Protein A as a fusion partner for the expression of heterologous proteins in Lactobacillus

An expression system based on the Staphylococcus aureus protein A gene (spa) was developed to allow the production and export of proteins in Lactobacillus. Plasmid shuttle vectors were constructed that carried the eZZ gene, a synthetic gene based on the Protein A gene (spa) but lacking the carboxy-terminal membrane-anchoring region. A gene fusion was created between the eZZ gene and the VD4 region of a chlamydial major outer-membrane protein gene. Expression studies demonstrated the recognition of the spa regulatory signals by several Lactobacillus, with the recombinant protein being expressed (from 0.1 μg of EZZVD4 fusion protein per ml in L. plantarum up to 10 μg of EZZ protein per ml in L. fermentum) and exported (levels up to 20% in L. fermentum) in several Lactobacillus strains. Received: 27 August 1996 / Received revision: 27 November 1996 / Accepted: 29 November 1996     

Cloning and expression of the Bacillus sphaericus 2362 mosquitocidal genes in a non-toxic unicellular cyanobacterium, Synechococcus PCC6301

Genes encoding the mosquitocidal binary toxin of Bacillus sphaericus 2362 were introduced into Synechococcus PCC6301, a cyanobacterium that can tolerate a number of potential variations in the mosquito breeding environment, and can serve as a food source for mosquito larvae. The toxin genes, preceded by a Synechococcus rbcL promoter, were located on a mobilizable Escherichia coli Synechococcus shuttle vector, which was introduced into Synechococcus PCC6301 at frequencies of 10⁻⁵–10⁻⁷ exconjugants/recipient, depending on the selective conditions used. Recombinant Synechococcus exhibited significant toxicity against 2-day-old and 6-day-old Culex quinquefasciatus larvae, the concentration required to kill 50 % of larvae (LC₅₀) being 2.1 × 10⁵ and 1.3 × 10⁵ cells/ml respectively. Mosquitocidal activity decreased tenfold after 20 generations of non-selective growth. Received: 23 July 1996 / Received revision: 11 November 1996 / Accepted: 15 November 1996     

A novel nuclear factor, SREB, binds to a cis-acting element, SRE, required for inducible expression of the Aspergillus oryzae Taka-amylase A gene in A. nidulans

The Taka-amylase A gene (taaG2) of Aspergillus oryzae is inducibly expressed in A. nidulans upon exposure to inducing carbon sources, such as starch and maltose. In order to identify nuclear factor(s) possibly involved in the induction of the taaG2 gene, gel mobility shift assays and DNase I footprinting analyses were carried out, and revealed a novel nuclear factor in A. nidulans extracts, which specifically bound to two sites in the taaG2 promoter region, −204 to −189 and −182 to −168, which share the common sequence GGAAATT. The nuclear factor was detected in nuclei from both induced and uninduced mycelia. Mutational analysis within and around the binding sequences demonstrated that only the upstream binding sequence, designated SRE (starch responsive element), was required for the inducible expression of the taaG2 gene, and thus we designated the nuclear factor SREB (SRE binding factor). The downstream binding site contained an inverted SRE (ISRE) and played no role in the induction of taaG2 expression. SREB was shown by gel retardation assays to have higher affinity for SRE than for ISRE. Received: 26 January 1999 / Accepted: 10 November 1999     

α-Amylase production in high cell density submerged cultivation of Aspergillus oryzae and A. nidulans

The effect of biomass concentration on the formation of Aspergillus oryzaeα-amylase during submerged cultivation with A. oryzae and recombinant A. nidulans strains has been investigated. It was found that the specific rate of α-amylase formation in chemostats decreased significantly with increasing biomass concentration in the range of approx. 2–12 g dry weight kg⁻¹. When using a recombinant A. nidulans strain in which the gene responsible for carbon catabolite repression of the A. oryzaeα-amylase gene (creA) was deleted, no significant decrease in the specific rate of α-amylase formation was observed. On the basis of the experimental results, it is suggested that the low value of the specific α-amylase productivity observed at high biomass concentration is caused by slow mixing of the concentrated feed solution in the viscous fermentation medium. Received: 13 January 2000 / Received revision: 30 June 2000 / Accepted: 1 July 2000     

Induction of xylanase in Aspergillus tamarii by methyl β-d-xyloside

Aspergillus tamarii produced extracellular xylanase and intracellular β-xylosidase inductively in washed glucose-grown mycelia incubated with xylan and methyl β-d-xyloside, a synthetic glycoside. Methyl β-d-xyloside was a more effective inducer than xylan at the same concentration for both enzymes. Glucose and cycloheximide were found to inhibit xylanase production by methyl β-d-xyloside. Methyl β-d-xyloside was hydrolyzed to xylose by mycelial extract in vitro. Received: 23 May 1996 / Received revision: 5 September 1996 / Accepted: 13 October 1996     

An aureobasidin A resistance gene isolated from Aspergillus is a homolog of yeast AUR1, a gene responsible for inositol phosphorylceramide (IPC) synthase activity

The AUR1 gene of Saccharomyces cerevisiae, mutations in which confer resistance to the antibiotic aureobasidin A, is necessary for inositol phosphorylceramide (IPC) synthase activity. We report the molecular cloning and characterization of the Aspergillus nidulans aurA gene, which is homologous to AUR1. A single point mutation in the aurA gene of A. nidulans confers a high level of resistance to aureobasidin A. The A. nidulans aurA gene was used to identify its homologs in other Aspergillus species, including A. fumigatus, A. niger, and A. oryzae. The deduced amino acid sequence of an aurA homolog from the pathogenic fungus A. fumigatus showed 87% identity to that of A. nidulans. The AurA proteins of A. nidulans and A. fumigatus shared common characteristics in primary structure, including sequence, hydropathy profile, and N-glycosylation sites, with their S. cerevisiae, Schizosaccharomyces pombe, and Candida albicans counterparts. These results suggest that the aureobasidin resistance gene is conserved evolutionarily in various fungi. Received: 27 August 1998 / Accepted: 19 November 1998     

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     

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. Received: 7 October 1997 / Received revision: 21 November 1997 / Accepted: 21 November 1997     

An Aspergillus nidulans nuclear protein, AnCP, involved in enhancement of Taka-amylase A gene expression, binds to the CCAAT-containing taaG2 , amdS , and gatA promoters

Using AnCP (Aspergillus nidulans CCAAT-binding protein) as a CCAAT-specific binding factor model, the possibility that one factor is able to recognize CCAAT sequences in several different genes in A.␣nidulans was examined. DNase I protection analysis showed that AnCP specifically bound to CCAAT sequence-containing regions comprising 21 to 36 bp of the taa, amdS and gatA genes. Furthermore, replacement of the CCAAT sequence with CGTAA was found to abolish the binding of AnCP and to have an inhibitory effect on taa promoter activity. This clearly demonstrates a positive function of the CCAAT element. However, amylase was induced by starch and repressed by glucose in a CCAAT-box disruptant, as in wild-type cells. Received: 28 June 1996 / Accepted: 7 October 1996     

Localization and characterization of inclusion bodies in recombinant Escherichia coli cells overproducing penicillin G acylase

Various concentrations of isopropyl β-d-thiogalactopyranoside (IPTG) were used to induce production of the enzyme penicillin G acylase by recom binant Escherichia coli harboring plasmid pQEA11. The plasmid pQEA11 carries a wild-type pga gene, which is under the control of the tac promoter and lacI ^q. At low IPTG concentrations (0.025 – 0.1 mM), enzyme activity increased with increasing IPTG concentrations. At higher IPTG concentrations (0.2 and 0.5 mM), enzyme activity declined progressively. Examination of induced recombinant E. coli cells by transmission electron microscopy showed the presence of only periplasmic inclusion bodies at low IPTG concentrations (up to 0.1 mM) and both periplasmic and cytoplasmic inclusion bodies at high IPTG concentrations (0.2 mM and 0.5 mM). Results from sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immunoblots of whole-cell proteins, membrane proteins and inclusion body proteins in these cells indicated that cytoplasmic inclusion bodies constituted an accumulation of preproenzyme (i.e., precursor polypeptide containing a signal peptide) and that periplasmic inclusion bodies constituted an accumulation of proenzyme (i.e., precursor polypeptide lacking a signal peptide). Received: 27 March 1996 / Received revision: 2 July 1996 / Accepted: 10 November 1996     

Molecular cloning and characterization of a gene encoding glutaminase from Aspergillus oryzae

 A glutaminase from Aspergillus oryzae was purified and its molecular weight was determined to be 82,091 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Purified glutaminase catalysed the hydrolysis not only of l-glutamine but also of d-glutamine. Both the molecular weight and the substrate specificity of this glutaminase were different from those reported previously [Yano et al. (1998) J Ferment Technol 66: 137–143]. On the basis of its internal amino acid sequences, we have isolated and characterized the glutaminase gene (gtaA) from A. oryzae. The gtaA gene had an open reading frame coding for 690 amino acid residues, including a signal peptide of 20 amino acid residues and a mature protein of 670 amino acid residues. In the 5′-flanking region of the gene, there were three putative CreAp binding sequences and one putative AreAp binding sequence. The gtaA structural gene was introduced into A. oryzae NS4 and a marked increase in activity was detected in comparison with the control strain. The gtaA gene was also isolated from Aspergillus nidulans on the basis of the determined nucleotide sequence of the gtaA gene from A. oryzae. Received: 23 August 1999 / Received last revision: 7 January 2000 / Accepted: 14 January 2000     

Molecular mass of poly[(R )-3-hydroxybutyric acid] produced in a recombinant Escherichia coli

 Poly[(R)-3-hydroxybutyric acid] (PHB) was produced at 37 °C by a recombinant Escherichia coli harboring the Alcaligenes eutrophus biosynthesis phbCAB genes in Luria-Bertani media containing glucose at 10–30 g/l at different pH values and the time-dependent changes in the molecular mass of PHB were studied. PHB polymers accumulated within cells while glucose was present in the medium. The number-average molecular mass of PHB decreased with time during the course of PHB accumulation, and the values for PHB were markedly dependent on the cultivation conditions of the E. coli, ranging from 0.5 MDa to 20 MDa. Under specific conditions (pH 6.0), E. coli produced PHB with an extremely high molecular mass (20 MDa). It has been suggested that a chain-transfer agent is generated in E. coli cells during the accumulation of PHB. Received: 18 July 1996 / Received revision: 4 November 1996 / Accepted: 4 November 1996     

The NADP-dependent glutamate dehydrogenase gene from Penicillium chrysogenum and the construction of expression vectors for filamentous fungi

The gdhA gene encoding the NADP-dependent glutamate dehydrogenase activity from Penicillium chrysogenum has been isolated and characterized for its use in gene expression. The nucleotide sequence of a 2816-bp genomic fragment was determined, showing an open reading frame of 1600 bp interrupted by two introns, of 160 bp and 57 bp respectively, with fungal consensus splice-site junctions. The predicted amino acid sequence revealed a high degree of identity to glutamate dehydrogenase enzymes, especially to those from the fungi Aspergillus nidulans (82%) and Neurospora crassa (78%). The gdhA gene was found to be present in a single copy in the genome of several P. chrysogenum strains with different penicillin productivity. The use of the gdhA promoter for homologous and heterologous gene expression in fungi and Escherichia coli was analyzed. Heterologous gene expression was ascertained by the construction of gene fusions with the lacZ gene from E. coli and the bleomycin-resistance determinant (ble ^R) from Streptoalloteichus hindustanus. Homologous gene expression was shown through the use of the penicillin-biosynthetic genes pcbC and penDE from P. chrysogenum and the cephalosporin biosynthetic genes cefEF and cefG from Acremonium chrysogenum. Received: 2 November 1998 / Received revision: 15 January 1999 / Accepted: 5 March 1999     

Induction of submerged conidiation of the biocontrol agent Penicillium oxalicum

Induction of submerged conidiation of Penicillium oxalicum has been examined using a range of synthetic and complex media and complex media supplemented with by-products of the brewing industry. Only one method (Morton's method), consisting of growth in a glucose/salts-based medium (C:N ratio 62.5, medium A) for 24 h and then transference to the same medium without a nitrogen source (medium B), induced conidiation. Levels of sporulation were significantly (P = 0.05) increased by addition of calcium or poly(ethylene glycol) 6000 to medium B. The optimum age for transference of the mycelium was 24 h and the optimum pH was 6. Calcium was an induction factor when added to medium A (C:N ratio 62.5) of Morton's method. It was concluded that nitrogen depletion and calcium addition to a medium with high C:N ratio are the factors inducing conidiation of P. oxalicum. Maximum levels of conidiation (35 × 10⁶ spores ml⁻¹) were obtained when the nitrogen level in medium A of Morton's method was further reduced (C:N ratio 142.9) and calcium (20 mM) was added. These results are the essential starting point to investigate liquid fermentation systems for the biocontrol agent P. oxalicum. Received: 19 November 1996 / Received revision: 25 March 1997 / Accepted: 27 March 1997     

Cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12

A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxymethylcellulose. Three plasmids conferring cellulase activity were purified and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan, but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a nickel nitrilotriacetate column. The enzyme had a pH optimum of 6–7 and its highest measured initial activity at 100 °C. The heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after 8 h at 90 °C. Received: 5 August 1997 / Received revision: 6 November 1997 / Accepted: 7 November 1997     

The Schizosaccharomyces pombe cdc6 gene encodes the catalytic subunit of DNA polymerase δ

The cdc6 mutants of Schizosaccharomyces pombe have been classified as being defective in progression through the G2 phase of the cell cycle. We cloned an S. pombe gene that could complement the temperature-sensitive growth of the cdc6-23 mutant. Unexpectedly, the cloned gene was allelic to pol3, which encodes the catalytic subunit of DNA polymerase δ. Integration mapping confirmed that cdc6 and pol3 are identical. The cdc6-23 mutant carries one amino acid substitution in the conserved N3 region of Pol3. Received: 17 October 1996 / Accepted: 19 November 1996     

A new arabinofuranohydrolase from Bifidobacterium adolescentis able to remove arabinosyl residues from double-substituted xylose units in arabinoxylan

An arabinofuranohydrolase (AXH-d3) was purified from a cell-free extract of Bifidobacterium adolescentis DSM 20083. The enzyme had a molecular mass of approximately 100 kDa as determined by gel filtration. It displayed maximum activity at pH 6 and 30 °C. Using an arabinoxylan-derived oligosaccharide containing double-substituted xylopyranosyl residues established that the enzyme specifically released terminal arabinofuranosyl residues linked to C-3 of double-substituted xylopyranosyl residues. In addition, this arabinofuranohydrolase released arabinosyl groups from wheat flour arabinoxylan polymer but showed no activity towards p-nitrophenyl α-l-arabinofuranoside or towards sugar-beet arabinan, soy arabinogalactan, arabino-oligosaccharides and arabinogalacto-oligosaccharides. Received: 15 July 1996 / Received revision: 18 October 1996 / Accepted: 18 October 1996     

Biodegradation of methyl violet by Pseudomonas mendocina MCM B-402

Pseudomonas mendocina MCM B-402 was found to utilize a triphenylmethane dye, methyl violet as the sole source of carbon when incorporated in synthetic medium. Almost complete decolorization of methyl violet by P. mendocina was observed within 48 h of incubation at ambient temperature (28 ± 2 °C) under aerated culture conditions, when the bacteria were inoculated into Davis Mingioli's synthetic medium at a concentration of 100 mg/l medium. Methyl violet was mineralized to CO₂ through three unknown intermediate metabolites and phenol. The decolorization of the dye involved demethylation. Received: 27 November 1998 / Received revision: 2 March 1999 / Accepted: 5 March 1999