Chieko Kumagai

Molecular Cloning and Characterization of a Transcriptional Activator Gene, amyR, Involved in the Amylolytic Gene Expression in Aspergillus oryzae

A gene, designated amyR, coding for a transcriptional activator involved in amylolytic gene expression has been cloned from Aspergillus oryzae by screening for a clone that enabled to reverse the reduced expression of the α-amylase gene (amyB) promoter. amyR encodes 604 amino acid residues of a putative DNA-binding protein carrying a zinc binuclear cluster motif (Zn(II)2Cys6) belonging to the GAL4 family of transcription factors. The amyR gene disruptants showed a significant restricted growth on starch medium and produced little of the amylolytic enzymes including α-amylase and glucoamylase compared with a non-disruptant, indicating that amyR is a transcriptional activator gene involved in starch/maltose-induced efficient expression of the amylolytic genes in A. oryzae. In addition, sequencing analysis found that amyR, agdA (encoding α-glucosidase), and amyA (encoding α-amylase), are clustered on a 12-kb DNA fragment of the largest chromosome in A. oryzae, and that amyR is about 1.5 kb upstream of agdA and transcribed in the opposite direction. Furthermore, transcriptional analysis revealed that the amyR gene was expressed in the presence of glucose comparable to the level in the presence of maltose, while the amylolytic genes were transcribed at high levels only in the presence of maltose.

Improvement of promoter activity by the introduction of multiple copies of the conserved region III sequence, involved in the efficient expression of Aspergillus oryzae amylase-encoding genes

Abstract The role of the conserved sequence region III in the promoter regions of the amylase-encoding genes amyB, glaA and agdA of Aspergillus oryzae was examined. Introduction of multiple copies of the region III fragment into the agdA promoter resulted in a significant increase in promoter activity at the transcriptional level. This result suggests that the fragment comprising region III consists of one or more cis-acting sequence(s). Moreover, expression of the agdA gene under the control of the improved agdA promoter resulted in efficient overproduction of α -glucosidase, even in the presence of glucose. Thus, overexpression of genes controlled by the improved promoter incorporating region III is possible. Interestingly, expression of the amyB and glaA genes in the transformant was strongly repressed. This result suggests that the trans-acting regulatory protein(s) that interact with region III are common to these amylase genes and that the titration of regulatory protein(s) reduced the expression of the amyB and glaA genes.

Functional elements of the promoter region of the Aspergillus oryzae glaA gene encoding glucoamylase

SummaryAnalysis was made of the promoter region of the Aspergillus oryzae glaA gene encoding glucoamylase. Northern blots using a glucoamylase cDNA as a probe indicated that the amount of mRNA corresponding to the glaA gene increased when expression was induced by starch or maltose. The promoter region of the glaA gene was fused to the Escherichia coli uidA gene, encoding β-glucuronidase (GUS), and the resultant plasmid was introduced into A. oryzae. Expression of GUS protein in the A. oryzae transformants was induced by maltose, indicating that the glaA-GUS gene was regulated at the level of transcription in the presence of maltose. The nucleotide sequence 1.1 kb upstream of the glaA coding region was determined. A comparison of the nucleotide sequence of the A. oryzae glaA promoter with those of A. oryzae amyB, encoding α-amylase, and A. niger glaA showed two regions with similar sequences. Deletion and site-specific mutation analysis of these homologous regions indicated that both are essential for direct high-level expression when grown on maltose.

High level expression of the synthetic human lysozyme gene in Aspergillus oryzae

SummaryAspergillus oryzae was transformed with a synthetic gene consisting of a chicken lysozyme signal sequence and a mature human lysozyme (HLY) sequence. The transformants secreted active HLY (about 1.2 mg/l) when the HLY gene was expressed under the control of the Taka-amylase A gene (amyB) promoter. Western blot analysis suggested that the secreted protein was immunoreactive with anti-human lysozyme antibody and the signal peptide was correctly cleavaged off in the A. oryzae transformants. The transcriptional level of the HLY gene was investigated by Northern blot analysis using a probe that was equivalently specific to both the HLY gene and the amyB gene. The HLY gene was expressed of a higher level compared with the amyB gene because of its multi-copy intergration. The efficient transcription of the HLY gene suggested that A. oryzae is a promising host for production of heterologous proteins from higher eukaryotes.

Secretion of calf chymosin from the filamentous fungus Aspergillus oryzae

Active calf chymosin was secreted from Aspergillus oryzae transformants when the chymosin cDNA was expressed under the control of glucoamylase gene (glaA) promoter. Secreted prochymosin was autocatalytically activated to the chymosin (0.07– 0.16 mg/l). Western blot analysis showed that a secreted protein immunoreactive with an anti-chymosin antibody was of similar size to authentic chymosin. Northern blot analysis revealed that mRNA of the chymosin cDNA was expressed at as high level as that of the glaA gene. The size and the level of the transcript were different among transformants, due to the intergration position of the plasmid on the chromosome.

Breeding of a new type of baker's yeast by δ-integration for overproduction of glucoamylase using a homothallic yeast

Abstract With the aim of developing a new type of bread that can be produced from raw materials such as rice, new recombinant yeast strains producing glucoamylase were constructed. The yeast OC-2, which is used for wine making, was selected as a host strain in a screening test because of its superiority in flavor formation in bread. OC-2 is a homothallic and diploid strain, and has no auxotrophic marker. First, tryptophan auxotrophic mutants (Δ trp1 /Δ trp1 ) were constructed from OC-2 by the gene disruption method. Conventional YEp- and YIp-type yeast expression plasmids containing glucoamylase cDNA from Aspergillus oryzae , and the TRP1 gene as a selective marker were introduced into the recipient strain. Both transformants with the YEp- or YIp-type plasmid showed low glucoamylase productivity in the culture broth, probably due to very low plasmid stability, and a low copy number, respectively. A new type of integrative plasmid was thus constructed by the δ-integration system using a δ sequence of the yeast retrotransposon Ty1 to obtain transformants with higher glucoamylase production ability. These transformants produced higher amounts of glucoamylase and could grow in synthetic medium containing starch as a carbon source. Furthermore, using the integrated transformants, duplication of copies of the integrated glucoamylase cDNA by spore formation and autodiploidization was carried out by utilizing the homothallic characteristics of the strain. By Southern blot analysis, the copy number was estimated to be about twice that of the original transformant, and the glucoamylase productivity was proportionally increased. The fact that the constructed strains could grow in synthetic medium containing soluble starch as a sole carbon source suggested that they will be useful for making new types bread.

Transformation of the industrial strain of Aspergillus oryzae with the homologous amdS gene as a dominant selectable marker

A homologous transformation system has been developed for Aspergillus oryzae industrial strains using the A. oryzae acetamidase-encoding gene (amdS) as a dominant selectable marker. Although the transformation efficiency is relatively low (0.4–1 transformants per μg DNA), the transformants contained significantly high copy numbers of the plasmid integrated into the genome. In addition, the promoter of the amdS gene was replaced by the strong expression promoter of the α-amylase-encoding gene (amyB) from A. oryzae, thereby enhancing the selection of the transformants.

Rapid and sensitive detection of hiochi bacteria by amplification of hiochi bacterial common antigen gene by PCR method and characterization of the antigen

Abstract A rapid and sensitive method of detection of the sake spoilage microorganisms hiochi bacteria was developed. The hiochi bacterial common antigen (hb-CA) was identified using a monoclonal antibody (MAb) and its gene was cloned. The nucleotide sequences of the hb-CA gene from four standard strains of hiochi bacteria were then determined. Two polymerase chain reaction (PCR) primers for use in the detection of hiochi bacteria were designed by homology analysis of these sequences. When genomic DNA from the hetero-fermentative true hiochi bacillus (he-T) Lactobacillus fructivorans HI was amplified by PCR using this set of primers and electrophoretically analyzed, a PCR product could be detected when as little as 10 fg of genomic DNA, which corresponds to the amount of DNA in a single cell, had been used. When he-T L. fructivorans H1 was inoculated into 300 ml of pasteurized sake, the cells recovered by filtration, and the DNA analyzed by this method, the detection limit was found to be about 10 cells. This method is much faster than the standard culture method. This PCR technique is a rapid and highly sensitive method of detection of hiochi bacteria which is applicable to quality control of sake. The results of a homology search performed using the deduced amino acid sequence of this common antigen suggest that this antigen is the hiochi bacterial elongation factor Tu (EF-Tu).

Mutant Isolation of Non-Urea Producing Sake Yeast by Positive Selection

Abstract Urea is reported to be a main precursor in wine and sake (Japanese rice wine) of ethyl carbamate (ECA), a suspected carcinogen. We constructed an arginase-deficient mutant (Δ car1 /Δ car1 ) from a diploid sake yeast, Kyokai no. 9, using a gene disruption method (Kitamoto, K. et al. , Appl. Environ. Microbiol., 57, 301, 1991). The car1 mutant thus constructed enabled us to brew sake containing no urea or ECA. In spite of their superior characteristics, industrial use of mutants for sake brewing has so far been difficult because guidelines for recombinant DNA utilization in the food and beverages industry have not yet been established. Using the genetically engineered car1 mutant, we have developed a new medium for the positive selection of car1 mutants. Many arginase-deficient mutants could be easily isolated from not only a laboratory haploid strain (X2180-1A), but also sake yeasts (Kyokai no. 9 and Kyokai no 10) and wine yeasts (Geisenheim 74 and Eperney). Sake with no urea could be brewed using the car1 mutants, and no ECA was detected in the resulting sake even after heat treatment ( hi-ire ) and storage.

Isolation of a New Lytic Enzyme for Hiochi Bacteria and Other Lactic Acid Bacteria.

A microorganism producing a lytic enzyme preparation that could rapidly lyse bacterial cells such as hiochi bacteria and other lactic acid bacteria was screened. The microorganism was identified as Streptomyces fulvissimus. The enzyme produced by this organism lysed boil-denatured cells quicker than intact cells of hiochi bacteria. A mutant strain of S. fulvissimus producing the enzyme exhibiting high activity against intact cells of hiochi bacteria was screened on plates, containing intact cells inactivated with UV irradiation. The optimal pH for lytic activity against intact cells of the hiochi bacterium Lactobacillus casei S-4 was from 3.5 to 4.0, and the optimum temperature was close to 50 degrees C. This enzyme activity was stable between pH 3.5 and pH 8.0 and up to 60 degrees C. The enzyme exhibits N-acetyl glucosaminidase and muramidase activities. The effects of adjusting the pH and using different inducers for enzyme production were investigated. Chitin was the most effective inducer of enzyme production. Intact DNA was easily isolated from the cells of many lactic acid bacteria following lysis with the enzyme. It is thought that this enzyme will be a good biotechnological tool.