Awamori Strain Research Articles

Development of schemes of induced mutagenesis for improving the productivity of Aspergillus strains producing amylolytic enzymes

In order to improve the biosynthesis of amylolytic enzymes by industrial Aspergillus strains, the efficiency of stepwise application of the following methods of induced mutagenesis was studied: ultraviolet (UV) irradiation, gamma irradiation, and treatment with N-methyl-N-nitro-N-nitrosoguanidine (NG). It was found that at the early stages of mutagenesis of the glucoamylase-producing A. awamori strain UV and NG, used either alone or in combination, were efficient enough as mutagenic agents, providing an increase of glucoamylase activity by 30–35 and 50–60%, respectively. At the later stages, serial UV mutagenesis and gamma-irradiation showed high efficiency for both A. awamori, and A. oryzae strains. Gamma-mutagenesis of Aspergillus strains using a cobalt source provided the most stable and highly active strains retaining 90–95% of their activity after five transfers on agar medium. The experiments resulted in significant improvement of the studied industrial strains, more than doubling activity of the target enzymes.

Multiplex PCR analysis of fumonisin biosynthetic genes in fumonisin-nonproducing Aspergillus niger and A. awamori strains

To determine the genetic basis for loss of fumonisin B2 (FB2) biosynthesis in FB2-nonproducing Aspergillus niger and A. awamori strains, we developed multiplex PCR primer sets to amplify fragments of eight fumonisin biosynthetic pathway (fum) genes. Fragments of all eight fum genes were amplified from FB2-producing A. niger and A. awamori strains; from FB2-nonproducing strains four amplification patterns arose in which one or more fum gene fragments were absent. Southern hybridization analysis of strains yielding patterns 2 and 3 confirmed that loss of FB2 production in A. awamori is associated with gene deletions within the fumonisin biosynthetic gene cluster. In addition, we observed a fifth multiplex amplification pattern in which all eight fum gene fragments appeared. Reverse transcription-PCR analysis of strains yielding pattern 5 showed that the expression of at least one fum gene was reduced relative to expression in FB2-producing A. niger. This suggests that in these strains loss of FB2 production is a result of structural or regulatory mutations that alter gene expression or function. These results demonstrate a diversity of genotypes within FB2-nonproducing A. niger and A. awamori populations and provide tools useful for identifying certain non-toxigenic strains for industrial or ecological applications.

Aspergillus niger contains the cryptic phylogenetic species A. awamori

Aspergillus section Nigri is an important group of species for food and medical mycology, and biotechnology. The Aspergillus niger ‘aggregate’ represents its most complicated taxonomic subgroup containing eight morphologically indistinguishable taxa: A. niger, Aspergillus tubingensis, Aspergillus acidus, Aspergillus brasiliensis, Aspergillus costaricaensis, Aspergillus lacticoffeatus, Aspergillus piperis, and Aspergillus vadensis. Aspergillus awamori, first described by Nakazawa, has been compared taxonomically with other black aspergilli and recently it has been treated as a synonym of A. niger. Phylogenetic analyses of sequences generated from portions of three genes coding for the proteins β-tubulin ( benA), calmodulin ( CaM), and the translation elongation factor-1 alpha ( TEF-1α) of a population of A. niger strains isolated from grapes in Europe revealed the presence of a cryptic phylogenetic species within this population, A. awamori. Morphological, physiological, ecological and chemical data overlap occurred between A. niger and the cryptic A. awamori, however the splitting of these two species was also supported by AFLP analysis of the full genome. Isolates in both phylospecies can produce the mycotoxins ochratoxin A and fumonisin B 2, and they also share the production of pyranonigrin A, tensidol B, funalenone, malformins, and naphtho-γ-pyrones. In addition, sequence analysis of four putative A. awamori strains from Japan, used in the koji industrial fermentation, revealed that none of these strains belong to the A. awamori phylospecies.

Increase in glucoamylase productivity of Aspergillus awamori strain by combination of radiation mutagenesis and plasmid transformation methods

Increase in the level of amylolytic genes activator protein encoded by amyR gene was shown to result in enhancement of glucoamylase productivity of A. awamori strain by 30%. However, the same effect equal to 30% increase can be achieved by introduction of extra copies of gla gene encoding glucoamylase. These two effects were not additive, which gave the possibility to suggest an additional limitation in the egulation mechanism of glucoamylase gene expression in Aspergillus family strains while introducing an additional copies of amyR and gla genes.

Optimizing culture conditions for the production of endo--1,4-glucanase by Aspergillus awamori strain Vietnam Type Culture Collection (VTCC)-F099

In the present study, twenty six strains of Aspergillus awamori from the Vietnam Type Culture Collection (Institute of Microbiology and Biotechnology, Vietnam University Hanoi) were used for the endoglucanase production by growing at 37°C in the growth medium. Result showed that A. awamori strain VTCC-F099 produced the highest level of endo β-1,4-glucanase in the growth medium, pH 6.5, at 30°C for 96 h, agitated at 200 rpm. The optimal concentration of the inducer CMC (carboxymethyl cellulose) for the endoglucanase production by A. awamori VTCC-F099 was 2%. Among tested carbon sources (coconut fiber, coffee shell, corncob, dried tangerine skin, peanut shell, rice bran, saw dust, sugar-cane bagasse as organic wasters and glucose, lactose sucrose as pure carbon sources), corncob showed the highest endoglucanase production by A. awamor i VTCC-F099 at the concentration of 3%. Ammonium acetate was the best among nitrogen source (casein, peptone, fish powder, soybean powder as organic sources and CH 3 COONH 4 , NH 4 NO 3 , (NH 4 ) 2 SO 4 , urea as inorganic sources) for the endoglucanase production by A. awamori VTCC-F099 at the concentration of 0.3%. Key words : Aspergillus awamor i, carboxymethyl cellulose, endoglucanase production, optimization of culture conditions.

Use of Mesophilic Fungal Amylases Produced by Solid-state Fermentation in the Cold Hydrolysis of Raw Babassu Cake Starch

Amylases constitute one of the most important groups of industrial enzymes, presenting several applications, such as in the food, textile, and ethanol manufacturing. In this work, a starchy residue from the Brazilian agroindustry, namely babassu cake, was used for the production of amylases by solid-state fermentation and for obtaining sugar hydrolysates, which can be used as building blocks for future bioconversions. Eight filamentous fungi from the genera Aspergillus and Penicillium were screened. Regarding amylase production, A. awamori strains showed well-balanced endoamylase and exoamylase activities, while A. wentii produced an amylolytic complex much richer in the endo-acting enzymes. Simultaneous liquefaction and saccharification using the crude enzyme extracts from the four most promising fungal strains was then investigated applying DOE techniques. The highest total reducing sugar content (24.70 g L(-1)) was obtained by the crude extract from A. awamori IOC-3914, corresponding to a hydrolysis yield of 52% of total starch in the cake, while A. awamori IOC-3915 produced the most appropriate extract in terms of glucose release (maximum of 5.52 g L(-1)). Multivariate analysis of the DOE studies indicated that these extracts showed their best performance at 50-57 degrees C under acid conditions (pH 3.6-4.5), but were also able to act satisfactorily under milder conditions (36 degrees C and pH 5.0), when TRS and glucose released were about 65% of the maximum values obtained. These data confirm the high potential of the enzyme extracts under study for cold hydrolysis of starch.

Production, partial purification and characterization of feruloyl esterase by Aspergillus awamori in submerged fermentation

Microbial feruloyl esterases acting on plant cell wall polymers represent key tools for the degradation of plant cell wall. In this paper, we describe in detail the microbial production, partial purification and characterization of feruloyl esterase from a culture medium of Aspergillus awamori strain IFO4033 obtained from a crude hemicellulose preparation of wheat straw, corncobs and wheat germ. Feruloyl esterase was extracted using centrifugation and dialysis, and then purified by ion exchange chromatography and microfiltration to homogeneity, which was checked by SDSPAGE and isoelectric focusing-PAGE. Protein content and activity of the enzyme were measured in each step of extraction and purification. Biomass was determined by the dry weight method. pH and temperature optima of feruloyl esterase enzyme were also determined. The effects of culturing time, and carbon and nitrogen sources on enzyme production were systematically investigated. Finally, enzyme activities under different storage conditions were examined.

Stimulatory effect of phosphate-solubilizing fungal strains ( Aspergillus awamori and Penicillium citrinum) on the yield of chickpea ( Cicer arietinum L. cv. GPF2)

The effect of six phosphate-solubilizing fungi (PSF, two strains of Aspergillus awamori, and four of Penicillium citrinum) isolated from rhizosphere of various crops, was observed on the growth and seed production of chickpea plants ( Cicer arietinum L. cv. GPF2) in pot experiments. The phosphate (P) solubilizing activity of PSF in liquid varied from 38 to 760 μg ml −1 for tricalcium phosphate (TCP) and 28–248 μg ml −1 for mussoorie rock phosphate (MRP). All PSF isolates were biocompatible and produced growth-promoting hormone, Indole acetic acid (IAA), varying in concentration from 2.5 to 9.8 μg ml −1. Of the various pot experiments carried out in green house, maximum stimulatory effect on chickpea plants growth was observed by inoculation of two A. awamori strains. This treatment resulted in 7–12% increase in shoot height, nearly three-fold increase in seed number and two-fold increase in seeds weight as compared to the control (un-inoculated) plants. Inoculation of four strains of P. citrinum exhibited lesser stimulatory effect. It showed 7% increase in shoot height, two-fold increase in seed number and 87% increase in seeds weight as compared to the control plants. However, a consortium of all the six fungal isolates showed no stimulatory effect on chickpea plants growth.

Thr/Ser-rich domain of Aspergillus glucoamylase is essential for secretion.

The recombinant Aspergillus awamori strain carrying the mutant glucoamylase-encoding gene in which the entire Thr/Ser-rich Gp-I domain was deleted abolished secretion of mutant glucoamylase. The transcription of the Bip-encoding bipA was low in the wild type (wt) strain, but elevated in the recombinant strain under the condition of glaA expression. The results indicate that the Gp-I domain is vital for glucoamylase secretion.

Transformation of Aspergillus awamori by Agrobacterium tumefaciens-mediated homologous recombination.

Agrobacterium tumefaciens is known to transfer part of its tumor-inducing (Ti) plasmid to the filamentous fungus Aspergillus awamori by illegitimate recombination with the fungal genome. Here, we show that when this Ti DNA shares homology with the A. awamori genome, integration can also occur by homologous recombination. On the basis of this finding, we have developed an efficient method for constructing recombinant mold strains free from bacterial DNA by A. tumefaciens-mediated transformation. Multiple copies of a gene can be integrated rapidly at a predetermined locus in the genome, yielding transformants free of bacterial antibiotic resistance genes or other foreign DNA. Recombinant A. awamori strains were constructed containing up to nine copies of a Fusarium solani pisi cutinase expression cassette integrated in tandem at the pyrG locus. This allowed us to study how mRNA and protein levels are affected by gene copy number, without the influence of chromosomal environmental effects. Cutinase mRNA and protein were maximal with four gene copies, indicating a limitation at the transcriptional level. This transformation system will potentially stimulate market acceptance of derived products by avoiding introduction of bacterial and other foreign DNA into the fungi.

Secretion of the sweet-tasting protein thaumatin by recombinant strains of Aspergillus niger var. awamori.

A recombinant form of the sweet-tasting protein thaumatin has been produced in the filamentous fungus Aspergillus niger var. awamori. Expression cassettes containing a synthetic gene encoding thaumatin II were prepared and used to transform Aspergillus niger var. awamori strain NRRL312. Several fungal strains capable of synthesizing and secreting thaumatin into the culture medium were generated, and their production capabilities were determined, first in shake flasks and later in a laboratory fermentor. We report the expression and secretion of thaumatin in concentrations of 5-7 mg/l. This recombinant thaumatin is sweet.

Influence of morphology on product formation in aspergillus awamori during submerged fermentations

The relationship between fungal morphology and heterologous protein production was examined for an Aspergillus awamori strain during a series of fermentations with a batch phase followed by a fed-batch phase. Agitation rate and inoculation concentration were used as controlled variables to generate different fungal morphologies in 20-dm3 stirred tank reactors. Morphology was quantitatively characterized using Image Analysis. The different agitation rates and inoculum concentrations had large effects on the development in hyphal length and number of tips during the fermentations. A reduced inoculum concentration resulted in a more branched mycelium. The different agitation rates affected the morphology after 30 h of fermentation significantly but did not affect the start time of fragmentation. A 3-fold increase in hyphal length increased the apparent viscosity by a factor of 7. The observed morphological differences had only a limited effect on product formation, suggesting that the structural features such as hyphal length and number of tips are of less importance for product formation. The primary effect of morphology on product formation is due to viscosity.

An Aspergillus awamori acetylesterase: purification of the enzyme, and cloning and sequencing of the gene.

An inducible acetylesterase was purified from the culture medium of Aspergillus awamori strain IFO4033 growing on wheat-bran culture by ion-exchange, gel-filtration and hydrophobic-interaction chromatographies. The purified enzyme had an Mr of 31000 and contained Asn-linked oligosaccharides. The enzyme liberated acetic acid from wheat bran, hydrolysed only alpha-naphthyl acetate and propionate when aromatic esters were used for the substrate, and was tentatively classified as a carboxylic esterase (EC 3.1.1.1). The gene encoding acetylesterase was cloned and sequenced. The deduced amino acid sequence showed that acetylesterase was produced as a 304-amino-acid-residue precursor, which was converted post-translationally into a 275-amino-acid-residue mature protein. Part of the sequence of acetylesterase was similar to the region near the active-site serine of lipases of Geotrichum candidum and Candida cylindracea. A unique site of putative Asn-linked oligosaccharides was presented.

Efficient production of secreted proteins by Aspergillus: progress, limitations and prospects.

Filamentous fungi are widely used for the production of homologous and heterologous proteins but, compared to homologous proteins, the levels of production of heterologous proteins are usually low. During the last 5 years, the levels of production of heterologous proteins have been drastically improved by fusing the corresponding gene to the 3' end of a homologous gene, encoding a well-secreted protein such as glucoamylase. Nevertheless, little research has been carried out to determine the limitations that hamper heterologous protein production. Recently we have carried out a detailed analysis of the levels of production of several proteins and glucoamylase fusion proteins in defined recombinant Aspergillus awamori strains. In this review we will focus on the use of filamentous fungi for the production of heterologous, especially non-fungal proteins. In particular, the effect of gene-fusion strategies will be reviewed. Furthermore, the remaining limitations in heterologous protein production and suggestions for improvement strategies for overproduction of these protein will be discussed.

Analysis of heterologous protein production in defined recombinant Aspergillus awamori strains.

A study was carried out to obtain more insight into the parameters that determine the secretion of heterologous proteins from filamentous fungi. A strategy was chosen in which the mRNA levels and protein levels of a number of heterologous genes of different origins were compared. All genes were under control of the Aspergillus awamori 1,4-beta-endoxylanase A (exlA) expression signals and were integrated in a single copy at the A. awamori pyrG locus. A Northern (RNA) analysis showed that large differences occurred in the steady-state mRNA levels obtained with the various genes; those levels varied from high values for genes of fungal origin (A. awamori 1,4-beta-endoxylanase A, Aspergillus niger glucoamylase, and Thermomyces lanuginosa lipase) to low values for genes of nonfungal origin (human interleukin 6 and Cyamopsis tetragonoloba [guar] alpha-galactosidase). With the C. tetragonoloba alpha-galactosidase wild-type gene full-length mRNA was even undetectable. Surprisingly, small amounts of full-length mRNA could be detected when a C. tetragonoloba alpha-galactosidase gene with an optimized Saccharomyces cerevisiae codon preference was expressed. In all cases except human interleukin 6, the protein levels corresponded to the amounts expected on basis of the mRNA levels. For human interleukin 6, very low protein levels were observed, whereas relatively high steady-state mRNA levels were obtained. Our data suggest that intracellular protein degradation is the most likely explanation for the low levels of secreted human interleukin 6.

Isolation and characterization of a 1,4-beta-endoxylanase gene of A. awamori.

An enzyme with a particular 1,4-beta-xylanase activity was identified and purified from wheat-bran culture medium of an Aspergillus awamori strain. With oligonucleotides based on the N-terminal amino-acid sequence of the enzyme, the exlA gene of A. awamori, encoding 1,4-beta-xylanase A, has been cloned. Based on the deduced amino-acid sequence, 1,4-beta-xylanase A is produced as a 211 amino-acid-residue-long precursor, which is converted post-translationally into a 184-aa residue-long mature protein. Transformation of the original A. awamori strain with multiple copies of the exlA gene resulted in a 40-fold overproduction of 1,4-beta-xylanase A. The overproduced enzyme has the same biochemical and enzymological properties as the wild-type enzyme.

The cloning and sequencing of the genes encoding phytase ( phy) and pH 2.5-optimum acid phosphatase ( aph) from Aspergillus niger var. awamori

The genes encoding phytase (EC 3.1.3.8) and pH 2.5-optimum acid phosphatase (EC 3.1.3.2) have been cloned and sequenced from Aspergillus niger var. awamori. The translated nucleotide sequences yielded polypeptides of 467 and 479 amino acids (aa) for phytase and acid phosphatase, respectively. The genes were isolated using oligodeoxyribonucleotide probes based on the aa sequences of the purified proteins. Recombinant A. niger var. awamori strains carrying additional copies of the gene sequences demonstrated elevated enzyme activities.

An aspergillus awamori mutant with enhanced glucoamylase activity

AbstractThe mutagenesis of a glucoamylase‐producing A. awamori strain yielded a mutant exhibiting an 80% increase in the level of the biosynthesis of this enzyme under liquid state fermentation conditions (LSF).