Secretion, purification, and characterisation of barley α-amylase produced by heterologous gene expression in Aspergillus niger

Efficient production of recombinant barley α-amylase has been achieved in Aspergillus niger. The cDNA encoding α-amylase isozyme 1 (AMY1) and its signal peptide was placed under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter and the A. nidulans trpC gene terminator. Secretion yields up to 60 mg/l were obtained in media optimised for α-amylase activity and low protease activity. The recombinant AMY1 (reAMY1) was purified to homogeneity and found to be identical to native barley AMY1 with respect to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the endogenous plant signal peptide is correctly processed in A. niger. Electrospray ionisation/mass spectrometry gave a molecular mass for the dominant form of 44 960 Da, in accordance with the loss of the LQRS C-terminal residues; glycosylation apparently did not occur. The activities of recombinant and native barley α-amylases are very similar towards insoluble and soluble starch as well as 2-chloro-4-nitrophenol β-d-maltoheptaoside and amylose (degree of polymerisation = 17). Barley α-amylase is the first plant protein efficiently secreted and correctly processed by A. niger using its own signal sequence. Received: 22 August 1997 / Received revision: 21 November 1997 / Accepted: 29 November 1997     

Improved α-amylase production by Aspergillus oryzae after a double deletion of genes involved in carbon catabolite repression

In filamentous fungi, the expression of secretory glycoside hydrolase encoding genes, such as those for amylases, cellulases, and xylanases, is generally repressed in the presence of glucose. CreA and CreB have been observed to be regulating factors for carbon catabolite repression. In this study, we generated single and double deletion creA and/or creB mutants in Aspergillus oryzae. The α-amylase activities of each strain were compared under various culture conditions. For the wild-type strain, mRNA levels of α-amylase were markedly decreased in the later stage of submerged culture under inducing conditions, whereas this reduced expression was not observed for single creA and double creA/creB deletion mutants. In addition, α-amylase activity of the wild-type strain was reduced in submerged culture containing high concentrations of inducing sugars, whereas all constructed mutants showed higher α-amylase activities. In particular, the α-amylase activity of the double deletion mutant in a medium containing 5 % starch was >10-fold higher than that of the wild-type strain under the same culture conditions. In solid-state cultures using wheat bran as a substrate, the α-amylase activities of single creA and double deletion mutants were >2-fold higher than that of the wild-type strain. These results suggested that deleting both creA and creB resulted in dramatic improvements in the production of secretory glycoside hydrolases in filamentous fungi.     

Formosusin A,a novel specific inhibitor of mammalian DNA polymerase β from the fungus Paecilomyces formosus

Variotin (1) and three novel compounds, formosusin A (2), B (3), and C (4), were isolated from the cultures of the fungus Paecilomyces formosus, and their structures were determined by spectroscopic analyses. Compound 2 is (6Z,8E,10E)-variotin, a new cis-olefin analog of compound 1. Compound 2 selectively inhibited the activity of mammalian DNA polymerase β (pol β) in vitro, with an IC₅₀ of 35.6 μM. By contrast, compounds 1, 3, and 4 did not influence the activity of pol β. These four compounds showed no effect on the activities of other 10 mammalian pols (i.e., pols α, γ, δ, ε, η, ι, κ, λ, and μ, and terminal deoxynucleotidyl transferase). These compounds also did not inhibit the activities of fish, insect, plant, and prokaryotic pols and other DNA metabolic enzymes tested. These results suggested that compound 2 could be a selective inhibitor of mammalian pol β. The compound 2-induced inhibition of rat pol β activity was competitive and non-competitive with respect to the DNA template–primer substrate and the dNTP substrate, respectively. On the basis of these results, the relationship between the three-dimensional structure and pol β inhibitory mechanism of compound 2 is discussed.     

Production of novel antioxidative phenolic amides through heterologous expression of the plant’s chlorogenic acid biosynthesis genes in yeast

Phenolic esters like chlorogenic acid play an important role in therapeutic properties of many plant extracts. We aimed to produce phenolic esters in baker’s yeast, by expressing tobacco 4CL and globe artichoke HCT. Indeed yeast produced phenolic esters. However, the primary product was identified as N-(E)-p-coumaroyl-3-hydroxyanthranilic acid by NMR. This compound is an amide condensation product of p-coumaric acid, which was supplied to the yeast, with 3-hydroxyanthranilic acid, which was unexpectedly recruited from the yeast metabolism by the HCT enzyme. N-(E)-p-coumaroyl-3-hydroxyanthranilic acid has not been described before, and it shows structural similarity to avenanthramides, a group of inflammation-inhibiting compounds present in oat. When applied to mouse fibroblasts, N-(E)-p-coumaroyl-3-hydroxyanthranilic acid induced a reduction of intracellular reactive oxygen species, indicating a potential therapeutic value for this novel compound.     

Indigestible dextrin is an excellent inducer for α-amylase, α-glucosidase and glucoamylase production in a submerged culture of Aspergillus oryzae

α-Amylase activities of Aspergillus oryzae grown on dextrin or indigestible dextrin were 7·8 and 27·7 U ml⁻¹, respectively. Glucoamylase activities of the cultures grown on dextrin or indigestible dextrin were 5·4 and 301 mU ml⁻¹, respectively. The specific glucoamylase production rate in indigestible dextrin batch culture reached 1·35 U g DW⁻¹ h⁻¹. In contrast, biomass concentration of A. oryzae in indigestible dextrin culture was 35% of that in dextrin culture. Thus, the culture method using indigestible dextrin has the potential to improve amylolytic enzyme production and fungal fermentation broth rheology.     

Contribution Ratios of amyA, amyB, amyC Genes to High-Level α-Amylase Expression in Aspergillus oryzae

Aspergillus oryzae strains express α-amylases abundantly, and the genome reference strain RIB40 has three α-amylase genes (amyA, amyB, and amyC). However, there is no information on the contribution ratios of individual α-amylase genes to total expression. In this study, we generated single, double, and triple disruptants of α-amylase genes by employing a strain (ΔligD) with high gene-targeting efficiency and pyrG marker recycling in A. oryzae. All the disruptants showed reduced activities of α-amylases, and the triple disruptant completely lost activity. Comparative analyses of the activities and mRNA amounts of the α-amylases suggest that the contribution of amyA to the α-amylase expression is smaller than those of amyB and amyC. The present study suggests that the ability to express a large amount of α-amylases in A. oryzae is attributed to gene duplication of genes such as amyB and amyC.     

Overproduction of an α-Amylase/Glucoamylase Fusion Protein in Aspergillus oryzae Using a High Expression Vector

The systemic immune response against orally administered antigens is suppressed (oral tolerance), and this has been postulated to avoid excess immunity against dietary constituents which are present in large amounts in the gastrointestinal tract. Taking into consideration that such orally administered protein antigens are subjected to enzymatic degradation in the gastrointestinal tract, we examined whether an enzymatic digest of milk proteins could induce oral tolerance. A tryptic digest of casein, containing mainly fragments smaller than 6000 Da, was fed to mice as a constituent of their diet. Mice fed with the casein-digest diet responded poorly to subsequent immunization with casein, indicating that oral tolerance to casein was induced in these animals. The results suggest the presence of immunosuppressive fragment(s) in the casein digest, which may be of use for preventing milk allergy.     

Further enhanced production of heterologous proteins by double-gene disruption (ΔAosedD ΔAovps10) in a hyper-producing mutant of Aspergillus oryzae

The filamentous fungus Aspergillus oryzae is used as one of the most favored hosts for heterologous protein production due to its ability to secrete large amounts of proteins into the culture medium. We previously generated a hyper-producing mutant strain of A. oryzae, AUT1, which produced 3.2- and 2.6-fold higher levels of bovine chymosin (CHY) and human lysozyme (HLY), respectively, compared with the wild-type strain. However, further enhancement of heterologous protein production by multiple gene disruption is difficult because of the low gene-targeting efficiency in strain AUT1. Here, we disrupted the ligD gene, which is involved in nonhomologous recombination, and the pyrG gene to create uridine/uracil auxotrophy in strain AUT1, to generate a hyper-producing mutant applicable to pyrG marker recycling with highly efficient gene targeting. We generated single and double disruptants of the tripeptidyl peptidase gene AosedD and vacuolar sorting receptor gene Aovps10 in the hyper-producing mutant background, and found that all disruptants showed significant increases in heterologous protein production. Particularly, double disruption of the Aovps10 and AosedD genes increased the production levels of CHY and HLY by 1.6- and 2.1-fold, respectively, compared with the parental strain. Thus, we successfully generated a fungal host for further enhancing the heterologous protein production ability by combining mutational and molecular breeding techniques.     

Buchnera aphidicola,the endosymbiont of aphids,contains genes for four ribosomal RNA proteins,initiation factor-3, and the α-subunit of RNA polymerase

Buchnera aphidicola is a prokaryotic endosymbiont of the aphidSchizaphis graminum. With the polymerase chain reaction (PCR) and oligonucleotide primers to conserved regions, two DNA fragments of the endosymbiont -operon and L20 operon were amplified, cloned intoEscherichia coli, and their sequences were determined. The results indicated that the organization of the endosymbiont genes on these fragments was identical with that of the corresponding operons ofE. coli. The 1032 base pair (bp) fragment of the -operon contained the genes for small ribosomal subunit proteins S11 and S4, followed by the gene for the -subunit of RNA polymerase (-RNAP). The 702-bp fragment of the L20-operon contained the genes for initiation factor-3 (IF3) and large ribosomal subunit proteins L35 and L20. As in other prokaryotes, the genes of the -operon and the L20-operon were present as single copies in the genome ofB. aphidicola. Comparisons of the amino acid sequences of these proteins were consistent with the previously established close relationship betweenB. aphidicola andE. coli and a distant relationship to species ofBacillus.     

High-level soluble expression, purification, and functional characterization of the recombinant human leukemia inhibitory factor: a potential general strategy for the recombinant expression of cytokines consisting of four α-helices in a bundle

The human leukemia inhibitory factor (hLIF) is one of the most important cytokines in the interleukin-6 (IL-6) cytokine family. Numerous studies have demonstrated that hLIF is a pleiotropic cytokine with multiple effects on different types of cells and tissues. The optimal chemical synthesis of the hLIF gene has been previously reported to increase the expression of the recombinant inclusion body protein in E. coli. However, the required refolding step limits the recovery rate. In this report, a novel strategy was designed to produce a soluble recombinant human LIF (rhLIF) in the prokaryotic system in order to obtain higher yields of the bioactive protein with simpler steps. This optimal hLIF gene was cloned, and it successfully expressed the soluble recombinant protein in E. coli using the thioredoxin (Trx) protein as a fusion partner. A simple purification procedure is established to purify the recombinant fusion protein from the soluble supernatant of the lysed culture cells. This procedure yields up to 5 mg/L rhLIF with above 95? purity. The strategy allows the protease to release target cytokines without additional N-terminus amino acids, which is an important consideration for maintaining its bioactivity. Functional analysis of the purified rhLIF by murine myeloblastic leukemia M1 cell proliferation assay demonstrates biological activity that is similar and comparable to that of hLIF. These results present a sound strategy for the soluble production of rhLIF and other homologous tertiary structure cytokines consisting of four α-helices in a bundle for basic research, as well as clinical applications.     

Site-directed mutagenesis and expression inEscherichia coli of WMAI-1, a wheat monomeric inhibitor of insect α-amylase

The wheat monomeric inhibitor WMAI-1 (syn. 0.28) produced inEscherichia coli using the pT7-7 expression ventor has the correct N-terminal sequence and the same electrophoretic mobility and specific activity towards the -amylase from the insectTenebrio molitor as the native WMAI-1 isolated from wheat. This confirms that the native inhibitor is not glycosylated and contradicts claims that a putative glycosyl moiety was essential for inhibition. Thirteen mutants have been obtained at six different sites. Substitution of the highly conserved N-terminal S by the sequence ARIRAR increased the pre-incubation time required for maximum activity. A similar result was obtained by insertion of GPRLPW after position 4, while insertion of EPRAPW at the same position rendered the inhibitor inactive. The substitution D/EGPRL and insertions DGP or D, at position 58, produced complete inactivation. All other mutations had only minor effects on activity.     

Cloning of cDNA,expression, and chromosomal location of genes encoding the three types of subunits of the barley tetrameric inhibitor of insect α-amylase

Three cDNA clones from barley developing endosperm, corresponding to proteins BTAI-CMa, BTAI-CMb and BTAI-CMd, which are the three types of subunits of the tetrameric inhibitor of insect -amylases, have been identified and sequenced. The deduced amino acid sequence of BTAI-CMb corresponds to the CM16/CM17 type of subunit in wheat (92/90% identical residues) and has one putative N-glycosylation site (NLT) and a possible kinase-C phosphorylation site (SCR). The BTAI-CMa sequence differs at four amino acid residues from a previously reported one from cv. Bomi and the sequence deduced for BTAI-CMd completes (11 N-terminal residues) and confirms previously available data. The gene for BTAI-CMa (Iat1) is located in the arm of barley chromosome 7H (syn.1), while genes for both BTAI-CMb (Iat2) and BTAI-CMd (Iat3) are in the long arm of chromosome 4H. The three genes are expressed in endosperm and their mRNAs are not detected in the other tissues tested, except Iat1, which seems to be expressed at a low level in coleoptile and roots, where it is switched off by 50 M methyl jasmonate.     

The effects of dehydroaltenusin,a novel mammalian DNA polymerase α inhibitor,on cell proliferation and cell cycle progression

As described previously, a natural product isolated from fungus (Acremonium sp.), dehydroaltenusin, is an inhibitor of mammalian DNA polymerase α in vitro [Y. Mizushina, S. Kamisuki, T. Mizuno, M. Takemura, H. Asahara, S. Linn, T. Yamaguchi, A. Matsukage, F. Hanaoka, S. Yoshida, M. Saneyoshi, F. Sugawara, K. Sakaguchi, Dehydroaltenusin, a mammalian DNA polymerase α inhibitor, J. Biol. Chem. 275 (2000) 33957_33961]. In this study, we investigated the interaction of dehydroaltenusin with lipid bilayers using an in vitro liposome system, which is a model of the cell membrane, and found that approximately 4% of dehydroaltenusin was incorporated into liposomes. We also investigated the influence of dehydroaltenusin on cultured cancer cells. Dehydroaltenusin inhibited the growth of HeLa cells with an LD₅₀ value of 38 μM, and as expected, S phase accumulation in the cell cycle. The total DNA polymerase activity of the extract of incubated cells with dehydroaltenusin was 23% lower than that of nontreated cells. Dehydroaltenusin increased cyclin E and cyclin A levels. In the analysis of the cell cycle using G1/S synchronized cells by employing hydroxyurea, the compound delayed both entry into the S phase and S phase progression. In a similar analysis using G2/M synchronized cells by employing nocodazole, the compound accumulated the cells at G1/S and inhibited entry into the S phase. Thus, the pharmacological abrogation of cell proliferation by dehydroaltenusin may prove to be an effective chemotherapeutic agent against tumors.     

Substantial decrease in cell wall α-1,3-glucan caused by disruption of the kexB gene encoding a subtilisin-like processing protease in Aspergillus oryzae

Disruption of the kexB encoding a subtilisin-like processing protease in Aspergillus oryzae (ΔkexB) leads to substantial morphological defects when the cells are grown on Czapek-Dox agar plates. We previously found that the disruption of kexB causes a constitutive activation of the cell wall integrity pathway. To understand how the disruption of the kexB affects cell wall organization and components, we analyzed the cell wall of ΔkexB grown on the plates. The results revealed that both total N-acetylglucosamine content, which constitutes chitin, and chitin synthase activities were increased. Whereas total glucose content, which constitutes β-1,3-glucan and α-1,3-glucan, was decreased; this decrease was attributed to a remarkable decrease in α-1,3-glucan. Additionally, the β-1,3-glucan in the alkali-insoluble fraction of the ΔkexB showed a high degree of polymerization. These results suggested that the loss of α-1,3-glucan in the ΔkexB was compensated by increases in the chitin content and the average degree of β-1,3-glucan polymerization.     

Increased enzyme production under liquid culture conditions in the industrial fungus Aspergillus oryzae by disruption of the genes encoding cell wall α-1,3-glucan synthase

Under liquid culture conditions, the hyphae of filamentous fungi aggregate to form pellets, which reduces cell density and fermentation productivity. Previously, we found that loss of α-1,3-glucan in the cell wall of the fungus Aspergillus nidulans increased hyphal dispersion. Therefore, here we constructed a mutant of the industrial fungus A. oryzae in which the three genes encoding α-1,3-glucan synthase were disrupted (tripleΔ). Although the hyphae of the tripleΔ mutant were not fully dispersed, the mutant strain did form smaller pellets than the wild-type strain. We next examined enzyme productivity under liquid culture conditions by transforming the cutinase-encoding gene cutL1 into A. oryzae wild-type and the tripleΔ mutant (i.e. wild-type-cutL1, tripleΔ-cutL1). A. oryzae tripleΔ-cutL1 formed smaller hyphal pellets and showed both greater biomass and increased CutL1 productivity compared with wild-type-cutL1, which might be attributable to a decrease in the number of tripleΔ-cutL1 cells under anaerobic conditions.     

Role of the side chain stereochemistry in the α-glucosidase inhibitory activity of kotalanol, a potent natural α-glucosidase inhibitor

Synthesis and evaluation of four diastereomers (9a, 9b, 9c and 9d) of kotalanol, a potent α-glucosidase inhibitor isolated from an Ayurvedic medicinal plant Salacia species, are described. Stereo-inversion at C-3' and C-4' of kotalanol (2) caused significant decrease of the inhibitory activities against maltase and sucrase, whereas inhibitory activity against isomaltase sustained, thus resulted in exerting selectivity against isomaltase.     

First synthesis of (S)-(+)-hymenoic acid,a DNA polymerase λ inhibitor isolated from Hymenochaetaceae sp

Hymenoic acid, isolated from cultures of the fungus, Hymenochaetaceae sp., is a specific inhibitor of DNA polymerase λ. The first synthesis of (S)-(+)-hymenoic acid was achieved by starting from trans-1,4-cyclohexanedimethanol and methyl (R)-(?)-3-hydroxyisobutyrate, and Julia–Kocienski olefination was employed as the key step.