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Applied Microbiology and Biotechnology (v.80, #4)

Lysostaphin: an antistaphylococcal agent by Jaspal K. Kumar (pp. 555-561).

Lysostaphin is a zinc metalloenzyme which has a specific lytic action against Staphylococcus aureus. Lysostaphin has activities of three enzymes namely, glycylglycine endopeptidase, endo-β-N-acetyl glucosamidase and N-acteyl muramyl-l-alanine amidase. Glycylglycine endopeptidase specifically cleaves the glycine–glycine bonds, unique to the interpeptide cross-bridge of the S. aureus cell wall. Due to its unique specificity, lysostaphin could have high potential in the treatment of antibiotic-resistant staphylococcal infections. This review article presents a current understanding of the lysostaphin and its applications in therapeutic agent as a treatment against antibiotic-resistant S. aureus and methicillin-resistant S. aureus (MRSA) infections, either alone or in combination with other antibiotics.

Keywords: Staphylococcus aureus ; MRSA; Lysostaphin; Therapeutics

Novel roles of Bacillus thuringiensis to control plant diseases by Yi Zhou; Yong-Lark Choi; Ming Sun; Ziniu Yu (pp. 563-572).

Bacillus thuringiensis is well known as an effective bio-insecticidal bacterium. However, the roles of B. thuringiensis to control plant diseases are not paid great attention to. In recent years, many new functions in protecting plants from pathogen infection have been discovered. For example, acyl homoserine lactone lactonase produced by B. thuringiensis can open the lactone ring of N-acyl homoserine lactone, a signal molecule in the bacterial quorum-sensing system. This in turn, significantly silences bacterial virulence. This finding resulted in the development of a new strategy against plant bacterial diseases by quenching bacterial quorum sensing. Another new discovery about B. thuringiensis function is zwittermicin A, a linear aminopolyol antibiotic with high activity against the Oomycetes and their relatives, as well as some gram-negative bacteria. This paper summarized the relative progresses of B. thuringiensis in plant disease control and its favorable application prospects.

Keywords: Bacillus thuringiensis ; Acyl homoserine lactonase (AHL lactonase); Zwittermicin A

Expression of ethylene-forming enzyme (EFE) of Pseudomonas syringae pv. glycinea in Trichoderma viride by Li Tao; Hong-Jun Dong; Xi Chen; San-Feng Chen; Tian-Hong Wang (pp. 573-578).

The efe gene encoding an ethylene-forming enzyme from Pseudomonas syringae pv. glycinea has been expressed for the first time under the control of Trichoderma reesei cbh1 promoter in Trichoderma viride. Reverse transcription polymerase chain reaction analysis showed that transformant Y2 produced mRNA of the efe gene. Southern blot analysis showed that there was one copy of efe gene which was integrated into the chromosomal DNA of T. viride. Ethylene production by transformant Y2 was efficiently induced by cellulose, while very low level of ethylene was produced when sodium carboxymethyl cellulose or lactose was used as carbon source. Peptone exerted a much greater stimulatory effect on ethylene production. A high level of ethylene was produced when transformant Y2 was cultured in solid fermentation medium containing wheat straw, indicating that plant wastes could be directly converted to ethylene by the recombinant filamentous fungus.

Keywords: Ethylene; Ethylene-forming enzyme (EFE); cbh1 promoter; Trichoderma viride ; Pseudomonas syringae

Production of the aroma chemicals 3-(methylthio)-1-propanol and 3-(methylthio)-propylacetate with yeasts by M. M. W. Etschmann; P. Kötter; J. Hauf; W. Bluemke; K.-D. Entian; J. Schrader (pp. 579-587).

Yeasts can convert amino acids to flavor alcohols following the Ehrlich pathway, a reaction sequence comprising transamination, decarboxylation, and reduction. The alcohols can be further derivatized to the acetate esters by alcohol acetyl transferase. Using l-methionine as sole nitrogen source and at high concentration, 3-(methylthio)-1-propanol (methionol) and 3-(methylthio)-propylacetate (3-MTPA) were produced with Saccharomyces cerevisiae. Methionol and 3-MTPA acted growth inhibiting at concentrations of >5 and >2 g L⁻¹, respectively. With the wild type strain S. cerevisiae CEN.PK113-7D, 3.5 g L⁻¹ methionol and trace amounts of 3-MTPA were achieved in a bioreactor. Overexpression of the alcohol acetyl transferase gene ATF1 under the control of a TDH3 (glyceraldehyde-3-phosphate dehydrogenase) promoter together with an optimization of the glucose feeding regime led to product concentrations of 2.2 g L⁻¹ 3-MTPA plus 2.5 g L⁻¹ methionol. These are the highest concentrations reported up to now for the biocatalytic synthesis of these flavor compounds which are applied in the production of savory aroma compositions such as meat, potato, and cheese flavorings.

Accumulation of prenyl alcohols by terpenoid biosynthesis inhibitors in various microorganisms by Masayoshi Muramatsu; Chikara Ohto; Shusei Obata; Eiji Sakuradani; Sakayu Shimizu (pp. 589-595).

Squalene synthase inhibitors significantly accelerate the production of farnesol by various microorganisms. However, farnesol production by Saccharomyces cerevisiae ATCC 64031, in which the squalene synthase gene is deleted, was not affected by the inhibitors, indicating that farnesol accumulation is enhanced in the absence of squalene synthase activity. The combination of diphenylamine as an inhibitor of carotenoid biosynthesis and a squalene synthase inhibitor increases geranylgeraniol production by a yeast, Rhodotorula rubra NBRC 0870. An ent-kauren synthase inhibitor also enhances the production of farnesol and geranylgeraniol by a filamentous fungus, Gibberella fujikuroi NBRC 30336. These results indicate that the inhibition of downstream enzymes from prenyl diphosphate synthase leads to the production of farnesol and geranylgeraniol.

Keywords: Farnesol; Farnesyl diphosphate; Geranylgeraniol; Prenyl alcohol; Terpenoid

Cloning and expression of the l-1-amino-2-propanol dehydrogenase gene from Rhodococcus erythropolis, and its application to double chiral compound production by M. Kataoka; T. Ishige; N. Urano; Y. Nakamura; E. Sakuradani; S. Fukui; S. Kita; K. Sakamoto; S. Shimizu (pp. 597-604).

The gene encoding NADP⁺-dependent l-1-amino-2-propanol dehydrogenase (AADH) of Rhodococcus erythropolis MAK154 was cloned and sequenced. A 780-bp nucleotide fragment was confirmed to be the gene encoding AADH by agreement of the N-terminal and internal amino acid sequences of the purified AADH. The gene (aadh) codes a total of 259 amino acid residues, and the deduced amino acid sequence shows similarity to several short-chain dehydrogenase/reductase family proteins. An expression vector, pKKAADH, which contains the full length aadh was constructed. Escherichia coli cells possessing pKKAADH exhibited a 10.4-fold increase in specific activity as to catalysis of the reduction of (S)-1-phenyl-2-methylaminopropan-1-one (MAK), as compared with that of R. erythropolis MAK154 induced by 1-amino-2-propanol (1 mg/ml). Coexpression of aadh with a cofactor regeneration enzyme (glucose dehydrogenase) gene was also performed, and a system for sufficient production of d-pseudoephedrine from racemic MAK was constructed.

Keywords: Rhodococcus erythropolis ; Asymmetric reduction; l-1-amino-2-propanol dehydrogenase; Ephedrine

Identification of archaeon-producing hyperthermophilic α-amylase and characterization of the α-amylase by Shujun Wang; Zhaoxin Lu; Mingsheng Lu; Song Qin; Hongfei Liu; Xiangyuan Deng; Qian Lin; Jianan Chen (pp. 605-614).

The extremely thermophilic anaerobic archaeon strain, HJ21, was isolated from a deep-sea hydrothermal vent, could produce hyperthermophilic α-amylase, and later was identified as Thermococcus from morphological, biochemical, and physiological characteristics and the 16S ribosomal RNA gene sequence. The extracellular thermostable α-amylase produced by strain HJ21 exhibited maximal activity at pH 5.0. The enzyme was stable in a broad pH range from pH 5.0 to 9.0. The optimal temperature of α-amylase was observed at 95°C. The half-life of the enzyme was 5 h at 90°C. Over 40% and 30% of the enzyme activity remained after incubation at 100°C for 2 and 3 h, respectively. The enzyme did not require Ca²⁺ for thermostability. This α-amylase gene was cloned, and its nucleotide sequence displayed an open reading frame of 1,374 bp, which encodes a protein of 457 amino acids. Analysis of the deduced amino acid sequence revealed that four homologous regions common in amylases were conserved in the HJ21 α-amylase. The molecular weight of the mature enzyme was calculated to be 51.4 kDa, which correlated well with the size of the purified enzyme as shown by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Keywords: Hyperthermophilic α-amylase; Thermococcus sp. Thermostability; Archaeon; Identification; Characterization

Identification of two novel esterases from a marine metagenomic library derived from South China Sea by Xinmin Chu; Haoze He; Changquan Guo; Baolin Sun (pp. 615-625).

The demand for novel biocatalysts is increasing in modern biotechnology, which greatly stimulates the development of powerful tools to explore the genetic resources in the environment. Metagenomics, a culture independent strategy, provides an access to valuable genetic resources of the uncultured microbes. In this study, two novel esterase genes designated as estA and estB, which encoded 277- and 328-amino-acid peptides, respectively, were isolated from a marine microbial metagenomic library by functional screening, and the corresponding esterases EstA and EstB were biochemically characterized. Amino acid sequence comparison and phylogenetic analysis indicated that EstA together with other putative lipolytic enzymes was closely related to family III, and EstB with its relatives formed a subfamily of family IV. Site-directed mutagenesis showed that EstA contained classical catalytic triad made up of S146–D222–H255, whereas EstB contained an unusual catalytic triad which consisted of S–E–H, an important feature of the subfamily. EstA exhibited habitat-specific characteristics such as its high level of stability in the presence of various divalent cations and at high concentrations of NaCl. EstB displayed remarkable activity against p-nitrophenyl esters and was highly stable in 30% methanol, ethanol, dimethylformamide, and dimethyl sulfoxide, making EstB a potential candidate for industrial applications.

Keywords: Metagenome; Esterase; BAC library; Phylogenetic tree; Marine

High-level expression of his-tagged clostridial collagenase in Clostridium perfringens by Eiji Tamai; Shigeru Miyata; Hiroaki Tanaka; Hirofumi Nariya; Motoo Suzuki; Osamu Matsushita; Naoya Hatano; Akinobu Okabe (pp. 627-635).

Clostridium histolyticum collagenase is used to isolate cells from various organs and tissues for tissue engineering, and also to treat destructive fibrosis; thus, the demand for high-grade enzyme preparations is increasing. In this study, we constructed a plasmid encoding C. histolyticum type II collagenase (ColH) with a C-terminal hexahistidine tag (ColH-his) to facilitate the purification of the enzyme through immobilized metal affinity chromatography (IMAC). When ColH-his was expressed in a protease-deficient mutant of Clostridium perfringens, it was produced in the culture supernatant more efficiently than the untagged ColH. ColH-his exhibited the same hydrolytic activity as ColH against 4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-d-Arg (Pz peptide), a synthetic collagenase substrate. From 100 ml of the culture supernatant, approximately 1 mg of ColH-his was purified by ammonium sulfate precipitation, IMAC, and high-performance liquid chromatography on a MonoQ column. When IMAC was performed on chelating Sepharose charged with Zn²⁺ instead of Ni²⁺, a potential carcinogenic metal, the specific activities against Pz peptide and type I collagen decreased slightly. However, they were comparable to those reported for other recombinant ColHs and a commercial C. histolyticum collagenase preparation, suggesting that this expression system is useful for large-scale preparation of high-grade clostridial collagenases.

Keywords: Collagenase; Clostridium histolyticum ; Clostridium perfringens ; Protein purification

Genetic organization of the putative salbostatin biosynthetic gene cluster including the 2-epi-5-epi-valiolone synthase gene in Streptomyces albus ATCC 21838 by Woo Sik Choi; Xiumei Wu; Yong-Hoon Choeng; Taifo Mahmud; Byeong Chul Jeong; Sang Hee Lee; Yong Keun Chang; Chang-Joon Kim; Soon-Kwang Hong (pp. 637-645).

The cyclization of sedoheptulose 7-phosphate to 2-epi-5-epi-valiolone, catalyzed by the 2-epi-5-epi-valiolone synthases, is the first committed step in the biosynthesis of C 7N-aminocyclitol-containing natural products, such as validamycin and acarbose. These natural products contain in their structures a valienamine unit, which is important for their biological activity. The same core unit is also found in salbostatin, a related pseudodisaccharide that has strong trehalase inhibitory activity. In silico analysis of the putative biosynthetic gene cluster of salbostatin from Streptomyces albus ATCC 21838 revealed 20 open reading frames, including an acbC homolog gene (salQ), which is believed to be involved in the biosynthesis of salbostatin. The salQ gene was overexpressed in Escherichia coli and the catalytic function of the recombinant protein was confirmed to be a 2-epi-5-epi-valiolone synthase. In addition, SalF, SalL, SalM, SalN, SalO, and SalR were found to be homologous to AcbR, AcbM, AcbL, AcbN, AcbO, and AcbP from the acarbose pathway, respectively, which suggests that the biosynthesis of C 7N-aminocyclitol moiety of salbostatin may be very similar to that of acarbose.

Keywords: Salbostatin; 2-epi-5-epi-valiolone synthase; Streptomyces albus ; C 7N-aminocyclitol

Carboxy-terminal half of Cry1C can help vegetative insecticidal protein to form inclusion bodies in the mother cell of Bacillus thuringiensis by Rong Song; Donghai Peng; Ziniu Yu; Ming Sun (pp. 647-654).

Vegetative insecticidal protein (VIP) is a class of insecticidal proteins produced by some strains of Bacillus thuringiensis during the vegetative stage of their growth and has toxicity against a wide spectrum of lepidopteran insects. Unlike insecticidal crystal proteins, which are produced as parasporal crystal proteins within the cell during sporulation, VIP is secreted into the culture medium. Here, we show that Vip3Aa7 protein can be relocated into the mother cell of B. thuringiensis by altering its synthesis using cry1C promoters, combined with a cry1C transcription termination sequence at the 3′ region and a STAB-SD sequence from cry1C promoters at the 5′ region of the gene. Further, when the carboxy-terminal half of Cry1C was included in the construct, the synthesis of Vip3Aa7 markedly increased, and its expression was relocated into the mother cell in the form of inclusion bodies. The expression of Vip3Aa7 with higher yields in the form of inclusion bodies demonstrated here would facilitate the development of a suitable formulation for the application of this class of insecticidal protein in the field, and the described system offers an additional method for potentially improving the efficacy of insecticides based on B. thuringiensis.

Keywords: Vegetative insecticidal protein; Bacillus thuringiensis ; Carboxy-terminal half; Relocation expression; Inclusion body

Electricity production from xylose in fed-batch and continuous-flow microbial fuel cells by Liping Huang; Bruce E. Logan (pp. 655-664).

A medium-scale (0.77 l) air-cathode, brush-anode microbial fuel cell (MFC) operated in fed-batch mode using xylose (20 mM) generated a maximum power density of 13 ± 1 W/m³ (673 ± 43 mW/m²). Xylose was rapidly removed (83.5%) within 8 h of a 60-h cycle, with 42.1% of electrons in intermediates (8.5 ± 0.2 mM acetate, 5.9 ± 0.01 mM ethanol, 4.3 ± 0.1 mM formate, and 1.3 ± 0.03 mM propionate), 9.1% captured as electricity, 16.1% in the remaining xylose, and 32.7% lost to cell storage, biomass, and other processes. The final Coulombic efficiency was 50%. At a higher initial xylose concentration (54 mM), xylose was again rapidly removed (86.9% within 24 h of a 116-h cycle), intermediates increased in concentration (18.4 ± 0.4 mM acetate, 7.8 ± 0.4 mM ethanol and 2.1 ± 0.2 mM propionate), but power was lower (5.2 ± 0.4 W/m³). Power was increased by operating the reactor in continuous flow mode at a hydraulic retention time of 20 h (20 ± 1 W/m³), with 66 ± 1% chemical oxygen demand removal. These results demonstrate that electricity generation is sustained over a cycle primarily by stored substrate and intermediates formed by fermentation and that the intermediates produced vary with xylose loading.

Keywords: Microbial fuel cell; Xylose; Degradation; Power production; Coulombic efficiency; Inhibition

The conversion of BTEX compounds by single and defined mixed cultures to medium-chain-length polyhydroxyalkanoate by Jasmina Nikodinovic; Shane T. Kenny; Ramesh P. Babu; Trevor Woods; Werner J. Blau; Kevin E. O’Connor (pp. 665-673).

Here, we report the use of petrochemical aromatic hydrocarbons as a feedstock for the biotechnological conversion into valuable biodegradable plastic polymers—polyhydroxyalkanoates (PHAs). We assessed the ability of the known Pseudomonas putida species that are able to utilize benzene, toluene, ethylbenzene, p-xylene (BTEX) compounds as a sole carbon and energy source for their ability to produce PHA from the single substrates. P. putida F1 is able to accumulate medium-chain-length (mcl) PHA when supplied with toluene, benzene, or ethylbenzene. P. putida mt-2 accumulates mcl-PHA when supplied with toluene or p-xylene. The highest level of PHA accumulated by cultures in shake flask was 26% cell dry weight for P. putida mt-2 supplied with p-xylene. A synthetic mixture of benzene, toluene, ethylbenzene, p-xylene, and styrene (BTEXS) which mimics the aromatic fraction of mixed plastic pyrolysis oil was supplied to a defined mixed culture of P. putida F1, mt-2, and CA-3 in the shake flasks and fermentation experiments. PHA was accumulated to 24% and to 36% of the cell dry weight of the shake flask and fermentation grown cultures respectively. In addition a three-fold higher cell density was achieved with the mixed culture grown in the bioreactor compared to shake flask experiments. A run in the 5-l fermentor resulted in the utilization of 59.6 g (67.5 ml) of the BTEXS mixture and the production of 6 g of mcl-PHA. The monomer composition of PHA accumulated by the mixed culture was the same as that accumulated by single strains supplied with single substrates with 3-hydroxydecanoic acid occurring as the predominant monomer. The purified polymer was partially crystalline with an average molecular weight of 86.9 kDa. It has a thermal degradation temperature of 350 °C and a glass transition temperature of −48.5 °C.

Expression of a heterologous xylose transporter in a Saccharomyces cerevisiae strain engineered to utilize xylose improves aerobic xylose consumption by Ronald E. Hector; Nasib Qureshi; Stephen R. Hughes; Michael A. Cotta (pp. 675-684).

The goal of this investigation was to determine the effect of a xylose transport system on glucose and xylose co-consumption as well as total xylose consumption in Saccharomyces cerevisiae. We expressed two heterologous transporters from Arabidopsis thaliana in recombinant xylose-utilizing S. cerevisiae cells. Strains expressing the heterologous transporters were grown on glucose and xylose mixtures. Sugar consumption rates and ethanol concentrations were determined and compared to an isogenic control strain lacking the A. thaliana transporters. Expression of the transporters increased xylose uptake and xylose consumption up to 46% and 40%, respectively. Xylose co-consumption rates (prior to glucose depletion) were also increased by up to 2.5-fold compared to the control strain. Increased xylose consumption correlated with increased ethanol concentration and productivity. During the xylose/glucose co-consumption phase, strains expressing the transporters had up to a 70% increase in ethanol production rate. It was concluded that in these strains, xylose transport was a limiting factor for xylose utilization and that increasing xylose/glucose co-consumption is a viable strategy for improving xylose fermentation.

Keywords: Saccharomyces cerevisiae ; Xylose fermentation; Xylose transport; Arabidopsis thaliana

Distribution of actinomycetes, their antagonistic behaviour and the physico-chemical characteristics of the world’s largest tidal mangrove forest by Anindita Mitra; Subhas Chandra Santra; Joydeep Mukherjee (pp. 685-695).

We examined the relationship between distribution of actinomycetes and antagonistic behaviour with the physico-chemical characteristics of the Sundarbans, off the Bay of Bengal, India. Soil/sediment samples were collected from three regions: near to the sea, intertidal regions and mangrove forests. For the enumeration of actinomycetes, four treatments combining dilution with distilled or sea water with or without heating followed by plating onto starch-casein, glycerol-arginine and starch-nitrate media were done. Dilution with seawater, heating and plating onto starch-casein yielded maximum number of actinomycetes. The highest number of actinomycetes was isolated from an intertidal region having alluvial soil and the lowest from a site containing sandy sediments. Antimicrobial activity was dependent upon seawater. Antimicrobial score of an actinomycetes strain was calculated allotting maximum points to the isolate showing activity against all the test bacteria, next lower point to the isolate showing activity against one less the total and so on. The antagonistic potential (AP) of a sampling site was the ratio of total antimicrobial score of the isolates and their number. The high AP sites were influenced by tides, while the low AP sites were not. Pearson’s correlation between soil chemical parameters and microbiological parameters revealed soil nitrogen as the key factor determining the antagonistic activity.

Keywords: Actinomycetes; Sundarbans; Intertidal; Antagonistic potential; Soil nitrogen

Deep-sea thermophilic Geobacillus bacteriophage GVE2 transcriptional profile and proteomic characterization of virions by Bin Liu; Xiaobo Zhang (pp. 697-707).

Thermophilic bacteria and viruses represent novel sources of genetic materials and enzymes with great potential for use in industry and biotechnology. In this study, GVE2, a virulent tailed Siphoviridae bacteriophage infecting deep-sea thermophilic Geobacillus sp. E263, was characterized. The bacteriophage contained a 40,863-bp linear double-stranded genomic deoxyribonucleic acid (DNA) with 62 presumptive open reading frames (ORFs). A viral DNA microarray was developed to monitor the viral gene transcription program. Microarray analysis indicated that 74.2% of the presumptive ORFs were expressed. The structural proteins of purified GVE2 virions were identified by mass spectrometric analysis. The purified virions contained six protein bands. Of the newly retrieved proteins, VP371 was further characterized. The immuno-electron microscopy indicated that the VP371 protein was a component of the viral capsid. Transcriptional analyses and proteomic characterization of GVE2 would be helpful to understand the complex host–virus interaction during virus infection.

Keywords: Thermophilic Geobacillus ; Bacteriophage; Transcription; Proteomics

Finding new pathway-specific regulators by clustering method using threshold standard deviation based on DNA chip data of Streptomyces coelicolor by Yung-Hun Yang; Ji-Nu Kim; Eunjung Song; Eunjung Kim; Min-Kyu Oh; Byung-Gee Kim (pp. 709-717).

In order to identify the regulators involved in antibiotic production or time-specific cellular events, the messenger ribonucleic acid (mRNA) expression data of the two gene clusters, actinorhodin (ACT) and undecylprodigiosin (RED) biosynthetic genes, were clustered with known mRNA expression data of regulators from S. coelicolor using a filtering method based on standard deviation and clustering analysis. The result identified five regulators including two well-known regulators namely, SCO3579 (WlbA) and SCO6722 (SsgD). Using overexpression and deletion of the regulator genes, we were able to identify two regulators, i.e., SCO0608 and SCO6808, playing roles as repressors in antibiotics production and sporulation. This approach can be easily applied to mapping out new regulators related to any interesting target gene clusters showing characteristic expression patterns. The result can also be used to provide insightful information on the selection rules among a large number of regulators.

Keywords: Streptomyces coelicolor; DNA chip; Clustering; Standard deviation; Pathway-specific regulator

Secretome analysis of Phanerochaete chrysosporium strain CIRM-BRFM41 grown on softwood by Holy Ravalason; Gwénaël Jan; Daniel Mollé; Maryvonne Pasco; Pedro M. Coutinho; Catherine Lapierre; Brigitte Pollet; Frédérique Bertaud; Michel Petit-Conil; Sacha Grisel; Jean-Claude Sigoillot; Marcel Asther; Isabelle Herpoël-Gimbert (pp. 719-733).

Proteomic analysis was performed to determine and differentiate the composition of the secretomes of Phanerochaete chrysosporium CIRM-BRFM41, a peroxidase hypersecretory strain grown under ligninolytic conditions and on softwood chips under biopulping conditions. Extracellular proteins from both cultures were analyzed by bidimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. A total of 37 spots were identified. The secretome in liquid synthetic medium comprised mainly peroxidases, while several wood-degrading enzymes and enzymes involved in fungal metabolism were detected in biopulping cultures on softwood. This prompted an analysis of the impact of secretome modulation in the presence of softwood chips. Biotreated wood was submitted to kraft cooking and chemical bleaching using chlorine dioxide. The fungal pre-treatment led to a significant increase in pulp yield and a better bleachability of the pulp. This bleachability improvement could be explained by the production of specific lignocellulose-degrading enzymes.

Keywords: Phanerochaete chrysosporium ; Secretome; Softwood; Chemical pulping

Development of a yeast protein fragment complementation assay (PCA) system using dihydrofolate reductase (DHFR) with specific additives by Seiji Shibasaki; Kozue Sakata; Jun Ishii; Akihiko Kondo; Mitsuyoshi Ueda (pp. 735-743).

A yeast protein fragment complementation assay (PCA) system based on dihydrofolate reductase (DHFR) is difficult to be operated because it is not as sensitive to trimethoprim (TMP) as the system using a prokaryotic microorganism. Here, the PCA system using DHFR, specific inhibitors, and a substrate in the yeast Saccharomyces cerevisiae was newly developed. As a model, the human oncoprotein Ras and the Ras-binding domain (RBD) of Raf-1 were individually and genetically fused to DHFR fragment, and each genetic construct was coexpressed under the control of the GAL1 promoter. An interaction between Ras and RBD could be evaluated on the basis of cell proliferation. To establish the experimental conditions for the yeast PCA system based on the DHFR reconstitution, we examined yeast host strains and the concentration of inhibitory additives to prevent endogenous DHFR activity, namely, TMP and sulfanilamide, and the substrate of DHFR, namely, folic acid. The transformant harboring wild-type Ras or its variants showed positive interaction signals, and the order of interactions for combination corresponded to the results of other in vitro assays. Moreover, combinatorial mutated Ras-binding domains were constructed, and the interaction of RBDs with Ras using this yeast PCA system was examined. As a result, various types of mutated clone for RBD were obtained. These demonstrations suggest that the yeast PCA system based on DHFR can be one of good, convenient, and inexpensive tools for investigating eukaryotic protein–protein interactions in vivo.

Keywords: Saccharomyces cerevisiae ; PCA; DHFR; Trimethoprim; Sulfanilamide

Partially saturated canthaxanthin purified from Aspergillus carbonarius induces apoptosis in prostate cancer cell line by Nallasamy Kumaresan; Konasur R. Sanjay; Kundumani S. Venkatesh; Ravi-Kumar Kadeppagari; Govindaswamy Vijayalakshmi; Sukumaran Umesh-Kumar (pp. 745-745).

Efficient production of active form of recombinant cassava hydroxynitrile lyase using Escherichia coli in low-temperature culture by Hisashi Semba; Eita Ichige; Tadayuki Imanaka; Haruyuki Atomi; Hideki Aoyagi (pp. 747-748).

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Applied Microbiology and Biotechnology (v.80, #4)

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Applied Microbiology and Biotechnology (v.80, #4)