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Applied Microbiology and Biotechnology (v.96, #1)

Microbial d-xylonate production by Mervi H. Toivari; Yvonne Nygård; Merja Penttilä; Laura Ruohonen; Marilyn G. Wiebe (pp. 1-8).

d-Xylonic acid is a versatile platform chemical with reported applications as complexing agent or chelator, in dispersal of concrete, and as a precursor for compounds such as co-polyamides, polyesters, hydrogels and 1,2,4-butanetriol. With increasing glucose prices, d-xylonic acid may provide a cheap, non-food derived alternative for gluconic acid, which is widely used (about 80 kton/year) in pharmaceuticals, food products, solvents, adhesives, dyes, paints and polishes. Large-scale production has not been developed, reflecting the current limited market for d-xylonate. d-Xylonic acid occurs naturally, being formed in the first step of oxidative metabolism of d-xylose by some archaea and bacteria via the action of d-xylose or d-glucose dehydrogenases. High extracellular concentrations of d-xylonate have been reported for various bacteria, in particular Gluconobacter oxydans and Pseudomonas putida. High yields of d-xylonate from d-xylose make G. oxydans an attractive choice for biotechnical production. G. oxydans is able to produce d-xylonate directly from plant biomass hydrolysates, but rates and yields are reduced because of sensitivity to hydrolysate inhibitors. Recently, d-xylonate has been produced by the genetically modified bacterium Escherichia coli and yeast Saccharomyces cerevisiae and Kluyveromyces lactis. Expression of NAD⁺-dependent d-xylose dehydrogenase of Caulobacter crescentus in either E. coli or in a robust, hydrolysate-tolerant, industrial Saccharomyces cerevisiae strain has resulted in d-xylonate titres, which are comparable to those seen with G. oxydans, at a volumetric rate approximately 30 % of that observed with G. oxydans. With further development, genetically modified microbes may soon provide an alternative for production of d-xylonate at industrial scale.

Keywords: d-Xylonate; d-Xylose; Oxidation; d-Xylose dehydrogenase; Lignocellulosic hydrolyzate

Soyfoods and soybean products: from traditional use to modern applications by Kuan-I Chen; Mei-Hui Erh; Nan-Wei Su; Wen-Hsiung Liu; Cheng-Chun Chou; Kuan-Chen Cheng (pp. 9-22).

Soybean products (soyfoods), reported as potential functional foods, are implicated in several health-enhancing properties, such as easing the symptoms of postmenopausal women, reducing the risk of osteoporosis, preventing cardiovascular disease, and antimutagenic effects. Isoflavone, for example, is one of the most important compounds abundantly found in soybean, mainly accounting for the health-enhancing properties as mentioned earlier. However, most biological activities of isoflavones are mainly attributed to their aglycone forms. It has also been demonstrated that isoflavone aglycones are absorbed faster and in greater amount than their glycosides in human intestines. Fortunately, deglycosylation of isoflavones can be achieved during fermentation process by several strains such as lactic acid bacteria, basidiomycetes, filamentous fungus, and Bacillus subtilis with their β-glucosidase activity. This article presents an overview of soybean’s chemistry, application, state-of-the-art advances in soybean fermentation processing and products as well as their applications in food and pharmaceutical industries. Different compounds, such as isoflavone, dietary fibers, and proteins which exhibit significant bioactivities, are summarized. The roles of different microorganisms in bioconversion and enhancement of bioactivities of fermented soybean are also discussed.

Keywords: Soyfoods; Soybean; Fermentation; Bioactive compounds; Food processing

Hyperstructure interactions influence the virulence of the type 3 secretion system in yersiniae and other bacteria by Vic Norris; Laurence Menu-Bouaouiche; Jean-Michel Becu; Rachel Legendre; Romain Norman; Jason A. Rosenzweig (pp. 23-36).

A paradigm shift in our thinking about the intricacies of the host–parasite interaction is required that considers bacterial structures and their relationship to bacterial pathogenesis. It has been proposed that interactions between extended macromolecular assemblies, termed hyperstructures (which include multiprotein complexes), determine bacterial phenotypes. In particular, it has been proposed that hyperstructures can alter virulence. Two such hyperstructures have been characterized in both pathogenic and nonpathogenic bacteria. Present within a number of both human and plant Gram-negative pathogens is the type 3 secretion system (T3SS) injectisome which in some bacteria serves to inject toxic effector proteins directly into targeted host cells resulting in their paralysis and eventual death (but which in other bacteria prevents the death of the host). The injectisome itself comprises multiple protein subunits, which are all essential for its function. The degradosome is another multiprotein complex thought to be involved in cooperative RNA decay and processing of mRNA transcripts and has been very well characterized in nonpathogenic Escherichia coli. Recently, experimental evidence has suggested that a degradosome exists in the yersiniae as well and that its interactions within the pathogens modulate their virulence. Here, we explore the possibility that certain interactions between hyperstructures, like the T3SS and the degradosome, can ultimately influence the virulence potential of the pathogen based upon the physical locations of hyperstructures within the cell.

Keywords: Molecular assembly; Degradation; Disease; Membrane; RNA; Enolase

Ecofriendly control of potato late blight causative agent and the potential role of lactic acid bacteria: a review by Claudia Axel; Emanuele Zannini; Aidan Coffey; Jiahui Guo; Deborah M. Waters; Elke K. Arendt (pp. 37-48).

In times of increasing societal pressure to reduce the application of pesticides on crops, demands for environmentally friendly replacements have intensified. In the case of late blight, a devastating potato plant disease, the historically most widely known plant destroyer has been the oomycete Phytophthora infestans. To date, the most important strategy for control of this pathogen has been the frequent application of fungicides. Due to the aforementioned necessity to move away from traditional chemical treatments, many studies have focused on finding alternative ecofriendly biocontrol systems. In general, due to the different modes of actions (i.e. antagonistic effects or induction of plant defence mechanisms), the use of microorganisms as biological control agents has a definite potential. Amongst them, several species of lactic acid bacteria have been recognised as producers of bioactive metabolites which are functional against a broad spectrum of undesirable microorganisms, such as fungi, oomycetes and other bacteria. Thus, they may represent an interesting tool for the development of novel concepts in pest management. This review describes the present situation of late blight disease and summarises current literature regarding the biocontrol of the phytopathogen P. infestans using antagonistic microorganisms.

Keywords: Phytophthora infestans ; Potato late blight; Lactic acid bacteria; Pesticides; Biological control

Biochemical aspects of red koji and tofuyo prepared using Monascus fungi by Masaaki Yasuda; Shinjiro Tachibana; Megumi Kuba-Miyara (pp. 49-60).

Red koji or red mold rice is prepared by growing a genus Monascus on steamed rice. For centuries, it has been used in Asia for the production of fermented foods including red rice wine and fermented tofu. Although red koji is an important source of various hydrolytic enzymes critical for food fermentation, information on the enzymatic properties in red koji has been limited. Hydrolytic enzymes produced by Monascus fungi may play important roles in ripening of tofuyo (Japanese fermented tofu) regarding the chemical and physical properties of the product. This review provides an introduction of red koji, its properties, and the application of hydrolytic enzymes, especially aspartic proteinases and carboxypeptidases from Monascus fungi. We also describe tofuyo and a novel fermented soybean protein food using a microbial action originating from red koji.

Keywords: Monascus ; Koji; Aspartic proteinases; Carboxypeptidases; Fermented tofu; Tofuyo

Anaerobic bacteria as producers of antibiotics by Swantje Behnken; Christian Hertweck (pp. 61-67).

Anaerobic bacteria are the oldest terrestrial creatures. They occur ubiquitously in soil and in the intestine of higher organisms and play a major role in human health, ecology, and industry. However, until lately no antibiotic or any other secondary metabolite has been known from anaerobes. Mining the genome sequences of Clostridium spp. has revealed a high prevalence of putative biosynthesis genes (PKS and NRPS), and only recently the first antibiotic from the anaerobic world, closthioamide, has been isolated from the cellulose degrading bacterium Clostridium cellulolyticum. The successful genetic induction of antibiotic biosynthesis in an anaerobe encourages further investigations of obligate anaerobes to tap their hidden biosynthetic potential.

Keywords: Anaerobes; Antibiotics; Closthioamide; Genome mining; Clostridium ; Secondary metabolites

Insight into microwave irradiation and enzyme catalysis in enantioselective resolution of dl-(±)-3-phenyllactic acid by Ganapati D. Yadav; Sandip V. Pawar (pp. 69-79).

Lipase catalyzed kinetic resolution of dl-(±)-3-phenyllactic acid (dl-(±)-3-PLA) was investigated to study the synergistic effect of microwave irradiation and enzyme catalysis. Lipases from different sources were employed for the transesterification of dl-(±)-3-PLA under otherwise similar conditions, among which Novozyme 435 efficiently catalyzed the resolution of dl-(±)-3-PLA to l-(−)-O-acetyl-3-PLA using vinyl acetate as the acyl donor, showing excellent conversion (49 %) and enantiomeric excess (>99 %). The effect of various parameters affecting the initial rate, conversion and enantiomeric excess of the reaction were studied to establish kinetics and mechanism. There is a synergism between enzyme catalysis and microwave irradiation; an increase in initial rates up to 1.8-fold was observed under microwave irradiation than that under conventional heating. The analysis of initial rate data showed that reaction obeys ternary complex (ordered bi-bi) mechanism with inhibition by dl-(±)-3-PLA. The calculated and simulated rates match very well showing the validity of the proposed kinetic model.

Keywords: Immobilized lipase; Microwave irradiation; Enzyme catalysis; Kinetic study; dl-(±)-3-phenyllactic acid

Efficient heterologous expression and secretion in Aspergillus oryzae of a llama variable heavy-chain antibody fragment V_HH against EGFR by Fumiyoshi Okazaki; Jun-ichi Aoki; Soichiro Tabuchi; Tsutomu Tanaka; Chiaki Ogino; Akihiko Kondo (pp. 81-88).

We have constructed a filamentous fungus Aspergillus oryzae that secretes a llama variable heavy-chain antibody fragment (V_HH) that binds specifically to epidermal growth factor receptor (EGFR) in a culture medium. A major improvement in yield was achieved by fusing the V_HH with a Taka-amylase A signal sequence (sTAA) and a segment of 28 amino acids from the N-terminal region of Rhizopus oryzae lipase (N28). The yields of secreted, immunologically active anti-EGFR V_HH reached 73.8 mg/1 in a Sakaguchi flask. The V_HH fragments were released from the sTAA or N28 proteins by an indigenous A. oryzae protease during cultivation. The purified recombinant V_HH fragment was specifically recognized and could bind to the EGFR with a high affinity.

Keywords: Aspergillus oryzae ; Antibody; V_HH ; EGFR

Production and characterization of human granulocyte–macrophage colony-stimulating factor (hGM-CSF) expressed in the oleaginous yeast Yarrowia lipolytica by Najla Gasmi; Rabeb Lassoued; Atef Ayed; Brigitte Tréton; Didier Chevret; Jean Marc Nicaud; Héla Kallel (pp. 89-101).

Since its isolation, the human granulocyte–macrophage colony-stimulating factor (hGM-CSF) has been proposed as a new class of therapeutic biological products in the treatment of various diseases. However, the toxicity of this cytokine towards its expression host constitutes a major obstacle to bioprocess development for large-scale production. In this work, the optimized gene encoding hGM-CSF was expressed in the yeast Yarrowia lipolytica in one and two copies under the control of the fatty acid-inducible POX2 promoter. Protein secretion was directed by the targeting sequence of the extracellular lipase (LIP2): preXALip2. After 48 h of induction, Western blot analysis revealed the presence of a nonglycosylated form of 14.5 kDa and a trail of hGM-CSF hyperglycosylated varying from 23 kDa to more than 60 kDa. The two-copy transformants produced hGM-CSF level which was sevenfold higher compared to the single-copy ones. Deglycosylation with PNGase F showed two forms: a mature form of 14.5 kDa and an unprocessed form of 18 kDa. The addition of two alanines to the signal sequence resulted in correct hGM-CSF processing. The production level was estimated at 250 mg/l after preliminary optimization studies of the cultivation and induction phases. The purified hGM-CSF was identified by N-terminal sequencing and LC-MS/MS analysis; its biological activity was confirmed by stimulating the proliferation of TF1 cell line. This study demonstrated that Y. lipolytica is a promising host for the efficient production of active toxic proteins like hGM-CSF.

Keywords: Yarrowia lipolytica ; hGM-CSF expression; Signal sequence; Cultivation strategy

SUMO fusion system facilitates soluble expression and high production of bioactive human fibroblast growth factor 23 (FGF23) by Xiaoju Liu; Yubin Chen; Xiaoping Wu; Haiyan Li; Chao Jiang; Haishan Tian; Lu Tang; Dezhong Wang; Ting Yu; Xiaokun Li (pp. 103-111).

As a key humoral regulator of phosphate homeostasis and its involvement in the pathogenesis of human disease, human fibroblast growth factor 23 (hFGF23) has become a particularly attractive therapeutic target. To prepare soluble and bioactive recombinant human FGF23 to meet the increasing demand in its pharmacological application, small ubiquitin-related modifier (SUMO)-FGF23 fusion gene and FGF23 non-fusion gene were amplified by standard PCR methods and cloned into vector pET-22b and pET-3c, then transformed into Escherichia coli Rosetta (DE3) and BL21 (DE3). The best combination of plasmid and host strain was screened, and only Rosetta (DE3)/pET-SUMO-FGF23 was screened for rhFGF23 protein expressed. The average bacterial yield and the soluble expression level of recombinant hFGF23 of three batches attained 687 ± 18 g and 30 ± 1.5%, respectively, after treatment with 0.4 mM isopropyl-thio-β-galactopyranoside for 19 h at 16 °C in a 30-L fermentor, after which it was purified by DEAE Sepharose FF and nickel nitrilotriacetic acid affinity chromatography. Once cleaved by the SUMO protease, the recombinant human FGF23 was released from the fusion protein. The purity of rFGF23 was shown by high performance liquid chromatography to be greater than 90% and the yield was 60 ± 1.5 mg/L. In vitro data showed that the purified rFGF23 can induce the phosphorylation of mitogen-activated protein kinases in the glioma U251 cell. The results of in vivo animal experiments also showed that rFGF23 could decrease the concentration in the plasma of normal rats fed with a fixed formula diet.

Keywords: SUMO fusion; Soluble expression; High production; Bioactive fibroblast growth factor 23 (FGF23)

Characterization of a new ScbR-like γ-butyrolactone binding regulator (SlbR) in Streptomyces coelicolor by Yung-Hun Yang; Eunjung Song; Ji-Nu Kim; Bo-Rahm Lee; Eun-Jung Kim; Sung-Hee Park; Woo-Seong Kim; Hyung-Yeon Park; Jong-Min Jeon; Thangamani Rajesh; Yun-Gon Kim; Byung-Gee Kim (pp. 113-121).

γ-Butyrolactones in Streptomyces are well recognized as bacterial hormones, and they affect secondary metabolism of Streptomyces. γ-Butyrolactone receptors are considered important regulatory proteins, and various γ-butyrolactone synthases and receptors have been reported in Streptomyces. Here, we characterized a new regulator, SCO0608, that interacted with SCB1 (γ-butyrolactone of Streptomyces coelicolor) and bound to the scbR/A and adpA promoters. The SCO0608 protein sequences are not similar to those of any known γ-butyrolactone binding proteins in Streptomyces such as ScbR from S. coelicolor or ArpA from Streptomyces griseus. Interestingly, SCO0608 functions as a repressor of antibiotic biosynthesis and spore formation in R5 complex media. We showed the existence of another type of γ-butyrolactone receptor in Streptomyces, and this SCO0608 was named ScbR-like γ-butyrolactone binding regulator (SlbR) in S. coelicolor.

Keywords: Streptomyces coelicolor ; DNA affinity capture assay; γ-Butyrolactone; scbR/A promoter; ScbR; Antibiotic production

Preparation and characterization of a thermostable enzyme (Mn-SOD) immobilized on supermagnetic nanoparticles by Chongfu Song; Liangquan Sheng; Xiaobo Zhang (pp. 123-132).

Superoxide dismutase (SOD) has been widely applied in medical treatments, cosmetic, food, agriculture, and chemical industries. In industry, the immobilization of enzymes can offer better stability, feasible continuous operations, easy separation and reusing, and significant decrease of the operation costs. However, little attention has focused on the immobilization of the SOD, as well as the immobilization of thermostable enzymes. In this study, the recombinant thermostable manganese superoxide dismutase (Mn-SOD) of Thermus thermophilus wl was purified and covalently immobilized onto supermagnetic 3-APTES-modified Fe₃O₄@SiO₂ nanoparticles using glutaraldehyde method to prepare the Mn-SOD bound magnetic nanoparticles. The Mn-SOD nanoparticles were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, X-ray diffraction, transmission electron microscopy, and vibrating sample magnetometer analysis. The results indicated that the diameter of Mn-SOD nanoparticles was 40 (± 5) nm, and its saturation magnetization value was 27.9 emu/g without remanence or coercivity. By comparison with the free Mn-SOD, it was found that the immobilized Mn-SOD on nanoparticles exhibited better resistance to temperature, pH, metal ions, enzyme inhibitors, and detergents. The results showed that the immobilized Mn-SOD on nanoparticles could be reused ten times without significant decrease of enzymatic activity. Therefore, our study presented a novel strategy for the immobilization of thermostable Mn-SOD and for the application of thermostable enzymes.

Keywords: Manganese superoxide dismutase; Superparamagnetic nanoparticles; Thermostable enzyme; Immobilization

Characterization and application of fusidane antibiotic biosynethsis enzyme 3-ketosteroid-∆¹-dehydrogenase in steroid transformation by Miao-Miao Chen; Feng-Qing Wang; Liang-Cai Lin; Kang Yao; Dong-Zhi Wei (pp. 133-142).

Microbial ∆ 1 -dehydrogenation is one of the most important transformations in the synthesis of steroid hormones. In this study, a 3-ketosteroid-∆¹-dehydrogenase (kstD_F) involved in fusidane antibiotic biosynthesis from Aspergillus fumigatus CICC 40167 was characterized for use in steroid transformation. KstD_F encodes a polypeptide consisting of 637 amino acid residues. It shows 51% amino acid identity with a kstD from Thermomicrobium roseum DSM 5159. Expression of kstD_F in Escherichia coli and Pichia pastoris showed that all kstD_F activity is located in the cytoplasm. This indicates that it is a soluble intracytoplasmic enzyme, unlike most kstDs from bacteria, which are membrane-bound. The expression of kstD_F was performed in P. pastoris, both intracellularly and extracelluarly. The intracellularly expressed protein displayed good activity in steroid transformation, while the extracellularly expressed protein showed nothing. Interestingly, the engineered P. pastoris KM71 (KM71_I) and GS115 (GS115_I) showed different transformation activities for 4-androstene-3,17-dione (AD) when kstD_F was expressed intracellularly. Under the same conditions, KM71_I was found capable of transforming 1.0 g/l AD to 1,4-androstadiene-3,17-dione (ADD), while GS115_I could transform 1.5 g/l AD to both ADD and boldenone (BD). The production of BD is attributed to a 17β-hydroxysteroid dehydrogenase in P. pastoris GS115_I, which catalyzes the reversible reaction between C17-one and C17-alcohol of steroids. The conversion of AD by GS115_I and KM71_I may provide alternative means of preparing ADD or BD. In brief, we show here that kstD_F is a promising enzyme in steroid ∆ 1 -dehydrogenation that is propitious to construct genetically engineered steroid-transforming recombinants by heterologous overexpression.

Keywords: 3-Ketosteroid-∆¹-dehydrogenase; Steroidal antibiotic fusidanes; Steroid transformation; 4-Androstene-3,17-dione; 1,4-Androstadiene-3,17-dione; Boldenone

Screening of enzymatic activities for the depolymerisation of the marine bacterial exopolysaccharide HE800 by Coraline Rigouin; Christine Delbarre-Ladrat; Jacqueline Ratiskol; Corinne Sinquin; Sylvia Colliec-Jouault; Michel Dion (pp. 143-151).

The exopolysaccharide (EPS) HE800 is a marine-derived polysaccharide (from 8 × 10⁵ to 1.5 × 10⁶ g mol⁻¹) produced by Vibrio diabolicus and displaying original structural features close to those of glycosaminoglycans. In order to confer new biological activities to the EPS HE800 or to improve them, structural modifications need to be performed. In particular, depolymerisation is required to generate low-molecular-weight derivatives. To circumvent the use of chemical methods that lack specificity and reproducibility, enzymes able to perform such reaction are sought. This study reports the screening for enzymes capable of depolymerising the EPS HE800. A large diversity of enzyme sources has been studied: commercially available glycoside hydrolases with broad substrate specificity, lyases, and proteases as well as growing microorganisms. Interestingly, we found that the genus Enterococcus and, more particularly, the strain Enterococcus faecalis were able to depolymerise the EPS HE800. Partial characterization of the enzymatic activity gives evidence for a random and incomplete depolymerisation pattern that yields low-molecular-weight products of 40,000 g mol⁻¹. Genomic analysis and activity assays allowed the identification of a relevant open reading frame (ORF) which encodes an endo-N-acetyl-galactosaminidase. This study establishes the foundation for the development of an enzymatic depolymerisation process.

Keywords: Polysaccharide; Enzymatic depolymerisation; Low-molecular-weight derivatives; Enterococcus faecalis ; Endo-N-acetyl-galactosaminidase

Specific adsorption of tungstate by cell surface display of the newly designed ModE mutant by Kouichi Kuroda; Takashi Nishitani; Mitsuyoshi Ueda (pp. 153-159).

By cell surface display of ModE protein that is a transcriptional regulator of operons involved in the molybdenum metabolism in Escherichia coli, we have constructed a molybdate-binding yeast (Nishitani et al., Appl Microbiol Biotechnol 86:641–648, 2010). In this study, the binding specificity of the molybdate-binding domain of the ModE protein displayed on yeast cell surface was improved by substituting the amino acids involved in oxyanion binding with other amino acids. Although the displayed S126T, R128E, and T163S mutant proteins adsorbed neither molybdate nor tungstate, the displayed ModE mutant protein (T163Y) abolished only molybdate adsorption, exhibiting the specific adsorption of tungstate. The specificity of the displayed ModE mutant protein (T163Y) for tungstate was increased by approximately 9.31-fold compared to the displayed wild-type ModE protein at pH 5.4. Therefore, the strategy of protein design and its cell surface display is effective for the molecular breeding of bioadsorbents with metal-specific adsorption ability based on a single species of microorganism without isolation from nature.

Keywords: Binding selectivity; Molybdate; Tungstate; ModE; Rare metals; Yeast cell surface engineering

A new locus affects cell motility, cellulose binding, and degradation by Cytophaga hutchinsonii by Xiaofei Ji; Yuanxi Xu; Cong Zhang; Ning Chen; Xuemei Lu (pp. 161-170).

Cytophaga hutchinsonii is a Gram-negative gliding bacterium, which can rapidly degrade crystalline cellulose via a novel strategy without any recognizable processive cellulases. Its mechanism of cellulose binding and degradation is still a mystery. In this study, the mutagenesis of C. hutchinsonii with the mariner-based transposon HimarEm3 and gene complementation with the oriC-based plasmid carrying the antibiotic resistance gene cfxA or tetQ were reported for the first time to provide valuable tools for mutagenesis and genetic manipulation of the bacterium. Mutant A-4 with a transposon mutation in gene CHU_0134, which encodes a putative thiol-disulfide isomerase exhibits defects in cell motility and cellulose degradation. The cellulose binding ability of A-4 was only half of that of the wild-type strain, while the endo-cellulase activity of the cell-free supernatants and on the intact cell surface of A-4 decreased by 40 %. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of proteins binding to cellulose in the outer membrane showed that most of them were significantly decreased or disappeared in A-4 including some Gld proteins and hypothetical proteins, indicating that these proteins might play an important role in cell motility and cellulose binding and degradation by the bacterium.

Keywords: Cytophaga hutchinsonii ; Gene complementation; Cellulose binding protein; Cellulose degradation

Fluorescent protein vectors for promoter analysis in lactic acid bacteria and Escherichia coli by Tomás García-Cayuela; Luz P. Gómez de Cadiñanos; M. Luz Mohedano; Pilar Fernández de Palencia; Daniel Boden; Jerry Wells; Carmen Peláez; Paloma López; Teresa Requena (pp. 171-181).

Fluorescent reporter genes are valuable tools for real-time monitoring of gene expression in living cells. In this study we describe the construction of novel promoter-probe vectors containing a synthetic mCherry fluorescent protein gene, codon-optimized for lactic acid bacteria, divergently linked, or not, to a gene encoding the S65T and F64L variant of the green fluorescent protein. The utility of the transcriptional fusion vectors was demonstrated by the cloning of a single or two divergent promoter regions and by the quantitative evaluation of fluorescence during growth of Lactococcus lactis, Enterococcus faecalis, and Escherichia coli.

Keywords: Lactic acid bacteria; mCherry; GFP; Divergent promoters; Expression vectors

Transcriptomic responses of Synechocystis sp. PCC 6803 encapsulated in silica gel by David J. Dickson; Markael D. Luterra; Roger L. Ely (pp. 183-196).

Global gene expression of Synechocystis sp. PCC 6803 encapsulated in silica gel was examined by microarray analysis. Cultures were encapsulated in gels derived from aqueous precursors or from alkoxide precursors and incubated under constant light for 24 h prior to RNA extraction. Cultures suspended in liquid media were exposed to 500 mM salt stress and incubated under identical conditions for comparison purposes. The expression of 414 genes was significantly altered by encapsulation in aqueous-derived gels (fold change ≥1.5 and P value < 0.01), the expression of 1,143 genes was significantly altered by encapsulation in alkoxide-derived gels, and only 243 genes were common to both encapsulation chemistries. Additional qRT-PCR analyses of four selected genes, ggpS, cpcG2, slr5055, and sll5057, confirmed microarray results for those genes. These results illustrate that encapsulation stress is quite different than salt stress in terms of gene expression response. Furthermore, a number of hypothetical and unknown proteins associated with encapsulation and alcohol stress have been identified with implications for improving encapsulation protocols and rationally engineering microorganisms for direct biofuel production.

Keywords: Synechocystis sp. PCC 6803; Silica sol–gel; Microarray; Transcriptomics; Iron stress; Ethanol stress

The metabolic burden of cellulase expression by recombinant Saccharomyces cerevisiae Y294 in aerobic batch culture by Eugéne van Rensburg; Riaan den Haan; Justin Smith; Willem H. van Zyl; Johann F. Görgens (pp. 197-209).

Two recombinant strains of Saccharomyces cerevisiae Y294 producing cellulase using different expression strategies were compared to a reference strain in aerobic culture to evaluate the potential metabolic burden that cellulase expression imposed on the yeast metabolism. In a chemically defined mineral medium with glucose as carbon source, S. cerevisiae strain Y294[CEL5] with plasmid-borne cellulase genes produced endoglucanase and β-glucosidase activities of 0.038 and 0.30 U mg dry cell weight⁻¹, respectively. Chromosomal expression of these two cellulases in strain Y294[Y118p] resulted in no detectable activity, although low levels of episomally co-expressed cellobiohydrolase (CBH) activity were detected. Whereas the biomass concentration of strain Y294[CEL5] was slightly greater than that of a reference strain, CBH expression by Y294[Y118p] resulted in a 1.4-fold lower maximum specific growth rate than that of the reference. Supplementation of the growth medium with amino acids significantly improved culture growth and enzyme production, but only partially mitigated the physiological effects and metabolic burden of cellulase expression. Glycerol production was decreased significantly, up to threefold, in amino acid-supplemented cultures, apparently due to redox balancing. Disproportionately higher levels of glycerol production by Y294[CEL5] indicated a potential correlation between the redox balance of anabolism and the physiological stress of cellulase production. With the reliance on cellulase expression in yeast for the development of consolidated bioprocesses for bioethanol production, this work demonstrates the need for development of yeasts that are physiologically robust in response to burdens imposed by heterologous enzyme production.

Keywords: Saccharomyces cerevisiae ; Cellulase; CBP; Metabolic burden; Redox

Impaired dendritic cell maturation and IL-10 production following H. pylori stimulation in gastric cancer patients by Lin-Li Chang; Sheng-Wen Wang; I-Chen Wu; Fang-Jung Yu; Yu-Chung Su; Ye-Pin Chen; Deng-Chyang Wu; Chang-Hung Kuo; Chih-Hsing Hung (pp. 211-220).

The current study was to investigate the interaction between Helicobacter pylori and human dendritic cells (DCs). Whether impaired DC function can influence the outcome of H. pylori infections. Human monocyte-derived DCs (MDDCs) from five gastric cancer patients and nine healthy controls were stimulated with H. pylori. Maturation markers of MDDC were examined by flow cytometry. IL-10 and TNF-α released by MDDCs and IL-17 produced by T cells were measured by ELISA. Regulatory signaling pathways of IL-10 were examined by ELISA, western blotting, and chromatin immunoprecipitation assay. The results showed that as compared with healthy individuals, the maturation marker CD40 in MDDCs, IL-17A expression from T cells, and IL-10 expression from MDDCs were significantly lower in gastric cancer patients. Blocking DC-SIGN, TLR2, and TLR4 could reverse H. pylori-associated IL-10 production. Activation of the p38 MAPK and NF-kB signaling pathways concomitant with decreased tri-methylated H3K9 and increased acetylated H3 accounted for the effect of H. pylori on IL-10 expression. Furthermore, upregulated IL-10 expression was significantly suppressed in H. pylori-pulsed MDDCs by histone acetyltransferase and methyltransferase inhibitors. Taken together, impaired DC function contributes to the less effective innate and adaptive immune responses against H. pylori seen in gastric cancer patients. H. pylori can regulate IL-10 production through Toll-like and DC-SIGN receptors, activates p-p38 MAPK signaling and the transcription factors NF-kB, and modulates histone modification.

Keywords: Dendritic cell; Epigenetics; Gastric cancer; Helicobacter pylori ; IL-10

Production of the ¹⁴C-labeled insecticidal protein Cry1Ab for soil metabolic studies using a recombinant Escherichia coli in small-scale batch fermentations by Petra Valldor; Rona Miethling-Graff; Susanne Dockhorn; Rainer Martens; Christoph C. Tebbe (pp. 221-229).

Insecticidal Cry proteins naturally produced by Bacillus thuringiensis are a major recombinant trait expressed by genetically modified crops. They are released into the soil during and after cropping. The objective of this study was to produce ¹⁴C-labeled Cry1Ab proteins for soil metabolic studies in scope of their environmental risk assessment. Cry1Ab was synthesized as a protoxin by Escherichia coli HB101 pMP in 200-mL liquid batch culture fermentations and purified from inclusion bodies after trypsin digestion. For cultivation, U-¹⁴C-glycerol was the main carbon source. Inclusion bodies were smaller and Cry1Ab yield was lower when the initial amount of total organic carbon in the cultivation broth was below 6.4 mg C L⁻¹. Concentrations of 12.6 g ¹⁴C-labeled glycerol L⁻¹ (1 % v/v) resulted in the production of 17.1 mg ¹⁴C-Cry1Ab L⁻¹ cultivation medium. ¹⁴C mass balances showed that approx. 50 % of the label was lost by respiration and 20 % remained in the growth media, while the residual activity was associated with biomass. Depending on the production batch, 0.01 to 0.05 % of the total ¹⁴C originated from Cry1Ab. In the presence of 2.04 MBq ¹⁴C-labeled carbon sources, a specific activity of up to 268 Bq mg⁻¹ ¹⁴C-Cry1Ab was obtained. A more than threefold higher specific activity was achieved with 4.63 MBq and an extended cultivation period of 144 h. This study demonstrates that ¹⁴C-labeled Cry1Ab can be obtained from batch fermentations with E. coli in the presence of a simple ¹⁴C-labeled carbon source. It also provides a general strategy to produce ¹⁴C-labeled proteins useful for soil metabolic studies.

Keywords: Cry1Ab protein; Bt toxin; ¹⁴C labeling; Recombinant gene expression; Protein biosynthesis; Escherichia coli HB101

Silica gel-encapsulated AtzA biocatalyst for atrazine biodegradation by Eduardo Reátegui; Erik Reynolds; Lisa Kasinkas; Amit Aggarwal; Michael J. Sadowsky; Alptekin Aksan; Lawrence P. Wackett (pp. 231-240).

Encapsulation of recombinant Escherichia coli cells expressing a biocatalyst has the potential to produce stable, long-lasting enzyme activity that can be used for numerous applications. The current study describes the use of this technology with recombinant E. coli cells expressing the atrazine-dechlorinating enzyme AtzA in a silica/polymer porous gel. This novel recombinant enzyme-based method utilizes both adsorption and degradation to remove atrazine from water. A combination of silica nanoparticles (Ludox TM40), alkoxides, and an organic polymer was used to synthesize a porous gel. Gel curing temperatures of 23 or 45 °C were used either to maintain cell viability or to render the cells non-viable, respectively. The enzymatic activity of the encapsulated viable and non-viable cells was high and extremely stable over the time period analyzed. At room temperature, the encapsulated non-viable cells maintained a specific activity between (0.44 ± 0.06) μmol/g/min and (0.66 ± 0.12) μmol/g/min for up to 4 months, comparing well with free, viable cell-specific activities (0.61 ± 0.04 μmol/g/min). Gels cured at 45 °C had excellent structural rigidity and contained few viable cells, making these gels potentially compatible with water treatment facility applications. When encapsulated, non-viable cells were assayed at 4 °C, the activity increased threefold over free cells, potentially due to differences in lipid membranes as shown by FTIR spectroscopy and electron microscopy.

Keywords: Atrazine; Silica; Bacteria; Biodegradation; AtzA; E. coli

A kinetic modeling for carbon metabolism in sequencing batch reactor under multiple aerobic/anoxic conditions by Ji Fan; Peter A. Vanrolleghem; Shuguang Lu (pp. 241-252).

In order to further investigate activated sludge system for better carbon metabolism and nitrogen removal with less energy consumption, a new kinetic model was established. The detailed description of the proposed model was introduced for understanding the mechanisms involved in the activated sludge system, especially simultaneous substrate storage and biomass growth (SSSG) processes and soluble microbial product generation. The evaluation of the proposed model was demonstrated by a lab-scale sequencing batch reactor (SBR) operated with three different sets, i.e., aeration/non-aeration (set 1), non-aeration/aeration/non-aeration (set 2), and alternating aeration/non-aeration (set 3) processes. The purpose was to investigate carbon metabolism under multiple aerobic/anoxic conditions. The calibrated results showed quite an acceptable model fit to the on-line measured dissolved oxygen (DO) data for the three SBR sets. Predictions of the calibrated model were successfully confirmed using off-line analyses of soluble chemical oxygen demands (COD) and nitrogen dynamic variations, respectively. The simulated results showed that more SMP was generated under aerobic condition than that under anoxic condition, and more nitrate (S _NO) consumption resulted in less SMP generation, i.e., approximately 7% and 57% less extra carbon source in sets 2 and 3 were required to remove 8% and 58% of S _NO, respectively, compared with set 1. And the kinetics of SSSG process in the proposed model was indirectly validated by comparisons between experimental DO profiles and simulations. Therefore, the new model provides an effective technique for better optimizing the effluent COD and nitrogen with low energy cost in biological wastewater treatment plants.

Keywords: Activated sludge modeling; SBR; Simultaneous substrate storage and biomass growth process (SSSG); SMPs; Biological wastewater treatment

The impact of dissolved organic carbon on the spatial variability of methanogenic archaea communities in natural wetland ecosystems across China by Deyan Liu; Weixin Ding; Zhongjun Jia; Zucong Cai (pp. 253-263).

Significant spatial variation in CH₄ emissions is a well-established feature of natural wetland ecosystems. To understand the key factors affecting CH₄ production, the variation in community structure of methanogenic archaea, in relation to substrate and external environmental influences, was investigated in selected wetlands across China, using denaturing gradient gel electrophoresis. Case study areas were the subtropical Poyang wetland, the warm-temperate Hongze wetland, the cold-temperate Sanjiang marshes, and the alpine Ruoergai peatland on the Qinghai–Tibetan Plateau. The topsoil layer in the Hongze wetland exhibited the highest population of methanogens; the lowest was found in the Poyang wetland. Maximum CH₄ production occurred in the topsoil layer of the Sanjiang Carex lasiocarpa marsh, the minimum was observed in the Ruoergai peatland. CH₄ production potential was significantly correlated with the dissolved organic carbon (DOC) concentration but not with the abundance or diversity indices of methanogenic archaea. Phylogenetic analysis and DOC concentration indicated a shift in the dominant methanogen from the hydrogenotrophic Methanobacteriales in DOC-rich wetlands to Methanosarcinaceae with a low affinity in wetlands with relatively high DOC and then to the acetotrophic methanogen Methanosaetaceae with a high affinity in wetlands with low DOC, or with high DOC but rich sulfate-reducing bacteria. Therefore, it is proposed that the dominant methanogen type in wetlands is primarily influenced by available DOC concentration. In turn, the variation in CH₄ production potential in the wetlands of eastern China is attributable to differences in the DOC content and the dominant type of methanogen present.

Keywords: Dissolved organic carbon; Methanogenic archaea communities; 16S rRNA gene; CH₄ production potential; Wetlands

Simultaneous removal of phosphorus and nitrogen in a sequencing batch biofilm reactor with transgenic bacteria expressing polyphosphate kinase by Hongwei Du; Liuyan Yang; Jun Wu; Lin Xiao; Xiaolin Wang; Lijuan Jiang (pp. 265-272).

To improve phosphorus removal from wastewater, we constructed a high-phosphate-accumulating microorganism, KTPPK, using Pseudomonas putida KT2440 as a host. The expression plasmid was constructed by inserting and expressing polyphosphate kinase gene (ppk) from Microcystis aeruginosa NIES-843 into broad-host-range plasmid, pBBR1MCS-2. KTPPK was then added to a sequencing batch biofilm reactor (SBBFR) using lava as a biological carrier. The results showed that SBBFR with KTPPK not only efficiently removed COD, NH₃–N, and NO ₃ ⁻ –N but also had a high removal capacity for PO ₄ ³⁻ –P, resulting in a low phosphorus concentration remaining in the outflow of the SBBFR. The biofilm increased by 30–53% on the lava in the SBBFR that contained KTPPK after 11 days when compared with the reactor that contained P. putida KT2440. Real-time quantitative polymerase chain reaction confirmed that the copy of ppk was maintained at about 3.5 × 10¹⁰ copies per μL general DNA in the biofilm after 20 days. Thus, the transgenic bacteria KTPPK could maintain a high density and promote phosphorus removal in the SBBFR. In summary, this study indicates that the use of SBBFR with transgenic bacteria has the potential to remove phosphorus and nitrogen from wastewater.

Keywords: Poly-phosphate kinase gene; Pseudomonas putida ; Sequencing batch biofilm reactor; Phosphorus removal; Nitrogen removal

Aerobic bioreduction of nickel(II) to elemental nickel with concomitant biomineralization by Guoqiang Zhan; Daping Li; Liang Zhang (pp. 273-281).

Although microorganisms have the potential to reduce metals, products with elementary forms are unusual. In the present study, a strain of Pseudomonas sp. MBR was tested for its ability to reduce metal ions to their elementary forms coupled to biomineralization under aerobic conditions. The Pseudomonas sp. MBR strain was able to reduce metals such as Fe(III), Mn(II), Cu(II), Ni(II), Cd(II), Co(II), Al(III), Se(IV), and Te(IV) as electron acceptors to elementary forms using citrate, lactate, pyruvate, succinate, malate, glucose, or ethanol as electron donors. Growth and reduction during biomineralization occurred within the pH range of 6.0 to 11.0 and temperature range of 4 to 40 °C, with an optimum growth temperature of 28 °C. The resistance of Ni(II) varied from 0.5 to 5 mM. Ni(II) reduction was still observed when nitrate was present in addition to oxygen as a potential electron acceptor. The Ni(II) reduction efficiency was related with the molar ratio of the electron donor to Ni(II). Unlike other dissimilatory metal-reducing bacteria, which oxidizes organic matter with Fe(III) or Mn(IV) as the sole electron acceptor coupled to energy production under facultative anaerobic conditions, this strain used oxygen as an electron acceptor combined with metal reduction. The aerobic metal reduction may relate to a co-metabolic reduction. Transmission electron microscopy images demonstrated that the cells had the ability to accumulate heavy metals, and that the detoxicity mechanism was intracellular metal reduction. These results suggested that the use of Pseudomonas sp. MBR could be promising for toxic heavy metal bioremediation and biological metallurgy.

Keywords: Bioreduction; Elemental nickel; Biomineralization; Aerobic conditions

Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha by Jingnan Lu; Christopher J. Brigham; Claudia S. Gai; Anthony J. Sinskey (pp. 283-297).

Wild-type Ralstonia eutropha H16 produces polyhydroxybutyrate (PHB) as an intracellular carbon storage material during nutrient stress in the presence of excess carbon. In this study, the excess carbon was redirected in engineered strains from PHB storage to the production of isobutanol and 3-methyl-1-butanol (branched-chain higher alcohols). These branched-chain higher alcohols can directly substitute for fossil-based fuels and be employed within the current infrastructure. Various mutant strains of R. eutropha with isobutyraldehyde dehydrogenase activity, in combination with the overexpression of plasmid-borne, native branched-chain amino acid biosynthesis pathway genes and the overexpression of heterologous ketoisovalerate decarboxylase gene, were employed for the biosynthesis of isobutanol and 3-methyl-1-butanol. Production of these branched-chain alcohols was initiated during nitrogen or phosphorus limitation in the engineered R. eutropha. One mutant strain not only produced over 180 mg/L branched-chain alcohols in flask culture, but also was significantly more tolerant of isobutanol toxicity than wild-type R. eutropha. After the elimination of genes encoding three potential carbon sinks (ilvE, bkdAB, and aceE), the production titer improved to 270 mg/L isobutanol and 40 mg/L 3-methyl-1-butanol. Semicontinuous flask cultivation was utilized to minimize the toxicity caused by isobutanol while supplying cells with sufficient nutrients. Under this semicontinuous flask cultivation, the R. eutropha mutant grew and produced more than 14 g/L branched-chain alcohols over the duration of 50 days. These results demonstrate that R. eutropha carbon flux can be redirected from PHB to branched-chain alcohols and that engineered R. eutropha can be cultivated over prolonged periods of time for product biosynthesis.

Keywords: Ralstonia eutropha ; Biofuel; Branched-chain alcohol; Isobutanol; 3-Methyl-1-butanol; Branched-chain amino acid

Erratum to: Lactobacillus reuteri CRL 1101 highly produces mannitol from sugarcane molasses as carbon source by Maria Eugenia Ortiz; María José Fornaguera; Raúl R. Raya; Fernanda Mozzi (pp. 299-300).

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Applied Microbiology and Biotechnology (v.96, #1)

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Applied Microbiology and Biotechnology (v.96, #1)