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Applied Microbiology and Biotechnology (v.85, #4)

Engineering of cyclodextrin glucanotransferases and the impact for biotechnological applications by Hans Leemhuis; Ronan M. Kelly; Lubbert Dijkhuizen (pp. 823-835).

Cyclodextrin glucanotransferases (CGTases) are industrially important enzymes that produce cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. Cyclodextrin glucanotransferases are also applied as catalysts in the synthesis of glycosylated molecules and can act as antistaling agents in the baking industry. To improve the performance of CGTases in these various applications, protein engineers are screening for CGTase variants with higher product yields, improved CD size specificity, etc. In this review, we focus on the strategies employed in obtaining CGTases with new or enhanced enzymatic capabilities by searching for new enzymes and improving existing enzymatic activities via protein engineering.

Keywords: Amylase; Biocatalysis; Directed evolution; Glycoside hydrolase; Protein engineering; Starch

Pyranose dehydrogenases: biochemical features and perspectives of technological applications by Clemens K. Peterbauer; Jindřich Volc (pp. 837-848).

Pyranose dehydrogenase is a fungal flavin-dependent sugar oxidoreductase which is structurally and catalytically related to fungal pyranose oxidase and cellobiose dehydrogenase and probably fulfills similar biological functions in lignocellulose breakdown. It is a monomeric secretory glycoprotein and is limited to a rather small group of litter-decomposing basidiomycetes. Compared with pyranose oxidase, it displays broader substrate specificity and a variable regioselectivity and is unable to utilize oxygen as electron acceptor using substituted benzoquinones and (organo) metallic ions instead. Depending on the structure of the sugar in pyranose form (mono/di/oligosaccharide or glycoside) and the enzyme source, selective monooxidations at C-1, C-2, C-3, or dioxidations at C-2,3 or C-3,4 of the molecule to the corresponding aldonolactones (C-1), or (di)dehydrosugars (aldos(di)uloses) can be performed. These features make pyranose dehydrogenase a promising and versatile biocatalyst for production of highly reactive, sometimes unique, di- and tri-carbonyl sugar derivatives that may serve as interesting chiral intermediates for the synthesis of rare sugars, novel drugs, and fine chemicals.

Keywords: Pyranose dehydrogenase; Sugar oxidoreductase; Regioselectivity; Biocatalysis

Biogas production: current state and perspectives by Peter Weiland (pp. 849-860).

Anaerobic digestion of energy crops, residues, and wastes is of increasing interest in order to reduce the greenhouse gas emissions and to facilitate a sustainable development of energy supply. Production of biogas provides a versatile carrier of renewable energy, as methane can be used for replacement of fossil fuels in both heat and power generation and as a vehicle fuel. For biogas production, various process types are applied which can be classified in wet and dry fermentation systems. Most often applied are wet digester systems using vertical stirred tank digester with different stirrer types dependent on the origin of the feedstock. Biogas is mainly utilized in engine-based combined heat and power plants, whereas microgas turbines and fuel cells are expensive alternatives which need further development work for reducing the costs and increasing their reliability. Gas upgrading and utilization as renewable vehicle fuel or injection into the natural gas grid is of increasing interest because the gas can be used in a more efficient way. The digestate from anaerobic fermentation is a valuable fertilizer due to the increased availability of nitrogen and the better short-term fertilization effect. Anaerobic treatment minimizes the survival of pathogens which is important for using the digested residue as fertilizer. This paper reviews the current state and perspectives of biogas production, including the biochemical parameters and feedstocks which influence the efficiency and reliability of the microbial conversion and gas yield.

Keywords: Anaerobic digestion; Biogas; Biogas upgrading; Biomethanation; Biomass; Co-digestion; Digestate; Dry fermentation; Energy crops; Methane potential; Wet fermentation

High-temperature fermentation: how can processes for ethanol production at high temperatures become superior to the traditional process using mesophilic yeast? by Babiker M. A. Abdel-Banat; Hisashi Hoshida; Akihiko Ano; Sanom Nonklang; Rinji Akada (pp. 861-867).

The process of ethanol fermentation has a long history in the production of alcoholic drinks, but much larger scale production of ethanol is now required to enable its use as a substituent of gasoline fuels at 3%, 10%, or 85% (referred to as E3, E10, and E85, respectively). Compared with fossil fuels, the production costs are a major issue for the production of fuel ethanol. There are a number of possible approaches to delivering cost-effective fuel ethanol production from different biomass sources, but we focus in our current report on high-temperature fermentation using a newly isolated thermotolerant strain of the yeast Kluyveromyces marxianus. We demonstrate that a 5°C increase only in the fermentation temperature can greatly affect the fuel ethanol production costs. We contend that this approach may also be applicable to the other microbial fermentations systems and propose that thermotolerant mesophilic microorganisms have considerable potential for the development of future fermentation technologies.

Keywords: Kluyveromyces marxianus ; Saccharomyces cerevisiae ; Fuel; Ethanol; Cost

Biotechnological production and applications of statins by Javier Barrios-González; Roxana U. Miranda (pp. 869-883).

Statins are a group of extremely successful drugs that lower cholesterol levels in blood; decreasing the risk of heath attack or stroke. In recent years, statins have also been reported to have other biological activities and numerous potential therapeutic uses. Natural statins are lovastatin and compactin, while pravastatin is derived from the latter by biotransformation. Simvastatin, the second leading statin in the market, is a lovastatin semisynthetic derivative. Lovastatin is mainly produced by Aspergillus terreus strains, and compactin by Penicillium citrinum. Lovastatin and compactin are produced industrially by liquid submerged fermentation, but can also be produced by the emerging technology of solid-state fermentation, that displays some advantages. Advances in the biochemistry and genetics of lovastatin have allowed the development of new methods for the production of simvastatin. This lovastatin derivative can be efficiently synthesized from monacolin J (lovastatin without the side chain) by a process that uses the Aspergillus terreus enzyme acyltransferase LovD. In a different approach, A. terreus was engineered, using combinational biosynthesis on gene lovF, so that the resulting hybrid polyketide synthase is able to in vivo synthesize 2,2-dimethylbutyrate (the side chain of simvastatin). The resulting transformant strains can produce simvastatin (instead of lovastatin) by direct fermentation.

Keywords: Statins; Biosynthesis and genetics; Biotechnological production

Spaceflight and modeled microgravity effects on microbial growth and virulence by Jason A. Rosenzweig; Ohunene Abogunde; Kayama Thomas; Abidat Lawal; Y-Uyen Nguyen; Ayodotun Sodipe; Olufisayo Jejelowo (pp. 885-891).

For unsuspecting bacteria, the difference between life and death depends upon efficient and specific responses to various stressors. Facing a much larger world, microbes are invariably challenged with ever-changing environments where temperature, pH, chemicals, and nutrients are in a constant state of flux. Only those that are able to rapidly reprogram themselves and express subsets of genes needed to overcome the stress will survive and outcompete neighboring microbes. Recently, low shear stress, emulating microgravity (MG) experienced in space, has been characterized in a number of microorganisms including fungi and prokaryotes ranging from harmless surrogate organisms to bona fide pathogens. Interestingly, MG appears to induce a plethora of effects ranging from enhanced pathogenicity in several Gram-negative enterics to enhanced biofilm formation. Furthermore, MG-exposed bacteria appeared better able to handle subsequent stressors including: osmolarity, pH, temperature, and antimicrobial challenge while yeast exhibited aberrant budding post-MG-exposure. This review will focus on MG-induced alterations of virulence in various microbes with the emphasis placed on bacteria.

Keywords: Space microbiology; Microgravity; Low-shear modeled microgravity; Virulence; Bacteria

Pentanol isomer synthesis in engineered microorganisms by Anthony F. Cann; James C. Liao (pp. 893-899).

Pentanol isomers such as 2-methyl-1-butanol and 3-methyl-1-butanol are a useful class of chemicals with a potential application as biofuels. They are found as natural by-products of microbial fermentations from amino acid substrates. However, the production titer and yield of the natural processes are too low to be considered for practical applications. Through metabolic engineering, microbial strains for the production of these isomers have been developed, as well as that for 1-pentanol and pentenol. Although the current production levels are still too low for immediate industrial applications, the approach holds significant promise for major breakthroughs in production efficiency.

Keywords: Pentanol; 2-Methyl-1-butanol; 3-Methyl-1-butanol; Biofuels

Genetic engineering of fungal biocontrol agents to achieve greater efficacy against insect pests by Raymond J. St. Leger; Chengshu Wang (pp. 901-907).

Molecular biology methods have elucidated pathogenic processes in several fungal biocontrol agents including two of the most commonly applied entomopathogenic fungi, Metarhizium anisopliae and Beauveria bassiana. In this review, we describe how a combination of molecular techniques has: (1) identified and characterized genes involved in infection; (2) manipulated the genes of the pathogen to improve biocontrol performance; and (3) allowed expression of a neurotoxin from the scorpion Androctonus australis. The complete sequencing of four exemplar species of entomopathogenic fungi including B. bassiana and M. anisopliae will be completed in 2010. Coverage of these genomes will help determine the identity, origin, and evolution of traits needed for diverse lifestyles and host switching. Such knowledge combined with the precision and malleability of molecular techniques will allow design of multiple pathogens with different strategies to be used for different ecosystems and avoid the possibility of the host developing resistance.

Keywords: Entomopathogenic fungi; Metarhizium anisopliae ; Beauveria bassiana ; Virulence gene; Genetic engineering

New perspective for phage display as an efficient and versatile technology of functional proteomics by Wei Li; Nora B. Caberoy (pp. 909-919).

Phage display with antibody libraries has been widely used with versatile applications. However, phage display with cDNA libraries is rare and inefficient. Because of uncontrollable reading frames and stop codons in cDNA repertoires, high percentage of phage clones identified from conventional cDNA libraries are non-open reading frames (non-ORFs) encoding unnatural short peptides with minimal implications in protein networks. Consequently, phage display has not been used as a technology of functional proteomics to elucidate protein–protein interactions like yeast two-hybrid system and mass spectrometry-based technologies. Several strategies, including C-terminal display and ORF cDNA libraries, have been explored to circumvent the technical problem. The accumulative endeavors eventually led to the efficient elucidation of a large number of tubby- and phosphatidylserine-binding proteins in recent studies by ORF phage display with minimal reading frame issue. ORF phage display inherits all the versatile applications of antibody phage display, but enables efficient identification of real endogenous proteins with efficiency, sensitivity, and accuracy comparable to other technologies of functional proteomics. Its ELISA-like procedure can be conveniently adapted by individual laboratories or fully automated for high-throughput screening. Thus, ORF phage display is an efficient, sensitive, versatile, and convenient technology of functional proteomics for elucidation of global and pathway-specific protein–protein interactions, disease mechanisms, or therapeutic targets.

Keywords: Phage display; Functional proteomics; Yeast two-hybrid system; Mass spectrometry; ORF phage display; Protein–protein interaction

Enzymatic and whole-cell synthesis of lactate-containing polyesters: toward the complete biological production of polylactate by Ken’ichiro Matsumoto; Seiichi Taguchi (pp. 921-932).

The importance of polylactic acid, a representative bio-based polyester, has been established on a worldwide scale in response to emerging global environmental problems such as green house gas emission and limited petroleum consumption. The current methods for generating this bio-based polymer involve biological synthesis and lactic acid (LA) fermentation, followed by chemical ring-opening polymerization. Among the research community working on polyhydroxyalkanoate polyesters, the prospect of direct biological synthesis of LA into a polymeric form is very attractive from the academic and industrial perspectives. In 2008, this challenge was met for the first time by the discovery of an “LA-polymerizing enzyme”. Using this novel enzyme, the metabolic engineering approach outlined here provided an entirely new, single organism generation of the polymer. This is a major breakthrough in the field. In this review, we provide an overview of the whole-cell synthesis of LA-containing polyesters in comparison with conventional lipase-catalyzed polymer synthesis in terms of both the concepts and strategies of their synthetic processes.

Keywords: Polylactide; Polyhydroxyalkanoate; Lactate-polymerizing enzyme; Lipase; PHA synthase

Biotransformation of steriodal saponins in Dioscorea zingiberensis C. H. Wright to diosgenin by Trichoderma harzianum by Lin Liu; Yue-Sheng Dong; Shan-Shan Qi; Hui Wang; Zhi-Long Xiu (pp. 933-940).

Diosgenin is an important starting material in the steroidal hormone industry. Traditionally, diosgenin is mainly produced by acid hydrolysis of Dioscorea zingiberensis C. H. Wright (DZW) tubers. This method yields numerous byproducts that can cause serious pollution. In this study, diosgenin was obtained by biotransformation of steroidal saponins in DZW afforded by Trichoderma harzianum CGMCC 2979. The medium was optimized for maximum diosgenin production. The addition of phosphate buffer, surfactant Tween-85, and Fe²⁺ increased the yield of diosgenin by 50.28%, 33.35%, and 22.07%, respectively. The optimum medium obtained by response surface methodology was composed of 60 mmol l⁻¹ phosphate buffer, 0.07% (w/v) Tween-85, and 0.93 mmol l⁻¹ Fe²⁺. Under these conditions, a maximum diosgenin yield of 30.05 ± 0.59 mg g⁻¹ was achieved, which was slightly higher than that obtained from traditional acid hydrolysis. By hydrolyzing the un-transformed steroidal saponins after biotransformation, the total diosgenin yield increased by 35% compared to traditional method. Moreover, chemical oxygen demand and residual reduced sugar in the wastewater produced by this integrated process were only 3.72% and 0.3%, respectively, that of the traditional acid hydrolysis method.

Keywords: Diosgenin; Biotransformation; Trichoderma harzianum ; Acid hydrolysis; Response surface methodology

Production of individual ganoderic acids and expression of biosynthetic genes in liquid static and shaking cultures of Ganoderma lucidum by Jun-Wei Xu; Yi-Ning Xu; Jian-Jiang Zhong (pp. 941-948).

Two-stage culture was efficient in enhancing total ganoderic acid (GA) production by Ganoderma lucidum (Fang and Zhong, Biotechnol Prog 18:51–54, 2002). As different GAs have different bioactivities, it is critical to understand the kinetics of individual GA production during fermentation, but no related information is yet available. To understand the regulation of GA biosynthesis, investigation of the accumulation of intermediate (lanosterol) and by-product (ergosterol) and of the expression of three important biosynthetic genes was also conducted in liquid shaking and static cultures of G. lucidum. The results showed that the content of individual GAs increased rapidly in the liquid static culture, and their maximum value was 6- to 25-fold that of shaking culture while lanosterol content in the former was lower than the latter. The transcript of squalene synthase (SQS), lanosterol synthase and 3-hydroxy-3-methylglutaryl coenzyme A reductase in liquid static culture was 4.3-, 2.1-, and 1.9-fold that of the shaking culture, respectively. Higher GA content in liquid static culture was related to increased transcription of those genes especially SQS. The work is helpful to the production of individual GAs and provided an insight into why the liquid static culture was superior to the shaking culture in view of biosynthetic gene expression.

Keywords: Ganoderma lucidum ; Individual ganoderic acid; Liquid static culture; Gene expression; Medicinal mushroom fermentation

Biosynthesis of novel terpolymers poly(lactate-co-3-hydroxybutyrate-co-3-hydroxyvalerate)s in lactate-overproducing mutant Escherichia coli JW0885 by feeding propionate as a precursor of 3-hydroxyvalerate by Fumi Shozui; Ken’ichiro Matsumoto; Takanori Nakai; Miwa Yamada; Seiichi Taguchi (pp. 949-954).

Novel lactate (LA)-based terpolymers, P[LA-co-3-hydroxybutyrate(3HB)-co-3-hydroxyvalerate(3HV)]s (PLBVs), were produced in LA-overproducing mutant, Escherichia coli JW0885, which was found to be a superior host for the efficient production of LA-based polyesters. Recombinant E. coli JW0885 harboring the genes encoding LA-polymerizing enzyme (Ser325Thr/Gln481Lys mutant of polyhydroxyalkanoate synthase from Pseudomonas sp. 61-3) and three monomer supplying enzymes [propionyl-CoA transferase, β-ketothiolase, and nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)-dependent acetoacetyl-CoA reductase] was aerobically grown on glucose with feeding of propionate as a precursor of 3-hydroxyvaleryl-CoA (3HV-CoA). Gas chromatography and nuclear magnetic resonance (NMR) analyses revealed that polymers accumulated in the cells were composed of LA, 3HB, and 3HV units, thus being identified as terpolymers, PLBVs. In addition, ¹H-NMR analysis suggested the existence of LA-3HV sequence in the terpolymer. When 100 mg/l of sodium propionate was added into the medium, 3HV fraction in the terpolymer linearly reached up to 7.2 mol%, while LA fraction was inversely decreased. This phenomenon could be due to the change in metabolic fluxes of lactyl-CoA (LA-CoA) and 3HV-CoA depending on the concentration of propionate fed into the medium.

Keywords: Microbial polymer production; Copolymer; Poly(LA-co-3HB-co-3HV); Substrate specificity; Metabolic engineering; PhaC

Simple adaptive pH control in bioreactors using gain-scheduling methods by S. Gnoth; A. Kuprijanov; R. Simutis; A. Lübbert (pp. 955-964).

A simple well-performing adaptive control technique for pH control in fermentations of recombinant protein production processes is described and its design procedure is explained. First, the entire control algorithm was simulated and parameterized. Afterwards it was tested in real cultivation processes. The results show that this simple technique leads to significant reductions in the fluctuations of the pH values in microbial cultures at a minimum of expenditures. The signal-to-noise ratio and thus the information captured by the pH signal were increased by about an order of magnitude. This leads to a substantial improvement in the noise of many other process signals that are used to monitor and control the process. For instance, respiratory off-gas data of CO₂ and its derived carbon dioxide production rate signals from the cultures carry much less noise as compared to those values obtained with conventional pH control. Detailed process analysis revealed that even very small pH jumps of 0.03 values during the fermentation were shown to result in pronounced deflections in CO₂-volume fraction of 8% (peak to peak). The proposed controller, maintaining the pH within the interval of 0.01 around the setpoint, reduces the noise considerably.

Keywords: pH control; CO₂ ; Bicarbonate; Gain scheduling; Fermentation

Influence of culture aeration on the cellulase activity of Thermobifida fusca by Yu Deng; Stephen S. Fong (pp. 965-974).

Currently, one of the hurdles hindering efficient production of cellulosic biofuel is the recalcitrant nature of cellulose to hydrolysis. A wide variety of cellulase enzymes are found natively in microorganisms that can potentially be used to effectively hydrolyze cellulose to fermentable sugars. In this study, phenomenological and mechanistic parameters affecting cellulase activity were studied using the moderately thermophilic, aerobic, and cellulolytic microogainsm Thermobifida fusca. Two major sets of experiments were conducted to (1) study the mechanistic differences in growth in a flask compared to a bioreactor and (2) study the cell culture parameters influencing cellulase activity using a series of bioreactor experiments. Specific cellulase and specific endoglucanase activities were found to be higher in the bioreactor as compared to flask growth. Measurements of messenger RNA transcript levels of 18 cellulase-related genes and intracellular ATP levels indicated that measured enzyme activity was likely more influenced by post-transcriptional energetics rather than transcriptional regulation. By delineating the effects of culture aeration and stir speed using a bioreactor, it was found that cellulase activity increased with increasing aeration and increasing stir speeds (highest K _l a) with a tradeoff of decreased cellular growth at the highest stir speeds tested (400 rpm). Overall, these results allude to a connection between aeration and oxidative respiration that lead to increased ATP allowing for increased cellulase synthesis as the primary constraint on overall cellulase activity.

Keywords: Cellulase; Gene expression; Thermobifida fusca ; Bioreactor

A new non-hydrophobic cell wall protein (CWP10) of Metarhizium anisopliae enhances conidial hydrophobicity when expressed in Beauveria bassiana by Jun Li; Sheng-Hua Ying; Le-Tian Shan; Ming-Guang Feng (pp. 975-984).

A cell wall protein, CWP10, resolved from the conidial formic acid extract of a Metarhizium anisopliae isolate, was characterized as a new 9.9-kDa protein with a 32-aa signal peptide with a central hydrophobic region (ca. 10 residues) at its N-terminus. This protein was proven neither to be hydrophobic nor glycosylated and encoded by a 363-bp, single-copy gene with three introns. CWP10 was existent in the conidial extracts of seven of 18 tested M. anisopliae isolates and much more abundant (immunogold-labeled) on conidial walls than in cytoplasm. Integrating the gene into a CWP10-absent strain of Beauveria bassiana led to excellent expression of CWP10 in aerial conidia, increasing net conidial hydrophobicity by 10.8% or adhesion to hydrophobic Teflon by 1.3-fold. However, the expressed protein had no effect on conidial tolerance to thermal and ultraviolet stresses. This is the first report on a non-hydrophobic cell-wall protein enhancing conidial hydrophobicity and adhesion of the fungal species.

Keywords: Metarhizium anisopliae ; Beauveria bassiana ; Cell wall protein; Gene cloning; Protein expression; Functional analysis

The mushroom ribosome-inactivating protein lyophyllin exerts deleterious effects on mouse embryonic development in vitro by W. Y. Chan; T. B. Ng; Joyce S. Y. Lam; Jack H. Wong; K. T. Chu; P. H. K. Ngai; S. K. Lam; H. X. Wang (pp. 985-993).

Earlier investigations disclose that some plant ribosome-inactivating proteins (RIPs) adversely affect mouse embryonic development. In the present study, a mushroom RIP, namely lyophyllin from Lyophyllum shimeji, was isolated, partially sequenced, and its translation inhibitory activity determined. Its teratogenicity was studied by using a technique entailing microinjection and postimplantation whole-embryo culture. It was found that embryonic abnormalities during the period of organogenesis from E8.5 to E9.5 were induced by lyophyllin at a concentration as low as 50 μg/ml, and when the lyophyllin concentration was raised, the number of abnormal embryos increased, the final somite number decreased, and the abnormalities increased in severity. The affected embryonic structures included the cranial neural tube, forelimb buds, branchial arches, and body axis, while optic and otic placodes were more resistant. Lyophyllin at a concentration higher than 500 μg/ml also induced forebrain blisters within the cranial mesenchyme. When the abnormal embryos were examined histologically, an increase of cell death was found to be associated with abnormal structures, indicating that cell death may be one of the underlying causes of teratogenicity of the mushroom RIP. This constitutes the first report on the teratogenicity of a mushroom RIP.

Keywords: Ribosome-inactivating proteins; Teratogenic effects; Mouse postimplantation embryos; Whole-embryo culture; Organogenesis; Hypsin; Lyophyllin; Velutin

In vitro evolution of styrene monooxygenase from Pseudomonas putida CA-3 for improved epoxide synthesis by Lucas J. Gursky; Jasmina Nikodinovic-Runic; K. Anton Feenstra; Kevin E. O’Connor (pp. 995-1004).

The styAB genes from Pseudomonas putida CA-3, which encode styrene monooxygenase, were subjected to three rounds of in vitro evolution using error-prone polymerase chain reaction with a view to improving the rate of styrene oxide and indene oxide formation. Improvements in styrene monooxygenase activity were monitored using an indole to indigo conversion assay. Each round of random mutagenesis generated variants improved in indigo formation with third round variants improved nine- to 12-fold over the wild type enzyme. Each round of in vitro evolution resulted in two to three amino acid substitutions in styrene monooxygenase. While the majority of mutations occurred in styA (oxygenase), mutations were also observed in styB (reductase). A mutation resulting in the substitution of valine with isoleucine at amino acid residue 303 occurred near the styrene and flavin adenine dinucleotide binding site of styrene monooxygenase. One mutation caused a shift in the reading frame in styA and resulted in a StyA variant that is 19 amino acids longer than the wild-type protein. Whole cells expressing the best styrene monooxygenase variants (round 3) exhibited eight- and 12-fold improvements in styrene and indene oxidation rates compared to the wild-type enzyme. In all cases, a single enantiomer, (S)-styrene oxide, was formed from styrene while (1S,2R)-indene oxide was the predominant enantiomer (e.e. 97%) formed from indene. The average yield of styrene oxide and indene oxide from their respective alkene substrates was 65% and 90%, respectively.

Keywords: Biocatalysis; Directed evolution; Epoxidation; Styrene monooxygenase

Characterization of endo-β-1,4-glucanase from a novel strain of Penicillium pinophilum KMJ601 by Marimuthu Jeya; Ah-Reum Joo; Kyoung-Mi Lee; Won-Il Sim; Deok-Kun Oh; Yeong-Suk Kim; In-Won Kim; Jung-Kul Lee (pp. 1005-1014).

A novel endo-β-1,4-glucanase (EG)-producing strain was isolated and identified as Penicillium pinophilum KMJ601 based on its morphology and internal transcribed spacer (ITS) rDNA gene sequence. When rice straw and corn steep powder were used as carbon and nitrogen sources, respectively, the maximal EG activity of 5.0 U mg protein⁻¹, one of the highest levels among EG-producing microorganisms, was observed. The optimum temperature and pH for EG production were 28°C and 5.0, respectively. The increased production of EG by P. pinophilum in culture at 28°C was confirmed by two-dimensional electrophoresis followed by MS/MS sequencing of the partial peptide. A partial EG gene (eng5) was amplified by degenerate polymerase chain reaction (PCR) based on the peptide sequence. A full-length eng5 was cloned by genome-walking PCR, and P. pinophilum EG was identified as a member of glycoside hydrolase family 5. The present results should contribute to improved industrial production of EG by P. pinophilum KMJ601.

Keywords: Penicillium pinophilum ; Endo-β-1,4-glucanase; Two-dimensional electrophoresis; Cloning; Genome-walking PCR

Gene cloning and expression of a new acidic family 7 endo-β-1,3-1,4-glucanase from the acidophilic fungus Bispora sp. MEY-1 by Huiying Luo; Jun Yang; Peilong Yang; Jiang Li; Huoqing Huang; Pengjun Shi; Yingguo Bai; Yaru Wang; Yunliu Fan; Bin Yao (pp. 1015-1023).

Most reported microbial β-1,3-1,4-glucanases belong to the glycoside hydrolase family 16. Here, we report a new acidic family 7 endo-β-1,3-1,4-glucanase (Bgl7A) from the acidophilic fungus Bispora sp. MEY-1. The cDNA of Bgl7A was isolated and over-expressed in Pichia pastoris, with a yield of about 1,000 U ml^–1 in a 3.7-l fermentor. The purified recombinant Bgl7A had three activity peaks at pH 1.5, 3.5, and 5.0 (maximum), respectively, and a temperature optimum at 60°C. The enzyme was stable at pH 1.0–8.0 and highly resistant to both pepsin and trypsin. Belonging to the group of non-specific endoglucanase, Bgl7A can hydrolyze not only β-glucan and cellulose but also laminarin and oat spelt xylan. The specific activity of Bgl7A against barley β-glucan and lichenan (4,040 and 2,740 U mg^–1) was higher than toward carboxymethyl cellulose sodium (395 U mg^–1), which was different from other family 7 endo-β-glucanases.

Keywords: Endo-β-1,3-1,4-glucanase; Acidophilic fungus; Bispora sp. MEY-1; Pichia pastoris

Analysis of aldehyde reductases from Gluconobacter oxydans 621H by Paul Schweiger; Uwe Deppenmeier (pp. 1025-1031).

Two cytosolic nicotinamide adenine dinucleotide phosphate-dependent aldehyde reductases, Gox1899 and Gox2253, from Gluconobacter oxydans 621H were overproduced and purified from Escherichia coli. The purified proteins exhibited subunit masses of 26.4 (Gox1899) and 36.7 kDa (Gox2253). Both proteins formed homo-octamers exhibiting native masses of 210 and 280 kDa, respectively. The substrate spectra, optimal reaction conditions, and kinetic constants were determined for Gox1899 and Gox2253. Both enzymes efficiently catalyzed the reduction of medium/long-chain aldehydes. However, Gox1899 had a wider substrate spectrum and was more catalytically efficient. The best activity with Gox1899 was found for aliphatic aldehydes of C6-C10. In contrast, Gox2253 had a limited substrate spectrum and reduced octanal, nonanal, and decanal. Both enzymes were unable to oxidize primary alcohols. Aldehyde removal may be of particular importance for Gluconobacter because the membrane-bound alcohol dehydrogenase rapidly oxidizes short to long-chain alcohols, and large quantities of aldehydes could enter the cell, making detoxification necessary.

Keywords: Detoxification; Alcohol dehydrogenase; Acetic acid bacteria; Vinegar; Genome sequence

Helicoverpa armigera cadherin fragment enhances Cry1Ac insecticidal activity by facilitating toxin-oligomer formation by Donghai Peng; Xiaohui Xu; Weixing Ye; Ziniu Yu; Ming Sun (pp. 1033-1040).

The interaction between Bacillus thuringiensis insecticidal crystal protein Cry1A and cadherin receptors in lepidopteran insects induces toxin oligomerization, which is essential for membrane insertion and mediates Cry1A toxicity. It has been reported that Manduca sexta cadherin fragment CR12-MPED and Anopheles gambiae cadherin fragment CR11-MPED enhance the insecticidal activity of Cry1Ab and Cry4Ba to certain lepidopteran and dipteran larvae species, respectively. This study reports that a Helicoverpa armigera cadherin fragment (HaCad1) containing its toxin binding region, expressed in Escherichia coli, enhanced Cry1Ac activity against H. armigera larvae. A binding assay showed that HaCad1 was able to bind to Cry1Ac in vitro and that this event did not block toxin binding to the brush border membrane microvilli prepared from H. armigera. When the residues ¹⁴²³GVLSLNFQ¹⁴³⁰ were deleted from the fragment, the subsequent mutation peptide lost its ability to bind Cry1Ac and the toxicity enhancement was also significantly reduced. Oligomerization tests showed that HaCad1 facilitates the formation of a 250-kDa oligomer of Cry1Ac-activated toxin in the midgut fluid environment. Oligomer formation was dependent upon the toxin binding to HaCad1, which was also necessary for the HaCad1-mediated enhancement effect. Our discovery reveals a novel strategy to enhance insecticidal activity or to overcome the resistance of insects to B. thuringiensis toxin-based biopesticides and transgenic crops.

Keywords: Helicoverpa armigera cadherin; Cry1Ac insecticidal protein; Toxicity enhancement; Oligomer formation

Expression of ribonuclease A and ribonuclease N₁ in the filamentous fungus Neurospora crassa by Silke Allgaier; Nancy Weiland; Ibtisam Hamad; Frank Kempken (pp. 1041-1049).

In this study, we investigated the ability of the fungus Neurospora crassa to produce and secrete two ribonucleases: the heterologous bovine RNase A and the endogenous RNase N₁. A set of expression vectors was constructed, each consisting of an RNase A open reading frame under the control of a specific promoter and each with a specific terminator. N. crassa transformants were analyzed at the transcriptional and protein levels. Irrespective of the promoter used, all transformants showed an RNase A-specific transcript in northern hybridization, but transcriptional strengths differed significantly. The strongest transcription was detected in transformants under the control of the cfp promoter. Western blot analysis and ELISA assays of selected transformants showed an effective secretion up to 356 ng/mL of recombinant RNase A protein. However, the highest ribonuclease activity could be detected in transformants carrying the endogenous RNase N₁ under the control of the ccg1 promoter. Expression and secretion of RNase N₁ thus represent an alternative to recombinant expression of RNase A protein. In conclusion, we have created a viable expression system for expression of homologous and heterologous proteins in N. crassa.

Keywords: Neurospora crassa ; Recombinant proteins; RNase A; RNase N₁

Soluble cytoplasmic expression, rapid purification, and characterization of cyanovirin-N as a His-SUMO fusion by Xianglei Gao; Wei Chen; Chaowan Guo; Chuiwen Qian; Ge Liu; Feng Ge; Yadong Huang; Kaio Kitazato; Yifei Wang; Sheng Xiong (pp. 1051-1060).

Cyanovirin-N (CVN) is a promising antiviral candidate that has an extremely low sequence homology with any other known proteins. The efficient and soluble expression of biologically functional recombinant CVN (rCVN) is still an obstacle due to insufficient yield, aggregation, and abnormal modification. Here, we describe an improved approach to preparing native rCVN from Escherichia coli more efficiently. A fusion gene consisting of cvn and sumo (small ubiquitin-related modifier) and a hexahistidine tag was constructed according to the codon bias of the host cell. This small ubiquitin-related modifier (SUMO)-fused CVN is expressed in the cytoplasm of E. coli in a folded and soluble form (>30% of the total soluble protein), yielding 3 to 4 mg of native rCVN from 1 g of wet cells to a purity up to 97.6%. Matrix-assisted laser desorption ionization coupled to time-of-flight mass spectrometry and reverse-phase high-performance liquid chromatographic analysis showed that the purified rCVN was an intact and homogeneous protein with a molecular weight of 11,016.68 Da. Potent antiviral activity of rCVN against herpes simplex virus type 1 and human immunodeficiency virus type 1/IIIB was confirmed in a dose-dependent manner at nanomolar concentrations. Thus, the His-SUMO double-fused CVN provides an efficient approach for the soluble expression of rCVN in the cytoplasm of E. coli, allowing an alternative system to develop bioprocess for the large-scale production of this antiviral candidate.

Keywords: Cyanovirin-N; SUMO; Escherichia coli ; Soluble expression; Anti HIV-1

Characterization of an adenylate cyclase gene (cyaB) deletion mutant of Corynebacterium glutamicum ATCC 13032 by Pu Hyeon Cha; Sun-Yung Park; Min-Woo Moon; Bindu Subhadra; Tae-Kwang Oh; Eungbin Kim; Jihyun F. Kim; Jung-Kee Lee (pp. 1061-1068).

Genome analysis of C. glutamicum ATCC 13032 has showed one putative adenylate cyclase gene, cyaB (cg0375) which encodes membrane protein belonging to class III adenylate cyclases. To characterize the function of cyaB, a deletion mutant was constructed, and the mutant showed decreased level of intracellular cyclic AMP compared to that of wild-type. Interestingly, the cyaB mutant displayed growth defect on acetate medium, and this effect was reversed by complementation with cyaB gene. Similarly, it showed growth defect on glucose-acetate mixture minimal medium, and the utilization of glucose was retarded in the presence of acetate. The deletion mutant retained the activity of glyoxylate bypass enzymes. Additionally, the mutant could grow on ethanol but not on propionate medium. The data obtained from this study suggests that adenylate cyclase plays an essential role in the acetate metabolism of C. glutamicum, even though detailed regulatory mechanisms involving cAMP are not yet clearly defined. The observation that glyoxylate bypass enzymes are derepressed in cyaB mutant indicates the involvement of cAMP in the repression of aceB and aceA.

Keywords: Corynebacterium glutamicum ; Adenylate cyclase; cyaB ; cAMP; Acetate metabolism

Characterization and analysis of the regulatory network involved in control of lipomycin biosynthesis in Streptomyces aureofaciens Tü117 by Liliya Horbal; Yuriy Rebets; Maria Rabyk; Andriy Luzhetskyy; Bogdan Ostash; Elisabeth Welle; Tatsunosuke Nakamura; Victor Fedorenko; Andreas Bechthold (pp. 1069-1079).

Analysis of the α-lipomycin biosynthesis gene cluster of Streptomyces aureofaciens Tü117 led to the identification of five putative regulatory genes, which are congregated into a subcluster. Analysis of the lipReg1–4 and lipX1 showed that they encode components of two-component signal transduction systems (LipReg1 and LipReg2), multiple antibiotics resistance-type regulator (LipReg3), large ATP-binding regulators of the LuxR family-type regulator (LipReg4), and small ribonuclease (LipRegX1), respectively. A combination of targeted gene disruptions, complementation experiments, lipomycin production studies, and gene expression analysis via RT-PCR suggests that all regulatory lip genes are involved in α-lipomycin production. On the basis of the obtained data, we propose that LipReg2 controls the activity of LipReg1, which in its turn govern the expression of the α-lipomycin pathway-specific regulatory gene lipReg4. The ribonuclease gene lipX1 and the transporter regulator lipReg3 appear to work independently of genes lipReg1, lipReg2, and lipReg4.

Keywords: Streptomyces ; Secondary metabolite; α-Lipomycin; Regulation

Homologous recombination in the antibiotic producer Penicillium chrysogenum: strain ΔPcku70 shows up-regulation of genes from the HOG pathway by Birgit Hoff; Jens Kamerewerd; Claudia Sigl; Ivo Zadra; Ulrich Kück (pp. 1081-1094).

In Penicillium chrysogenum, the industrial producer of the β-lactam antibiotic penicillin, generating gene replacements for functional analyses is very inefficient. Here, we constructed a recipient strain that allows efficient disruption of any target gene via homologous recombination. Following isolation of the Pcku70 (syn. hdfA) gene encoding a conserved eukaryotic DNA-binding protein involved in non-homologous end joining (NHEJ), a Pcku70 knockout strain was constructed using a novel nourseothricin-resistance cassette as selectable marker. In detailed physiological tests, strain ΔPcku70 showed no significant reduction in vegetative growth due to increased sensitivity to different mutagenic substances. Importantly, deletion of the Pcku70 gene had no effect on penicillin biosynthesis. However, strain ΔPcku70 exhibits higher sensitivity to osmotic stress than the parent strain. This correlated well with comparative data from microarray analyses: Genes related to the stress response are significantly up-regulated in the Pcku70 deletion mutant. To demonstrate the applicability of strain ΔPcku70, three genes related to β-lactam antibiotic biosynthesis were efficiently disrupted, indicating that this strain shows a low frequency of NHEJ, thus promoting efficient homologous recombination. Furthermore, we discuss strategies to reactivate Pcku70 in strains successfully used for gene disruptions.

Keywords: β-Lactam biosynthesis; Penicillium chrysogenum ; Gene targeting; Non-homologous end joining; Pcku70 ; Velvet ; pcbC ; Pcrfx1 ; Array analysis; HOG-signalling pathway

Monitoring of diguanylate cyclase activity and of cyclic-di-GMP biosynthesis by whole-cell assays suitable for high-throughput screening of biofilm inhibitors by Davide Antoniani; Paola Bocci; Anna Maciąg; Nadia Raffaelli; Paolo Landini (pp. 1095-1104).

In Gram-negative bacteria, production of bis-(3′,5′)-cyclic diguanylic acid (c-di-GMP) by diguanylate cyclases (DGCs) is the main trigger for production of extracellular polysaccharides and for biofilm formation. Mutants affected in c-di-GMP biosynthesis are impaired in biofilm formation, thus making DGCs interesting targets for new antimicrobial agents with anti-biofilm activity. In this report, we describe a strategy for the screening for DGC inhibitors consisting of a combination of three microbiological assays. The primary assay utilizes an Escherichia coli strain overexpressing the adrA gene, encoding the DGC protein AdrA, and relies on detection of AdrA-dependent cellulose production as red colony phenotype on solid medium supplemented with the dye Congo red (CR). Presence of DGC inhibitors blocking AdrA activity would result in a white phenotype on CR medium. The CR assay can be performed in 96-well microtiter plates, making it suitable for high-throughput screenings. To confirm specific inhibition of c-di-GMP biosynthesis, chemical compounds positive in the CR assay are tested for their ability to inhibit biofilm formation and in a reporter gene assay which monitors expression of curli-encoding genes as a function of DGC activity. Screening of a chemical library using the described approach allowed us to identify sulfathiazole, an antimetabolite drug, as an inhibitor of c-di-GMP biosynthesis. Sulfathiazole probably affects c-di-GMP biosynthesis in an indirect fashion rather than by binding to DGCs; however, sulfathiazole represents the first example of drug able to affect biofilm formation by interfering with c-di-GMP metabolism.

Keywords: c-di-GMP; Diguanylate cyclase; Biofilm formation; High-throughput screening; Antimicrobial drugs; Sulfathiazole

Gene transcription profiling of Fusarium graminearum treated with an azole fungicide tebuconazole by Xin Liu; Jinhua Jiang; Jiaofang Shao; Yanni Yin; Zhonghua Ma (pp. 1105-1114).

Using a deep serial analysis of gene expression (DeepSAGE) sequencing approach, we profiled the transcriptional response of Fusarium graminearum to tebuconazole, a most widely used azole fungicide. By comparing the expression of genes in F. graminearum treated and untreated with tebuconazole, we identified 324 and 155 genes showing more than a 5-fold increase and decrease, respectively, in expression upon tebuconazole treatment. These genes are involved in a variety of cell functions including egrosterol biosynthesis, transcription, and cellular metabolism. The validity of DeepSAGE results were confirmed by real-time PCR analysis of expression of 20 genes with different expression levels in the DeepSAGE analysis. The results from this study provide useful information in understanding the mechanisms for the responses of F. graminearum to azole fungicides.

Keywords: DeepSAGE; Fusarium graminearum ; Real-time PCR; Tebuconazole; Transcriptome

Antibacterial activity and mechanism of silver nanoparticles on Escherichia coli by Wen-Ru Li; Xiao-Bao Xie; Qing-Shan Shi; Hai-Yan Zeng; You-Sheng OU-Yang; Yi-Ben Chen (pp. 1115-1122).

The antibacterial activity and acting mechanism of silver nanoparticles (SNPs) on Escherichia coli ATCC 8739 were investigated in this study by analyzing the growth, permeability, and morphology of the bacterial cells following treatment with SNPs. The experimental results indicated 10 μg/ml SNPs could completely inhibit the growth of 10⁷ cfu/ml E. coli cells in liquid Mueller–Hinton medium. Meanwhile, SNPs resulted in the leakage of reducing sugars and proteins and induced the respiratory chain dehydrogenases into inactive state, suggesting that SNPs were able to destroy the permeability of the bacterial membranes. When the cells of E. coli were exposed to 50 μg/ml SNPs, many pits and gaps were observed in bacterial cells by transmission electron microscopy and scanning electron microscopy, and the cell membrane was fragmentary, indicating the bacterial cells were damaged severely. After being exposed to 10 μg/ml SNPs, the membrane vesicles were dissolved and dispersed, and their membrane components became disorganized and scattered from their original ordered and close arrangement based on TEM observation. In conclusion, the combined results suggested that SNPs may damage the structure of bacterial cell membrane and depress the activity of some membranous enzymes, which cause E. coli bacteria to die eventually.

Keywords: Silver nanoparticles (SNPs); Escherichia coli ; Antibacterial mechanism; Permeability; Bacterial membrane

Using regulatory information to manipulate glycerol metabolism in Saccharomyces cerevisiae by Jin Hou; Goutham N. Vemuri (pp. 1123-1130).

Metabolic engineering has emerged as an attractive alternative to random mutagenesis and screening to design cell factories for industrial fermentation processes. The design of metabolic networks has been realized by gene deletions or strong overexpression of heterologous genes. There is an increasing body of evidence that indicates complete inactivation of native genes and high-level activity of heterologous enzymes may be deleterious to the cell. To moderately implement their expression, genes of interest are expressed under the control of promoters with different strengths. Constructing a promoter library is labor-intensive and requires precise quantification of the promoter strength. However, when the mechanisms of pathway regulation are known, it is possible to exploit this information to effect genetic changes efficiently. We report the implementation of this concept to reducing glycerol production during aerobic growth of Saccharomyces cerevisiae. Glycerol is produced to dispose excess cytosolic reduced nicotinamide adenine dinucleotide (NADH), and the regulating step in the pathway is mediated by glycerol 3-phosphate dehydrogenase (encoded by GPD1 and GPD2 genes). We expressed NADH oxidase in S. cerevisiae under the control of the GPD2 promoter to modulate the decrease in cytosolic NADH to the right level where the heterologous enzyme does not compete with oxidative phosphorylation while at the same time, decreasing glycerol production. This metabolic design resulted in substantially decreasing glycerol production and indeed, the excess carbon was redirected to biomass, resulting in a 14% increase in the specific growth rate. We believe that such strategies are more efficient than conventional methods and will find applications in bioprocesses.

Keywords: Redox metabolism; Glycerol; Saccharomyces cerevisiae ; Glycerol 3-phosphate dehydrogenase; Promoter engineering

Effect of two types of biosurfactants on phenanthrene availability to the bacterial bioreporter Burkholderia sartisoli strain RP037 by Robin Tecon; Jan Roelof van der Meer (pp. 1131-1139).

Biosurfactants are tensio-active agents that have often been proposed as a means to enhance the aqueous solubility of hydrophobic organic contaminants, such as polycyclic aromatic hydrocarbons (PAHs). Biosurfactant-producing bacteria such as those belonging to the genus Pseudomonas might therefore enhance PAH availability to PAH-degrading bacteria. We tested the effects of two types of biosurfactants produced by Pseudomonas sp., cyclic lipopeptides and rhamnolipids, on phenanthrene bioavailability. Bioavailability was judged from growth rates on phenanthrene and from specific induction of a phenanthrene-responsive GFP-reporter in Burkholderia sartisoli strain RP037. Co-culturing of strain RP037 with the lipopeptide-producing bacterium Pseudomonas putida strain PCL1445 enhanced GFP expression compared to a single culture, but this effect was not significantly different when strain RP037 was co-cultivated with a non-lipopeptide-producing mutant of P. putida. The addition of partially purified supernatant extracts from the P. putida lipopeptide producer equally did not unequivocally enhance phenanthrene bioavailability to strain RP037 compared to controls. In contrast, a 0.1% rhamnolipid solution strongly augmented RP037 growth rates on phenanthrene and led to a significantly larger proportion of cells in culture with high GFP expression. Our data therefore suggest that biosurfactant effects may be strongly dependent on the strain and type of biosurfactant.

Keywords: Bioreporter; GFP; Rhamnolipid; Lipopeptide; Polycyclic aromatic hydrocarbons (PAH)

Electricity generation at high ionic strength in microbial fuel cell by a newly isolated Shewanella marisflavi EP1 by Jiexun Huang; Baolin Sun; Xiaobo Zhang (pp. 1141-1149).

Increasing the ionic strength of the electrolyte in a microbial fuel cell (MFC) can remarkably increase power output due to the reduction of internal resistance. However, only a few bacterial strains are capable of producing electricity at a very high ionic strength. In this report, we demonstrate a newly isolated strain EP1, belonging to Shewanella marisflavi based on polyphasic analysis, which could reduce Fe(III) and generate power at a high ionic strength of up to 1,488 mM (8% NaCl) using lactate as the electron donor. Using this bacterium, a measured maximum power density of 3.6 mW/m² was achieved at an ionic strength of 291 mM. The maximum power density was increased by 167% to 9.6 mW/m² when ionic strength was increased to 1,146 mM. However, further increasing the ionic strength to 1,488 mM resulted in a decrease in power density to 5.2 mW/m². Quantification of the internal resistance distribution revealed that electrolyte resistance was greatly reduced from 1,178 to 50 Ω when ionic strength increased from 291 to 1,488 mM. These results indicate that isolation of specific bacterial strains can effectively improve power generation in some MFC applications.

Keywords: Microbial fuel cell; Shewanella marisflavi ; Ionic strength; Internal resistance

Linking performance to microbiology in biofilters treating dimethyl sulphide in the presence and absence of methanol by Alexander C. Hayes; Yuefeng Zhang; Steven N. Liss; D. Grant Allen (pp. 1151-1166).

The performance and microbiology of two inorganic biofilters treating dimethyl sulphide (DMS) in the presence and absence of methanol was investigated. Addition of methanol was shown to result in an increase in DMS removal for methanol loadings below 90 g MeOH per cubic metre per hour with the optimal methanol loading around 10–15 g MeOH per cubic metre per hour for a DMS loading of 3.4 g DMS per cubic metre per hour, a fivefold increase in the DMS removal rate compared to the biofilter treating DMS alone. Microbial community analysis revealed that the addition of methanol led to a significant increase of up to an order of magnitude in the abundance of Hyphomicrobium spp. in the biofilter co-treating DMS and methanol compared to the biofilter treating DMS alone, whilst there was no significant difference in the abundance of Thiobacillus spp. between the two biofilters. Given the behaviour of the biofilter co-treating DMS and methanol, the magnitude of the increase in Hyphomicrobium spp. in the biofilter co-treating DMS and methanol and the ability of Hyphomicrobium spp. to use both methanol and DMS as growth substrates, it was concluded that Hyphomicrobium spp. were the microorganisms responsible for the bulk of the DMS degradation in the biofilter co-treating DMS and methanol.

Keywords: Biofiltration; Dimethyl sulphide; Methanol; Microbial community analysis; 16S rDNA clone library; Denatured gradient gel electrophoresis; Real-time PCR

Biodegradation of thiocyanate using co-culture of Klebsiella pneumoniae and Ralstonia sp. by Ashvini U. Chaudhari; Kisan M. Kodam (pp. 1167-1174).

Thiocyanate-degrading microbial co-culture was isolated from thiocyanate-contaminated site and tested for thiocyanate degradation potential and thiocyanate-toxicity tolerance and identified as Klebsiella pneumoniae and Ralstonia sp. by 16S rDNA sequencing. The co-culture was able to degrade thiocyanate with degradation rate of 500 mg L⁻¹d⁻¹ at 2,500 mg L⁻¹ thiocyanate concentration at pH 6.0 and 37ºC following thiocyanate hydrolase pathway. The Haldane kinetic model elucidates the growth and thiocyanate biodegradation kinetics of the co-culture with Ki value of 1,876 mg L⁻¹. The thiocyanate biodegradation kinetics was not affected by the additional supply of glucose. The very high activities of thiocyanate hydrolase, cyanide oxygenase, and cytochrome P-450 content during growth on thiocyanate were observed, showing the induction mechanism.

Keywords: Co-culture; Klebsiella ; Ralstonia ; Thiocyanate; Thiocyanate hydrolase

Characterization of corrosive bacterial consortia isolated from petroleum-product-transporting pipelines by Aruliah Rajasekar; Balakrishnan Anandkumar; Sundaram Maruthamuthu; Yen-Peng Ting; Pattanathu K. S. M. Rahman (pp. 1175-1188).

Microbiologically influenced corrosion is a problem commonly encountered in facilities in the oil and gas industries. The present study describes bacterial enumeration and identification in diesel and naphtha pipelines located in the northwest and southwest region in India, using traditional cultivation technique and 16S rDNA gene sequencing. Phylogenetic analysis of 16S rRNA sequences of the isolates was carried out, and the samples obtained from the diesel and naphtha-transporting pipelines showed the occurrence of 11 bacterial species namely Serratia marcescens ACE2, Bacillus subtilis AR12, Bacillus cereus ACE4, Pseudomonas aeruginosa AI1, Klebsiella oxytoca ACP, Pseudomonas stutzeri AP2, Bacillus litoralis AN1, Bacillus sp., Bacillus pumilus AR2, Bacillus carboniphilus AR3, and Bacillus megaterium AR4. Sulfate-reducing bacteria were not detected in samples from both pipelines. The dominant bacterial species identified in the petroleum pipeline samples were B. cereus and S. marcescens in the diesel and naphtha pipelines, respectively. Therefore, several types of bacteria may be involved in biocorrosion arising from natural biofilms that develop in industrial facilities. In addition, localized (pitting) corrosion of the pipeline steel in the presence of the consortia was observed by scanning electron microscopy analysis. The potential role of each species in biofilm formation and steel corrosion is discussed.

Keywords: Carbon steel API 5 L-X60; Petroleum product pipeline; Bacterial community; 16S rDNA analysis; Microbiologically influenced corrosion

Characterisation of hexane-degrading microorganisms in a biofilter by stable isotope-based fatty acid analysis, FISH and cultivation by Michèle M. Friedrich; André Lipski (pp. 1189-1199).

The hexane-degrading bacterial community of a biofilter was characterised by a combination of stable isotope-based phospholipid fatty acid analyses, fluorescence in situ hybridisation and cultivation. About 70 bacterial strains were isolated from a full-scale biofilter used for treatment of hexane containing waste gas of an oil mill. The isolation approach led to 16 bacterial groups, which were identified as members of the Alpha-, Beta- and Gammaproteobacteria, Actinobacteria and Firmicutes. Three groups showed good growth on hexane as the sole source of carbon. These groups were allocated to the genera Gordonia and Sphingomonas and to the Nevskia-branch of the Gammaproteobacteria. Actively degrading populations in the filter material were characterised by incubation of filter material samples with deuterated hexane and subsequent phospholipid fatty acid analysis. Significant labelling of the fatty acids 16:1 cis10, 18:1 cis9 and 18:0 10methyl affiliated the hexane-degrading activity of the biofilter with the isolates of the genus Gordonia. In vitro growth on hexane and in situ labelling of characteristic fatty acids confirmed the central role of these organisms in the hexane degradation within the full-scale biofilter.

Keywords: Biofilter; Hexane; PLFA; FISH

A simple method to introduce marker-free genetic modifications into the chromosome of naturally nontransformable Bacillus amyloliquefaciens strains by Natalia P. Zakataeva; Oksana V. Nikitina; Sergey V. Gronskiy; Dmitriy V. Romanenkov; Vitaliy A. Livshits (pp. 1201-1209).

A simple method to introduce marker-free deletions, insertions, and point mutations into the chromosomes of naturally nontransformable Bacillus amyloliquefaciens strains has been developed. The method is efficient and fast, and it allows for the generation of genetic modifications without the use of a counter-selectable marker or a special prerequisite strain. This method uses the combination of the following: the effective introduction of a delivery plasmid into cells for gene replacement; a two-step replacement recombination procedure, which occurs at a very high frequency due to the use of a thermosensitive rolling-circle replication plasmid; and colony polymerase chain reaction (PCR) analysis for screening. Using PCR primers with mismatches at the 3′ end enables the selection of strains that contain a single nucleotide substitution in the target gene. This approach can be used as a routine method for the investigation of complex physiological pathways and for the metabolic engineering of food-grade industrial B. amyloliquefaciens and other Bacillus strains.

Keywords: Naturally nontransformable Bacillus amyloliquefaciens ; Thermosensitive rolling-circle plasmid; Replacement recombination; Marker-free genetic modifications; Point mutation; Colony PCR analysis

Isolation of functional single cells from environments using a micromanipulator: application to study denitrifying bacteria by Naoaki Ashida; Satoshi Ishii; Sadakazu Hayano; Kanako Tago; Takashi Tsuji; Yoshitaka Yoshimura; Shigeto Otsuka; Keishi Senoo (pp. 1211-1217).

We developed a novel method to isolate functionally active single cells from environmental samples and named it the functional single-cell (FSC) isolation method. This method is based on a combination of substrate-responsive direct viable counts, live-cell staining with 5-carboxyfluorescein diacetate acetoxymethyl ester, and micromanipulation followed by cultivation in a medium. To evaluate this method, we applied it to study a denitrifying community in rice paddy soil. Similar denitrifier counts were obtained by the conventional most probable number analysis and our FSC isolation method. Using the FSC isolation method, 37 denitrifying bacteria were isolated, some of which harbored copper-containing nitrite reductase gene (nirK). The 16S rRNA gene analysis showed that members belonging to the genera Azospirillum and Ochrobactrum may be the major denitrifiers in the rice paddy soil. These results indicate that the FSC isolation method is a useful tool to obtain functionally active single cells from environmental samples.

Keywords: Single-cell isolation; Micromanipulation; Direct viable counts; Live-cell staining; Denitrification; Rice paddy soil

Identification of avermectin-high-producing strains by high-throughput screening methods by Hong Gao; Mei Liu; Xianlong Zhou; Jintao Liu; Ying Zhuo; Zhongxuan Gou; Bing Xu; Wenquan Zhang; Xiangyang Liu; Aiqun Luo; Chuansen Zheng; Xiaoping Chen; Lixin Zhang (pp. 1219-1225).

Avermectins produced by Streptomyces avermitilis are potent against a broad spectrum of nematode and arthropod parasites with low-level side effects on the host organisms. This study was designed to investigate a high-throughput screening strategy for the efficient identification of avermectin high-yield strains. The production protocol was miniaturized in 96 deep-well microplates. UV absorbance at 245 nm was used to monitor avermectin production. A good correlation between fermentation results in both 96 deep-well microplates and conventional Erlenmeyer flasks was observed. With this protocol, the production of avermectins was determined in less than 10 min for a full plate without compromising accuracy. The high-yield strain selected through this protocol was also tested in 360 m³ batch fermentation with 1.6-fold improved outcome. Thus, the development of this protocol is expected to accelerate the selection of superior avermectin-producing strains.

Keywords: Avermectin; High-throughput screening; Strain improvement; Miniaturized production

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Applied Microbiology and Biotechnology (v.85, #4)

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Applied Microbiology and Biotechnology (v.85, #4)