Combined Expression of Aspergillus nidulans Endoxylanase X24 and Aspergillus oryzae (alpha)-Amylase in Industrial Baker's Yeasts and Their Use in Bread Making

The Aspergillus nidulans endoxylanase X24 and the Aspergillus oryzae (alpha)-amylase cDNAs were placed under the control of the Saccharomyces cerevisiae actin promoter (pACT1) and introduced into baker's yeast. Bread made with transformants expressing both enzymes (YEpACT-AMY-ACT-X24) showed a 30% increase in volume and reduced firmness in comparison with that produced with a commercial strain. Endoxylanase X24 and (alpha)-amylase seem to act synergistically to improve the quality of bread in terms of volume and density.     

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     

The wide-domain carbon catabolite repressor CreA indirectly controls expression of the Aspergillus nidulans xlnB gene, encoding the acidic endo-beta-(1,4)-xylanase X(24)

The Aspergillus nidulans xlnB gene, which encodes the acidic endo-beta-(1,4)-xylanase X(24), is expressed when xylose is present as the sole carbon source and repressed in the presence of glucose. That the mutation creA(d)30 results in considerably elevated levels of xlnB mRNA indicates a role for the wide-domain repressor CreA in the repression of xlnB promoter (xlnBp) activity. Functional analyses of xlnBp::goxC reporter constructs show that none of the four CreA consensus target sites identified in xlnBp are functional in vivo. The CreA repressor is thus likely to exert carbon catabolite repression via an indirect mechanism rather than to influence xlnB expression by acting directly on xlnB.     

Cloning and characterization of the nagA gene that encodes beta-n-acetylglucosaminidase from Aspergillus nidulans and its expression in Aspergillus oryzae

We isolated a beta-N-acetylglucosaminidase encoding gene and its cDNA from the filamentous fungus Aspergillus nidulans, and designated it nagA. The nagA gene contained no intron and encoded a polypeptide of 603 amino acids with a putative 19-amino acid signal sequence. The deduced amino acid sequence was very similar to the sequence of Candida albicans Hex1 and Trichoderma harzianum Nag1. Yeast cells containing the nagA cDNA under the control of the GAL1 promoter expressed beta-N-acetylglucosaminidase activity. The chromosomal nagA gene of A. nidulans was disrupted by replacement with the argB marker gene. The disruptant strains expressed low levels of beta-N-acetylglucosaminidase activity and showed poor growth on a medium containing chitobiose as a carbon source. Aspergillus oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of beta-N-acetylglucosaminidase in a wheat bran solid culture.     

Construction of an Aspergillus nidulans multicopy transformant for the xlnB gene and its use in purifying the minor X24 xylanase

 Using recombinant DNA techniques, an Aspergillus nidulans multicopy transformant for the gene xlnB coding for the minor X₂₄ xylanase has been constructed. When grown on glucose as sole carbon source this transformant secretes 114 U of xylanase (mg protein)^-1. In this culture condition, X₂₄ is the only xylanase secreted and the predominant protein in the culture filtrate. This strategy has been used to purify the X₂₄ enzyme to homogeneity. The purified xylanase showed a single band on sodium dodecyl sulphate/ polyacrylamide gel electrophoresis with a molecular mass of 24 kDa and had an isoelectric point of approximately 3.5. The enzyme was a non-debranching endo-1,4-β-xylan xylanohydrolase highly specific for xylans and showed optimal activity at pH 5.5 and 52°C. The X₂₄ xylanase had a Michaelis constant, K _m, of 12.43 mg oat spelt xylan ml^-1 and a V _max of 1639 μmol min^-1 (mg protein)^-1. Received: 17 May 1995/Received last revision: 25 September 1995/Accepted: 29 September 1995     

The Formation, General Characteristics and Production of α-Amylase in Heterocaryons and Diploids of Aspergillus oryzae

S ummary . Heterocaryons and diploids from Aspergillus oryzae were investigated with respect to nuclear number/conidium and to conidial size. Heterocaryons usually had larger conidia and more nuclei/conidium than diploids and the haploid parent mutants. Diploids contained significantly fewer nuclei/conidium than haploids. However, they could not be distinguished from haploids by measurement of conidial size. The strains were examined for the production of α-amylase. All auxotrophic mutants produced less α-amylase than the prototrophic wild type. Heterocaryons gave yields which were intermediate between that of their parent mutants or the same as the best producing parent. Diploids which produced more α-amylase than the best producing parent strain were synthesized. The highest yield from a diploid was of the same order of magnitude as the yield from the wild type.     

Starch digestion and adsorption by β-amylase of Emericella nidulans (Aspergillus nidulans)

B. CHATTERJEE, A. GHOSH AND A. DAS. 1992. A mutant strain of Emericella nidulans MNU 82 was isolated by multistep mutation. The β-amylase produced by the mutant was able to digest raw starch. It was readily and strongly adsorbed onto raw starch at pH 5.0. The enzyme to starch ratio was 1950 U/g starch. The enzyme showed no correlation between the capacity of raw starch digestion and adsorption of the enzyme.     

Analysis of acetate non-utilizing (acu) mutants in Aspergillus nidulans.

Genetic analysis of 119 acetate non-utilizing (acu) mutants in Aspergillus nidulans revealed ten new loci affecting acetate metabolism in addition to the three previously recognized on the basis of resistance to fluoroacetate and acetate non-utilization. The enzyme lesions associated with mutations at seven of the acu loci are described. These are: facA (= acuA), acetyl-CoA synthase; acuD, isocitrate lyase; acuE, malate synthase; acuF, phosphoenolpyruvate carboxykinase; acuG, fructose 1,6-diphosphatase; acuK and acuM, malic enzyme. The acu loci have been mapped and are widely distributed over the genome of A. nidulans. Close linkage has only been found between acuA and acuD (less than 1% recombination). There is no evidence for any pleiotropic mutation in that region affecting the expression of both these genes. Poor induction of the enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase in mutants lacking acetyl-CoA synthase, and also in the other two classes of fluoroacetate-resistant mutants, indicates that the inducer, acetate, may be metabolized to a true metabolic inducer, perhaps acetyl-CoA, to effect formation of the enzymes. There is no evidence of any other class of pleiotropic recessive acu mutations affecting the expression of the acuD and acuE genes, which are therefore thought to be subject to negative rather than positive control.     

Visualizing nuclear migration during conidiophore development in Aspergillus nidulans and Aspergillus oryzae: multinucleation of conidia occurs through direct migration of plural nuclei from phialides and confers greater viability and early germination in Aspergillus oryzae

Nuclear migration is indispensable for normal growth, differentiation, and development, and has been studied in several fungi including Aspergillus nidulans and Neurospora crassa. To better characterize nuclear movement and its consequences during conidiophore development, conidiation, and conidial germination, we performed confocal microscopy and time-lapse imaging on A. nidulans and Aspergillus oryzae strains expressing the histone H2B-EGFP fusion protein. Active trafficking of nuclei from a vesicle to a phialide and subsequently into a conidium provided the mechanistic basis for the formation of multinucleate conidia in A. oryzae. In particular, the first direct visual evidence on multinucleate conidium formation by the migration of nuclei from a phialide into the conidium, rather than by mitotic division in a newly formed conidium, was obtained. Interestingly, a statistical analysis on conidial germination revealed that conidia with more nuclei germinated earlier than those with fewer nuclei. Moreover, multinucleation of conidia conferred greater viability and resistance to UV-irradiation and freeze-thaw treatment.     

Conidium differentiation in Aspergillus nidulans wild-type and wet-white (wetA) mutant strains

Conidium (asexual spore) differentiation in wild-type and the wet-white (wetA) mutant of Aspergillus nidulans was compared in intact chains of successively older conidia. Carbohydrate cytochemistry helped define three stages (Stages I, II, and III) of wild-type conidium maturation on the basis of changes in the ultrastructure and composition of the conidium wall. Conidia of the wetA6 mutant strain formed normally but failed to mature during Stages II and III. Specifically, the inner wall layer of wetA6 conidia did not condense during Stage II and two wall layers that stained for carbohydrates did not form during the transition to Stage III. Concomitantly, wetA6 conidia formed large cytoplasmic vacuoles and underwent lysis. The wetA gene appears to have a conidium-specific function for the modification of the conidium wall during Stages II and III. These modifications of the conidium wall are essential for the stability of mature, dormant conidia.     

Insertion analysis of putative functional elements in the promoter region of the Aspergillus oryzae Taka-amylase A gene (amyB) using a heterologous Aspergillus nidulans amdS-lacZ fusion gene system

Expression of the Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose. The A. oryzae amyB gene promoter contains three highly conserved sequences, designated Regions I, II, and III, compared with promoter regions of the A. oryzae glaA encoding glucoamylase and the agdA encoding alpha-glucosidase. To identify the function of these sequences within the amyB promoter, various fragments containing conserved sequences in the amyB promoter were introduced into the upstream region of the heterologous A. nidulans amdS gene (encoding acetamidase) fused to the Escherichia coli lacZ gene as a reporter. Introduction of the sequence between -290 to -233 (the number indicates the distance in base pairs from the translation initiation point (+1)) containing Region III significantly increased the expression of the lacZ reporter gene in the presence of maltose. The sequence between -377 to -290 containing Region I also increased the lacZ activity, but its maltose inducibility was less than that of Region III. The sequence between -233 to -181 containing Region II had no effect on the expression. These results indicated that Region III is most likely involved in the maltose induction of the amyB gene expression.     

Cloning and nucleotide sequence of one of the most highly expressed genes, a pdcA homolog of Aspergillus nidulans, in Aspergillus oryzae

A homolog of Aspergillus nidulans pdcA that is probably one of the most highly expressed in Aspergillus oryzae ATCC 22788 was isolated, as measured by the frequency among randomly selected 324 expressed sequence tags. It has an 1,632 bp open reading frame for a polypeptide of about 60 kDa. Its amino acid sequence revealed 74% identity and 84% similarity to that of A. nidulans pyruvate decarboxylase.     

The heterotrimeric G-protein GanB(alpha)-SfaD(beta)-GpgA(gamma) is a carbon source sensor involved in early cAMP-dependent germination in Aspergillus nidulans

The role of heterotrimeric G-proteins in cAMP-dependent germination of conidia was investigated in the filamentous ascomycete Aspergillus nidulans. We demonstrate that the G alpha-subunit GanB mediates a rapid and transient activation of cAMP synthesis in response to glucose during the early period of germination. Moreover, deletion of individual G-protein subunits resulted in defective trehalose mobilization and altered germination kinetics, indicating that GanB(alpha)-SfaD(beta)-GpgA(gamma) constitutes a functional heterotrimer and controls cAMP/PKA signaling in response to glucose as well as conidial germination. Further genetic analyses suggest that GanB plays a primary role in cAMP/PKA signaling, whereas the SfaD-GpgA (G betagamma) heterodimer is crucial for proper activation of GanB signaling sensitized by glucose. In addition, the RGS protein RgsA is also involved in regulation of the cAMP/PKA pathway and germination via attenuation of GanB signaling. Genetic epistatic analyses led us to conclude that all controls exerted by GanB(alpha)-SfaD(beta)-GpgA(gamma) on conidial germination are mediated through the cAMP/PKA pathway. Furthermore, GanB may function in sensing various carbon sources and subsequent activation of downstream signaling for germination.     

Spray-Dried Gliadin Powders Inclusive of Linoleic Acid (Microcapsules): Their Preservability,Digestibility and Application to Bread Making

An aqueous-alcohol solution containing gliadin (or zein) and linoleic acid was pulverized by spray-drying and the powders were examined for their stability, digestibility and application. Linoleic acid in the powders was kept stable during three months’ storage in each case of low, moderate and high water activity. When the spray-dried powders were treated with such digestive enzymes as pepsin and pancreatin, linoleic acid was effectively changed into the hexane-extractable form concomitantly with the digestion of gliadin. The powders were administered to rats, instead of gluten and essential fatty acid in a semi-purified diet, and there was no abnormal excretion of fatty acid into the faeces. These facts indicate that two components of the powders could be digested and absorbed in the gut as usual. On the other hand, supplementation of bread with gliadin and linoleic acid in the form of spray-dried powders led to a considerable increase in bulk after baking, without affecting the taste and flavor. Linoleic acid in a loaf of bread seemed to be fairly resistlant to peroxidation during baking in an oven and during storage under aerobic conditions for a week.     

米曲霉木聚糖酶基因的克隆及其在毕赤酵母中的表达

目的:构建米曲霉木聚糖酶基因的真核表达载体,并转化巴斯德毕赤酵母,进行分泌表达。方法:以米曲霉总RNA为模板,根据已知的米曲霉木聚糖酶基因序列设计引物,采用RT-PCR技术克隆木聚糖酶基因cDNA序列,将其与pPIC9K质粒连接构建表达载体后转化毕赤酵母,经MM/MD快慢斑筛选,得到Muts型重组子,进行甲醇诱导表达。结果:克隆得到的cDNA序列全长666 bp,连续编码221个氨基酸;阳性克隆子在诱导培养数天后,将菌液点于RBB-木聚糖平板上,产生了明显的透明圈,表明重组木聚糖酶在毕赤酵母中获得表达。结论:木聚糖酶基因的真核表达载体构建成功,并能够在毕赤酵母中表达。