Translational control of alpha-amylase gene expression in Aspergillus awamori

Regulation of alpha-amylase gene expression in Aspergillus awamori was studied by analyzing the enzyme activity levels, rate of protein synthesis, and alpha-amylase-specific mRNA levels under various conditions of growth. alpha-Amylase synthesis was sensitive to catabolite repression as glucose repressed its synthesis by about fourfold. The stimulation of alpha-amylase synthesis in the presence of its substrate starch was shown to be due to derepression rather than induction as the enzyme was synthesized at similar rates in both starch and starvation media. Repression and derepression of enzyme synthesis was found to be mediated at the translational level. The cellular levels of alpha-amylase-specific mRNA as measured by an in vitro translation assay system, were almost identical under all conditions of enzyme synthesis. Relative in vivo and in vitro alpha-amylase mRNA template activities suggest that alpha-amylase mRNA is translated much more efficiently during the derepression than under the conditions of repressed synthesis.     

High level expression in Escherichia coli of a fungal gene under the control of strong promoters

A recent report (Patino et al., (1989) FEMS Microbiol. Lett. 58, 139-144) described the low level expression, in Escherichia coli, of the Isopenicillin N Synthase (IPNS) gene from Cephalosporium acremonium under the control of strong promoters. We report here our work on the expression of the IPNS gene. Plasmids containing the IPNS gene under the control of the trp or trc promoters directed synthesis of high levels of active IPNS in E. coli. Constitutive and inductive high level IPNS expression systems have been developed. Importantly, the expression vectors do not encode beta-lactamase so IPNS activity can be determined directly by biological assays. Analysis by nmr verified that the IPNS produced from these expression systems catalysed the conversion of delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (LLD-ACV) to isopenicillin N in high yield.     

Efficient production of Arthromyces ramosus peroxidase by Aspergillus awamori

The heterologous production of Arthromyces ramosus peroxidase (ARP) was analysed in the filamentous fungus Aspergillus awamori under control of the inducible endoxylanase promoter. Secretion of active ARP was achieved up to 800 mg l(-1) in shake flask cultures. Western blot analysis showed that an rARP product of the correct molecular weight was produced. In contrast to several other studies about heterologous production of heme containing peroxidases, our results suggest that in A. awamori no heme limitation exists during overproduction of ARP.     

Polygalacturonase production by Aspergillus awamori on wheat in solid-state fermentation

The production of exo-polygalacturonase (exo-PG) and endo-PG by Aspergillus awamori grown on wheat in solid-state fermentation was studied. Endo- and exo-PG activities were detected after 24 h of inoculation. Glucose released from starch hydrolysis acted as a catabolite repressor for the exo-PG enzyme. In contrast, endo-PG production was not affected by glucose repression. When milled grains were used, the particle-size distribution and the chemical composition of the medium influenced the rate of micro-organism growth and therefore the trend followed by endo- and exo-PG production. However, these two parameters did not affect the maximum production of exo-PG and endo-PG. For one of the milled samples, three different moisture contents were used (50, 55, 60%). Moisture contents of 60% provide a higher yield of pectinases by A. awamori.     

Cloning and heterologous expression of gene encoding A polygalacturonase from Aspergillus awamori

A polygalacturonase gene of Aspergillus awamori IFO 4033 was cloned by genomic Southern hybridization with a probe of a DNA fragment synthesized by PCR. This was done using primers constructed based on the N-terminal amino acid sequence of a polygalacturonase, protopectinase-AS, produced by the strain and the consensus internal amino acid sequence of fungal polygalacturonases. The cloned polygalacturonase gene, containing an ORF, encodes 362 amino acids, including a 52-bp intron. It contains the consensus nucleotide sequence of PacC binding sites, and its expression was appeared to be regulated by ambient pH. After the intron was excised, the cloned gene was inserted into an expression plasmid for yeast, pMA91, and introduced into Saccharomyces cerevisiae to be expressed. The expressed gene product was purified to a homogeneous preparation, and this confirmed that the polygalacturonase produced was the product of the cloned gene.     

Enhancement of acid amylase production by an isolated Aspergillus awamori

AIMS: Evaluation of the influence of fermentation components on extracellular acid amylase production by an isolated fungal strain Aspergillus awamori. METHODS AND RESULTS: Eight fungal metabolic influential factors, viz. soluble starch, corn steep liquor (CSL), casein, potassium dihydrogen phosphate (KH(2)PO(4)) and magnesium sulfate (MgSO(4) x 7H(2)O), pH, temperature and inoculum level were selected to optimize amylase production by A. awamori using fractional factorial design of Taguchi methodology. Significant improvement in acid amylase enzyme production (48%) was achieved. The optimized medium composition consisted of soluble starch--3%; CSL--0.5%; KH(2)PO(4)--0.125%; MgSO(4) x 7H(2)O--0.125%; casein--1.5% at pH 4.0 and temperature at 31 degrees C. CONCLUSION: Optimization of the components of the fermentation medium was carried out using fractional factorial design of Taguchi's L-18 orthogonal array. Based on the influence of interaction components of fermentation, these could be classified as the least significant and the most significant at individual and interaction levels. Least significant factors of individual level have higher interaction severity index and vice versa at enzyme production in this fungal strain. The pH of the medium and substrate (soluble starch) showed maximum production impact (60%) at optimized environment. Temperature and CSL were the least influential factors for acid amylase production. SIGNIFICANCE AND IMPACT OF THE STUDY: Acid amylase production by isolated A. awamori is influenced by the interaction of fermentation factors with fungal metabolism at individual and interaction levels. The pH of the fermentation medium and substrate concentration regulates maximum enzyme production process in this fungal strain.     

Cloning of a phenol oxidase gene from Acremonium murorum and its expression in Aspergillus awamori

Fungal multicopper oxidases have many potential industrial applications, since they perform reactions under mild conditions. We isolated a phenol oxidase from the fungus Acremonium murorum var. murorum that was capable of decolorizing plant chromophores (such as anthocyanins). This enzyme is of interest in laundry-cleaning products because of its broad specificity for chromophores. We expressed an A. murorum cDNA library in Saccharomyces cerevisiae and subsequently identified enzyme-producing yeast colonies based on their ability to decolor a plant chromophore. The cDNA sequence contained an open reading frame of 1,806 bp encoding an enzyme of 602 amino acids. The phenol oxidase was overproduced by Aspergillus awamori as a fusion protein with glucoamylase, cleaved in vivo, and purified from the culture broth by hydrophobic-interaction chromatography. The phenol oxidase is active at alkaline pH (the optimum for syringaldazine is pH 9) and high temperature (optimum, 60 degrees C) and is fully stable for at least 1 h at 60 degrees C under alkaline conditions. These characteristics and the high production level of 0.6 g of phenol oxidase per liter in shake flasks, which is equimolar with the glucoamylase protein levels, make this enzyme suitable for use in processes that occur under alkaline conditions, such as laundry cleaning.     

Xylanase and pectinase production by Aspergillus awamori on grape pomace in solid state fermentation

The feasibility of using grape pomace for the production of xylanase and exo-polygalacturonase by Aspergillus awamori in solid state fermentation has been evaluated. Solid state fermentation experiments indicated that the particle size did not influence the enzyme production. The addition of extra carbon sources and the initial moisture content of the grape pomace were found to have a marked influence on the enzymes yields. Xylanase and exo-PG activities were high at 65% (w/w) initial moisture content and glucose supplementation.     

Improved tryprostatin B production by heterologous gene expression in Aspergillus nidulans

Tryprostatin B, a prenylated diketopiperazine with anti-tubulin activity, has been overproduced in fungal culture by expression of genes of the fumitremorgin cluster from Aspergillus fumigatus in the naïve host Aspergillus nidulans using the alcA promoter. The products of the expressed genes catalyse the first two steps of fumitremorgin biosynthesis, namely the formation of brevianamide F and its conversion to tryprostatin B. Yields of tryprostatin B were up to 250 mg/l, a significant improvement in previously reported levels. This approach illustrates how the availability of fungal genome sequences and knowledge of gene function can be used to achieve the efficient production of biologically active secondary metabolites by genetic manipulation.     

Substrate-induced lipase gene expression and aflatoxin production in Aspergillus parasiticus and Aspergillus flavus

AIMS: To establish a relationship between lipase gene expression and aflatoxin production by cloning the lipA gene and studying its expression pattern in several aflatoxigenic and nontoxigenic isolates of Aspergillus flavus and A. parasiticus. METHODS AND RESULTS: We have cloned a gene, lipA, that encodes a lipase involved in the breakdown of lipids from aflatoxin-producing A. flavus, A. parasiticus and two nonaflatoxigenic A. flavus isolates, wool-1 and wool-2. The lipA gene was transcribed under diverse media conditions, however, no mature mRNA was detected unless the growth medium was supplemented with 0.5% soya bean or peanut oil or the fungus was grown in lipid-rich medium such as coconut medium. The expression of the lipase gene (mature mRNA) under substrate-induced conditions correlated well with aflatoxin production in aflatoxigenic species A. flavus (SRRC 1007) and A. parasiticus (SRRC 143). CONCLUSIONS: Substrate-induced lipase gene expression might be indirectly related to aflatoxin formation by providing the basic building block 'acetate' for aflatoxin synthesis. No direct relationship between lipid metabolism and aflatoxin production can be ascertained, however, lipase gene expression correlates well with aflatoxin formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Lipid substrate induces and promotes aflatoxin formation. It gives insight into genetic and biochemical aspects of aflatoxin formation.     

Creation of a heterologous gene expression system on the basis of Aspergillus awamori recombinant strain

A heterologous gene expression system was created in a domestic Aspergillus awamori Co-6804 strain, which is a producer of the glucoamylase gene. Vector pGa was prepared using promoter and terminator areas of the glucoamylase gene, and A. niger phytase, Trichoderma reesei endoglucanase, and Penicillium canescens xylanase genes were then cloned into pGa vector. Separation of enzyme samples using FPLC showed the amount of the recombinant proteins to be within the 0.6-14% range of total protein.     

Ethanol production from starch by immobilized Aspergillus awamori and Saccharomyces pastorianus using cellulose carriers

A simultaneous saccharification and fermentation (SSF) process was investigated to produce ethanol using two kinds of cellulose carriers that were respectively suitable for immobilization of Aspergillus awamori and Saccharomyces pastorianus. The maximum ethanol concentration attained by the batch operation was 25.5 g l⁻¹. Under suitable conditions, both cellulose carriers with immobilized cells could be reused efficiently for three cycles. The total amount of ethanol production was 66.0 g (per 1 l working volume) after the repeated operation. Ethanol productivity mainly depends on a saccharification process. There is a limit in durability in the repeated batch operation, and it is important to maintain high activity of the fungus in order to produce ethanol efficiently. Journal of Industrial Microbiology & Biotechnology (2001) 27, 52–57. Received 11 December 2000/ Accepted in revised form 02 June 2001     

Expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori

We report the expression and production of llama variable heavy-chain antibody fragments (V_HHs) by Aspergillus awamori. Fragments encoding V_HHs were cloned in a suitable Aspergillus expression vector and transformants secreting V_HH fragments were analysed for integrated gene copy-numbers, mRNA levels and protein production. Functional V_HHs were detected in the culture medium, indicating the feasibility of producing this type of protein in a fungal expression system. Secreted V_HHs were subjected to (extracellular) degradation, which could be partially prevented by the addition of BSA to the culture medium.An erratum to this article can be found at     

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.