DNA-mediated transformation system in a bipolar basidiomycete, <Emphasis Type="Italic">Pholiota microspora</Emphasis> (<Emphasis Type="Italic">P. nameko</Emphasis>)

We cloned a gene encoding the succinate dehydrogenase iron-sulfur protein subunit (sip) from a bipolar mushroom, Pholiota microspora, and introduced a point mutation that confers carboxin resistance into this gene. Using this homologous selective marker and also a heterologous drug selective marker, the hygromycin B phosphotransferase gene (hph), we successfully constructed a DNA-mediated transformation system in P. microspora. Both these selection markers have high transformation efficiency: the efficiency of carboxin resistance transformation was about 88.8 transformants/μg pMBsip2 DNA using 5 × 10⁶ protoplasts in regeneration plates containing 1.0 μg/ml carboxin, and the efficiency of hygromycin B resistance transformation was about 122.4 transformants/μg pMBhph1 DNA using 5 × 10⁶ protoplasts in regeneration plates containing 150 μg/ml hygromycin B. Southern hybridization analysis showed that the introduced sequence (mutant sip or hph) was integrated into the chromosomal DNA in these transformants with a copy number of one or more.     

Expression and Secretion of Bifidobacterium adolescentis Amylase by Bifidobacterium Longum

Bifidobacterium adolescentis Int-57 (INT57), isolated from human feces, secretes an amylase. We have shot-gun cloned, sequence analyzed and expressed the gene encoding this amylase in B. longum. The sequenced 2477 bp fragment was homologous to other extracellular amylases. The encoded protein was predicted to be composed of 595 amino acids with a molecular weight of 64 kDa, and was designated AmyB. Highly conserved amylase domains were found in AmyB. The signal sequence and cleavage site was predicted by sequence analysis. AmyB was subcloned into pBES2, a novel E. coli–Bifidobacterium shuttle vector, to construct pYBamy59. Subsequently, B. longum, with no apparent amylase activity, was transformed with pYBamy59. More than 90% of the amylase activity was detected in the culture broth. This approach may open the way for the development of more efficient expression and secretion systems for Bifidobacterium. Both authors contributed equally Received 17 June 2005; Revisions requested 13 July 2005 and 26 September 2005; Revisions received 12 September 2005 and 8 November 2005; Accepted 11 November 2005     

Production of the Artemisinin Precursor Amorpha-4,11-diene by Engineered Saccharomyces cerevisiae

The gene encoding for amorpha-4,11-diene synthase from Artemisia annua was transformed into yeast Saccharomyces cerevisiae in two fundamentally different ways. First, the gene was subcloned into the galactose-inducible, high-copy number yeast expression vector pYeDP60 and used to transform the Saccharomyces cerevisiae strain CEN·PK113-5D. Secondly, amorpha-4,11-diene synthase gene, regulated by the same promoter, was introduced into the yeast genome by homologous recombination. In protein extracts from galactose-induced yeast cells, a higher activity was observed for yeast expressing the enzyme from the plasmid. The genome-transformed yeast grows at the same rate as wild-type yeast while plasmid-carrying yeast grows somewhat slower than the wild-type yeast. The plasmid and genome-transformed yeasts produced 600 and 100 μg/l of the artemisinin precursor amorpha-4,11-diene, respectively, during 16-days’ batch cultivation. Revisions requested 14 November 2005; Revisions received 17 January 2006     

Stable RNA Interference in Spodoptera frugiperda Cells by a DNA Vector-based Method

Double-stranded RNA (dsRNA)-mediated interference (RNAi) is a powerful tool for silencing of gene expression in many organisms. To establish a DNA vector-based method for stable RNAi in Spodoptera frugiperda cells (Sf9), we created a stably transfected Sf9 cell line to express large dsRNA fragment targeting to silence the firefly luciferase gene (luc). The luc dsRNA specifically and stably suppressed the baculovirus-mediated luciferase expression. Thus, gene silencing in Sf9 cells was achieved using DNA vectors similar to the facile design described in this study. Received 21 September 2005; Revisions requested 5 October 2005; Revisions received 22 November 2005; Accepted 25 November 2005     

Improvement of Bacillus thuringiensis δ-endotoxins Synthesis Yields Through Acquisition of Erythromycin Resistance

The acquisition of the erythromycin resistance by Bacillus thuringiensis kurstaki improved yields of δ-endotoxins in sporulating cells ranging from 134 to 215%. Resistance to erythromycin decreased the final spore count by at least 50%. Consequently, erythromycin resistance is an efficient tool for the improvement of bioinsecticides yields with a high ratio of δ-endotoxins to spores. Revisions requested 31 October 2005; Revisions received 28 November 2005     

Cloning and Sequence Analysis of the Gene Encoding (6-4)photolyase from Dunaliella salina

DNA photolyase can repair UV-induced DNA damage in a light-dependent manner. A cDNA of (6-4)photolyase from Dunaliella salina (GenBank accession number: AY845324) was cloned, sequenced and its amino acid sequence was deduced. The derived amino acid sequence showed high homology with other (6-4)photolyases and a predicted 3D model was constructed by homology modeling. Revisions requested 20 May 2005 and 18 August 2005; Revisions received 2 August 2005 and 28 November 2005     

Development of Transformation System of Verticillium lecanii (Lecanicillium spp.) (Deuteromycotina: Hyphomycetes) Based on Nitrate Reductase Gene of Aspergillus nidulans

A heterologous transformation system was developed for V. lecanii based on the complementation of a nitrate reductase mutant. Nitrate reductase mutants were obtained by resistance to chlorate in a rate of 23.24% when compared to other mutations that lead to the chlorate resistance. Mutant no. 01 and 04 was chosen for the transformation experiments. Plasmid pBT was used as transformation vector containing the Aspergillus nidulans nitrate reductase gene. A frequency of approximately 3 transformants/μg DNA was obtained using the circular vector pBT. The establishment of a transformation system for V. lecanii is fundamental for genetic manipulation of this microorganism.     

Transformation of a methionine auxotrophic mutant of Mucor circinelloides by direct cloning of the corresponding wild type gene

Summary A transformation system has been developed for Mucor circinelloides, by direct cloning of a wild-type methionine gene that complements the auxotrophic mutation. The marker gene isolated was associated with an autonomous replication sequence (ARS) functional in this zygomycete. Southern hybridisation analyses of transformants showed sequence homology both with vector DNA and with Mucor wild-type DNA. The transformation frequency (up to 6000 per g DNA) and the mitotic instability of the transformed cells were studied. The hybridisation pattern of undigested DNA from the transformants suggests that the inserts contain a novel autonomous replication element for this filamentous fungus.     

Properties of Laccases Produced by Pycnoporus sanguineus Induced by 2,5-xylidine

Two isoforms of laccase produced from the culture supernatant of Pycnoporus sanguineus were partially purified by phenyl-Sepharose chromatography. Molecular masses of the enzymes were 80 kDa (Lac I) and 68 kDa (Lac II). Optimum activity of Lac I was at pH 4.8 and 30 °C, and Lac II was at pH 4.2 and 50 °C over 5 min reaction. The K_m values of enzymes toward syringaldazine were 10 μm (Lac I) and 8 μm (Lac II). Sodium azide inhibited Lac I (85%) and Lac II (75%) activities. Revisions requested 30 November 2005; Revisions received 26 January 2006     

Cloning and Production of a Novel Bacteriocin, Lactococcin K, from Lactococcus lactis Subsp. lactis MY23

A gene encoding the antimicrobial peptide, lactococcin K, was isolated from Lactococcus lactis subsp. lactis MY23 then cloned and expressed in Escherichia coli. Because the expressed lactococcin K was formed as an inclusion body in recombinant E. coli, a fusion protein containing lactococcin K and maltose-binding protein (MBP) was produced in a soluble form. For high-level production of lactococcin K, we performed a pH-stat fed-batch culture to produce 43,000 AU lactococcin K ml⁻¹ in 12 h. Revisions requested 3 November 2005; Revisions received 7 December 2005     

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     

Reduction of Cr(VI) by a Bacillus sp.

A Bacillus sp. RE was resistant to chromium and reduced Cr(VI) without accumulating chromium inside the cell. When Cr(VI) was 10 and 40 μg ml⁻¹, >95% of the total Cr(VI) was reduced in 24 and 72 h of growth, respectively, whereas at 80 μg Cr(VI) ml⁻¹ only 50% of Cr(VI) was reduced. However growth was not affected; the cell mass was 0.7–0.8 mg ml⁻¹ in all cases. The cell-free extract showed Cr(VI) reducing enzyme activity which was enhanced (>5 fold) by NADH and NADPH. Like whole cells the enzyme also reduced Cr(VI) with decreasing efficiency on increasing Cr(VI) concentration. The enzyme activity was optimal at pH 6.0 and 30 °C. The enzyme was stable up to 30 °C and from pH 5.5 to 8, but from pH 4 to 5 the enzyme was severely destabilized. Its K_m and V_max were 14 μm and 3.8 nmol min⁻¹ mg⁻¹ respectively. The enzyme activity was enhanced by Cu²⁺ and Ni²⁺ and inhibited by Hg²⁺. Received 21 September 2005; Revisions requested 5 October 2005; Revisions received 16 November 2005; Accepted 16 November 2005     

A Simple and Green Procedure for the Microbial Effective Synthesis of 1-phenylethyl Alcohol in Both Enantiomeric Forms

Both R- and S-phenylethyl alcohol of high enantiomeric purity (98%) and with a satisfactory yield (40–80%) were obtained by bioreduction of acetophenone, catalyzed by whole cells of baker’s yeast. Revisions requested 29 November 2005; Revisions received 9 January 2006     

Lactose-induced Production of Human Soluble B Lymphocyte Stimulator (hsBLyS) in E. coli with Different Culture Strategies

Over-production of human soluble B lymphocyte stimulator (hsBLyS) was carried out with four different fed-batch culture strategies using lactose as inducer, instead of IPTG, in a fed-batch culture of Escherichia coli. As lactose acted as both inducer and carbon source, the best and simplest culture strategy was direct feeding of lactose after batch culture, thereby giving hsBLyS at 3.7 g l⁻¹ and a productivity of 0.11 g l⁻¹ h⁻¹. Revisions requested 1 September 2005 and 11 November 2005; Revisions received 7 November 2005 and 4 January 2006     

Development of a homologous transformation system for Aspergillus niger based on the pyrG gene

Summary The development of a homologous transformation system for Aspergillus niger is described. The system is based on the use of an orotidine-5-phosphate decarboxylase deficient mutant (pyrG) and a vector, pAB4-1, which contains the functional A. niger pyrG gene as a selection marker. Transformation of the A. niger pyrG mutant with pAB4-1 resulted in the appearance of stable Pyr⁺ transformants at a frequency of 40 transformants per g of DNA. In 90% of these transformants integration had occurred at the resident pyrG locus, resulting either in replacement of the mutant allele by the wild-type allele (60%) or in insertion of one or two copies of the vector (40%). The A. niger pyrG mutant could also be transformed with the vector pDJB2 containing the pyr4 gene of Neurospora crassa, at a frequency of 2 transformants per g of DNA. Integration at the resident pyrG locus was not found with this vector. The vector pAB4-1 is also capable of transforming an Aspergillus nidulans pyrG mutant to Pyr⁺. The pyrG transformation system was used for the introduction of a non-selectable gene into A. niger.     

Molecular cloning of the promoter region of the glyceraldehyde-3-phosphate dehydrogenase gene that contributes to the construction of a new transformation system in <Emphasis Type="Italic">Coriolus versicolor</Emphasis>

The white-rot basidiomycete Coriolus versicolor secretes several enzymes that participate in the degradation of lignin and various persistent organic pollutants. In this study, we attempted to establish a genetic transformation system with a homogenous promoter sequence for driving the gene for antibiotic resistance. We succeeded in cloning the promoter sequence of the gene for glyceraldehyde-3-phosphate dehydrogenase (gpd), which is expressed at high levels in C. versicolor. The expression vector pT7GPTHPT was constructed, which included a gene for resistance to hygromycin B under control of the gpd promoter. The successful selection of transformants on medium that contained hygromycin B indicated that the system should be useful not only for the genetic transformation of C. versicolor, but also for the overproduction of useful fungal enzymes such as laccase and peroxidase.     

Transformation of Aspergillus oryzae using the A. niger pyrG gene

Summary A transformation system for Aspergillus oryzae based on the orotidine-5-phosphate decarboxylase gene (pyrG) was developed. Transformation frequencies of up to 16 transformants per g of DNA were obtained with the vector pAB4-1, which carries the pyrG gene of A. niger. Southern blotting analysis showed that vector DNA sequences were integrated into the chromosomal DNA, in various copy numbers and presumably at different sites. Efficient cotransformation of an unselectable gene was also shown. Under the conditions used no transformants were obtained with the equivalent pyr4 gene of Neurospora crassa.     

Heterologous expression of Fusarium oxysporum tomatinase in Saccharomyces cerevisiae increases its resistance to saponins and improves ethanol production during the fermentation of Agave tequilana Weber var. azul and Agave salmiana must

This paper describes the effect of the heterologous expression of tomatinase from Fusarium oxysporum f. sp lycopersici in Saccharomyces cerevisiae. The gene FoTom1 under the control of the S. cerevisiae phosphoglycerate kinase (PGK1) promoter was cloned into pYES2. S. cerevisiae strain Y45 was transformed with this vector and URA3 transformant strains were selected for resistance to α-tomatine. Two transformants were randomly selected for further study (designated Y45-1 and Y45-2). Control strain Y45 was inhibited at 50 μM α-tomatine, in contrast, transformants Y45-1 and Y45-2 did not show inhibition at 200 μM. Tomatinase activity was detected by HPLC monitoring tomatine disappearance and tomatidine appearance in the supernatants of culture medium. Maximum tomatinase activity was observed in the transformants after 6 h, remaining constant during the following 24 h. No tomatinase activity was detected in the parental strain. Moreover, the transformants were able to grow and produce ethanol in a mix of Agave tequilana Weber var. azul and Agave salmiana must, contrary to the Y45 strain which was unable to grow and ferment under these conditions.     

Purification and Properties of a β-1,3-glucanase from Chaetomium sp. that is Involved in Mycoparasitism

A β-1,3-glucanase was detected, using laminarin as substrate, in the culture broth of Chaetomium sp. Major activity was associated with a 70 kDa protein band visualized on a polyacrylamide gel. β-1,3-Glucanase was purified by a one-step, native gel purification procedure. Optimal activity was observed at pH 6.0 and 30 °C (over 30 min). It could degrade cell walls of plant pathogens including Rhizoctonia solani, Gibberella zeae, Fusarium sp., Colletotrichum gloeosporioides and Phoma sp. The N-terminal amino acid residues of the purified β-1,3-glucanase are PYQLQTP, which do not exhibit homology to other fungal β-1,3-glucanases suggesting it may be a novel enzyme. Received 20 July 2005; Revisions requested 2 August 2005 and 27 September 2005; Revisions received 16 September 2005 and 3 November 2005; Accepted 6 November 2005     

Inducible nickel resistance in a river isolate of India phylogenetically ascertained as a novel strain of Acinetobacter junii

Summary A gram negative, motile, short rod-shaped, and nickel resistant (tolerating 6.5 mM Ni²⁺) bacterium, strain BB1A, was isolated from the waters of the River Torsa in Hashimara, Jalpaiguri district, West Bengal, India. The isolate BB1A was identified as a strain of Acinetobacter junii following detailed analysis of morphological, physio-biochemical and 16S rRNA gene sequence. The expression of nickel resistance in BB1A was inducible by exposure to nickel chloride at a concentration as low as 50 μM Ni²⁺. The other metal ions, Cu²⁺, Zn²⁺, or Pb²⁺ at a concentration range of 20–30 μM, also induced the nickel resistance system in this bacterium. Southern hybridizations of BB1A genomic DNA with digoxigenin-dUTP labeled DNA probes specific for well known nickel resistance determinants, cnr, ncc or nre, resulted in no detectable signal, but nir specific probe yielded weak hybridization signal with restricted genomic DNA of BB1A. The isolate BB1A, therefore, carries out a novel induction phenomenon of nickel resistance and presumably with a nickel resistance genetic system different from that previously characterized in other bacteria.