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Applied Microbiology and Biotechnology (v.76, #1)

Tagatose: properties, applications, and biotechnological processes by Deok-Kun Oh (pp. 1-8).

d-Tagatose has attracted a great deal of attention in recent years due to its health benefits and similar properties to sucrose. d-Tagatose can be used as a low-calorie sweetener, as an intermediate for synthesis of other optically active compounds, and as an additive in detergent, cosmetic, and pharmaceutical formulation. Biotransformation of d-tagatose has been produced using several biocatalyst sources. Among the biocatalysts, l-arabinose isomerase has been mostly applied for d-tagatose production because of the industrial feasibility for the use of d-galactose as a substrate. In this article, the characterization of many l-arabinose isomerases and their d-tagatose production is compared. Protein engineering and immobilization of the enzyme for increasing the conversion rate of d-galactose to d-tagatose are also reviewed.

Keywords: Application; l-Arabinose isomerase; Biotransformation; Properties; Tagatose

Applications of RNA interference: current state and prospects for siRNA-based strategies in vivo by Achim Aigner (pp. 9-21).

Within the recent years, RNA interference (RNAi) has become an almost-standard method for in vitro knockdown of any target gene of interest. Now, one major focus is to further explore its potential in vivo, including the development of novel therapeutic strategies. From the mechanism, it becomes clear that small interfering RNAs (siRNAs) play a pivotal role in triggering RNAi. Thus, the efficient delivery of target gene-specific siRNAs is one major challenge in the establishment of therapeutic RNAi. Numerous studies, based on different modes of administration and various siRNA formulations and/or modifications, have already accumulated promising results. This applies to various animal models covering viral infections, cancer and multiple other diseases. Continuing efforts will lead to the development of efficient and “double-specific” drugs, comprising of siRNAs with high target gene specificity and of nanoparticles enhancing siRNA delivery and target organ specificity.

Keywords: RNA interference; RNAi; siRNA; Gene-targeting; Gene knockdown; Nonviral siRNA delivery; Nanoplexes

Microbial production and application of sophorolipids by Inge N. A. Van Bogaert; Karen Saerens; Cassandra De Muynck; Dirk Develter; Wim Soetaert; Erick J. Vandamme (pp. 23-34).

Sophorolipids are surface-active compounds synthesized by a selected number of yeast species. They have been known for over 40 years, but because of growing environmental awareness, they recently regained attention as biosurfactants due to their biodegradability, low ecotoxicity, and production based on renewable resources. In this paper, an overview is given of the producing yeast strains and various aspects of fermentative sophorolipid production. Also, the biochemical pathways and regulatory mechanisms involved in sophorolipid biosynthesis are outlined. To conclude, a summary is given on possible applications of sophorolipids, either as native or modified molecules.

Keywords: Sophorolipids; Candida bombicola ; Candida apicola ; Biosurfactant; Yeast

The twin-arginine translocation system and its capability for protein secretion in biotechnological protein production by Thomas Brüser (pp. 35-45).

The biotechnological production of recombinant proteins is challenged by processes that decrease the yield, such as protease action, aggregation, or misfolding. Today, the variation of strains and vector systems or the modulation of inducible promoter activities is commonly used to optimize expression systems. Alternatively, aggregation to inclusion bodies may be a desired starting point for protein isolation and refolding. The discovery of the twin-arginine translocation (Tat) system for folded proteins now opens new perspectives because in most cases, the Tat machinery does not allow the passage of unfolded proteins. This feature of the Tat system can be exploited for biotechnological purposes, as expression systems may be developed that ensure a virtually complete folding of a recombinant protein before purification. This review focuses on the characteristics that make recombinant Tat systems attractive for biotechnology and discusses problems and possible solutions for an efficient translocation of folded proteins.

Keywords: Twin-arginine translocation; Protein secretion; Protein production; Tat translocons; Chaperones; PspA

Microbial tannases: advances and perspectives by Cristóbal N. Aguilar; Raúl Rodríguez; Gerardo Gutiérrez-Sánchez; Christopher Augur; Ernesto Favela-Torres; Lilia A. Prado-Barragan; Ascensión Ramírez-Coronel; Juan C. Contreras-Esquivel (pp. 47-59).

In the last years, tannase has been the subject of a lot of studies due to its commercial importance and complexity as catalytic molecule. Tannases are capable of hydrolyzing complex tannins, which represent the main chemical group of natural anti-microbials occurring in the plants. The general outline of this work includes information of the substrates, the enzyme, and the applications. This review considers in its introduction the concepts and history of tannase and explores scientific and technological aspects. The “advances” trace the route from the general, molecular, catalytic, and functional information obtained under close to optimal conditions for microbial production through purification, description of the enzyme properties, and the commercial applications to the “perspectives” including expression studies, regulation, and potential uses; aspects related to the progress in our understanding of tannin biodegradation are also included.

Keywords: Tannase; Tannins biodegradation; Microbial sources; Advances; Perspectives

Microbial asymmetric oxidation of 2-butyl-1,3-propanediol by K. Mitsukura; T. Uno; T. Yoshida; T. Nagasawa (pp. 61-65).

Microbial asymmetric oxidation of 2-butyl-1,3-propanediol was investigated for an efficient synthesis of S- and R-enantiomers of 2-hydroxymethylhexanoic acid (2-HMHA). From an intensive survey of the stocked bacterial strains, Acetobacter pasteurianus IAM 12073 and Pseudomonas putida IFO 3738 were found to show the highest S- and R-2-HMHA-producing activity, respectively. Under optimized conditions, A. pasteurianus (351 mg dry cell weight) and P. putida (642 mg dry cell weight) cells produced 12.0 g l⁻¹ S-2-HMHA with 89% enantiomeric excess (e.e.) at 24 h of incubation and 5.1 g l⁻¹ R-2-HMHA with 94% e.e. at 35 h of incubation from 2-butyl-1,3-propanediol.

Keywords: Acetobacter pasteurianus ; Asymmetric oxidation; 2-Butyl-1,3-propanediol; 2-Hydroxymethylhexanoic acid; Pseudomonas putida

Repeated pH-stat fed-batch fermentation for rhamnolipid production with indigenous Pseudomonas aeruginosa S2 by Shan-Yu Chen; Yu-Hong Wei; Jo-Shu Chang (pp. 67-74).

Rhamnolipid is one of the most commonly used biosurfactants with the ability to reduce the surface tension of water from 72 to 30 mN/m. An indigenous isolate Pseudomonas aeruginosa S2 possessing excellent ability to produce rhamnolipid was used as a model strain to explore fermentation technology for rhamnolipid production. Using optimal medium and operating conditions (37°C, pH 6.8, and 250 rpm agitation) obtained from batch fermentation, P. aeruginosa S2 was able to produce up to 5.31 g/l of rhamnolipid from glucose-based medium. To further improve the rhamnolipid yield, a pH-stat fed-batch culture was performed by maintaining a constant pH of 6.8 through manipulating glucose feeding. The effect of influent glucose concentration on rhamnolipid yield and productivity was investigated. Using the pH-stat culture, a maximum rhamnolipid concentration (6.06 g/l) and production rate (172.5 ml/h/l) was obtained with 6% glucose in the feed. Moreover, combining pH-stat culture with fill-and-draw operation allowed a stable repeated fed-batch operation for approximately 500 h. A marked increase in rhamnolipid production was achieved, leading to the best rhamnolipid yield of approximately 9.4 g/l during the second repeated run.

Keywords: Biosurfactant; Fed-batch fermentation; Fill-and-draw; pH-stat culture; Pseudomonas aeruginosa ; Rhamnolipid

Kinetic studies on autohydrogenotrophic growth of Ralstonia eutropha with nitrate as terminal electron acceptor by Armin Tiemeyer; Hannes Link; Dirk Weuster-Botz (pp. 75-81).

Autohydrogenotrophic batch growth of Ralstonia eutropha H16 was studied in a stirred-tank reactor with nitrate and nitrite as terminal electron acceptors and the sole limiting substrates. Assuming product inhibition by nitrite, saturation kinetics with the two limiting substrates and a simple switching function, which allows growth on nitrite only at low nitrate concentrations, resulted in a kinetic growth model with nine model parameters. The data of two batch experiments were used to identify the kinetic model. The kinetic model was validated with two additional batch experiments. The model predictions are in very good agreement with the experimental data. The maximum nitrite concentration was estimated to be 30.7 mM (total inhibition of growth). After complete reduction of nitrate, the growth rate decreases almost to zero before it increases again because of the following nitrite respiration. The maximum autohydrogenotrophic growth rate of Ralstonia eutropha with nitrate as a final electron acceptor (0.509 d⁻¹) was found to be reduced by 90–95% compared to the so far reported autohydrogenotrophic growth rates with oxygen.

Keywords: Ralstonia eutropha ; Autohydrogenotrophic; Kinetic; Nitrate respiration; Nitrite respiration; Cupriavidus necator

Effect of culture temperature on follicle-stimulating hormone production by Chinese hamster ovary cells in a perfusion bioreactor by Sung Kwan Yoon; Yong-Ho Ahn; Myeong Hyeon Jeong (pp. 83-89).

Follicle-stimulating hormone (FSH) was produced in Chinese hamster ovary (CHO) cells using a perfusion bioreactor. Perfusion culture at 37°C yielded a high cell density but a low FSH production. To investigate the effect of culture temperature in the range of 26–37°C on cell growth and FSH production, batch cultures were performed. Lowering culture temperature below 32°C resulted in growth suppression. However, specific productivity of FSH, q _FSH, increased as culture temperature decreased, and the maximum q _FSH of 43.4 ng/10⁶ cells/h was obtained at 28°C, which is 13-fold higher than that at 37°C. Based on the results obtained from batch cultures, we performed perfusion cultures with two consecutive temperatures. CHO cells were grown up to 3.2 × 10⁷ cells/ml at 37°C and culture temperature shifted down to 28°C to obtain a high FSH titer. Soon after the maximum FSH titer of 21 μg/ml was achieved, a rapid loss of not only viable cell concentration but also cell viability was observed, probably due to the low activities of enzymes related to cell growth. Thus, the extension of production period at 28°C is critical for the enhancement of FSH production, and the use of antiapoptotic genes seems to be promising.

Keywords: Perfusion; CHO cells; Low culture temperature; Follicle-stimulating hormone

Quantification of total and bioavailable lysine in feed protein sources by a whole-cell green fluorescent protein growth-based Escherichia coli biosensor by V. I. Chalova; W. K. Kim; C. L. Woodward; S. C. Ricke (pp. 91-99).

Using a fluorescent whole-cell Escherichia coli biosensor previously developed in our laboratory, we determined total and bioavailable lysine in four feed ingredients (soybean, cottonseed, meat and bone meal, and sorghum) and three complete feeds (chick starter and finisher, and swine starter). The same feed sources were analyzed for total lysine by high performance liquid chromatography (HPLC) and bioavailable lysine by chick bioassay. No significant differences were found between bioavailable lysine estimates for soybean, cottonseed, meat and bone meal, chick starter and finisher, and swine starter obtained by the fluorescent E. coli biosensor and chick bioassay. Except for sorghum, the E. coli biosensor estimates for total lysine were highly comparable to those obtained by HPLC. Comparisons were also conducted between conventionally performed optical density-based and the newly developed fluorescence-based lysine assay. The lack of significant differences in data obtained for total and bioavailable lysine by both detection modes indicated reliance and accuracy of the fluorescent E. coli biosensor. Overall results suggest that the microbial assay based on green fluorescent protein fluorescence represents a promising alternative method for lysine quantification.

Keywords: Escherichia coli ; Lysine bioavailability; Biosensor; Green fluorescent protein

Medium optimization of antifungal lipopeptide, iturin A, production by Bacillus subtilis in solid-state fermentation by response surface methodology by Shinji Mizumoto; Makoto Shoda (pp. 101-108).

Iturin A, a lipopeptide antibiotic produced by Bacillus subtilis RB14-CS, suppresses the growth of various plant pathogens. Here, enhancement of iturin A production in solid-state fermentation (SSF) on okara, a soybean curd residue produced during tofu manufacturing, was accomplished using statistical experimental design. Primary experiments showed that the concentrations of carbon and nitrogen sources were the main factors capable of enhancing iturin A production, whereas initial pH, initial water content, temperature, relative humidity, and volume of inoculum were only minor factors. Glucose and soybean meal were the most effective among tested carbon and nitrogen sources, respectively. Based on these preliminary findings, response surface methodology was applied to predict the optimum amounts of the carbon and nitrogen sources in the medium. The maximum iturin A concentration was 5,591 μg/g initial wet okara under optimized condition. Subsequent experiments confirmed that iturin A production was significantly improved under the predicted optimal medium conditions. The SSF product generated under the optimized conditions exhibited significantly higher suppressive effect on the damping-off of tomato caused by Rhizoctonia solani K-1 compared with the product generated under the non-optimized conditions.

Keywords: Optimization; Iturin A; Bacillus subtilis ; Solid-state fermentation; Response surface methodology

Controlling the sucrose concentration increases Coenzyme Q10 production in fed-batch culture of Agrobacterium tumefaciens by Suk-Jin Ha; Sang-Yong Kim; Jin-Ho Seo; Hee-Jung Moon; Kyoung-Mi Lee; Jung-Kul Lee (pp. 109-116).

The production yield of Coenzyme Q₁₀ (CoQ₁₀) from the sucrose consumed by Agrobacterium tumefaciens KCCM 10413 decreased, and high levels of exopolysaccharide (EPS) accumulated after switching from batch culture to fed-batch culture. Therefore, we examined the effect of sucrose concentration on the fermentation profile by A. tumefaciens. In the continuous fed-batch culture with the sucrose concentration maintained constantly at 10, 20, 30, and 40 g l⁻¹, the dry cell weight (DCW), specific CoQ₁₀ content, CoQ₁₀ production, and the production yield of CoQ₁₀ from the sucrose consumed increased, whereas EPS production decreased as maintained sucrose concentration decreased. The pH-stat fed-batch culture system was adapted for CoQ₁₀ production to minimize the concentration of the carbon source and osmotic stress from sucrose. Using the pH-stat fed-batch culture system, the DCW, specific CoQ₁₀ content, CoQ₁₀ production, and the product yield of CoQ₁₀ from the sucrose consumed increased by 22.6, 13.7, 39.3, and 39.3%, respectively, whereas EPS production decreased by 30.7% compared to those of fed-batch culture in the previous report (Ha SJ, Kim SY, Seo JH, Oh DK, Lee JK, Appl Microbiol Biotechnol, 74:974–980, 2007). The pH-stat fed-batch culture system was scaled up to a pilot scale (300 l), and the CoQ₁₀ production results obtained (626.5 mg l⁻¹ of CoQ₁₀ and 9.25 mg g DCW⁻¹ of specific CoQ₁₀ content) were similar to those obtained at the laboratory scale. Thus, an efficient and highly competitive process for microbial CoQ₁₀ production is available.

Keywords: Agrobacterium tumefaciens ; Coenzyme Q₁₀ ; Exopolysaccharide; pH-stat fed-batch culture; Scale-up

Altering the substrate specificity site of Aspergillus niger PhyB shifts the pH optimum to pH 3.2 by Jeremy D. Weaver; Edward J. Mullaney; Xin Gen Lei (pp. 117-122).

Phytases are of biotechnological importance as animal feed additives for their ability to catalyze the hydrolysis of phosphate from phytate for absorption by simple-stomached animals, and to reduce their fecal phosphorus excretion. Aspergillus niger PhyB has high catalytic activity at low pHs around 2.5, but has little activity at the commonly observed gastric pH of young animals (3.0–3.5). Our objective was to determine if the pH optima of PhyB could be broadened to a more characteristic pH range in the stomach of young animals through site-directed mutagenesis. We created two mutants, E272K and E272Q, each with a single amino acid substitution of the same residue in the substrate specificity site. Mutants were designed to replace an acidic amino acid, with either a neutral amino acid (E272Q) or basic amino acid (E272K), and were overexpressed in the yeast Pichia pastoris. While the wild-type (WT) pH optimum was 2.5, mutant E272K shifted to a new optimum of pH 3.2. E272K had a concomitant reduction in K _m of 36-fold at pH 2.5 and 6-fold at pH 3.2 compared to the WT. Our results indicate that the pH optimum of PhyB can be altered to match the stomach pH, along with an improved substrate affinity.

Keywords: Phytases; Animal feed additive; Aspergillus niger ; pH

New role of C-terminal 30 amino acids on the insoluble chitin hydrolysis in actively engineered chitinase from Vibrio parahaemolyticus by Hsu-Han Chuang; Fu-Pang Lin (pp. 123-133).

A chitinase (VpChiA) and its C-terminal truncated G589 mutant (VpChiAG589) of Vibrio parahaemolyticus were cloned by polymerase chain reaction (PCR) techniques. To study the role of the C-terminal 30 amino acids of VpChiA in the enzymatic hydrolysis of chitin, both the recombinant VpChiA and VpChiAG589 encoded in 1,881 and 1,791 bp DNA fragments, respectively, were expressed in Escherichia coli using the pET-20b(+) expression system. The His–Tag affinity purified VpChiA and VpChiAG589 enzymes had a calculated molecular mass of 65,713 and 62,723 Da, respectively. The results of biochemical characterization including kinetic parameters, spectroscopy of fluorescence and circular dichroism, chitin-binding and hydrolysis, and thermostability, both VpChiA and VpChiAG589, had very similar physicochemical properties such as the optimum pH (6), temperature (40°C), and kinetic parameters of Km and kcat against the 4MU–(GlcNAc)₂ or 4MU–(GlcNAc)₃ soluble substrates. The significant increase of thermostability and the drastic decrease of the hydrolyzing ability of VpChiAG589 toward the insoluble α-chitin substrate suggested that a new role could be played by the C-terminal 30 amino acids.

Keywords: Vibrio parahaemolyticus ; Chitinase; Site-directed mutagenesis; C-terminal truncation; Crystalline chitin

Directed evolution for increased chitinase activity by Yanhua Fan; Weiguo Fang; Yuehua Xiao; Xingyong Yang; Yongjun Zhang; Michael J. Bidochka; Yan Pei (pp. 135-139).

Directed evolution through DNA shuffling and screening was used to enhance the catalytic ability of a fungal, Beauveria bassiana, chitinase, Bbchit1. The Bbchit gene was first linked to various prokaryotic signal sequences and expressed in Escherichia coli. The signal peptide, PelB, from Erwinia carotovora resulted in greatest chitinase secretion into broth. The nucleotide sequence expressing PelB signal peptide was then incorporated into an E. coli vector to express Bbchit1 variants generated by three rounds of DNA shuffling. A Bbchit1 library with 150,000 variants was constructed with a nucleotide point mutation frequency of 0.6% and screened for chitinolytic activity. Two Bbchit1 variants (SHU-1 and SHU-2) were selected that showed increased chitinolytic activity compared to the wild type. Sequence analysis of these variants revealed mutations in amino acid residues that would not normally be considered for rational design of improved chitinase activity. The amino acid substitutions occurred outside of the two putative substrate-binding sites and the catalytic region.

Keywords: Beauveria bassiana ; Chitinase; Catalytic activity; DNA shuffling

Construction and characterization of two versions of bifunctional EGFP–sTRAIL fusion proteins by Jiayin Shen; Yifan Wu; Lijun Shi; Junhong Liu; Shunyi Liu; Zhengbing Guan; Zhimin Yin (pp. 141-149).

The extracellular portion (amino acids 95–281 or 114–281) of the human tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) was genetically linked to the C terminus of the fluoresce-enhanced green fluorescent protein variant (EGFP) to generate two versions of EGFP–sTRAIL fusion proteins, designated EGFP–sTR95 and EGFP–sTR114, respectively. The two versions of EGFP–sTRAIL fusion proteins both induce extensive apoptosis in lymphoid as well as nonlymphoid tumor cell lines. In addition, the two versions of fusion proteins retain similar fluorescence spectra to those of EGFP and have shown the specific binding to TRAIL receptor-positive cells; thus, the stained cells could be analyzed with flow cytometry. Hence, the two versions of fusion proteins represent a readily obtainable source of biologically active sTRAIL that may prove useful in exploit fully the characteristics of both the soluble TRAIL and its receptor system.

Keywords: Enhanced fluorescent protein; Human TNF-related apoptosis-inducing ligand; Apoptosis; Receptor-binding

High-efficiency recovery of target cells using improved yeast display system for detection of protein–protein interactions by Nobuo Fukuda; Jun Ishii; Seiji Shibasaki; Mitsuyoshi Ueda; Hideki Fukuda; Akihiko Kondo (pp. 151-158).

We constructed a high-throughput screening (HTS) system for target cells based on the detection of protein–protein interactions by flow cytometric sorting due to the improvement in the yeast cell surface display system. Interaction model proteins, which are the ZZ domain derived from Staphylococcus aureus and the Fc part of human immunoglobulin G (IgG), were displayed on the yeast cell surface. We achieved a rapid and enhanced expression of these proteins as a result of adopting an appropriate yeast strain and a suitable promoter. The displayed ZZ domain had an ability to bind to rabbit IgG and the displayed Fc part to protein A. These were confirmed by flow cytometry and fluorescence microscopy. Furthermore, the cells displaying the ZZ domain or Fc part were isolated from the model libraries constructed by mixing the control yeast cells with the target yeast cells. The ratio of the target cells was increased from 0.0001% to more than 70% by two cycles of cell sorting. These results indicate that we can achieve a rapid and highly efficient isolation method for the target cells with FACSCalibur and that this method will further extend the application of flow cytometric sorting to library selections.

Keywords: Improved yeast display system; Flow cytometric sorting; High-efficiency recovery; Protein-protein interactions

Analysis of catRABC operon for catechol degradation from phenol-degrading Rhodococcus erythropolis by M. Veselý; M. Knoppová; J. Nešvera; M. Pátek (pp. 159-168).

The gene cluster catRABC, involved in catechol degradation, was isolated from Rhodococcus erythropolis CCM2595. The genes catA, catB, catC, and the divergently transcribed catR code for catechol 1,2-dioxygenase, cis,cis-muconate cycloisomerase, muconolactone isomerase, and an IclR-type transcriptional regulator, respectively. Measurements of catechol 1,2-dioxygenase activity showed that the expression of catA is induced by phenol but not by catechol or cis,cis-muconate. The activity of catechol 1,2-dioxygenase was repressed by succinate, but no repression by glucose was observed. The transcription start points of catA and catR were determined by primer extension analysis, and the respective promoters (P-catA and P-catR) were thus localized. Measurements of promoter activity during batch cultivation using transcriptional fusion with the gfpuv reporter gene showed that expression of the catR-catABC operon is regulated at the level of transcription. Both P-catR and P-catA are repressed by CatR, and the induction of P-catA by phenol is maintained in the absence of the repressor (in R. erythropolis ΔcatR). Two different potential binding sites for the IclR-type regulator and a recognition site for the cyclic AMP receptor protein (CRP) were identified within the intergenic region between catR and catA.

In vivo transport of the intermediates of the penicillin biosynthetic pathway in tailored strains of Penicillium chrysogenum by Carlos García-Estrada; Inmaculada Vaca; Mónica Lamas-Maceiras; Juan Francisco Martín (pp. 169-182).

Penicillium chrysogenum npe10 (Δpen; lacking the 56.8-kbp amplified region containing the penicillin gene cluster), complemented with one, two, or three penicillin biosynthetic genes, was used for in vivo studies on transport of benzylpenicillin intermediates. 6-Aminopenicillanic acid (6-APA) was taken up efficiently by P. chrysogenum npe10 unlike exogenous δ(l-α-aminoadipyl)-l-cysteinyl-d-valine or isopenicillin N (IPN), which were not taken up or were taken up very poorly. Internalization of exogenous IPN and 6-APA inside peroxisomes was tested by quantifying their peroximal conversion into benzylpenicillin in strains containing only the penDE gene. Exogenous 6-APA was transformed efficiently into benzylpenicillin, whereas IPN was converted very poorly into benzylpenicillin due to its weak uptake. IPN was secreted to the culture medium. IPN secretion decreased when increasing levels of phenylacetic acid were added to the culture medium. The P. chrysogenum membrane permeability to exogenous benzylpenicillin was tested in the npe10 strain. Penicillin is absorbed by the cells by an unknown mechanism, but its intracellular concentration is kept low.

Keywords: Penicillin biosynthesis; Intermediates; Isopenicillin N; 6-APA; Peroxisomes; Membranes; Transport; Uptake

Increased production of pyruvic acid by Escherichia coli RNase G mutants in combination with cra mutations by Taro Sakai; Naoko Nakamura; Genryou Umitsuki; Kazuo Nagai; Masaaki Wachi (pp. 183-192).

The Escherichia coli RNase G is known as an endoribonuclease responsible for the 5′-end maturation of 16S rRNA and degradation of several specific mRNAs such as adhE and eno mRNAs. In this study, we found that an RNase G mutant derived from the MC1061 strain did not grow on a glucose minimal medium. Genetic analysis revealed that simultaneous defects of cra and ilvIH, encoding a transcriptional regulator of glycolysis/gluconeogenesis and one of isozymes of acetohydroxy acid synthase, respectively, were required for this phenomenon to occur. The results of additional experiments presented here indicate that the RNase G mutation, in combination with cra mutation, caused the increased production of pyruvic acid from glucose, which was then preferentially converted to valine due to the ilvIH mutation, resulting in depletion of isoleucine. In fact, the rng cra double mutant produced increased amount of pyruvate in the medium. These results suggest that the RNase G mutation could be applied in the breeding of producer strains of pyruvate and its derivatives such as valine.

Keywords: RNase G; Pyruvic acid; Valine; Glycolysis

Anti-proliferative effects of recombinant iron superoxide dismutase on HepG2 cells via a redox-dependent PI3k/Akt pathway by Min Lu; Chong-shan Bi; Xing-guo Gong; Han-min Chen; Xie-huang Sheng; Tong-le Deng; Ke-di Xu (pp. 193-201).

The coding sequence for an iron superoxide dismutase (fe-sod) was amplified from the Nostoc commune genome. Recombinant Fe-SOD was overexpressed in Escherichia coli, accounting for ∼76% of total bacterial protein. Fe-SOD was purified from bacterial lysate by Ni-NTA column chromatography and used to generate an anti-SOD antibody. The purified Fe-SOD was encapsulated in liposomes and delivered to HepG2 liver tumor cells to eliminate cellular superoxide anions. The SOD-loaded cells exhibited lower reactive oxygen species (ROS) levels and higher reduced glutathione (GSH) levels. In Fe-SOD-treated cells, the cell cycle was delayed in the G₁ phase, and HepG2 cell growth slowed in association with dephosphorylation of the serine–threonine kinase Akt. Low-dose H₂O₂ stimulated Akt phosphorylation, implying that Akt activation in HepG2 cells is redox-sensitive. Akt phosphorylation was abrogated by phosphatidylinositol 3-kinase (PI3K) inhibitors, suggesting that PI3K is an upstream mediator of Akt activation in HepG2 cells. This study provides insight into recombinant Fe-SOD-induced signaling mechanisms in liver tumor cells and suggests the feasibility of using Fe-SOD as an antitumor agent.

Keywords: Nostoc commune ; Iron superoxide dismutase; Superoxide anions; Oxidative stress; PI3k/Akt pathway

SKPDT is a signaling peptide that stimulates sporulation and cry1Aa expression in Bacillus thuringiensis but not in Bacillus subtilis by Angel E. Aceves-Diez; Refugio Robles-Burgueño; Mayra de la Torre (pp. 203-209).

We have identified and characterized in the supernatant of the transition phase of Bacillus thuringiensis var. kurstaki the peptide SKPDT. This peptide was previously identified by in silico analysis by Pottathil and Lazazzera (Front Biosci 8:32–45 2003) as a putative signaling peptide (NprRB) of the Phr family in B. thuringiensis. The chemically synthesized NprRB did not affect the growth kinetics of B. thuringiensis var. kurstaki but stimulated the sporulation, spore release, and transcription of cry1Aa when added to cultures during the transition phase. In fact, when the peptide (100 nM) was added to a culture in transition phase, the transcription of cry1Aa was stimulated almost threefold, mainly from the late promoter BtII, which requires the late-stage sporulation-specific transcription factor σ ^K. On the other hand, NprRB did not have any effect on B. subtilis. Thus, SKPDT seems to be a signaling peptide specific for B. thuringiensis.

Keywords: Signaling peptides; Bacillus thuringiensis ; Sporulation; cry1Aa expression

Starvation is not a prerequisite for the formation of aerobic granules by Yong-Qiang Liu; Wei-Wei Wu; Joo-Hwa Tay; Jian-Long Wang (pp. 211-216).

Activated sludge with sludge volume index (SVI)₃₀ of 77 ml g⁻¹ and SVI₃₀ of 433 ml g⁻¹ was inoculated to start up reactors R1 and R2, respectively. In both R1 and R2, cycle time of 1 h and the influent chemical oxygen demand (COD) concentrations of 1,000 mg l⁻¹ were employed. Initial settling time of 2 min resulted in the loss of a substantial amount of biomass as wash-out and high effluent COD concentrations within the first week of operation. This implied that there was no starvation phase in each cycle of R1 and R2 during the first week of operation. However, aerobic granules with a size above 400 μm formed by day 7. Thus, it was concluded that starvation was not a prerequisite for the formation of aerobic granules. When cycle time was 1 h, the instability of aerobic granules was observed. When cycle time was prolonged to 1.5 h and granular sludge of 200 ml was used to start up reactor R3, the reactor R3 reached steady state within 1 week. SVI, size, and the morphology of granular sludge in R3 remained stable during the 47-day operation, which indicated that prolonged starvation time had positive effects on the stability of aerobic granules.

Keywords: Aerobic granule; Sequencing batch reactor; Starvation; Cycle time; Settling time; Hydraulic selection pressure

Formulation of stable Bacillus subtilis AH18 against temperature fluctuation with highly heat-resistant endospores and micropore inorganic carriers by Soohee Chung; Hyung Mi Lim; Sang-Dal Kim (pp. 217-224).

To survive the commercial market and to achieve the desired effect of beneficial organisms, the strains in microbial products must be cost-effectively formulated to remain dormant and hence survive through high and low temperatures of the environment during transportation and storage. Dormancy and stability of Bacillus subtilis AH18 was achieved by producing endospores with enhanced heat resistance and using inorganic carriers. Heat stability assays, at 90°C for 1 h, showed that spores produced under a sublethal temperature of 57°C was 100 times more heat-resistant than the ones produced by food depletion at the growing temperature of 37°C. When these highly heat-resistant endospores were formulated with inorganic carriers of natural and synthetic zeolite or kaolin clay minerals having substantial amount of micropores, the dormancy of the endospores was maintained for 6 months at 15–25°C. Meanwhile, macroporous perlite carriers with average pore diameter larger than 3.7 μm stimulated the germination of the spores and rapid proliferation of the bacteria. These results indicated that a B. subtilis AH18 product that can remain dormant and survive through environmental temperature fluctuation can be formulated by producing heat-stressed endospores and incorporating inorganic carriers with micropores in the formulation step.

Keywords: Bacillus ; Formulation; Endospore; Dormancy; Micropore carriers; Temperature fluctuation

Development and application of an oligonucleotide microarray for the detection of food-borne bacterial pathogens by Xin-Wei Wang; Liang Zhang; Lian-Qun Jin; Min Jin; Zhi-Qiang Shen; Shuang An; Fu-Huan Chao; Jun-Wen Li (pp. 225-233).

The rapid and accurate detection and identification of food-borne pathogenic bacteria is critical for food safety. In this paper, we describe a rapid (<4 h) high-throughput detection and identification system that uses universal polymerase chain reaction (PCR) primers to amplify a variable region of bacterial the 16S rRNA gene, followed by reverse hybridization of the products to species-specific oligonucleotide probes on a chip. This procedure was successful in discriminating 204 strains of bacteria from pure culture belonging to 13 genera of bacteria. When this method was applied directly to 115 strains of bacteria isolated from foods, 112/115 (97.4%) were correctly identified; two strains were indistinguishable due to weak signal, while one failed to produce a PCR product. The array was used to detect and successfully identify two strains of bacteria from food poisoning outbreak samples, giving results through hybridization that were identical to those obtained by traditional methods. The sensitivity of the microarray assay was 10² CFU of bacteria. Thus, the oligonucleotide microarray is a powerful tool for the detection and identification of pathogens from foods.

Keywords: Oligonucleotide microarray; Detection; Bacterial pathogens; Food-borne

Phospholipases and their industrial applications by L. De Maria; J. Vind; K. M. Oxenbøll; A. Svendsen; S. Patkar (pp. 235-235).

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Applied Microbiology and Biotechnology (v.76, #1)

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Applied Microbiology and Biotechnology (v.76, #1)