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Applied Microbiology and Biotechnology (v.93, #2)

Nitric oxide elicitation for secondary metabolite production in cultured plant cells by Ben Zhang; Li Ping Zheng; Jian Wen Wang (pp. 455-466).

Nitric oxide (NO) is an important signal molecule in stress responses. Accumulation of secondary metabolites often occurs in plants subjected to stresses including various elicitors or signal molecules. NO has been reported to play important roles in elicitor-induced secondary metabolite production in tissue and cell cultures of medicinal plants. Better understanding of NO role in the biosynthesis of such metabolites is very important for optimizing the commercial production of those pharmaceutically significant secondary metabolites. This paper summarizes progress made on several aspects of NO signal leading to the production of plant secondary metabolites, including various abiotic and biotic elicitors that induce NO production, elicitor-triggered NO generation cascades, the impact of NO on growth development and programmed cell death in medicinal plants, and NO-mediated regulation of the biosynthetic pathways of such metabolites. Cross-talks among NO signaling and reactive oxygen species, salicylic acid, and jasmonic acid are discussed. Some perspectives on the application of NO donors for induction of the secondary metabolite accumulation in plant cultures are also presented.

Keywords: Nitric oxide; Elicitation; Secondary metabolites; Biosynthesis; Plant tissue cultures

Biosynthesis and function of gliotoxin in Aspergillus fumigatus by Daniel H. Scharf; Thorsten Heinekamp; Nicole Remme; Peter Hortschansky; Axel A. Brakhage; Christian Hertweck (pp. 467-472).

Gliotoxin (GT) is the prototype of the epidithiodioxopiperazine (ETP)-type fungal toxins. GT plays a critical role in the pathobiology of Aspergillus fumigatus. It modulates the immune response and induces apoptosis in different cell types. The toxicity has been attributed to the unusual intramolecular disulfide bridge, which is the functional motif of all ETPs. Because of the extraordinary structure and activity of GT, this fungal metabolite has been the subject of many investigations. The biosynthesis of GT involves unprecedented reactions catalysed by recently discovered enzymes. Here, we summarize the recent progress in elucidating the GT biosynthetic pathway and its role in virulence.

Keywords: Aspergillus fumigatus ; Non-ribosomal peptide biosynthesis; Gliotoxin; ETP toxins

Applications of microbial fermentations for production of gluten-free products and perspectives by Emanuele Zannini; Erica Pontonio; Deborah M. Waters; Elke K. Arendt (pp. 473-485).

A gluten-free (GF) diet is recognised as being the only accepted treatment for celiac disease—a permanent autoimmune enteropathy triggered by the ingestion of gluten-containing cereals. The bakery products available in today’s gluten-free market are characterised by lower palatability than their conventional counterparts and may lead to nutritional deficiencies of vitamins, minerals and fibre. Thus, the production of high-quality gluten-free products has become a very important socioeconomical issue. Microbial fermentation by means of lactic acid bacteria and yeast is one of the most ecological/economical methods of producing and preserving food. In this review, the role of a fermentation process for improving the quality of GF products and for developing a new concept of GF products with nutraceutical and health-promoting characteristics will be examined.

Keywords: Microbial fermentation; Gluten-free foods; Gluten-free beverages; Lactic acid bacteria; Functional foods

Biosynthesis of phloroglucinol compounds in microorganisms—review by Fang Yang; Yujin Cao (pp. 487-495).

Phloroglucinol derivatives are a major class of secondary metabolites of wide occurrence in biological systems. In the bacteria kingdom, these compounds can only be synthesized by some species of Pseudomonads. Pseudomonas spp. could produce 2,4-diacetylphloroglucinol (DAPG) that plays an important role in the biological control of many plant pathogens. In this review, we summarize knowledge about synthesis of phloroglucinol compounds based on the DAPG biosynthetic pathway. Recent advances that have been made in understanding phloroglucinol compound biosynthesis and regulation are highlighted. From these studies, researchers have identified the biosynthesis pathway of DAPG. Most of the genes involved in the biosynthesis pathway have been cloned and characterized. Additionally, heterologous systems of the model microorganism Escherichia coli are constructed to produce phloroglucinol. Although further work is still required, a full understanding of phloroglucinol compound biosynthesis is almost within reach. This review also suggests new directions and attempts to gain some insights for better understanding of the biosynthesis and regulation of DAPG. The combination of traditional biochemistry and molecular biology with new systems biology and synthetic biology tools will provide a better view of phloroglucinol compound biosynthesis and a greater potential of microbial production.

Keywords: 2,4-Diacetylphloroglucinol; Biosynthesis; Phloroglucinol; Pseudomonas fluorescens

Processive and nonprocessive cellulases for biofuel production—lessons from bacterial genomes and structural analysis by David B. Wilson (pp. 497-502).

Cellulases are key enzymes used in many processes for producing liquid fuels from biomass. Currently there many efforts to reduce the cost of cellulases using both structural approaches to improve the properties of individual cellulases and genomic approaches to identify new cellulases as well as other proteins that increase the activity of cellulases in degrading pretreated biomass materials. Fungal GH-61 proteins are important new enzymes that increase the activity of current commercial cellulases leading to lower total protein loading and thus lower cost. Recent work has greatly increased our knowledge of these novel enzymes that appear to be oxido-reductases that target crystalline cellulose and increase its accessibility to cellulases. They appear to carry out the C1 activity originally proposed by Dr Reese. Cellobiose dehydrogenase appears to interact with GH-61 proteins in this function, providing a role for this puzzling enzyme. Cellulase research is making considerable progress and appears to be poised for even greater advances.

Keywords: Synergism; Processivity; Molecular modeling; Swollenin; Oxidoreductase; Cellobiose dehydrogenase

l-Aspartate dehydrogenase: features and applications by Yinxia Li; Henry Joseph Oduor Ogola; Yoshihiro Sawa (pp. 503-516).

l-Amino acid dehydrogenases are a group of enzymes that catalyze the reversible oxidative deamination of l-amino acids to their corresponding 2-oxoacids, using either nicotinamide adenine dinucleotide (NAD⁺) or nicotinamide adenine dinucleotide phosphate (NADP⁺) as cofactors. These enzymes have been studied widely because of their potential applications in the synthesis of amino acids for use in production of pharmaceutical peptides, herbicides and insecticides, in biosensors or diagnostic kits, and development of coenzyme regeneration systems for industrial processes. This article presents a review of the currently available data about the recently discovered amino acid dehydrogenase superfamily member l-aspartate dehydrogenase (l-AspDH), their relevant catalytic properties and speculated physiological roles, and potential for biotechnological applications. The proposed classification of l-AspDH on the basis of bioinformatic information and potential role in vivo into NadB (NAD biosynthesis-related) and non-NadB type is unique. In particular, the mesophilic non-NadB type l-AspDH is a novel group of amino acid dehydrogenases with great promise as potential industrial biocatalysts owing to their relatively high catalytic properties at room temperature. Considering that only a few l-AspDH homologs have been characterized so far, identification and prodigious enzymological research of the new members will be necessary to shed light on the gray areas pertaining to these enzymes.

Keywords: l-Amino acid dehydrogenase; NAD biosynthesis; l-Aspartate dehydrogenase; Characteristics; Applications; Functions

Engineering bacteria toward tumor targeting for cancer treatment: current state and perspectives by Che-Hsin Lee (pp. 517-523).

One of the primary limitations of cancer therapy is lack of selectivity of therapeutic agents to tumor cells. Current efforts are focused on discovering and developing anticancer agents that selectively target only tumor cells and spare normal cells to improve the therapeutic index. The use of preferentially replicating bacteria as an oncolytic agent is one of the innovative approaches for the treatment of cancer. This is based on the observation that some obligate or facultative anaerobic bacteria are capable of multiplying selectively in tumors and inhibiting their growth. Meanwhile, bacteria have been demonstrated to colonize and destroy tumor, and have emerged as biological gene vectors to tumor microenvironment. To improve the efficacy and safety of the bacterial therapy, a further understanding of bacteria between with immune system is required. Furthermore, we want to evaluate how bacterial infection facilitates the “bystander effect” of chemotherapeutic agent and assess if it can be used for additional antitumor effect when combined with chemotherapy. This study may not only evaluate therapeutic efficacy of bacteria for the treatment of cancer but also elucidate the mechanisms underlying antitumor activities mediated by bacteria, which involve host immune responses and the cellular molecular responses.

Keywords: Bacteria; Oncolytic therapy; Tumor targeting; Immunity

Recent advances in curdlan biosynthesis, biotechnological production, and applications by Xiao-Bei Zhan; Chi-Chung Lin; Hong-Tao Zhang (pp. 525-531).

Curdlan is a water-insoluble β-(1,3)-glucan produced by Agrobacterium species under nitrogen-limited condition. Its heat-induced gelling properties render curdlan to be very useful in the food industry initially. Recent advances in the understanding of the role curdlan plays in both innate and adaptive immunity lead to its growing applications in biomedicine. Our review focuses on the recent advances on curdlan biosynthesis and the improvements of curdlan fermentation production both from our laboratory and many others as well as the latest advances on the new applications of curdlan and its derivatives particularly in their immunological functions in biomedicine.

Keywords: Curdlan; Oligosaccharides; Biosynthesis; Production improvement; New applications

Fungal chitinases: diversity, mechanistic properties and biotechnological potential by Lukas Hartl; Simone Zach; Verena Seidl-Seiboth (pp. 533-543).

Chitin derivatives, chitosan and substituted chito-oligosaccharides have a wide spectrum of applications ranging from medicine to cosmetics and dietary supplements. With advancing knowledge about the substrate-binding properties of chitinases, enzyme-based production of these biotechnologically relevant sugars from biological resources is becoming increasingly interesting. Fungi have high numbers of glycoside hydrolase family 18 chitinases with different substrate-binding site architectures. As presented in this review, the large diversity of fungal chitinases is an interesting starting point for protein engineering. In this review, recent data about the architecture of the substrate-binding clefts of fungal chitinases, in connection with their hydrolytic and transglycolytic abilities, and the development of chitinase inhibitors are summarized. Furthermore, the biological functions of chitinases, chitin and chitosan utilization by fungi, and the effects of these aspects on biotechnological applications, including protein overexpression and autolysis during industrial processes, are discussed in this review.

Keywords: Chitinase; Chitin; Chitosan; Cell wall; Fungi; GH family 18

Physiology, biochemistry and possible applications of microbial caffeine degradation by Sathyanarayana N. Gummadi; B. Bhavya; Nandhini Ashok (pp. 545-554).

Caffeine, a purine alkaloid is a constituent of widely consumed beverages. The scientific evidence which has proved the harm of this alkaloid has paved the way for innumerable research in the area of caffeine degradation. In addition to this, the fact that the by-products of the coffee and tea industry pollute the environment has called for the need of decaffeinating coffee and tea industry’s by-products. Though physical and chemical methods for decaffeination are available, the lack of specificity for removal of caffeine in these techniques and their non-eco-friendly nature has opened the area of microbial and enzymatic degradation of caffeine. Another important application of microbial caffeine degradation apart from its advantages like specificity, eco-friendliness and cost-effectiveness is the fact that this process will enable the production of industrially and medically useful components of the caffeine degradation pathway like theobromine and theophylline. This is a comprehensive review which mainly focuses on caffeine degradation, large-scale degradation of the same and its applications in the industrial world.

Keywords: Caffeine degradation; Microbial and enzymatic methods; Solid-state fermentation; Large-scale degradation; Applications

Bioproduction of vanillin using an organic solvent-tolerant Brevibacillus agri 13 by Nuttawat Wangrangsimagul; Kunticha Klinsakul; Alisa S. Vangnai; Jirarut Wongkongkatep; Pranee Inprakhon; Kohsuke Honda; Hisao Ohtake; Junichi Kato; Thunyarat Pongtharangkul (pp. 555-563).

Nowadays, majority of vanillin supplied to the world market is chemically synthesized from a petroleum-based raw material, raising a concern among the consumers regarding the product safety. In this study, an organic solvent-tolerant Brevibacillus agri 13 previously reported for a strong predilectic property was utilized as a whole-cell biocatalyst for bioproduction of vanillin from isoeugenol (IG). B. agri 13 is the first biocatalyst reported for bioproduction of vanillin at a temperature as high as 45°C. Both pH and temperature were found to affect vanillin production significantly. An extreme level of organic solvent tolerance of B. agri 13 allowed us to utilize it in a biphasic system using organic solvents generally considered as highly toxic to most bacteria. With an addition of butyl acetate at 30% (v/v) as an organic second phase, toxicity of IG exerted onto the biocatalyst was reduced dramatically while faster and more efficient vanillin production was obtained (1.7 g/L after 48 h with 27.8% molar conversion).

Keywords: Brevibacillus ; Vanillin production; Isoeugenol; Biphasic system

Kinetics of styrene biodegradation by Pseudomonas sp. E-93486 by Agnieszka Gąszczak; Grażyna Bartelmus; Izabela Greń (pp. 565-573).

The research into kinetics of styrene biodegradation by bacterial strain Pseudomonas sp. E-93486 coming from VTT Culture Collection (Finland) was presented in this work. Microbial growth tests in the presence of styrene as the sole carbon and energy source were performed both in batch and continuous cultures. Batch experiments were conducted for initial concentration of styrene in the liquid phase changed in the range of 5–90 g m⁻³. The Haldane model was found to be the best to fit the kinetic data, and the estimated constants of the equation were: μ _m = 0.1188 h⁻¹, K _S = 5.984 mg l⁻¹, and K _i = 156.6 mg l⁻¹. The yield coefficient mean value $$ Y_{ ext{xs}}^{ ext{app}} $$ for the batch culture was 0.72 g_{dry cells weight} (g_substrate)⁻¹. The experiments conducted in a chemostat at various dilution rates (D = 0.035–0.1 h⁻¹) made it possible to determine the value of the coefficient for maintenance metabolism m _d = 0.0165 h⁻¹ and the maximum yield coefficient value $$ Y_{ ext{xs}}^{ ext{M}} = 0.913 $$ . Chemostat experiments confirmed the high value of yield coefficient $$ Y_{ ext{xs}}^{ ext{app}} $$ observed in the batch culture. The conducted experiments showed high activity of the examined strain in the styrene biodegradation process and a relatively low sensitivity to inhibition of its growth at higher concentrations of styrene in the solution. Such exceptional features of Pseudomonas sp. E-93486 make this bacterial strain the perfect candidate for technical applications.

Keywords: Styrene; Pseudomonas ; Kinetics; Chemostat; Batch experiment

Investigation of the potential of biocalorimetry as a process analytical technology (PAT) tool for monitoring and control of Crabtree-negative yeast cultures by Moira Monika Schuler; Senthilkumar Sivaprakasam; Brian Freeland; Adel Hama; Katie-Marie Hughes; Ian W. Marison (pp. 575-584).

Biological reaction calorimetry, also known as biocalorimetry, has led to extensive applications in monitoring and control of different bioprocesses. A simple real-time estimator for biomass and growth rate was formulated, based on in-line measured metabolic heat flow values. The performance of the estimator was tested in a unique bench-scale calorimeter (BioRC1), improved to a sensitivity range of 8 mW l^− 1 in order to facilitate the monitoring of even weakly exothermic biochemical reactions. A proportional–integral feedback control strategy based on these estimators was designed and implemented to control the growth rate of Candida utilis, Kluyveromyces marxianus and Pichia pastoris by regulating an exponential substrate feed. Maintaining a particular specific growth rate throughout a culture is essential for reproducible product quality in industrial bioprocesses and therefore a key sequence for the step from quality by analysis to quality by design. The potential of biocalorimetry as a reliable biomass monitoring tool and as a key part of a robust control strategy for aerobic fed-batch cultures of Crabtree-negative yeast cells in defined growth medium was investigated. Presenting controller errors of less than 4% in the best cases, the approach paves the way for the development of a generally applicable process analytical technology platform for monitoring and control of microbial fed-batch cultures.

Keywords: Biocalorimetry; On-line biomass estimation; Specific growth rate; Fed-batch; Candida utilis ; Kluyveromyces marxianus ; Pichia pastoris

Laccase-catalysed protein–flavonoid conjugates for flax fibre modification by Suyeon Kim; Artur Cavaco-Paulo (pp. 585-600).

The introduction of flavonoid compounds into proteins can improve the natural properties of proteins, being promising products which essentially require antioxidant property. The oxidative conjugation of protein–flavonoids was processed by laccase catalysis resulting in the synthesis of biologically functional polymers. The new reaction products were detected in terms of sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionisation-time of flight mass spectra, showing a greater molecular weight formation. Their characterisations were further carried out in terms of UV–Vis spectroscopy, photon correlation spectroscopy, differential scanning calorimetry and Fourier transform infrared (FT-IR) spectroscopy analysis. In addition, their application of protein–flavonoid conjugates onto flax fibres was exploited to supplement a suitable microorganism environment of protein-possessed fibres. The anchoring of conjugates onto cationised fibres was successfully performed by ionic interaction with negatively charged proteins. The level of anchoring efficiency was quantified in terms of measuring colour strength (k/s) and fluorescence microscopy analysis. The conjugates onto fibres presented acceptable durability in terms of washing resistance and the surface became hydrophilic when α-casein–catechin was applied (lower contact angle 48°). By the anchoring of protein–flavonoid conjugates onto flax fibres, the final products with new colour generation and antioxidant activity (>93%) were obtained.

Keywords: Laccase; Protein–flavonoid conjugate; Surface modification; O-quinones; Antioxidant activity; Flax fibres

Trypsin promotes efficient influenza vaccine production in MDCK cells by interfering with the antiviral host response by Claudius Seitz; Britta Isken; Björn Heynisch; Maria Rettkowski; Timo Frensing; Udo Reichl (pp. 601-611).

Trypsin is commonly used in Madin–Darby canine kidney (MDCK) cell culture-based influenza vaccine production to facilitate virus infection by proteolytic activation of viral haemagglutinin, which enables multi-cycle replication. In this study, we were able to demonstrate that trypsin also interferes with pathogen defence mechanisms of host cells. In particular, a trypsin concentration of 5 BAEE U/mL (4.5 μg/mL porcine trypsin) used in vaccine manufacturing strongly inhibited interferon (IFN) signalling by proteolytic degradation of secreted IFN. Consequently, absence of trypsin during infection resulted in a considerably stronger induction of IFN signalling and apoptosis, which significantly reduced virus yields. Under this condition, multi-cycle virus replication in MDCK cells was not prevented but clearly delayed. Therefore, incomplete infection can be ruled out as the reason for the lower virus titres. However, suppression of IFN signalling by overexpression of viral IFN antagonists (influenza virus PR8-NS1, rabies virus phosphoprotein) partially rescued virus titres in the absence of trypsin. In addition, virus yields could be almost restored by using the influenza strain A/WSN/33 in combination with fetal calf serum (FCS). For this strain, FCS enabled trypsin-independent fast propagation of virus infection, probably outrunning cellular defence mechanisms and apoptosis induction in the absence of trypsin. Overall, addition of trypsin provided optimal conditions for high yield vaccine production in MDCK cells by two means. On the one hand, proteolytic degradation of IFN keeps cellular defence at a low level. On the other hand, enhanced virus spreading enables viruses to replicate before the cellular response becomes fully activated.

Keywords: Influenza; Vaccine production; MDCK cells; Trypsin; Interferon

Large-scale expression, purification, and glucose uptake activity of recombinant human FGF21 in Escherichia coli by Minjing Zhang; Xuebin Jiang; Zhijian Su; Jiancong Lin; Qi Xiang; Zhan Yang; Yadong Huang; Xiaokun Li (pp. 613-621).

As a novel important regulator of glucose and lipid metabolism homeostasis, human fibroblast growth factor 21 (hFGF21) has become a potential drug candidate for the treatment of metabolic diseases including obesity, and type 2 diabetes, as well as non-alcoholic fatty liver disease. To improve the production of recombinant hFGF21 to meet the increasing demand in clinical applications, an artificial gene encoding its mature peptide sequence was constructed, cloned into vector pET-3c and then expressed in Escherichia coli Origami B (DE3). Under optimal conditions in a 50-L fermentor, the average bacterial yield and the soluble expression level of recombinant hFGF21 of six batches attained 1750 ± 185 g and 32 ± 1.5%, respectively. The target protein was purified by the combination of nickel-nitrilotriacetic acid affinity chromatography and Sephadex S-100 resin. 5% (w/v) trehalose solution was able to prevent rhFGF21 from degradation effectively. The purity of rhFGF21 was higher than 97%, and the yield was 213 ± 17 mg/L. The preliminary biochemical characterization of rhFGF21 was confirmed using Western blot and peptide map finger analysis. Based on the glucose oxidase–peroxidase assay, the EC50 of glucose uptake activity of the purified rhFGF21 was 22.1 nM.

Keywords: Human fibroblast growth factor 21; Expression; Purification; Glucose uptake activity

Identification of a new subfamily of salt-tolerant esterases from a metagenomic library of tidal flat sediment by Jeong Ho Jeon; Hyun Sook Lee; Jun Tae Kim; Sang-Jin Kim; Sang Ho Choi; Sung Gyun Kang; Jung-Hyun Lee (pp. 623-631).

To search for novel lipolytic enzymes, a metagenomic library was constructed from the tidal flat sediment of Ganghwa Island in South Korea. By functional screening using tributyrin agar plates, 3 clones were selected from among the 80,050 clones of the fosmid library. The sequence analysis revealed that those clones contained different open reading frames, which showed 50–57% amino acid identity with putative lipolytic enzymes in the database. Based on the phylogenetic analysis, they were identified to encode novel members, which form a distinct and new subfamily in the family IV of bacterial lipolytic enzymes. The consensus sequence, GT(S)SA(G)G, encompassing the active site serine of the enzymes was different from the GDSAG motif, conserved in the other subfamily. The genes were expressed in Escherichia coli and recombinant proteins were purified as active soluble forms. The enzymes showed the highest activity toward p-nitrophenyl valerate (C5) and exhibited optimum activities at mesophilic temperature ranges and slightly alkaline pH. In particular, the enzymes displayed salt tolerance with over 50% of the maximum activity remained in the presence of 3 M NaCl (or KCl). In this study, we demonstrated that the metagenomic approach using marine tidal flat sediment as a DNA source expanded the diversity of lipolytic enzyme-encoding genes.

Keywords: Tidal flat sediment; Metagenome; Lipolytic enzymes; Family IV; Salt tolerance

Listeria bacteriophage peptidoglycan hydrolases feature high thermoresistance and reveal increased activity after divalent metal cation substitution by Mathias Schmelcher; Florian Waldherr; Martin J. Loessner (pp. 633-643).

The ability of the bacteriophage-encoded peptidoglycan hydrolases (endolysins) to destroy Gram-positive bacteria from without makes these enzymes promising antimicrobials. Recombinant endolysins from Listeria monocytogenes phages have been shown to rapidly lyse and kill the pathogen in all environments. To determine optimum conditions regarding application of recombinant Listeria phage endolysins in food or production equipments, properties of different Listeria endolysins were studied. Optimum NaCl concentration for the amidase HPL511 was 200 nM and 300 mM for the peptidases HPL118, HPL500, and HPLP35. Unlike most other peptidoglycan hydrolases, all four enzymes exhibited highest activity at elevated pH values at around pH 8–9. Lytic activity was abolished by EDTA and could be restored by supplementation with various divalent metal cations, indicating their role in catalytic function. While substitution of the native Zn²⁺ by Ca²⁺ or Mn²⁺ was most effective in case of HPL118, HPL500, and HPLP35, supplementation with Co²⁺ and Mn²⁺ resulted in an approximately 5-fold increase in HPL511 activity. Interestingly, the glutamate peptidases feature a conserved SxHxxGxAxD zinc-binding motif, which is not present in the amidases, although they also require centrally located divalent metals for activity. The endolysins HPL118, HPL511, and HPLP35 revealed a surprisingly high thermostability, with up to 35% activity remaining after 30 min incubation at 90°C. The available data suggest that denaturation at elevated temperatures is reversible and may be followed by rapid refolding into a functional state.

Keywords: Peptidoglycan hydrolase; Lytic enzyme; Listeria; Bacteriophage; Food safety

Enzymatic degradation of granular potato starch by Microbacterium aurum strain B8.A by Fean D. Sarian; Rachel M. van der Kaaij; Slavko Kralj; Dirk-Jan Wijbenga; Doede J. Binnema; Marc J. E. C. van der Maarel; Lubbert Dijkhuizen (pp. 645-654).

Microbacterium aurum strain B8.A was isolated from the sludge of a potato starch-processing factory on the basis of its ability to use granular starch as carbon- and energy source. Extracellular enzymes hydrolyzing granular starch were detected in the growth medium of M. aurum B8.A, while the type strain M. aurum DSMZ 8600 produced very little amylase activity, and hence was unable to degrade granular starch. The strain B8.A extracellular enzyme fraction degraded wheat, tapioca and potato starch at 37 °C, well below the gelatinization temperature of these starches. Starch granules of potato were hydrolyzed more slowly than of wheat and tapioca, probably due to structural differences and/or surface area effects. Partial hydrolysis of starch granules by extracellular enzymes of strain B8.A resulted in large holes of irregular sizes in case of wheat and tapioca and many smaller pores of relatively homogeneous size in case of potato. The strain B8.A extracellular amylolytic system produced mainly maltotriose and maltose from both granular and soluble starch substrates; also, larger maltooligosaccharides were formed after growth of strain B8.A in rich medium. Zymogram analysis confirmed that a different set of amylolytic enzymes was present depending on the growth conditions of M. aurum B8.A. Some of these enzymes could be partly purified by binding to starch granules.

Keywords: Microbacterium aurum ; Alpha-amylase; Scanning electron microscopy; Granular starch; Potato starch

Enzymatic properties of the glycine d-alanine aminopeptidase of Aspergillus oryzae and its activity profiles in liquid-cultured mycelia and solid-state rice culture (rice koji) by Junichiro Marui; Mayumi Matsushita-Morita; Sawaki Tada; Ryota Hattori; Satoshi Suzuki; Hitoshi Amano; Hiroki Ishida; Youhei Yamagata; Michio Takeuchi; Ken-Ichi Kusumoto (pp. 655-669).

The gdaA gene encoding S12 family glycine–d-alanine aminopeptidase (GdaA) was found in the industrial fungus Aspergillus oryzae. GdaA shares 43% amino acid sequence identity with the d-aminopeptidase of the Gram-negative bacterium Ochrobactrum anthropi. GdaA purified from an A. oryzae gdaA-overexpressing strain exhibited high d-stereospecificity and efficiently released N-terminal glycine and d-alanine of substrates in a highly specific manner. The optimum pH and temperature were 8 to 9 and 40°C, respectively. This enzyme was stable under alkaline conditions at pH 8 to 11 and relatively resistant to acidic conditions until pH 5.0. The chelating reagent EDTA, serine protease inhibitors such as AEBSF, benzamidine, TPCK, and TLCK, and the thiol enzyme inhibitor PCMB inhibited the enzyme. The aminopeptidase inhibitor bestatin did not affect the activity. GdaA was largely responsible for intracellular glycine and d-alanine aminopeptidase activities in A. oryzae during stationary-phase growth in liquid media. In addition, the activity increased in response to the depletion of nitrogen or carbon sources in the growth media, although the GdaA-independent glycine aminopeptidase activity highly increased simultaneously. Aminopeptidases of A. oryzae attract attention because the enzymatic release of a variety of amino acids and peptides is important for the enhancement of the palatability of fermented foods. GdaA activity was found in extracts of a solid-state rice culture of A. oryzae (rice koji), which is widely used as a starter culture for Japanese traditional fermented foods, and was largely responsible for the glycine and d-alanine aminopeptidase activity detected at a pH range of 6 to 9.

Keywords: Aspergillus oryzae ; Aminopeptidase; Glycine; d-alanine; d-stereospecificity; Solid-state rice culture

Enhanced production of nukacin D13E in Lactococcus lactis NZ9000 by the additional expression of immunity genes by Tijo Varghese Puramattathu; Mohammad R. Islam; Mami Nishie; Sae Yanagihara; Jun-ichi Nagao; Ken-ichi Okuda; Takeshi Zendo; Jiro Nakayama; Kenji Sonomoto (pp. 671-678).

Nukacin D13E (D13E) is a variant of type-A(II) lantibiotic nukacin ISK-1 produced by Staphylococcus warneri ISK-1. D13E exhibited a twofold higher specific antimicrobial activity than nukacin ISK-1 against a number of Gram-positive bacteria. We previously reported the heterologous production of D13E in Lactococcus lactis NZ9000 under the control of nisin-controlled gene expression system. In this study, we demonstrated enhanced production of D13E by the additional expression of immunity genes, nukFEG. The nukacin ISK-1 immunity, conferred by the ABC transporter complex, NukFEG, and the lantibiotic-binding protein, NukH, was not overwhelmed by D13E. The additional NukFEG resulted in a fourfold increase in the immunity level of the strain and a 5.2-fold increase in D13E production. The additional NukFEGH-expressing strain with the highest D13E immunity showed reduced level of production. Further improvement in D13E production was achieved by using pH-controlled batch fermentation.

Keywords: Lantibiotic; Nukacin ISK-1; Nisin-controlled gene expression; Immunity genes; Fermentation

Selection of optimal reference genes for expression analysis in the entomopathogenic fungus Beauveria bassiana during development, under changing nutrient conditions, and after exposure to abiotic stresses by Yong-Hong Zhou; Yong-Jun Zhang; Zhi-Bing Luo; Yan-Hua Fan; Gui-Rong Tang; Li-Ji Liu; Yan Pei (pp. 679-685).

The selection of suitable reference genes is crucial for accurate quantification of gene expression. To identify suitable reference genes in Beauveria bassiana, the expression of 14 candidates (18S, 28S, β-Tub, GAPD, γ-Act, TEF, HGPT, His3, His2A, TBP, CypA, CypB, PP1, and CrzA) was measured by quantitative polymerase chain reaction at different development stages and under various nutritional and stress conditions. Expression stability, as evaluated by the geNorm and NormFinder programs, revealed that His2A/γ-Act/CrzA was the most stably expressed set of genes throughout development, while 28S/PP1/CypA and His2A/γ-Act/CypA were the most stably expressed gene sets under a variety of nutritional and stress conditions, respectively. Overall, the most stably expressed genes under all conditions examined were PP1, γ-Act, and CypA.

Keywords: Beauveria bassiana ; Selection; Reference genes; Quantitative PCR

Biosynthesis of the nargenicin A₁ pyrrole moiety from Nocardia sp. CS682 by Sushila Maharjan; Niraj Aryal; Saurabh Bhattarai; Dinesh Koju; Janardan Lamichhane; Jae Kyung Sohng (pp. 687-696).

A number of structurally diverse natural products harboring pyrrole moieties possess a wide range of biological activities. Studies on biosynthesis of pyrrole ring have shown that pyrrole moieties are derived from l-proline. Nargenicin A₁, a saturated alicyclic polyketide from Nocardia sp. CS682, is a pyrrole-2-carboxylate ester of nodusmicin. We cloned and identified a set of four genes from Nocardia sp. CS682 that show sequence similarity to the respective genes involved in the biosynthesis of the pyrrole moieties of pyoluteorin in Pseudomonas fluorescens, clorobiocin in Streptomyces roseochromogenes subsp. Oscitans, coumermycin A₁ in Streptomyces rishiriensis, one of the pyrrole rings of undecylprodigiosin in Streptomyces coelicolor, and leupyrrins in Sorangium cellulosum. These genes were designated as ngnN4, ngnN5, ngnN3, and ngnN2. In this study, we presented the evidences that the pyrrole moiety of nargenicin A₁ was also derived from l-proline by the coordinated action of three proteins, NgnN4 (proline adenyltransferase), NgnN5 (proline carrier protein), and NgnN3 (flavine-dependent acyl-coenzyme A dehydrogenases). Biosynthesis of pyrrole moiety in nargenicin A₁ is initiated by NgnN4 that catalyzes ATP-dependent activation of l-proline into l-prolyl-AMP, and the latter is transferred to NgnN5 to create prolyl-S-peptidyl carrier protein (PCP). Later, NgnN3 catalyzes the two-step oxidation of prolyl-S-PCP into pyrrole-2-carboxylate. Thus, this study presents another example of a pyrrole moiety biosynthetic pathway that uses a set of three genes to convert l-proline into pyrrole-2-carboxylic acid moiety.

Keywords: Nargenicin A₁ ; Nocardia sp. CS682; Proline adenyltransferase; Proline carrier protein; FAD-dependent dehydrogenase

Development of replicative oriC plasmids and their versatile use in genetic manipulation of Cytophaga hutchinsonii by Yuanxi Xu; Xiaofei Ji; Ning Chen; Pengwei Li; Weifeng Liu; Xuemei Lu (pp. 697-705).

Cytophaga hutchinsonii is a Gram-negative aerobic soil bacterium which can digest crystalline cellulose completely through a strategy different from that of the well-studied cellulolytic aerobic fungi and anaerobic bacteria. However, despite the availability of a published genome sequence, studies on this organism have been very limited because of the lack of a genetic manipulation system. This paper describes the development of replicative oriC plasmids, carrying the replication origin of the C. hutchinsonii chromosome, and an electroporation method for Escherichia coli–C. hutchinsonii shuttle vectors based on oriC plasmids with an efficiency of about 2 × 10⁴ transformants per microgram plasmid DNA. Heterologous proteins, including green fluorescent protein and β-galactosidase, were expressed successfully and proved functional in C. hutchinsonii under the control of the CHU_1284 promoter in oriC plasmids. Finally, the gene CHU_0344, encoding the main extracellular protein, was targeted by homologous recombination based on the oriC plasmid. These genetic techniques and tools will provide a means to study the novel cellulose degradation system of C. hutchinsonii.

Keywords: Cytophaga hutchinsonii ; oriC plasmid; Genetic manipulation; Genetic tools

A combined physiological and proteomic approach to reveal lactic-acid-induced alterations in Lactobacillus casei Zhang and its mutant with enhanced lactic acid tolerance by Chongde Wu; Juan Zhang; Wei Chen; Miao Wang; Guocheng Du; Jian Chen (pp. 707-722).

Lactobacillus casei has traditionally been recognized as a probiotic and frequently used as an adjunct culture in fermented dairy products, where acid stress is an environmental condition commonly encountered. In the present study, we carried out a comparative physiological and proteomic study to investigate lactic-acid-induced alterations in Lactobacillus casei Zhang (WT) and its acid-resistant mutant. Analysis of the physiological data showed that the mutant exhibited 33.8% higher glucose phosphoenolpyruvate:sugar phosphotransferase system activity and lower glycolytic pH compared with the WT under acidic conditions. In addition, significant differences were detected in both cells during acid stress between intracellular physiological state, including intracellular pH, H⁺-ATPase activity, and intracellular ATP pool. Comparison of the proteomic data based on 2D-DIGE and i-TRAQ indicated that acid stress invoked a global change in both strains. The mutant protected the cells against acid damage by regulating the expression of key proteins involved in cellular metabolism, DNA replication, RNA synthesis, translation, and some chaperones. Proteome results were validated by Lactobacillus casei displaying higher intracellular aspartate and arginine levels, and the survival at pH 3.3 was improved 1.36- and 2.10-fold by the addition of 50-mM aspartate and arginine, respectively. To our knowledge, this is the first demonstration that aspartate may be involved in acid tolerance in Lactobacillus casei. Results presented here may help us understand acid resistance mechanisms and help formulate new strategies to enhance the industrial applications of this species.

Keywords: Acid stress; Lactobacillus casei ; 2D-DIGE; i-TRAQ; Physiological analysis

The effect of acetylated xylan and sugar beet pulp on the expression and secretion of enzymes by Penicillium purpurogenum by Mario Navarrete; Eduardo Callegari; Jaime Eyzaguirre (pp. 723-741).

Sugar beet pulp is a natural carbon source composed mainly of pectin and cellulose, which is utilized and degraded by the ascomycete Penicillium purpurogenum. The fungus also grows on and degrades acetylated xylan which lacks cellulose and pectin. Both carbon sources have been used in our laboratory to grow the fungus and to purify different enzymes secreted to the medium. The enzymes involved in the complex process of degradation of these carbon sources by the fungus have been explored previously under non-denaturing conditions; multienzyme complexes were separated and some subunits identified by Western blots and mass spectrometry. In this work, proteomic profiles show that the secretome is composed of numerous proteins varying in pI and molecular weight. Some enzymes are common to both growth conditions, while others are specific for each carbon source. The results show that the carbon sources utilized exert strong regulatory control over the proteins secreted. This is the first secretome study from a lignocellulolytic Penicillium.

Keywords: Penicillium purpurogenum ; Secretome; Sugar beet pulp; Hemicellulolytic enzymes

Antibacterial activity and interaction mechanism of electrospun zinc-doped titania nanofibers by Touseef Amna; M. Shamshi Hassan; Nasser A. M. Barakat; Dipendra Raj Pandeya; Seong Tshool Hong; Myung-Seob Khil; Hak Yong Kim (pp. 743-751).

In this study, a biological evaluation of the antimicrobial activity of Zn-doped titania nanofibers was carried out using Escherichia coli ATCC 52922 (Gram negative) and Staphylococcus aureus ATCC 29231 (Gram positive) as model organisms. The utilized Zn-doped titania nanofibers were prepared by the electrospinning of a sol–gel composed of zinc nitrate, titanium isopropoxide, and polyvinyl acetate; the obtained electrospun nanofibers were vacuum dried at 80°C and then calcined at 600°C. The physicochemical properties of the synthesized nanofibers were determined by X-ray diffraction pattern, field emission scanning electron microscopy, energy-dispersive X-ray spectroscopy, electron probe microanalysis, thermogravimetry, and transmission electron microscopy (TEM). The antibacterial activity and the acting mechanism of Zn-doped titania nanofibers against bacteria were investigated by calculation of minimum inhibitory concentration and analyzing the morphology of the bacterial cells following the treatment with nanofibers solution. Our investigations reveal that the lowest concentration of Zn-doped titania nanofibers solution inhibiting the growth of S. aureus ATCC 29231 and E. coli ATCC 52922 strains is found to be 0.4 and 1.6 μg/ml, respectively. Furthermore, Bio-TEM analysis demonstrated that the exposure of the selected microbial strains to the nanofibers led to disruption of the cell membranes and leakage of the cytoplasm. In conclusion, the combined results suggested doping promotes antimicrobial effect; synthesized nanofibers possess a very large surface-to-volume ratio and may damage the structure of the bacterial cell membrane, as well as depress the activity of the membranous enzymes which cause bacteria to die in due course.

Keywords: Pathogenic bacteria; Antibacterial mechanism; Zn-doped titania nanofibers; Transmission electron microscopy (TEM); X-ray diffraction (XRD)

Biodegradation of 2-mercaptobenzothiazolyl-(Z)-(2-aminothiazol-4-yl)-2-(tert-butoxycarbonyl) isopropoxyiminoacetate by Pseudomonas desmolyticum NCIM 2112 by Jai S. Ghosh; Kedar B. Rokade (pp. 753-761).

2-Mercaptobenzothiazolyl-(Z)-(2-aminothiazol-4-yl)-2-(tert-butoxycarbonyl) isopropoxyiminoacetate is used as supplementary additives in commercial-grade insecticides to compensate for the time factor needed for the actual pesticide chemical to start its action. This investigation describes the biodegradation of 2-mercaptobenzothiazolyl-(Z)-(2-aminothiazol-4-yl)-2-(tert-butoxycarbonyl) isopropoxyiminoacetate by Pseudomonas desmolyticum NCIM 2112. The biodegradation is influenced by other carbon and nitrogen sources and indicates that glucose and lactose are effective at 0.5% concentration whereas NaNO₃ and NaNO₂ at 0.05%. The percent degradation of 2-mercaptobenzothiazolyl-(Z)-(2-aminothiazol-4-yl)-2-(tert-butoxycarbonyl) isopropoxyiminoacetate was found to be 40%.The pH and temperature optima were found to be 7.0°C and 30°C, respectively. The effect on soil parameters was observed in treated soil and indicates remarkable decrease in soil fertility; the phytotoxicity indicates retarded growth and germination inhibition of treated seeds of Sorghum bicolor and Triticum aestivum. In paddy field the inhibition of germination of Oryza sativa was observed.

Keywords: Insecticide; 2-Mercaptobenzothiazolyl-(Z)-(2-aminothiazol-4-yl)-2-(tert-butoxycarbonyl) isopropoxyiminoacetate; Pseudomonas ; Phytotoxicity

Evolution of biofilms during the colonization process of pyrite by Acidithiobacillus thiooxidans by Dulce M. González; René H. Lara; Keila N. Alvarado; Donato Valdez-Pérez; Hugo R. Navarro-Contreras; Roel Cruz; Jessica Viridiana García-Meza (pp. 763-775).

We have applied epifluorescence principles, atomic force microscopy, and Raman studies to the analysis of the colonization process of pyrite (FeS₂) by sulfuroxidizing bacteria Acidithiobacillus thiooxidans after 1, 15, 24, and 72 h. For the stages examined, we present results comprising the evolution of biofilms, speciation of S _n ²⁻ /S⁰ species, adhesion forces of attached cells, production and secretion of extracellular polymeric substances (EPS), and its biochemical composition. After 1 h, highly dispersed attached cells in the surface of the mineral were observed. The results suggest initial non-covalent, weak interactions (e.g., van der Waal’s, hydrophobic interactions), mediating an irreversible binding mechanism to electrooxidized massive pyrite electrode (eMPE), wherein the initial production of EPS by individual cells is determinant. The mineral surface reached its maximum cell cover between 15 to 24 h. Longer biooxidation times resulted in the progressive biofilm reduction on the mineral surface. Quantification of attached cell adhesion forces indicated a strong initial mechanism (8.4 nN), whereas subsequent stages of mineral colonization indicated stability of biofilms and of the adhesion force to an average of 4.2 nN. A variable EPS (polysaccharides, lipids, and proteins) secretion at all stages was found; thus, different architectural conformation of the biofilms was observed during 120 h. The main EPS produced were lipopolysaccharides which may increase the hydrophobicity of A. thiooxidans biofilms. The highest amount of lipopolysaccharides occurred between 15–72 h. In contrast with abiotic surfaces, the progressive depletion of S _n ²⁻ /S⁰ was observed on biotic eMPE surfaces, indicating consumption of surface sulfur species. All observations indicated a dynamic biooxidation mechanism of pyrite by A. thiooxidans, where the biofilms stability and composition seems to occur independently from surface sulfur species depletion.

Keywords: Acidithiobacillus thiooxidans ; Pyrite; Biofilms evolution; Electrooxidation; Interfacial analysis; Proteins quantification

The cytotoxicity and anticancer mechanisms of alterporriol L, a marine bianthraquinone, against MCF-7 human breast cancer cells by Caihuan Huang; Hua Jin; Bing Song; Xun Zhu; Hongxia Zhao; Jiye Cai; Yongjun Lu; Bin Chen; Yongcheng Lin (pp. 777-785).

Alterporriol L, a new bianthraquinone derivative, was isolated from a marine fungus Alternaria sp. ZJ9-6B. The cytotoxic activity and anticancer mechanisms of alterporriol L towards breast cancer cells lines were detected using MTT assay, immunofluorescence, and flow cytometry. Simultaneously, the changes in morphological properties of cells were detected before and after treatment with alterporriol L by atomic force microscope (AFM) at a nanometer scale. MTT assay showed that alterporriol L could effectively inhibit the growth and proliferation, and there was a dose-dependent manner of cell death. Moreover, the alterporriol L could induce cancer cell apoptosis or necrosis. Furthermore, the reactive oxygen species, mitochondrial membrane potential, and cytosolic free calcium level were changed after treatment with alterporriol L, suggesting that alterporriol L played vital roles in breast cancer cells through destroying the mitochondrial. And all these alterations are in accord with changes of morphology detected by AFM, which suggested that the AFM is a useful tool to detect the morphological changes of the cancer cells.

Keywords: Marine bianthraquinone; Breast cancer; Apoptosis; Atomic force microscopy

Distribution of glyphosate and methylphosphonate catabolism systems in soil bacteria Ochrobactrum anthropi and Achromobacter sp by Alexey V. Sviridov; Tatyana V. Shushkova; Nina F. Zelenkova; Natalya G. Vinokurova; Igor G. Morgunov; Inna T. Ermakova; Alexey A. Leontievsky (pp. 787-796).

Bacterial strains capable of utilizing methylphosphonic acid (MP) or glyphosate (GP) as the sole sources of phosphorus were isolated from soils contaminated with these organophosphonates. The strains isolated from MP-contaminated soils grew on MP and failed to grow on GP. One group of the isolates from GP-contaminated soils grew only on MP, while the other one grew on MP and GP. Strains Achromobacter sp. MPS 12 (VKM B-2694), MP degraders group, and Ochrobactrum anthropi GPK 3 (VKM B-2554D), GP degraders group, demonstrated the best degradative capabilities towards MP and GP, respectively, and were studied for the distribution of their organophosphonate catabolism systems. In Achromobacter sp. MPS 12, degradation of MP was catalyzed by C–P lyase incapable of degrading GP (C–P lyase I). Adaptation to growth on GP yielded the strain Achromobacter sp. MPS 12A, which retained its ability to degrade MP via C–P lyase I and was capable of degrading GP with formation of sarcosine, thus suggesting the involvement of a GP-specific C–P lyase II. O. anthropi GPK 3 also degraded MP via C–P lyase I, but degradation of GP in it was initiated by glyphosate oxidoreductase, which was followed by product transformation via the phosphonatase pathway.

Keywords: Phosphonates; Glyphosate; Methylphosphonic acid; C–P lyase; Glyphosate Oxidoreductase; Phosphonatase

Applicability of a sensitive duplex real-time PCR assay for identifying B/Yamagata and B/Victoria lineages of influenza virus from clinical specimens by Naixing Zhang; Shisong Fang; Ting Wang; Jianxiong Li; Xiaowen Cheng; Cunyou Zhao; Xin Wang; Xing Lv; Chunli Wu; Renli Zhang; Jinquan Cheng; Hong Xue; Zuxun Lu (pp. 797-805).

Type B influenza virus is one of the major epidemic strains and responsible for considerable mortality and morbidity. Rapidly and accurately identifying different influenza B virus lineages, i.e., B/Yamagata (B/Y) and B/Victoria (B/V), is desirable during the flu season. However, the available rapid techniques lack sensitivity, and the usual methods for identifying influenza viruses require expansion of virus in tissue culture or embryonated hen’s eggs. Thus, we developed several sets of primer pairs that were able to detect and distinguish B/Y and B/V in a single real-time PCR assay. Used in conjunction with two sets of specific primers that exhibited purine at 3′ end of at least one primer targeting on HA gene of B/Y and B/V lineages allows us to accurately identify approximately 10² copies per microliter for B/Y and B/V with intra- and inter-assay coefficient of variation (CV) <4%. When it was used to test 17,765 throat swab specimens obtained in the 2006–2010 influenza surveillance season, this method was comparable to hemagglutination inhibition assay in detection, typing and subtyping of influenza viruses with 100% true-negative (specificity) and 100% true-positive (sensitivity). Taken together, this method provides sensitive and robust tool for routine diagnosis and on-time epidemiological examination for WHO decisions on vaccine composition.

Keywords: Influenza virus; Real-time RT-PCR; Yamagata lineage; Victoria lineage

Tandem repeat-tRNA (TRtRNA) PCR method for the molecular typing of non-Saccharomyces subspecies by Marianne Barquet; Valentina Martín; Karina Medina; Gabriel Pérez; Francisco Carrau; Carina Gaggero (pp. 807-814).

There is a worldwide trend to understand the impact of non-Saccharomyces yeast species on the process of winemaking. Although the predominant species at the end of the fermentation is Saccharomyces cerevisiae, several non-Saccharomyces species present during the first days of the process can produce and/or release aromas that improve the bouquet and complexity of the final wine. Since no genomic sequences are available for the predominant non-Saccharomyces species selected from grapes or musts (Hanseniaspora uvarum, Hanseniaspora vineae, Hanseniaspora opuntiae, Metschnikowia pulcherrima, Candida zemplinina), a reproducible PCR method was devised to discriminate strains at the subspecies level. The method combines different oligonucleotides based on tandem repeats with a second oligonucleotide based on a conserved tRNA region, specific for ascomycetes. Tandem repeats are randomly dispersed in all eukaryotic genomes and tRNA genes are conserved and present in several copies in different chromosomes. As an example, the method was applied to discriminate native M. pulcherrima strains but it could be extended to differentiate strains from other non-Saccharomyces species. The biodiversity of species and strains found in the grape ecosystem is a potential source of new enzymes, fungicides and/or novel sustainable methods for biological control of phytopathogens.

Keywords: Molecular typing; Tandem repeat; tRNA; Non-Saccharomyces grape yeasts; Metschnikowia pulcherrima

A convenient method for multiple insertions of desired genes into target loci on the Escherichia coli chromosome by Daisuke Koma; Hayato Yamanaka; Kunihiko Moriyoshi; Takashi Ohmoto; Kiyofumi Sakai (pp. 815-829).

We developed a method to insert multiple desired genes into target loci on the Escherichia coli chromosome. The method was based on Red-mediated recombination, flippase and the flippase recognition target recombination, and P1 transduction. Using this method, six copies of the lacZ gene could be simultaneously inserted into different loci on the E. coli chromosome. The inserted lacZ genes were functionally expressed, and β-galactosidase activity increased in proportion to the number of inserted lacZ genes. This method was also used for metabolic engineering to generate overproducers of aromatic compounds. Important genes of the shikimate pathway (aroF fbr and tyrA fbr or aroF fbr and pheA fbr) were introduced into the chromosome to generate a tyrosine or a phenylalanine overproducer. Moreover, a heterologous decarboxylase gene was introduced into the chromosome of the tyrosine or phenylalanine overproducer to generate a tyramine or a phenethylamine overproducer, respectively. The resultant strains selectively overproduced the target aromatic compounds. Thus, the developed method is a convenient tool for the metabolic engineering of E. coli for the production of valuable compounds.

Keywords: Escherichia coli ; Chromosome; Insertion; T7 promoter; Recombination; P1 transduction; Metabolic engineering

Biotransformation of acetamiprid by the white-rot fungus Phanerochaete sordida YK-624 by Jianqiao Wang; Hirofumi Hirai; Hirokazu Kawagishi (pp. 831-835).

Acetamiprid (ACE) belongs to the neonicotinoid class of systemic broad-spectrum insecticides, which are the most highly effective and largest-selling insecticides worldwide for crop protection. As neonicotinoid insecticides persist in crops, biotransformation of these insecticides represents a promising approach for improving the safety of foods. Here, the elimination of ACE from a liquid medium by the white-rot fungus Phanerochaete sordida YK-624 was examined. Under ligninolytic and non-ligninolytic conditions, 45% and 30% of ACE were eliminated, respectively, after 15 days of incubation. High-resolution electrospray ionization mass spectra and nuclear magnetic resonance analyses of a metabolite identified in the culture supernatant suggested that ACE was N-demethylated to (E)-N ¹-[(6-chloro-3-pyridyl)-methyl]-N ²-cyano-acetamidine, which has a much lower toxicity than ACE. In addition, we investigated the effect of the cytochrome P450 inhibitor piperonyl butoxide (PB) on the elimination of ACE. The elimination rate of ACE by P. sordida YK-624 was markedly reduced by the addition of either 0.01 or 0.1 mM PB to the culture medium. These results suggest that cytochrome P450 plays an important role in the N-demethylation of ACE by P. sordida YK-624.

Keywords: Acetamiprid; Detoxification; Phanerochaete sordida YK-624; N-demethylation; White-rot fungi

Alkanols and chlorophenols cause different physiological adaptive responses on the level of cell surface properties and membrane vesicle formation in Pseudomonas putida DOT-T1E by Thomas Baumgarten; José Vazquez; Christian Bastisch; Wilfried Veron; Marc G. J. Feuilloley; Sandor Nietzsche; Lukas Y. Wick; Hermann J. Heipieper (pp. 837-845).

In order to cope with the toxicity imposed by the exposure to environmental hydrocarbons, many bacteria have developed specific adaptive responses such as modifications in the cell envelope. Here we compared the influence of n-alkanols and chlorophenols on the surface properties of the solvent-tolerant bacterium Pseudomonas putida DOT-T1E. In the presence of toxic concentrations of n-alkanols, this strain significantly increased its cell surface charge and hydrophobicity with changes depending on the chain length of the added n-alkanols. The adaptive response occurred within 10 min after the addition of the solvent and was demonstrated to be of physiological nature. Contrary to that, chlorophenols of similar hydrophobicity and potential toxicity as the corresponding alkanols caused only minor effects in the surface properties. To our knowledge, this is the first observation of differences in the cellular adaptive response of bacteria to compound classes of quasi equal hydrophobicity and toxicity. The observed adaptation of the physico-chemical surface properties of strain DOT-T1E to the presence of alkanols was reversible and correlated with changes in the composition of the lipopolysaccharide content of the cells. The reaction is explained by previously described reactions allowing the release of membrane vesicles that was demonstrated for cells affected by 1-octanol and heat shock, whereas no membrane vesicles were released after the addition of chlorophenols.

Keywords: Pseudomonas putida DOT-T1E; Adaptation; Cell surface properties; Solvent stress; Membrane vesicles; Water contact angle; Zeta potential

Characterization of sulfide-oxidizing microbial mats developed inside a full-scale anaerobic digester employing biological desulfurization by Takuro Kobayashi; Yu-You Li; Kengo Kubota; Hideki Harada; Takeki Maeda; Han-Qing Yu (pp. 847-857).

The microbial mats responsible for biological desulfurization from biogas in a full-scale anaerobic digester were characterized in terms of their structure, as well as their chemical and microbial properties. Filament-shaped elemental sulfur 100–500 μm in length was shown to cover the mats, which cover the entire headspace of the digester. This is the first report on filamentous sulfur production in a non-marine environment. The results of the analysis of the mats suggest that the key players in the sulfide oxidation and sulfur production in the bio-desulfurization in the headspace of the digester were likely to be two sulfide-oxidizing bacteria (SOB) species related to Halothiobacillus neapolitanus and Sulfurimonas denitrificans, and that the microbial community, cell density, activity for sulfide oxidation varied according to the environmental conditions at the various locations of the mats. Since the water and nutrients necessary for the SOB were provided by the digested sludge droplets deposited on the mats, and our results show that a higher rate of sulfide oxidation occurred with more frequent digested sludge deposition, the habitat of the SOB needs to be made in the lower part of the headspace near the liquid level of the digested sludge to maintain optimal conditions.

Keywords: Biological desulfurization; Anaerobic digestion; Sulfide-oxidizing bacteria; Sulfur formation; Microbial community

Anaerobic digestibility of marine microalgae Phaeodactylum tricornutum in a lab-scale anaerobic membrane bioreactor by Carlos Zamalloa; Jo De Vrieze; Nico Boon; Willy Verstraete (pp. 859-869).

The biomass of industrially grown Phaeodactylum tricornutum was subjected in a novel way to bio-methanation at 33°C, i.e., in an anaerobic membrane bioreactor (AnMBR) at a hydraulic retention time of 2.5 days, at solid retention times of 20 to 10 days and at loading rates in the range of 2.6–5.9 g biomass-COD L⁻¹ day⁻¹ with membrane fluxes ranging from 1 to 0.8 L m⁻² h⁻¹. The total COD recovered as biogas was in the order of 52%. The input suspension was converted to a clear effluent rich in total ammonium nitrogen (546 mg TAN L⁻¹) and phosphate (141 mg PO₄-P L⁻¹) usable as liquid fertilizer. The microbial community richness, dynamics, and organization in the reactor were interpreted using the microbial resource management approach. The AnMBR communities were found to be moderate in species richness and low in dynamics and community organization relative to UASB and conventional CSTR sludges. Quantitative polymerase chain reaction analysis revealed that Methanosaeta sp. was the dominant acetoclastic methanogen species followed by Methanosarcina sp. This work demonstrated that the use of AnMBR for the digestion of algal biomass is possible. The fact that some 50% of the organic matter is not liquefied means that the algal particulates in the digestate constitute a considerable fraction which should be valorized properly, for instance as slow release organic fertilizer. Overall, 1 kg of algae dry matter (DM) could be valorized in the form of biogas (€2.07), N and P in the effluent (€0.02) and N and P in the digestate (€0.04), thus totaling about €2.13 per kilogram algae DM.

Keywords: Algae biomass; Mesophilic; Green energy; Biogas; Methane; Microbial resource management

Enhanced performance and mechanism study of microbial electrolysis cells using Fe nanoparticle-decorated anodes by Shoutao Xu; Hong Liu; Yanzhen Fan; Rebecca Schaller; Jun Jiao; Frank Chaplen (pp. 871-880).

Anode properties are critical for the performance of microbial electrolysis cells (MECs). In the present study, Fe nanoparticle-modified graphite disks were used as anodes to investigate the effects of nanoparticles on the performance of Shewanella oneidensis MR-1 in MECs. Results demonstrated that the average current densities produced with Fe nanoparticle-decorated anodes up to 5.89-fold higher than plain graphite anodes. Whole genome microarray analysis of the gene expression showed that genes encoding biofilm formation were significantly up-regulated as a response to nanoparticle-decorated anodes. Increased expression of genes related to nanowires, flavins, and c-type cytochromes indicates that enhanced mechanisms of electron transfer to the anode may also have contributed to the observed increases in current density. The majority of the remaining differentially expressed genes associated with electron transport and anaerobic metabolism demonstrate a systemic response to increased power loads.

Keywords: Microbial electrochemical system; Microbial fuel cell; Microbial electrolysis cell; Nanotechnology; Differential gene expression; DNA microarray

Effects of different replicons in conjugative plasmids on transformation efficiency, plasmid stability, gene expression and n-butanol biosynthesis in Clostridium tyrobutyricum by Mingrui Yu; Yinming Du; Wenyan Jiang; Wei-Lun Chang; Shang-Tian Yang; I-Ching Tang (pp. 881-889).

Clostridium tyrobutyricum ATCC 25755 can produce butyric acid, acetic acid, and hydrogen as the main products from various carbon sources. In this study, C. tyrobutyricum was used as a host to produce n-butanol by expressing adhE2 gene under the control of a native thiolase promoter using four different conjugative plasmids (pMTL82151, 83151, 84151, and 85151) each with a different replicon (pBP1 from C. botulinum NCTC2916, pCB102 from C. butyricum, pCD6 from Clostridium difficile, and pIM13 from Bacillus subtilis). The effects of different replicons on transformation efficiency, plasmid stability, adhE2 expression and aldehyde/alcohol dehydrogenase activities, and butanol production by different mutants of C. tyrobutyricum were investigated. Among the four plasmids and replicons studied, pMTL82151 with pBP1 gave the highest transformation efficiency, plasmid stability, gene expression, and butanol biosynthesis. Butanol production from various substrates, including glucose, xylose, mannose, and mannitol were then investigated with the best mutant strain harboring adhE2 in pMTL82151. A high butanol titer of 20.5 g/L with 0.33 g/g yield and 0.32 g/L h productivity was obtained with mannitol as the substrate in batch fermentation with pH controlled at ~6.0.

Keywords: Butanol; Clostridium tyrobutyricum ; Conjugative plasmid; Replicon

Bioconversion of lignin model compounds with oleaginous Rhodococci by Matyas Kosa; Arthur J. Ragauskas (pp. 891-900).

Although economically efficient biomass conversion depends on the utilization of the complete cell wall (biorefinery concept), including polysaccharides and lignin, current biofuels research concentrate mostly on cellulose conversion, while lignin is viewed as a side-product that is used primarily as a thermal resource. Microbiological conversion of lignin is almost exclusive to fungi, usually resulting in increased cell mass and lignolytic enzymes. Some bacteria can also degrade lignin-related compounds using the β-ketoadipate pathway; for example, Rhodococcus opacus DSM 1069 can degrade coniferyl alcohol and grow on it as sole carbon source. Moreover, this strain belongs to the actinomycetes group that is also known for oleaginous species with lipid accumulation over 20%. Present work shows that R. opacus DSM 1069 and PD630 strains under nitrogen limiting conditions can convert lignin model compounds into triacylglycerols, also known as neutral lipids. 4-Hydroxybenzoic and vanillic acid lignin model compounds were used as sole carbon sources, and after brief adaptation periods, the cells not only began growing but accumulated lipids to the level of oleaginicity. These lipids were extracted for transesterification and analysis of fatty acid methyl esters showed good composition for biodiesel applications with no aromatics. Furthermore, the two strains showed distinct substrate metabolism and product profiles.

Keywords: Lignin; Lipid; Triacylglycerol; Oleaginous; Rhodococcus; β-ketoadipate

Erratum to: Enzymatic properties of the glycine d-alanine aminopeptidase of Aspergillus oryzae and its activity profiles in liquid-cultured mycelia and solid-state rice culture (rice koji) by Junichiro Marui; Mayumi Matsushita-Morita; Sawaki Tada; Ryota Hattori; Satoshi Suzuki; Hitoshi Amano; Hiroki Ishida; Youhei Yamagata; Michio Takeuchi; Ken-Ichi Kusumoto (pp. 901-901).

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Applied Microbiology and Biotechnology (v.93, #2)

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Applied Microbiology and Biotechnology (v.93, #2)