2, slightly increased production of both cellobiohydrolases. Based on our results, endoglucanase II accounts for most of the endoglucanase activity produced by the hypercellulolytic host strain. Furthermore, loss of the egl2, gene causes a significant drop in the filter paper-hydrolysing activity, indicating that endoglucanase II has an important role in the total hydrolysis of cellulose.     

Construction and analysis of recombinant glucanolytic brewer's yeast strains

Summary Four different types of endo--1,4-glucanase active bottom-fermenting brewer's yeast strains were constructed using recombinant DNA technology. To study the effects of different promoters, copy numbers and integration sites, the egl1 gene of the filamentous fungus Trichoderma reesei was inserted between the promoter and terminator regions of either the PGK1 or ADH1 gene of yeast. The egl1 gene was transferred to the industrial brewer's yeast on a multicopy plasmid or alternatively integrated into the LEU2, PGK1 or ADH1 locus of the yeast. Integration into the PGK1 or ADH1 locus did not affect the brewing properties of the yeast or the quality of the finished beer. Integration into the LEU2 locus, however, decreased the metabolic activity of yeast and prolonged fermentation was needed. In pilot brewing conditions the PGK1 promoter was stronger than that of ADH1. Even a single copy of the egl1 gene in the PGK1 integrant strains gave rise to sufficient enzyme activity for the hydrolysis even of unusually high total amounts of -glucans in worts. Offprint requests to: M.-L. Suihko     

High frequency one-step gene replacement in Trichoderma reesei. I. Endoglucanase I overproduction

The chromosomal cellobiohydrolase 1 locus (cbh1) of the biotechnologically important filamentous fungus Trichoderma reesei was replaced in a single-step procedure by an expression cassette containing an endoglucanase I cDNA (egl1) under control of the cbh1 promoter. CBHI protein was missing from 37–63% of the transformants, showing that targeting of the linear expression cassette to the cbh1 locus was efficient. Studies of expression of the intact cbh1-egl1 cassette at the cbh1 locus revealed that egl1 cDNA is expressed from the cbh1 promoter as efficiently as cbh1 itself. Furthermore, a strain carrying two copies of the cbh1-egl1 expression cassette produced twice as much EG I as the amount of CBHI, the major cellulase protein, produced by the host strain. The level of egl1-specific mRNA in the single-copy transformant was about 10-fold higher than that found in the non transformed host strain, indicating that the cbh1 promoter is about 10 times stronger than the egl1 promoter. The 10-fold increase in the secreted EG I protein, measured with an enzyme-linked immunosorbent assay (ELISA), correlated well with the increase in egl1-specific mRNA.     

High frequency one-step gene replacement in Trichoderma reesei. II. Effects of deletions of individual cellulase genes

Four cellulase genes of Trichoderma reesei, cbh1, cbh2, egl1 and egl2, have been replaced by the amdS marker gene. When linear DNA fragments and flanking regions of the corresponding cellulase locus of more than 1 kb were used, the replacement frequencies were high, ranging from 32 to 52%. Deletion of the major cellobiohydrolase 1 gene led to a 2-fold increase in the production of cellobiohydrolase II; however, replacement of the cbh2 gene did not affect the final cellulase levels and deletion of egl1 or egl2, slightly increased production of both cellobiohydrolases. Based on our results, endoglucanase II accounts for most of the endoglucanase activity produced by the hypercellulolytic host strain. Furthermore, loss of the egl2, gene causes a significant drop in the filter paper-hydrolysing activity, indicating that endoglucanase II has an important role in the total hydrolysis of cellulose.     

Regulation of expression of a cDNA from barley roots encoding a high affinity sulphate transporter

A cDNA encoding a high-affinity sulphate transporter has been isolated from barley by complementation of a yeast mutant. The cDNA, designated HVST1, encodes a polypeptide of 660 amino acids (M_r = 72 550), which is predicted to have 12 membrane-spanning domains and has extensive sequence homology with other identified eukaryotic sulphate transporters. The K_m for sulphate was 6.9 µM when the HVST1 cDNA was expressed in a yeast mutant deficient in the gene encoding for the yeast SUL1 sulphate transporter. The strong pH-dependency of sulphate uptake when HVST1 was expressed heterologously in yeast suggests that the HVST1 polypeptide is a proton/sulphate co-transporter. The gene encoding HVST1 is expressed specifically in root tissues and the abundance of the mRNA is strongly influenced by sulphur nutrition. During sulphur-starvation of barley, the abundance of mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, both increase. Upon re-supply of sulphate, the abundance of the mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, decrease rapidly, concomitant with rises in tissue sulphate, cysteine and glutathione contents. Addition of the cysteine precursor, O-acetylserine, to plants grown with adequate sulphur supply, leads to increases in sulphate transporter mRNA, sulphate uptake rates and tissue contents of glutathione and cysteine. It is suggested, that whilst sulphate, cysteine and glutathione may be candidates for negative metabolic regulators of sulphate transporter gene expression, this regulation may be overridden by O-acetylserine acting as a positive regulator.     

Isolation and Identification of Novel Terpene Lactones from Quince Fruit (Cydonia oblonga Mill., Marmelo)

A glucoamylase gene has been cloned from a Rhizopus genomic DNA library using synthetic oligonucleotides corresponding to the amino acid sequence of the glucoamylase. Since this glucoamylase gene was not expressed in yeast cells, we have cloned a glucoamylase gene from a cDNA library prepared from Rhizopus mRNA. Sequence analysis of both glucoamylase genes revealed that the genomic gene contained 4 intervening sequences and the cDNA gene lacked 145 nucleotides corresponding to the N-terminal region. The glucoamylase consists of 604 amino acids including a putative signal peptide and its molecular weight was calculated to be 65,000. The glucoamylase gene to be expressed in yeast cells was constructed by recombination of both genes. The yeast cells containing this constructed glucoamylase gene secreted the glucoamylase into the culture fluid and grew at almost the normal rate on a medium containing starch as the sole carbon source.     

Functional complementation of a yeast vesicular transport mutation ypt1-1 by a Brassica napus cDNA clone encoding a small GTP-binding protein

A cDNA clone (bra) encoding a small GTP-binding protein was isolated from Brassica napus by screening a root cDNA library with a degenerate oligonucleotide probe that corresponds to a highly conserved GTP-binding domain of the Ras superfamily. Sequence analysis shows that the clone contains an open reading frame of 219 amino acid residues with the estimated molecular mass of 24379 Da and this coding region contains all the conserved motifs of the Ras superfamily. The deduced amino acid sequence of the bra gene is most closely related to the Ypt/Rab family that functions in the vesicular transport (46% and 47% amino acid identity to the yeast Ypt1 and to the human Rab1, respectively) and is more distantly related to the other Ras-related families. The protein encoded by the bra gene, when expressed in Escherichia coli, shows the ability to bind GTP. Furthermore, when the bra gene is introduced into Saccharomyces cerevisiae under the regulation of the yeast GAL1 promoter, the gene can complement the temperature-sensitive yeast mutation ypt1-1 that has defects in vesicular transport function. The amino acid sequence similarity and the functional complementation of the yeast mutation suggest that this gene is likely to be involved in the vesicular transport in plants. Genomic Southern analysis shows that this gene is a member of a small gene family in Brassica napus.     

白番红花CrCBF1基因的克隆及功能分析

该研究选取新疆地区耐寒植物白番红花为研究材料,采用RT-PCR方法,从白番红花中克隆得到白番红花CBF1的全长cDNA序列,命名为CrCBF1(GenBank登录号MF681787)。结果表明,CrCBF1基因完整开放阅读框ORF长642bp,编码213个氨基酸,分子量为23.8kD,理论等电点为4.99,具有CBF家族典型的AP2保守结构域;亚细胞定位分析显示,CrCBF1基因编码的蛋白定位于细胞核;酵母自激活分析显示,CrCBF1转录因子具有转录激活活性;酵母功能验证分析显示,过表达CrCBF1基因可以明显提高酵母的抗寒性。     

High frequency one-step gene replacement in Trichoderma reesei. I. Endoglucanase I overproduction

The chromosomal cellobiohydrolase 1 locus (cbh1) of the biotechnologically important filamentous fungus Trichoderma reesei was replaced in a single-step procedure by an expression cassette containing an endoglucanase I cDNA (egl1) under control of the cbh1 promoter. CBHI protein was missing from 37–63% of the transformants, showing that targeting of the linear expression cassette to the cbh1 locus was efficient. Studies of expression of the intact cbh1-egl1 cassette at the cbh1 locus revealed that egl1 cDNA is expressed from the cbh1 promoter as efficiently as cbh1 itself. Furthermore, a strain carrying two copies of the cbh1-egl1 expression cassette produced twice as much EG I as the amount of CBHI, the major cellulase protein, produced by the host strain. The level of egl1-specific mRNA in the single-copy transformant was about 10-fold higher than that found in the non transformed host strain, indicating that the cbh1 promoter is about 10 times stronger than the egl1 promoter. The 10-fold increase in the secreted EG I protein, measured with an enzyme-linked immunosorbent assay (ELISA), correlated well with the increase in egl1-specific mRNA.     

Expression of fungal thermotolerant endo-1,4-β-glucanase in transgenic barley seeds during germination

The malting quality of two barley cultivars, Kymppi and Golden Promise, was modified to better meet the requirements of the brewing process. The egl1 gene, coding for fungal thermotolerant endo-1,4--glucanase (EGI, cellulase), was transferred to the cultivars using particle bombardment, and transgenic plants were regenerated on bialaphos selection. Integration of the egl1 gene was confirmed by Southern blot hybridization. The transgenic seeds were screened for the expression of the heterologous EGI. Under the high-pI -amylase promoter, the egl1 gene was expressed during germination. The heterologous enzyme was thermotolerant at 65 °C for 2 h, thus being suitable for mashing conditions. The amount of heterologous EGI produced by the seeds (ca. 0.025% of soluble seed protein), has been shown to be sufficient to reduce wort viscosity by decreasing the soluble -glucan content. A decrease in the soluble -glucan content in the wort improves the filtration rate of beer.     

A proline-rich protein, verprolin, involved in cytoskeletal organization and cellular growth in the yeast Saccharomyces cerevisiae

A gene (VRP1) encoding a novel proline-rich protein (verprolin) has been isolated from the yeast Saccharomyces cerevisiae as a result of its hybridization to a chick vinculin cDNA probe. The deduced protein sequence contains 24% proline residues present as proline-rich motifs throughout the verprolin sequence. Several of these motifs resemble recently identified sequences shown to bind Src homology 3 (SH3) domains in vitro. Replacement of the wild-type VRP1 allele with a mutant allele results in strains that grow slower than wild-type strains and are temperature sensitive. The vrp1 mutants are impaired in both cell shape and size and display aberrant chitin and actin localization. We propose that verprolin is involved in the maintenance of the yeast actin cytoskeleton, through interactions with other proteins, possibly containing SH3 domains.     

Isolation and characterization of OsDMC1, the rice homologue of the yeast DMC1 gene essential for meiosis

Yeast DMC1 is a meiosis-specific gene required for homologous chromosome pairing in meiosis. Using degenerate primers designed according to amino acid motifs conserved in yeast Dmc1 and Arabidopsis AtDmc1, we obtained full-length cDNA of a rice homologue of the DMC1 gene (OsDMC1) by RT-PCR and rapid amplification of cDNA ends (RACEs). OsDmc1 exhibited 53% amino acid sequence identity to yeast Dmc1 and 81% to AtDmc1. OsDMC1 was expressed at high-levels in reproductive organs, low-levels in roots, and undetectable levels in leaves and seedlings. Southern blot analyses revealed that OsDMC1 is one of two DMC1 homologues present in rice. Received: 18 December 2000 / Accepted: 22 December 2000     

Cloning of the α-Amylase cDNA of Aspergillus shirousamii and Its Expression in Saccharomyces cerevisiae

α-Amylase cDNA was cloned and sequenced from Aspergillus shirousamii RIB2504. The putative protein deduced from the cDNA open reading frame (ORF) consisted of 499 amino acids with a molecular weight of 55,000. The amino acid sequence was identical to that of the ORF of the Taka-amylase A gene of Aspergillus oryzae, while the nucleotide sequence was different at two and six positions in the cDNA ORF and 3? non-coding regions, respectively, so far determined. The α-amylase cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast ADH1 promoter using a YEp-type plasmid, pYcDE1. The cDNA of glucoamylase, which was previously cloned from the same organism, was also expressed under the same conditions. Consequently, active α-amylase and glucoamylase were efficiently secreted into the culture medium. The amino acid sequence of the N-terminal regions of these enzymes purified from the yeast culture medium confirmed that the signal sequences of these enzymes were cleaved off at the same positions as those of the native enzymes of A. shirousamii.     

Transfer of genes for utilization of starch (sta2) and melibiose (mel) to industrial strains of Saccharomyces cerevisiae by single-chromosome transfer,using a kar1 mutant as vector

Summary A method has been developed for the transfer of genes from other yeast strains and species to industrial yeast strains, using a haploid, kar1-1 mutant strain of Saccharomyces cerevisiae as a vector. The sta2 gene, conferring the ability to metabolize starch was transferred from an autotrophic haploid strain of S. cerevisiae (S. diastaticus) and the melibiose-metabolism (mel) gene(s), from S. kluyveri, to the kar1-1 mutant [K5-5A; ( ade2 his4 can1 gal) by normal mating and protoplast fusion. From this strain, the genes were transferred to baker's yeast and brewing yeast strains, which did not utilize starch, and to baker's yeast strains, which did not utilize melibiose, by protoplast fusion, spore-cell pairing, or rare-mating. Strains that utilized starch or melibiose were obtained by all three methods. Pulsed-field gel electrophoresis preparations showed little change in the mobility of the chromosomes of the hybrids. The most probable explanation for the results obtained is that single chromosomes were transferred, first, from the donor strains to the kar1-1 haploid mutant strain, and then from the kar1-1 vector to the recipient industrial strain of S. cerevisiae. The transfer of the genes is probably accomplished through formation of disomic strains and the, in the case of the hybrids that metabolize starch, by integration of the sta2 gene into the genome of the industrial yeast strains.     

Cloning of an Arabidopsis thaliana cDNA coding for farnesyl diphosphate synthase by functional complementation in yeast

A cDNA encoding farnesyl diphosphate synthase, an enzyme that synthesizes C15 isoprenoid diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate, was cloned from an Arabidopsis thaliana cDNA library by complementation of a mutant of Saccharomyces cerevisiae deficient in this enzyme. The A. thaliana cDNA was also able to complement the lethal phenotype of the erg20 deletion yeast mutant. As deduced from the full-length 1.22 kb cDNA nucleotide sequence, the polypeptide contains 343 amino acids and has a relative molecular mass of 39689. The predicted amino acid sequence presents about 50% identity with the yeast, rat and human FPP synthases. Southern blot analyses indicate that A. thaliana probably contains a single gene for farnesyl diphosphate synthase.     

Protein phosphatase 2A: identification in Oryza sativa of the gene encoding the regulatory A subunit

A 2225 bp cDNA, designated RPA1, was isolated from an Oryza sativa cDNA library. Analysis revealed a 1761 bp coding sequence with 15 non-identical repeat units. The ORF encoded the A regulatory subunit of protein phosphatase 2A (PP2A-A) as ascertained by complementation of the yeast tpd3 mutant defective in this gene. The corresponding genomic DNA from a rice genome BAC library revealed that the gene contains eleven introns. The rice genome contains only a single copy of this gene as judged by Southern blot analysis. The PP2A protein is highly conserved in nature; the rice protein shows 88% amino acid identity with its counterparts in Arabidopsis or Nicotiana tabacum.