Check out our New Publishers' Select for Free Articles

Applied Microbiology and Biotechnology (v.73, #6)

A sincere thank you to all reviewers of AMB and to Professor Karl Esser by Alexander Steinbüchel (pp. 1223-1223).

The role of lipids in the biogenesis of integral membrane proteins by Roger Schneiter; Alexandre Toulmay (pp. 1224-1232).

Most integral membrane proteins are cotranslationally inserted into the lipid bilayer. In prokaryotes, membrane insertion of the nascent chain takes place at the plasma membrane, whereas in eukaryotes insertion takes place into the endoplasmatic reticulum. In both kingdoms of life, however, the same membrane that acquaints the newly born membrane protein also synthesizes the bilayer lipids and thus ensures the balanced growth of the membrane as a whole. Recent evidence indicates that the lipid composition of the host membrane can determine the fate of the newborn membrane protein, as it can affect (1) the efficiency of translocation, (2) the topology of the resulting membrane protein, (3) its stability, (4) its assembly into oligomeric complexes, (5) its transport and sorting along the secretory pathway, and (6) its enzymatic activity. The lipid composition of the membrane thus can affect the biogenesis and function of integral membrane proteins at multiple steps along its biogenetic pathway. While understanding this interdependence between bilayer lipids and protein biogenesis is interesting in its own right, careful consideration of a potential host’s membrane lipid composition may also allow optimization of the yield and activity of membrane proteins that are expressed in a heterologous organism. Here, we review and discuss some examples that illustrate the interdependence between bilayer lipids and the biogenesis of integral membrane proteins.

Keywords: Protein translocation and transport; Membrane microdomains (rafts); Secretory pathway; Plasma membrane; Lipids; Sterols; Sphingolipids; Fatty acids; Saccharomyces cerevisiae

Taxanes: perspectives for biotechnological production by Dieter Frense (pp. 1233-1240).

Taxol is a valuable plant-derived drug showing activity against various cancer types. Worldwide efforts had been made to overcome the supply problem, because the supply by isolation from the bark of the slow-growing yew trees is limited. Plant cell cultures as well as chemical and biotechnological semisynthesis are processes, which are intensively investigated for the production of taxanes paclitaxel (Taxol) and docetaxel (Taxotere) in the last few years. This article provides a comparison of the current research on taxane biosynthesis and production in yew cell cultures.

Overview of some theoretical approaches for derivation of the Monod equation by Yu Liu (pp. 1241-1250).

The Monod equation has been widely applied to describe microbial growth, but it has no mechanistic basis and is purely empirical. Extensive efforts have been dedicated to develop theoretical approaches for derivation of the Monod equation, which can be classified into three major groups, i.e., kinetic, thermodynamic, and substance transport approaches. In this review, four representative approaches are thus discussed. Due to the fact that different assumptions are made in each approach, no universal physical meaning of the Monod constant (K _s) can be revealed. However, it seems that the Monod constant would be free energy-dependent and have nonequilibrium thermodynamic characteristics.

Keywords: Monod equation; Derivation; Thermodynamics; Kinetics; Metabolism; Mass transport

Applications of whole-cell bacterial sensors in biotechnology and environmental science by Kiyohito Yagi (pp. 1251-1258).

Biosensors have major advantages over chemical or physical analyses with regard to specificity, sensitivity, and portability. Recently, many types of whole-cell bacterial biosensors have been developed using recombinant DNA technology. The bacteria are genetically engineered to respond to the presence of chemicals or physiological stresses by synthesizing a reporter protein, such as luciferase, β-galactosidase, or green fluorescent protein. In addition to an overview of conventional biosensors, this minireview discusses a novel type of biosensor using a photosynthetic bacterium as the sensor strain and the crtA gene, which is responsible for carotenoid synthesis, as the reporter. Since bacteria possess a wide variety of stress-response mechanisms, including antioxidation, heat-shock responses, nutrient-starvation, and membrane-damage responses, DNA response elements for several stress-response proteins can be fused with various reporter genes to construct a versatile set of bacterial biosensors for a variety of analytes. Portable biosensors for on-site monitoring have been developed using a freeze-dried biosensing strain, and cell array biosensors have been designed for high-throughput analysis. Moreover, in the future, the use of single-cell biosensors will permit detailed analyses of samples. Signals from such sensors could be detected with digital imaging, epifluorescence microscopy, and/or flow cytometry.

Keywords: Whole-cell biosensor; Reporter gene; Luciferase; β-galactosidase; Green fluorescent protein; crtA

Outdoor cultivation of lutein-rich cells of Muriellopsis sp. in open ponds by Antonio M. Blanco; José Moreno; José A. Del Campo; Joaquín Rivas; Miguel G. Guerrero (pp. 1259-1266).

The growth performance of the chlorophycean microalga Muriellopsis sp. outdoors in open tanks agitated with a paddlewheel and its ability to accumulate carotenoids have been evaluated throughout the year. The cells grown in the open system had free lutein as the main carotenoid, with violaxanthin, β-carotene, and neoxanthin also present. Lutein content of the dry biomass ranged from 0.4 to 0.6%, depending on the growth and environmental conditions. In addition, the biomass of Muriellopsis sp. had a high content in both protein and lipids with about half of the fatty acids being of the polyunsaturated type, with α-linolenic acid accounting for almost 30% of the total fatty acids. The effect of determinant parameters on the performance of the cultures in open tanks was evaluated. Operating conditions that allow the maintenance of productive cultures were established under semicontinuous regime for 9 months throughout the year. Biomass and lutein yields in the open system were not far from those in closed tubular photobioreactors, and reached productivity values of 20 g dry biomass, containing around 100 mg lutein m⁻² day⁻¹ in summer. The outdoor culture of Muriellopsis sp. in open ponds thus represents a real alternative to established systems for the production of lutein.

Keywords: Lutein; Carotenoids; Microalgae; Muriellopsis; Open tanks; Outdoor culture

A chemostat study of Streptomyces peucetius var. caesius N47 by Kristiina Kiviharju; Ulla Moilanen; Matti Leisola; Tero Eerikäinen (pp. 1267-1274).

The behavior of Streptomyces peucetius var. caesius N47 was studied in a glucose limited chemostat with a complex cultivation medium. The steady-state study yielded the characteristic constants μ _max over 0.10 h⁻¹, Y _XS 0.536 g g⁻¹, and m_S 0.54 mg g⁻¹ h⁻¹. The product of secondary metabolism, ɛ-rhodomycinone, was produced with characteristics Y _PX 12.99 mg g⁻¹ and m _P 1.20 mg g⁻¹ h⁻¹. Significant correlations were found for phosphate and glucose consumption with biomass and ɛ-rhodomycinone production. Metabolic flux analysis was conducted to estimate intracellular fluxes at different dilution rates. TCA, PPP, and shikimate pathway fluxes exhibited bigger values with production than with growth. Environmental perturbation experiments with temperature, airflow, and pH changes on a steady-state chemostat implied that an elevation of pH could be the most effective way to shift the cells from growing to producing, as the pH change induced the biggest transient increase to the calculated ɛ-rhodomycinone flux.

Keywords: Chemostat; ɛ-rhodomycinone; Metabolic flux analysis; Streptomyces peucetius var. caesius

Preparation of 3-ketovalidoxylamine A C–N lyase substrate: N-p-nitrophenyl-3-ketovalidamine by Stenotrophomonas maltrophilia CCTCC M 204024 by Jian-Fen Zhang; Yu-Guo Zheng; Zhi-Qiang Liu; Yin-Chu Shen (pp. 1275-1281).

3-Ketovalidoxylamine A C–N lyase is one of three key enzymes in the production of valienamine, which is a potent glucosidase inhibitor from validamycin A. N-p-Nitrophenyl-3-ketovalidamine, used as the substrate of 3-ketovalidoxylamine A C–N lyase, was prepared from N-p-nitrophenylvalidamine with free cells of Stenotrophomonas maltrophilia CCTCC M 204024. The yield and selectivity of N-p-nitrophenyl-3-ketovalidamine from cells were improved by treatment with 10 mM ethylenediaminetetraacetic acid. The optimal pH and temperature for N-p-nitrophenyl-3-ketovalidamine formation was pH 6.0 and 30°C, respectively. N-p-Nitrophenyl-3-ketovalidamine was formed with a yield of 0.68 in the first batch.

Keywords: N-p-Nitrophenyl-3-ketovalidamine; 3-Ketovalidoxylamine A C–N lyase; Ethylenediaminetetraacetic acid; Glucoside 3-dehydrogenase

Functional expression of the γ-isoenzyme of pig liver carboxyl esterase in Escherichia coli by Dominique Böttcher; Elke Brüsehaber; Kai Doderer; Uwe T. Bornscheuer (pp. 1282-1289).

The previously reported functional expression of the γ-isoenzyme of pig liver carboxylesterase (γ-rPLE) in Pichia pastoris is hampered by the small amount of active enzyme formed. Earlier attempts for expression in Escherichia coli failed completely and not even inactive protein was detected. The lack of glycosylation ability of E. coli was ruled out as a possible reason, as it could be shown in this work that deglycosylated PLE also is active. Expression of γ-rPLE was studied using a range of E. coli strains with careful design of the constructs used and control of the cultivation conditions. Indeed, expression in E. coli strains Rosetta, Origami and Rosetta-gami was successful, but the majority of enzymes was present as inclusion bodies and only little soluble but inactive protein was detected. Denaturation and refolding of inclusion bodies failed. However, with the E. coli strain Origami, coexpressing the molecular chaperones GroEL und GroES, a functional expression of γ-rPLE was possible. The recombinant enzyme was released by cell disruption and subjected to His-tag purification. The purified esterase had a specific activity of 92 U mg⁻¹ protein and a V _max/K _m value of 10.8×10⁻³ min⁻¹ towards p-nitrophenyl acetate. Activity staining of native polyacrylamide gels gave a single band at 175 kDa with esterolytic activity indicating a trimeric form of γ-rPLE (∼60 kDa per monomer). γ-rPLE was biochemically characterized and its properties were compared to the enzyme previously expressed in P. pastoris. pH and temperature profiles were identical and highest activity was found at pH 8–8.5 and 60 °C, respectively. In the kinetic resolution of (R,S)-1-phenyl-2-butyl acetate with esterase from both expression hosts, similar enantioselectivities (E=50) were found.

Keywords: Enzyme catalysis; Hydrolases; Inclusion bodies; Pig liver esterase

Effect of aniline coupling on kinetic and thermodynamic properties of Fusarium solani glucoamylase by Haq Nawaz Bhatti; M. Hamid Rashid; Rakhshanda Nawaz; A. Mukhtar Khalid; Muhammad Asgher; A. Jabbar (pp. 1290-1298).

Purified glucoamylase (GA) from Fusarium solani was chemically modified by cross-linking with aniline hydrochloride in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) for 1 [aniline-coupled glucoamylase-1 (ACG-1)], 7 (ACG-7), and 13 min (ACG-13). The aniline coupling of GA had a profound enhancing effect on temperature, pH optima, and pK _a’s of active site residues. The specificity constants (K _cat/K _m) of native, ACG-1, ACG-7, and ACG-13 were 136, 244, 262, and 208 at 55°C for starch, respectively. The enthalpy of activation (ΔH*) and free energy of activation (ΔG*) for soluble starch hydrolysis were lower for the chemically modified forms compared to native GA. Proteolysis of ACGs by α-chymotrypsin and subtilisin resulted in activation.

Keywords: Glucoamylase; Carboxylic group modification; Fusarium solani ; Enthalpy; Free energy; Thermostability

Synthesis of dl-tryptophan by modified broad specificity amino acid racemase from Pseudomonas putida IFO 12996 by Kuniki Kino; Masaru Sato; Mariko Yoneyama; Kohtaro Kirimura (pp. 1299-1305).

Broad specificity amino acid racemase (E.C. 5.1.1.10) from Pseudomonas putida IFO 12996 (BAR) is a unique racemase because of its broad substrate specificity. BAR has been considered as a possible catalyst which directly converts inexpensive l-amino acids to dl-amino acid racemates. The gene encoding BAR was cloned to utilize BAR for the synthesis of d-amino acids, especially d-Trp which is an important intermediate of pharmaceuticals. The substrate specificity of cloned BAR covered all of the standard amino acids; however, the activity toward Trp was low. Then, we performed random mutagenesis on bar to obtain mutant BAR derivatives with high activity for Trp. Five positive mutants were isolated after the two-step screening of the randomly mutated BAR. After the determination of the amino acid substitutions in these mutants, it was suggested that the substitutions at Y396 and I384 increased the Trp specific racemization activity and the racemization activity for overall amino acids, respectively. Among the positive mutants, I384M mutant BAR showed the highest activity for Trp. l-Trp (20 mM) was successfully racemized, and the proportion of d-Trp was reached 43% using I384M mutant BAR, while wild-type BAR racemized only 6% of initial l-Trp.

Keywords: Broad specificity amino acid racemase; d-Tryptophan; Random mutagenesis

Purification and identification of cutinases from Colletotrichum kahawae and Colletotrichum gloeosporioides by Zhenjia Chen; Catarina F. Franco; Ricardo P. Baptista; Joaquim M. S. Cabral; Ana V. Coelho; Carlos J. Rodrigues Jr.; Eduardo P. Melo (pp. 1306-1313).

Colletotrichum kahawae is the causal agent of the coffee berry disease, infecting leaves and coffee berries at any stage of their development. Colletotrichum gloeosporioides is the causal agent of brown blight, infecting ripe berries only. Both fungi secrete the same pattern of carboxylesterases to the fermentation broth when cutin is used as carbon source. By using two different strategies composed of two precipitation steps (ammonium sulphate and acetic acid precipitation) and two chromatographic steps, two proteins displaying carboxylesterase activity were purified to electrophoretic homogeneity. One, with a molecular weight (MW) of 21 kDa, has a blocked N terminus and was identified as cutinase by peptide mass fingerprint and mass spectrometry/mass spectrometry data acquired after peptide derivatization with 4-sulphophenyl isothiocyanate. The second, with a MW of 40 kDa, displays significant carboxylesterase activity on tributyrin but low activity on p-nitrophenyl butyrate. N-terminal sequencing for this protein does not reveal any homology to other carboxylesterases. These two enzymes, which were secreted by both fungi, appear homologous.

Keywords: Colletotrichum kahawae ; Colletotrichum gloeosporioides ; Carboxylesterase; Cutinase; Protein purification; Mass spectrometry

Biochemical characterization of 1-Cys peroxiredoxin from Antrodia camphorata by Lisa Wen; Hui-Ming Huang; Rong-Huay Juang; Chi-Tsai Lin (pp. 1314-1322).

Antrodia camphorata is a unique medicinal mushroom found only in Taiwan. It has been used as a remedy for various diseases in folk medicine. Antrodia camphorata has been shown to exhibit antioxidative effects. Peroxiredoxins play important roles in antioxidation and cell signaling. A gene encoding an antioxidant enzyme, 1-cysteine peroxiredoxin (1-Cys Prx), was identified in an expressed sequence tag database of the A. camphorata and cloned by polymerase chain reaction. The 1-Cys Prx cDNA (837 bp, accession no. AY870325) contains an open reading frame encoding a protein of 223 amino acid residues with calculated molecular mass of 25,081 Da. The deduced protein shared 44–58% identity with 1-Cys Prx from Homo sapiens, Bos taurus, and Saccharomyces cerevisia. The sequence surrounding the conserved cysteine DFTPVCTTE is conserved. The coding sequence was subcloned into a vector, pET-20b (+), and transformed into Escherichia coli. The recombinant 1-Cys Prx was purified by Ni²⁺-nitrilotriacetic acid (Sepharose). The purified enzyme was characterized under various conditions. The enzyme is thermostable because its half-life of inactivation was 15.5 min at 60°C. It was stable under alkaline pH range from 7.8 to 10.2. The enzyme showed decreased activity with increasing concentration of imidazole. The enzyme is sensitive to trypsin and chymotrypsin treatment.

Keywords: Mushroom; Antrodia camphorata ; Expression; 1-Cys peroxiredoxin (1-Cys Prx)

A type B feruloyl esterase from Aspergillus nidulans with broad pH applicability by Hyun-Dong Shin; Rachel Ruizhen Chen (pp. 1323-1330).

A hypothetical protein AN1772.2 of Aspergillus nidulans was found to have a 56% identity with a known type C ferulic acid esterase (FAE) from Talaromyces stipitatus. In addition, it contained a 13-amino acid conserved region flanking the characteristic G-X-S-X-G motif of a serine esterase, suggesting a FAE function for the protein. The putative FAE was successfully cloned from the genomic DNA and expressed in Saccharomyces cerevisiae. The recombinant protein exhibited high FAE activities. Therefore, its function as an FAE was unequivocally determined. About 86% of the enzyme activity was found in the growth medium, indicating that the native signal peptide was effective in the yeast expression system. The recombinant FAE was purified to its homogeneity, and subsequently characterized. The FAE is stable over an unusually wide range of pH (4.0–9.5), has a pH optimum of 7.0, and a temperature optimum of 45°C. A substrate specificity profiling reveals that the enzyme is a type B FAE, despite its strong sequence homology with type C FAEs, raising an interesting question on the role of the conserved region in substrate specificity.

Keywords: Feruloyl esterase; Aspergillus nidulans ; pH stability; Enzyme discovery; Biomass utilization; Ferulic acid

Cloning and functional expression of thermostable β-glucosidase gene from Thermoascus aurantiacus by Jiong Hong; Hisanori Tamaki; Hidehiko Kumagai (pp. 1331-1339).

A thermostable β-glucosidase (BGLI) was purified from Thermoascus aurantiacus IFO9748, and the gene (bgl1) encoding this enzyme was cloned and expressed in yeast Pichia pastoris. The deduced amino acid sequence encoded by bgl1 showed high similarity with the sequence of glycoside hydrolase family 3. The recombinant enzyme was purified and subjected to enzymatic characterization. Recombinant BGLI retained more than 70% of its initial activity after 1 h of incubation at 60°C and was stable in the pH range 3–8. The optimal temperature for enzyme activity was about 70°C and the optimal pH was about 5. P. pastoris expressing recombinant BGLI became able to utilize cellobiose as a carbon source.

Targeted deletion of the uvrBA operon and biological containment in the industrially important Bacillus licheniformis by Jens Waldeck; Heike Meyer-Rammes; Hannes Nahrstedt; Renée Eichstädt; Susanne Wieland; Friedhelm Meinhardt (pp. 1340-1347).

From a Bacillus licheniformis wild type as well as a defined asporogenous derivative, stable UV hypersensitive mutants were generated by targeted deletion of the uvrBA operon, encoding highly conserved key components of the nucleotide excision repair. Comparative studies, which included the respective parental strains, revealed no negative side effects of the deletion, neither on enzyme secretion nor on vegetative propagation. Thus, the uvrBA locus proved to be a useful deletion target for achieving biological containment in this industrially exploited bacterium. In contrast to recA mutants, which also display UV hypersensitivity, further strain development via homologous recombination techniques will be still possible in such uvr mutants.

Keywords: Industrial Bacillus ; Targeted gene deletion; Biological containment; SOS response; UV hypersensitivity; Batch fermentation; Extracellular enzymes

Agrobacterium-mediated transformation (AMT) of Trichoderma reesei as an efficient tool for random insertional mutagenesis by Yao Hua Zhong; Xiao Li Wang; Tian Hong Wang; Qiao Jiang (pp. 1348-1354).

Filamentous fungus Trichoderma reesei QM9414 was successfully transformed with Agrobacterium tumefaciens AGL-1 for random integration of transforming DNA (T-DNA). Co-cultivation of T. reesei conidia or protoplasts with A. tumefaciens in the presence of acetosyringone resulted in the formation of hygromycin B-resistant fungal colonies with high transformation frequency. Nine randomly selected resistant clones were proved to be stable through mitotic cell division. The integration of the hph gene into T. reesei genome was determined by PCR and dot blot analysis. Transgenic T. reesei strains were analyzed using TAIL-PCR for their T-DNA contents. The results showed that T-DNA inserts occurred evidently by fusing DNA at T-DNA borders via random recombination, which suggests that Agrobacterium-mediated transformation is a potentially powerful tool towards tagged mutagenesis and gene transfer technology for T. reesei.

Keywords: Agrobacterium-mediated transformation; Trichoderma reesei; T-DNA; Insertional mutagenesis; hygromycin B resistance; TAIL-PCR

High level expression of human endostatin in Pichia pastoris using a synthetic gene construct by Zhijian Su; Xiaoping Wu; Ya Feng; Changcai Ding; Yechen Xiao; Lu Cai; Wenke Feng; Xiaokun Li (pp. 1355-1362).

Endostatin, a 20-kDa C-terminal fragment derived from type XVIII collagen, is a potent angiogenesis inhibitor and an antitumor factor. To improve the production of recombinant human endostatin on increasing demand in clinical practice, we constructed an artificial gene encoding its mature peptide sequence in human collagen XVIII. The synthetic gene consisted of 20 codons in preference in methylotropic yeast—Pichia pastoris and was cloned into expression vector pPICZαA; and the recombinant protein was expressed in P. pastoris strain SMD1168 and purified to near homogeneity using heparin affinity chromatography. The amount of expressed recombinant protein in cultural media using described strategy was 80 mg/l in shake flask cultivation and 435 mg/l in high-density bioreactor fermentation. Methylthiazolium assay demonstrated that human endostatin expressed in P. pastoris using artificial synthetic gene of preference in P. pastoris was able to inhibit the acidic fibroblast growth factor-induced proliferation of endothelial cells in vitro.

Keywords: Endostatin; Pichia pastoris ; Artificial synthetic gene; High-density fermentation

Reducing haziness in white wine by overexpression of Saccharomyces cerevisiae genes YOL155c and YDR055w by Shauna L. Brown; Vanessa J. Stockdale; Filomena Pettolino; Kenneth F. Pocock; Miguel de Barros Lopes; Patrick J. Williams; Antony Bacic; Geoffrey B. Fincher; Peter B. Høj; Elizabeth J. Waters (pp. 1363-1376).

Grape proteins aggregate in white wine to form haze. A novel method to prevent haze in wine is the use of haze protective factors (Hpfs), specific mannoproteins from Saccharomyces cerevisiae, which reduce the particle size of the aggregated proteins. Hpf1p was isolated from white wine and Hpf2p from a synthetic grape juice fermentation. Putative structural genes, YOL155c and YDR055w, for these proteins were identified from partial amino acid sequences of Hpf1p and Hpf2p, respectively. YOL155c also has a homologue, YIL169c, in S. cerevisiae. Comparison of the partial amino acid sequence of deglycosylated-Hpf2p with the deduced protein sequence of YDR055w, confirmed five of the 15 potential N-linked glycosylation sites in this sequence were occupied. Methylation analysis of the carbohydrate moieties of Hpf2p indicated that this protein contained both N- and O-linked mannose chains. Material from fermentation supernatant of deletion strains had significantly less activity than the wild type. Moreover, YOL155c and YIL169c overexpressing strains and a strain overexpressing 6xHis-tagged Hpf2p produced greater haze protective activity than the wild type strains. A storage trial demonstrated the short to midterm stability of 6xHis-tagged Hpf2p in wine.

Keywords: Wine; Protein haze; Mannoproteins; Haze protective factors; Yeast

Expression and secretion of a single-chain sweet protein monellin in Bacillus subtilis by sacB promoter and signal peptide by Zhongjun Chen; Cai Heng; Zhengying Li; Xinle Liang; Shangguan Xinchen (pp. 1377-1381).

The sweet protein monellin gene was expressed in Bacillus subtilis under the control of the Bacillus subtilis sacB promoter and signal peptide sequence. A 294-bp DNA fragment, coding for sweet protein monellin, was ligated into the Escherichia coli/B. subtilis shuttle vector pHPC, producing pHPMS, which was subsequently transformed into B. subtilis QB1098, DB104, and DB403. The peptide efficiently directed the secretion of monellin from the recombinant B. subtilis cells. A maximum yield of monellin of 0.29 g protein l⁻¹ was obtained from the supernatant of B. subtilis DB403 harboring pHPMS. SDS-PAGE confirmed the purity of the recombinant product.

Keywords: Bacillus subtilis ; Expression; Monellin; Proteases deficiency

Overexpressing GLT1 in gpd1Δ mutant to improve the production of ethanol of Saccharomyces cerevisiae by Qing-Xue Kong; Li-Min Cao; Ai-Li Zhang; Xun Chen (pp. 1382-1386).

To improve ethanol production in Saccharomyces cerevisiae, two yeast strains were constructed. In the mutant, KAM-4, the GPD1 gene, which encodes a glycerol 3-phosphate dehydrogenase of S. cerevisiae to synthesize glycerol, was deleted. The mutant KAM-12 had the GLT1 gene (encodes glutamate synthase) placed under the PGK1 promoter while harboring the GPD1 deletion. Notably, overexpression of GLT1 by the PGK1 promoter along with GPD1 deletion resulted in a 10.8% higher ethanol production and a 25.0% lower glycerol formation compared to the wild type in anaerobic fermentations. The growth rate of KAM-4 was slightly lower than that of the wild type under the exponential phase whereas KAM-12 and the wild type were indistinguishable in the biomass concentration at the end of growth period. Meanwhile, dramatic reduction of formation of acetate and pyruvic acid was observed in all the mutants compared to the wild type.

Keywords: Ethanol production; Glutamate-synthase gene; Glycerol formation; Glycerol 3-phosphate dehydrogenase; S. cerevisiae

Genome shuffling of Streptomyces sp. U121 for improved production of hydroxycitric acid by Hiroyuki Hida; Takashi Yamada; Yasuhiro Yamada (pp. 1387-1393).

(2S, 3R)-Hydroxycitric acid (HCA) from Hibiscus subdariffa inhibits pancreatic α-amylase and intestine α-glucosidase, leading to reduction of carbohydrate metabolism. In our previous study, Streptomyces sp. U121 was identified as a producer of (2S, 3R)-HCA [Hida et al. (2005) Bioscience, Biotechnology, and Biochemistry 69:1555–1561]. Here, we applied genome shuffling of Streptomyces sp. U121 to achieve rapid improvement of HCA production. The initial mutant population was generated by nitrosoguanidine treatment of the spores, and an improved population producing fivefold more HCA over wild type was obtained by three rounds of genome shuffling. For efficient screening of the mutant library, trans-epoxyaconitic acid (EAA), an antibiotic analog of HCA, was utilized. EAA inhibited the regeneration of nonfused protoplasts, resulting in selective screening of shuffled strains. Mutant strains with enhanced EAA resistance exhibited significantly higher HCA production in liquid media. Furthermore, the best mutant showed increased cell growth in flask culture, as well as increased HCA production.

Keywords: Genome shuffling; Hydroxycitric acid; Streptomyces

Increase of organic solvent tolerance by overexpression of manXYZ in Escherichia coli by Mina Okochi; Masaki Kurimoto; Kazunori Shimizu; Hiroyuki Honda (pp. 1394-1399).

Transcriptional analysis has been investigated to detect the genes involved in organic solvent tolerance. A time course of gene expression profiles of Escherichia coli after exposure to organic solvents revealed that the expression levels of manX, manY, and manZ genes were strongly upregulated by 13.2-, 10.0-, and 7.0-folds, respectively, after 2 min and then decreased after 10 min. Organic solvent tolerance of E. coli was investigated by inducing overexpression of manX, manY, and manZ genes and manXYZ operon that encode a sugar transporter of the phosphotransferase system. Although the expression of manX, manY, and manZ alone was not effective, the organic solvent tolerance level was increased by the expression of manXYZ. The intracellular hexane level was kept lower in E. coli cells overexpressing manXYZ after 4 h of incubation with hexane.

Keywords: Organic solvent tolerance; DNA microarray; manXYZ

Factors affecting tyramine production in Enterococcus durans IPLA 655 by María Fernández; Daniel M. Linares; Ana Rodríguez; Miguel A. Alvarez (pp. 1400-1406).

The decarboxylation of tyrosine by certain lactic acid bacteria leads to the undesirable presence of tyramine in fermented foods. Tyramine is the most frequent biogenic amine found in cheese and is also commonly found in other fermented foods and beverages. The tyramine-producing strain Enterococcus durans IPLA 655 was grown in a bioreactor under different conditions to determine the influence of carbon source, tyrosine and tyramine concentrations, and pH on tyramine production. The carbon source appeared to have no significant effect on the production of tyramine. In contrast, tyrosine was necessary for tyramine production, while the presence of tyramine itself in the growth medium inhibited such production. pH showed by far the greatest influence on tyramine synthesis; tyramine was produced in the greatest quantities at pH 5.0, although this was accompanied by a reduced growth rate.

Keywords: Biogenic amines; Tyramine; Lactic acid bacteria; Enterococcus durans

Isolation of bacteria able to grow on both polyethylene glycol (PEG) and polypropylene glycol (PPG) and their PEG/PPG dehydrogenases by Xiaoping Hu; Akira Fukutani; Xin Liu; Kazuhide Kimbara; Fusako Kawai (pp. 1407-1413).

Two bacterial consortia growing on a random copolymer of ethylene glycol and propylene glycol units were obtained by enrichment cultures from various microbial samples. Six major strains included in both consortia were purified and identified as Sphingomonads, Pseudomonas sp. and Stenotrophomonas maltophilia. Three of them (Sphingobium sp. strain EK-1, Sphingopyxis macrogoltabida strain EY-1, and Pseudomonas sp. strain PE-2) utilized both PEG and polypropylene glycol (PPG) as a sole carbon source. Four PEG-utilizing bacteria had PEG dehydrogenase (PEG-DH) activity, which was induced by PEG. PCR products from DNA of these bacteria generated with primers designed from a PEG-DH gene (AB196775 for S. macrogoltabida strain 103) indicated the presence of a sequence that is the homologous to the PEG-DH gene (99% identity). On the other hand, five PPG-utilizing bacteria had PPG dehydrogenase (PPG-DH) activity, but the activity was constitutive. PCR of a PPG-DH gene was performed using primers designed from a polyvinyl alcohol dehydrogenase (PVA-DH) gene (AB190288 for Sphingomonas sp. strain 113P3) because a PPG-DH gene has not been cloned yet, but both PPG-DH and PVA-DH were active toward PPG and PVA (Mamoto et al. 2006). PCR products of the five strains did not have similarity to each other or to oxidoreductases including PVA-DH.

Keywords: Random polymer of ethylene oxide and propylene oxide; Polyethylene gycol (PEG); Polypropylene glycol (PPG); PEG dehydrogenase; PPG dehydrogenase; Bacterial assimilation

Xenoestrogenic short ethoxy chain nonylphenol is oxidized by a flavoprotein alcohol dehydrogenase from Ensifer sp. strain AS08 by Xin Liu; Akio Tani; Kazuhide Kimbara; Fusako Kawai (pp. 1414-1422).

The ethoxy chains of short ethoxy chain nonylphenol (NPEO_av2.0, containing average 2.0 ethoxy units) were dehydrogenated by cell-free extracts from Ensifer sp. strain AS08 grown on a basal medium supplemented with NPEO_av2.0. The reaction was coupled with the reduction in 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide and phenazine methosulfate. The enzyme (NPEO_av2.0 dehydrogenase; NPEO-DH) was purified to homogeneity with a yield of 20% and a 56-fold increase in specific activity. The molecular mass of the native enzyme was 120 kDa, consisting of two identical monomer units (60 kDa). The gene encoding NPEO-DH was cloned, which consisted of 1,659 bp, corresponding to a protein of 553 amino acid residues. The deduced amino acid sequence agreed with the N-terminal amino acid sequence of the purified NPEO-DH. The presence of a flavin adenine dinucleotide (FAD)-binding motif and glucose–methanol–choline (GMC) oxidoreductase signature motifs strongly suggested that the enzyme belongs to the GMC oxidoreductase family. The protein exhibited homology (40–45% identity) with several polyethylene glycol dehydrogenases (PEG-DHs) of this family, but the identity was lower than those (approximately 58%) among known PEG-DHs. The substrate-binding domain was more hydrophobic compared with those of glucose oxidase and PEG-DHs. The recombinant protein had the same molecular mass as the purified NPEO-DH and dehydrogenated PEG400-2000, NPEO_av2.0 and its components, and NPEOav10, but only slight or no activity was found using diethylene glycol, triethylene glycol, and PEG200.

Keywords: Short ethoxy chain nonylphenol; Flavoprotein alcohol dehydrogenase; Ensifer sp. strain AS08

Hybridoma Ped-2E9 cells cultured under modified conditions can sensitively detect Listeria monocytogenes and Bacillus cereus by Pratik Banerjee; Mark T. Morgan; Jenna L. Rickus; Kathy Ragheb; Carlos Corvalan; J. Paul Robinson; Arun K. Bhunia (pp. 1423-1434).

Lymphocyte origin hybridoma Ped-2E9 cell-based cytotoxicity assay can detect virulent Listeria or Bacillus species, and its application in a cell-based biosensor for onsite use would be very attractive. However, maintaining enough viable cells on a sensor platform for a prolonged duration is a challenging task. In this study, key factors affecting the survival and growth of Ped-2E9 cells under modified conditions were investigated. When the Ped-2E9 cells were grown in media containing 5% fetal bovine serum in sealed tubes without any replenishment of nutrients or exogenous CO₂ supply, a large portion of the cells remained viable for 6 to 7 days and cells entered into G0/G1 resting phase. The media pH change was negligible and no cell death was observed in the first 4 days, then cells sequentially underwent apoptotic (fourth day onward) phase until day 7 after which a majority was dead. Subsequent cytotoxicity testing of 3- to 7-day stored Ped-2E9 cells sensitively detected virulent Listeria and Bacillus species. These data strongly suggest that Ped-2E9 cells can be maintained in viable state for 6 days in a sealed tube mimicking the environment in a potential sensor device for onsite use without the need for expensive cell culture facilities.

Keywords: Ped-2E9; Hybridoma; Listeria monocytogenes ; Bacillus ; Cytotoxicity; Cell-based sensor

Analysis of bacterial communities on aging flue-cured tobacco leaves by 16S rDNA PCR–DGGE technology by Mingqin Zhao; Baoxiang Wang; Fuxin Li; Liyou Qiu; Fangfang Li; Shumin Wang; Jike Cui (pp. 1435-1440).

Many microorganisms, growing on aging flue-cured tobacco leaves, play a part in its fermentation process. These microflora were identified and described by culture-dependent methods earlier. In this study we report the identity of the microflora growing on the tobacco leaf surface by employing culture-independent methods. We have amplified microbial 16S rDNA sequences directly from the leaf surface and used denaturing gradient gel electrophoresis (DGGE) to identify bacterial community on the tobacco leaves. Our culture-independent methods for the study of microbial community on tobacco leaves showed that microbial community structures on leaves of variety Zhongyan 100, NC89 and Zhongyan 101 were similar between 0 and 6 months aging, and between 9 and 12 months aging, while the similarity is low between 0 and 6, and between 9 and 12 months aging, respectively. There were certain similarities of bacterial communities (similarity up to 63%) among the three tobacco varieties for 0 to 6 months aging. Five dominant 16S rDNA DGGE bands A, B, C, D and E were isolated, cloned, and sequenced. They were most similar to two cultured microbial species Bacteriovorax sp. EPC3, Bacillus megaterium, and three uncultured microbial species, respectively.

Keywords: Flue-cured tobacco; Fermentation; 16S rDNA; PCR–DGGE

Effect of the concentration of suspended solids on the enzymatic activities and biodiversity of a submerged membrane bioreactor for aerobic treatment of domestic wastewater by M. Molina-Muñoz; J. M. Poyatos; R. Vílchez; E. Hontoria; B. Rodelas; J. González-López (pp. 1441-1451).

A pilot-scale submerged membrane bioreactor was used for the treatment of domestic wastewater in order to study the influence of the variations in the concentration of volatile suspended solids (VSS) on the enzymatic activities (acid and alkaline phosphatases, glucosidase, protease, esterase, and dehydrogenase) and biodiversity of the bacterial community in the sludge. The influence of VSS concentration was evaluated in two separated experiments, which were carried out in two different seasons of the year (experiment 1 through spring–summer and experiment 2 through autumn–winter). Cluster analysis of the temperature gradient gel electrophoresis (TGGE) profiles demonstrated that the community composition was significantly different in both experiments. Within the same experiment, the bacterial community experienced sequential shifts as the biomass accumulated, as shown by the evolution of the population profiles through time as VSS concentration increased. All enzymatic activities studied were significantly lower during experiment 2, except for glucosidase. Concentrations of VSS over 8 g/l induced a strong descent of all enzymatic activities, which overlapped with a significant modification of the community composition. Sequences of the major TGGE bands were identified as representatives of the Alpha-proteobacteria, filamentous bacteria (Thiotrix), and nitrite oxidizers (Nitrospira). Some sequences which were poorly related to any validated bacterial taxon were obtained.

Keywords: Submerged membrane bioreactor; Domestic wastewater; Wastewater treatment; Biodiversity; Enzymatic activities; TGGE

Biodegradation of methyl parathion and p-nitrophenol: evidence for the presence of a p-nitrophenol 2-hydroxylase in a Gram-negative Serratia sp. strain DS001 by Suresh B. Pakala; Purushotham Gorla; Aleem Basha Pinjari; Ravi Kumar Krovidi; Rajasekhar Baru; Mahesh Yanamandra; Mike Merrick; Dayananda Siddavattam (pp. 1452-1462).

A soil bacterium capable of utilizing methyl parathion as sole carbon and energy source was isolated by selective enrichment on minimal medium containing methyl parathion. The strain was identified as belonging to the genus Serratia based on a phylogram constructed using the complete sequence of the 16S rRNA. Serratia sp. strain DS001 utilized methyl parathion, p-nitrophenol, 4-nitrocatechol, and 1,2,4-benzenetriol as sole carbon and energy sources but could not grow using hydroquinone as a source of carbon. p-Nitrophenol and dimethylthiophosphoric acid were found to be the major degradation products of methyl parathion. Growth on p-nitrophenol led to release of stoichiometric amounts of nitrite and to the formation of 4-nitrocatechol and benzenetriol. When these catabolic intermediates of p-nitrophenol were added to resting cells of Serratia sp. strain DS001 oxygen consumption was detected whereas no oxygen consumption was apparent when hydroquinone was added to the resting cells suggesting that it is not part of the p-nitrophenol degradation pathway. Key enzymes involved in degradation of methyl parathion and in conversion of p-nitrophenol to 4-nitrocatechol, namely parathion hydrolase and p-nitrophenol hydroxylase component “A” were detected in the proteomes of the methyl parathion and p-nitrophenol grown cultures, respectively. These studies report for the first time the existence of a p-nitrophenol hydroxylase component “A”, typically found in Gram-positive bacteria, in a Gram-negative strain of the genus Serratia.

Keywords: Parathion hydrolase; p-Nitrophenol hydroxylase component A; Serratia sp; Biodegradation; Catabolomics

Distribution of extracellular polymeric substances in aerobic granules by M. Y. Chen; D. J. Lee; J. H. Tay (pp. 1463-1469).

Extracellular matrix provides an architectural structure and mechanical stability for aerobic granules. Distributions of cells and extracellular polymeric substances (EPS), including proteins, α- and β-d-glucopyranose polysaccharides, in acetate-fed granules and phenol-fed granules were probed using a novel quadruple staining scheme. In acetate-fed granules, protein and β-d-glucopyranose polysaccharides formed the core, whereas, the cells and α-d-glucopyranose polysaccharides accumulated in the granule outer layers. Based on these experimental findings, this study indicated that different conclusions can be obtained regarding EPS distributions when granules were stained differently. The core of phenol-fed granules, conversely, was formed principally by proteins; whereas, the cells and α- and β-d-glucopyranose polysaccharides were accumulated at an outer filamentous layer. Using a series of confocal laser scanning microscope (CLSM) images whose threshold values were determined via Otsu’s scheme, the three-dimensional distributions of cells and EPS were produced using a polygonal surface model. Structural information extracted can be applied in further development of comprehensive granule models.

Keywords: Aerobic granule; EPS; Staining; Structure; Model

Cryopreservation of transgenic rice suspension cells producing recombinant hCTLA4Ig by Ji-Suk Cho; Seok-Mi Hong; Sung-Yeon Joo; Ji-Seon Yoo; Dong-Il Kim (pp. 1470-1476).

Transgenic suspension cells of Oryza sativa L. cv. Dongjin utilized as a host for producing recombinant human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) were preserved in liquid nitrogen (−196 °C) after slow prefreezing in a deep freezer (−70 °C). The development of an optimal procedure for long-term storage was investigated by the addition of various concentrations of cryoprotectant mixture and osmoticum in preculture media before cooling. A pre-deep-freezing time of 120 min was the most effective for maintaining cell viability. Compared with mannitol, sorbitol, trehalose, and NaCl under the same osmotic conditions, 0.5 M sucrose was found to be the best osmoticum for preculture media. The cryoprotectant comprising sucrose, glycerol, and dimethylsulfoxide (DMSO) was applied to the precultured cells, and a combination of 1 M sucrose, 1 M glycerol, and 1 M DMSO provided the best result. The viability with this optimized condition was 88% after cryocell-banking for 1 day. The expression of hCTLA4Ig in recovered callus from cryopreservation was also kept stable, and the production level was similar to that observed in noncryopreserved cultures.

Keywords: Cryopreservation; Transgenic plant cell cultures; hCTLA4Ig; Oryza sativa

Sections

Personal tools

Applied Microbiology and Biotechnology (v.73, #6)

Sections

Personal tools

Local resources

Applied Microbiology and Biotechnology (v.73, #6)