Check out our New Publishers' Select for Free Articles

Applied Microbiology and Biotechnology (v.80, #5)

Perspectives of microbial oils for biodiesel production by Qiang Li; Wei Du; Dehua Liu (pp. 749-756).

Biodiesel has become more attractive recently because of its environmental benefits, and the fact that it is made from renewable resources. Generally speaking, biodiesel is prepared through transesterification of vegetable oils or animal fats with short chain alcohols. However, the lack of oil feedstocks limits the large-scale development of biodiesel to some extent. Recently, much attention has been paid to the development of microbial, oils and it has been found that many microorganisms, such as algae, yeast, bacteria, and fungi, have the ability to accumulate oils under some special cultivation conditions. Compared to other plant oils, microbial oils have many advantages, such as short life cycle, less labor required, less affection by venue, season and climate, and easier to scale up. With the rapid expansion of biodiesel, microbial oils might become one of potential oil feedstocks for biodiesel production in the future, though there are many works associated with microorganisms producing oils need to be carried out further. This review is covering the related research about different oleaginous microorganisms producing oils, and the prospects of such microbial oils used for biodiesel production are also discussed.

Keywords: Biodiesel; Oleaginous microorganisms; Microbial oil; Sustainability

Bacterial CMP-sialic acid synthetases: production, properties, and applications by Rahman M. Mizanur; Nicola L. Pohl (pp. 757-765).

Sialic acids are abundant nine-carbon sugars expressed terminally on glycoconjugates of eukaryotic cells and are crucial for a variety of cell biological functions such as cell–cell adhesion, intracellular signaling, and in regulation of glycoproteins stability. In bacteria, N-acetylneuraminic acid (Neu5Ac) polymers are important virulence factors. Cytidine 5′-monophosphate (CMP)-N-acetylneuraminic acid synthetase (CSS; EC 2.7.7.43), the key enzyme that synthesizes CMP-N-acetylneuraminic acid, the donor molecule for numerous sialyltransferase reactions, is present in both prokaryotes and eukaryotic systems. Herein, we emphasize the source, function, and biotechnological applications of CSS enzymes from bacterial sources. To date, only a few CSS from pathogenic bacterial species such as Neisseria meningitidis, Escherichia coli, group B streptococci, Haemophilus ducreyi, and Pasteurella hemolytica and an enzyme from nonpathogenic bacterium, Clostridium thermocellum, have been described. Overall, the enzymes from both Gram-positive and Gram-negative bacteria share common catalytic properties such as their dependency on divalent cation, temperature and pH profiles, and catalytic mechanisms. The enzymes, however, can be categorized as smaller and larger enzymes depending on their molecular weight. The larger enzymes in some cases are bifunctional; they have exhibited acetylhydrolase activity in addition to their sugar nucleotidyltransferase activity. The CSSs are important enzymes for the chemoenzymatic synthesis of various sialooligosaccharides of significance in biotechnology.

Keywords: Sialic acid; CMP-sialic acid synthetases; Sugar nucleotide

Three trehalose synthetic pathways in the acarbose-producing Actinoplanes sp. SN223/29 and evidence for the TreY role in biosynthesis of component C by Jin-Sook Lee; Tran Hai; Hermann Pape; Tae-Jong Kim; Joo-Won Suh (pp. 767-778).

In this study, three trehalose gene clusters, treX-Y-Z, tpS1, and treS, of the acarbose-producing strain, Actinoplanes sp. SN223/29, have been identified. In particular, five trehalose synthetic genes were sequenced and characterized in detail. They were cloned and expressed in Escherichia coli BL21(DE3)pLysS using the His-tag vector pET19b. The recombinant proteins were purified by Ni²⁺-nitrilotriacetic acid agarose affinity chromatography, and their functions were characterized biochemically. Both the maltooligosyltrehalose synthase (TreY–TreZ) pathway and the trehalose synthase (TreS) pathway have maximum activity at 40°C and at pH 7.5 and 7.0, respectively, in 100-mM phosphate buffer. Meanwhile, the trehalose-6-phosphate synthase (TpS1) showed maximum activity at 35°C and at pH 7.5 in 100 mM Tris–HCl. As a cofactor candidate, Mg²⁺ enhanced the activities of all three trehalose synthetic reactions significantly. TreY produced component C from acarbose by its proposed isomerase activity, but TreS did not. This study suggests that the mutation of treY can improve acarbose production by repressing component C production. Based on the data obtained in this study, a model for component C production in Actinoplanes sp. SN 223/29 is proposed.

Keywords: Actinoplanes sp.; TreX-Y-Z ; TpS1 ; TreS ; Trehalose biosynthesis; Acarbose; Component C

High cell density cultivation of recombinant E. coli for hirudin variant 1 production by temperature shift controlled by pUC18-based replicative origin by Toru Matsui; Hiroaki Sato; Hotaka Yamamuro; Naoya Shinzato; Hitoshi Matsuda; Satoru Misawa; Seigo Sato (pp. 779-783).

The copy number of a plasmid, pUC-based vector, was previously shown to be affected by culture temperature. In this study, intracellular hirudin variant 1 (f-HV1) fused to porcine adenylate kinase protein was produced using recombinant Escherichia coli by temperature shift cultivation coupled with a high cell density cultivation technique for E. coli JM109. The optimal temperature for cellular growth suppressing f-HV1 production was 33°C, resulting in a final dried cell concentration of 45.7 g/l, with a specific growth rate of 0.54 1/h. Optimizing the temperature-shift conditions (temperature shifted to an OD660 nm of 15 from 33°C to 37°C) resulted in the production of f-HV1 up to 4763 mg/l as an inclusion body with dried cell concentration of 44 g/l in 18 h.

Keywords: Escherichia coli JM109; Hirudin; HCDC; Temperature shift

Characterization of cold-active uracil-DNA glycosylase from Bacillus sp. HJ171 and its use for contamination control in PCR by Gun A. Kim; Mi Sun Lee; Younguk Sun; Byung Doo Lee; Jong Il Lee; Jung-Hyun Lee; Suk-Tae Kwon (pp. 785-794).

In this study, the gene encoding Bacillus sp. HJ171 uracil-DNA glycosylase (Bsp HJ171 UDG) was cloned and sequenced. The Bsp HJ171 UDG gene consists of a 738-bp DNA sequence, which encodes for a protein that is 245-amino-acid residues in length. The deduced amino acid sequence of the Bsp HJ171 UDG had a high sequence similarity with other bacterial UDGs. The molecular mass of the protein derived from this amino acid sequence was 27.218 kDa. The Bsp HJ171 UDG gene was expressed under the control of a T7lac promoter in the pTYB1 plasmid in Escherichia coli BL21 (DE3). The expressed enzyme was purified in one step using the Intein Mediated Purification with an Affinity Chitin-binding Tag purification system. The optimal temperature range, pH, NaCl concentration, and KCl concentration of the purified enzyme was 20–25°C, 8.0, 25 and 25 mM, respectively. The half-life of the enzyme at 40°C and 50°C were approximately 131 and 45 s, respectively. These heat-labile characteristics enabled Bsp HJ171 UDG to control carry-over contamination in the polymerase chain reaction product (PCR) without losing the PCR product.

Keywords: Bacillus sp. HJ171 (Bsp HJ171); Uracil-DNA glycosylase (UDG); Bsp HJ171 UDG; Psychrophile; Carry-over contamination

Acid stabilization of Bacillus licheniformis alpha amylase through introduction of mutations by Yi-han Liu; Fu-ping Lu; Yu Li; Jian-ling Wang; Chen Gao (pp. 795-803).

This paper provided further understanding of the relationships between acid resistance and structural features of different mutants in Bacillus licheniformis alpha amylase (BLA) due to the changes of two crucial positions Leu134 and Ser320. In order to investigate effect of the two positions on the acid stability, we described the detailed characterization of wild-type and the single mutants L134R and S320A as well as the double mutant L134R/S320A. The highest k cat/Km with pH 4.5, approximately 14 times that of wild type, was observed in L134R/S320A. The k cat/Km corresponding to L134R and S320A were at an intermediate values between those for wild type and L134R/S320A. In addition, compared with wild type, which had a rapid decline of the activity, L134R/S320A could maintain its activity strongly in low pH. Meanwhile, lower tolerance of L134R and S320A in acidic conditions than that of L134R/S320A was determined. Surprisingly, the acid-resistant capability of L134R/S320A was significantly enhanced by directed evolution. These results, combined with three-dimensional structure analysis, show that the electrostatic effects play a significant role in determining the stability of BLA at two crucial positions, 134 and 320.

Keywords: Bacillus licheniformis alpha amylase; Acid stability; Kinetics; Electrostatic field; Protein structure

Engineering of NADPH-dependent aldo-keto reductase from Penicillium citrinum by directed evolution to improve thermostability and enantioselectivity by Hiroyuki Asako; Masatoshi Shimizu; Nobuya Itoh (pp. 805-812).

Penicillium citrinum β-keto ester reductase (KER) can catalyze the reduction of methyl 4-bromo-3-oxobutyrate (BAM) to methyl (S)-4-bromo-3-hydroxybutyrate with high optical purity. To improve the thermostability of KER, protein engineering was performed using error-prone polymerase chain reaction-based random mutagenesis. Variants with the highest levels of thermostability contained the single amino acid substitutions L54Q, K245R, and N271D. The engineered L54Q variant of KER retained 62% of its initial activity after heat treatment at 30°C for 6 h, whereas wild-type KER showed only 15% activity. The L54Q substitution also conferred improved enantioselectivity by KER. An Escherichia coli cell biocatalyst that overproduced the L54Q mutant of KER and glucose dehydrogenase as a cofactor regeneration enzyme showed the highest level of BAM reduction in a water/butyl acetate two-phase system.

Keywords: Aldo-keto reductase (NADPH); Thermostability; Enantioselectivity; Directed evolution; Mutagenesis; Penicillium citrinum

Plasmid DNA is released from nanosized acicular material surface by low molecular weight oligonucleotides: exogenous plasmid acquisition mechanism for penetration intermediates based on the Yoshida effect by N. Yoshida; K. Ide (pp. 813-821).

When a colloidal solution consisting of nanosized acicular material and bacterial cells is stimulated with sliding friction at the interface between the hydrogel and interface-forming material where the frictional coefficient increases rapidly, the nanosized acicular material accompanying the bacterial cells forms a penetration intermediate. This effect is known as the Yoshida effect in honor of its discoverer. Through the Yoshida effect, a novel property in which penetration intermediates incorporate exogenous plasmid DNA has been identified. This report proposes a possible mechanism for exogenous plasmid acquisition by penetration intermediates in the Yoshida effect. Escherichia coli cells, pUC18, and chrysotile were used as recipient cells, plasmid DNA, and nanosized acicular material, respectively. Even when repeatedly washing the mixture consisting of pUC18 and chrysotile, transformation efficiency by pUC18 was stable. Accordingly, pUC18 adsorbed onto chrysotile was introduced into recipient E. coli cells. At saturation, the amount of pUC18 adsorbed onto chrysotile was 0.8–1.2 µg/mg. To investigate whether pUC18 adsorbed on chrysotile is replicated by polymerase, polymerase chain reaction (PCR) was carried out with the chrysotile. Amplification of the β-lactamase gene coded in pUC18, which was adsorbed onto chrysotile, was strongly inhibited. This suggests that DNA adsorbed onto chrysotile is not replicated in vivo. When we searched for substances to release pUC18 adsorbed onto chrysotile, we found that a 300-bp single- or double-stranded segment of DNA releases pUC18 from chrysotile. Competitive adsorption onto chrysotile between double-stranded DNA and pUC18 was then examined through the Yoshida effect. The 310- and 603-bp double-stranded nucleotides caused 50% competitive inhibition at the same molar ratio with pUC18. Hence, the adsorbed region of pUC18 is about 300 bp in length. As the culture period for recipient cells increases, transformation efficiency decreases while the expression levels of small RNA of 300–600 bp also decrease. These results suggest that pUC18 adsorbed onto chrysotile can be released by 300-bp small RNA, replicated by DNA polymerase, and transferred to daughter cells.

Keywords: Chrysotile; Plasmid; Sliding friction; sRNA; Yoshida effect

Cloning and enzymatic characterization of a xylanase gene from a soil-derived metagenomic library with an efficient approach by Yong Hu; Guimin Zhang; Aiying Li; Jing Chen; Lixin Ma (pp. 823-830).

Screening interesting biocatalysts directly from soil samples is a more convenient and applicable approach than conventional cultivation-dependent ones. In our present work, a soil-derived metagenomic library containing 24,000 transformants was constructed with an efficient strategy for cloning xylanase genes. A gene encoding the enzyme (XynH) able to hydrolyze xylan was obtained. Similarity analysis revealed that this enzyme is a new member in the family 10 of xylanases. The molecular mass of XynH purified from Escherichia coli was estimated to be 39 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. It was found to display the maximal activity at lower temperature, under weakly alkaline conditions, different from most of xylanases. The K m and V_max values of XynH with birchwood xylan as substrate are 7.5 mg/ml and 190 μmol min⁻¹mg⁻¹, respectively. It is greatly interesting to note that the activity of XynH was not reduced significantly by Mn²⁺, Zn²⁺, Co²⁺, Ag⁺, and Cu²⁺, even at the concentration of 5 mM, which strongly inhibits most of the other xylanases studied previously.

Keywords: Soil microorganism DNA; Metagenomic library construction; Xylanase; Gene cloning; Heterologous expression

Application of comparative proteome analysis to reveal influence of cultivation conditions on asymmetric bioreduction of β-keto ester by Saccharomyces cerevisiae by Jinping Lin; Qinghai Liu; Erzheng Su; Dongzhi Wei; Shengli Yang (pp. 831-839).

Industrial bakers’ yeast strain Saccharomyces cerevisiae LH1 was selected for asymmetric reduction of ethyl benzoylacetate to (S)-ethyl 3-hydroxy-3-phenylpropionate. Higher reductive efficiency and higher cofactor availability were obtained with the alternation of cultivation condition (mainly growth medium). Compared to the bioreduction by yeast cells grown in malt extract (ME) medium, the concentration of substrate was increased 25-fold (up to 15.6 g/l) in the yeast peptone dextrose (YPD)-grown cells mediated bioreduction with 97.5% of enantioselective excess of (S)-product. The proteomic responses of S. cerevisiae LH1 cells to growth in aerobic batch cultures fed with either YPD or ME medium were examined and compared. Among the relative quantities of 550 protein spots in each gel, changes were shown in the expression level of 102 intracellular proteins when comparing YPD gel to ME gel. Most of the identified proteins were involved in energy metabolism and several cellular molecular biosynthetic pathway and catabolism. For YPD-grown yeast cells, not only enzymes involved in nicotinamide adenine dinucleotide phosphate regeneration, especially 6-phosphogluconate dehydrogenase, but also alcohol dehydrogenase 1 and d-arabinose 1-dehydrogenase which had been demonstrated activity toward ethyl benzoylacetate to (S)-hydroxy ester were significantly upregulated. These changes provided us insight in the way the yeast cells adapted to a change in cultivation medium and regulated its catalytic efficiency in the bioreduction.

Keywords: Asymmetric bioreduction; Saccharomyces cerevisiae ; Growth medium; Ethyl benzoylacetate; Proteome analysis

The GC and window-averaged DNA curvature profile of secondary metabolite gene cluster in Aspergillus fumigatus genome by Jin Hwan Do; Satoru Miyano (pp. 841-847).

An immense variety of complex secondary metabolites is produced by filamentous fungi including Aspergillus fumigatus, a main inducer of invasive aspergillosis. The identification of fungal secondary metabolite gene cluster is essential for the characterization of fungal secondary metabolism in terms of genetics and biochemistry through recombinant technologies such as gene disruption and cloning. Most of the prediction methods for secondary metabolite gene cluster severely depend on homology searches. However, homology-based approach has intrinsic limitation to unknown or novel gene cluster. We analyzed the GC and window-averaged DNA curvature profile of 26 secondary metabolite gene clusters in the A. fumigatus genome to find out potential conserved features of secondary metabolite gene cluster. Fifteen secondary metabolite gene clusters showed a conserved pattern in window-averaged DNA curvature profile, that is, the DNA regions including secondary metabolic signature genes such as polyketide synthase, nonribosomal peptide synthase, and/or dimethylallyl tryptophan synthase consisted of window-averaged DNA curvature values lower than 0.18 and these DNA regions were at least 20 kb. Forty percent of secondary metabolite gene clusters with this conserved pattern were related to severe regulation by a transcription factor, LaeA. Our result could be used for identification of other fungal secondary metabolite gene clusters, especially for secondary metabolite gene cluster that is severely regulated by LaeA or other proteins with similar function to LaeA.

Keywords: secondary metabolite; gene cluster; LaeA

Genome-scale reconstruction and in silico analysis of the Clostridium acetobutylicum ATCC 824 metabolic network by Joungmin Lee; Hongseok Yun; Adam M. Feist; Bernhard Ø. Palsson; Sang Yup Lee (pp. 849-862).

To understand the metabolic characteristics of Clostridium acetobutylicum and to examine the potential for enhanced butanol production, we reconstructed the genome-scale metabolic network from its annotated genomic sequence and analyzed strategies to improve its butanol production. The generated reconstructed network consists of 502 reactions and 479 metabolites and was used as the basis for an in silico model that could compute metabolic and growth performance for comparison with fermentation data. The in silico model successfully predicted metabolic fluxes during the acidogenic phase using classical flux balance analysis. Nonlinear programming was used to predict metabolic fluxes during the solventogenic phase. In addition, essential genes were predicted via single gene deletion studies. This genome-scale in silico metabolic model of C. acetobutylicum should be useful for genome-wide metabolic analysis as well as strain development for improving production of biochemicals, including butanol.

Keywords: Genome-scale metabolic network; In silico ; Metabolic flux analysis; Clostridium acetobutylicum ; Butanol

Comparative metabolomic analysis of Sinorhizobium sp. C4 during the degradation of phenanthrene by Young Soo Keum; Jong Su Seo; Qing X. Li; Jeong Han Kim (pp. 863-872).

Comparative metabolic responses of Sinorhizobium sp. C4 were investigated. Comprehensive metabolites profiles, including polar metabolites, fatty acids, and polyhydroxyalkanoates were evaluated through untargeted metabolome analyses. Intracellular metabolomes during the degradation of phenanthrene were compared with those from natural carbon sources. Principal component analysis showed a clear separation of metabolomes of phenanthrene degradation from other carbon sources. Shift to more hydrophobic fatty acid was observed from the analysis of fatty acid methyl ester. Polyhydroxyalkanoate from strain C4 was composed mainly with 3-hydroxybutyric acid and small amount of 3-hydroxypentanoic acid, while the monomeric composition was independent on carbon sources. However, the amount of polyhydroxyalkanoates during degradation of phenanthrene was 50–210% less than those from other carbon sources. Among 207 gas chromatography–mass spectrometry peaks from the polar metabolite fraction, 60% of the peaks were identified and compared. Several intermediates in tricarboxylic acid cycles and glycolysis were increased during phenanthrene degradation. Accumulation of trehalose was also evident in the phenanthrene-treated bacterium. Some amino acid, including branched amino acids, glycine, homoserine, and valine, were also increased, while more than 70% of identified metabolites were decreased during the phenanthrene metabolism. Accumulation of sulfur amino acids and nicotinic acid suggested the possible oxidative stress conditions during phenanthrene metabolism.

Keywords: Sinorhizobium ; Polycyclic aromatic hydrocarbon; Metabolomics; Polyhydroxyalkanoate; Fatty acids

Growth of Salinispora tropica strains CNB440, CNB476, and NPS21184 in nonsaline, low-sodium media by Ginger Tsueng; Kin S. Lam (pp. 873-880).

We recently described the development of a potassium-chloride-based salt formulation containing low sodium concentration (5.0 mM) to support the growth of Salinispora tropica strain NPS21184 and its production of salinosporamide A (NPI-0052). In order to determine whether the above low-sodium salt formulation can also support the growth of other S. tropica strains, we examined the growth of the type strain CNB440 and the parent strain CNB476, from which strain NPS21184 was derived as a single colony isolate. We demonstrated that good growth rate and yield of S. tropica strains CNB440 and CNB476, similar to S. tropica strain NPS21184 reported earlier, were detected in both agar and liquid media containing the potassium-chloride-based salt formulation with sodium concentration of 5.0 mM. Furthermore, we also detected good growth rate and yield of all three S. tropica strains on potassium-sulfate-based salt formulation agar medium containing both low-sodium (5.7 mM) and low-chloride (14 mM) content. This finding confirms the observation that the species of S. tropica does not have a seawater growth requirement but requirement for a specific combination of salts to provide a balance of salts and maintain a high enough ionic strength for growth.

Keywords: Salinosporamide A; NPI-0052; Salinispora tropica ; Low-sodium salt formulation; Nonsaline fermentation; Marine actinomycete

Reduction of volatile acidity of wines by selected yeast strains by A. Vilela-Moura; D. Schuller; A. Mendes-Faia; M. Côrte-Real (pp. 881-890).

Herein, we isolate and characterize wine yeasts with the ability to reduce volatile acidity of wines using a refermentation process, which consists in mixing the acidic wine with freshly crushed grapes or musts or, alternatively, in the incubation with the residual marc. From a set of 135 yeast isolates, four strains revealed the ability to use glucose and acetic acid simultaneously. Three of them were identified as Saccharomyces cerevisiae and one as Lachancea thermotolerans. Among nine commercial S. cerevisiae strains, strains S26, S29, and S30 display similar glucose and acetic acid initial simultaneous consumption pattern and were assessed in refermentation assays. In a medium containing an acidic wine with high glucose–low ethanol concentrations, under low oxygen availability, strain S29 is the most efficient one, whereas L. thermotolerans 44C is able to decrease significantly acetic acid similar to the control strain Zygosaccharomyces bailii ISA 1307 but only under aerobic conditions. Conversely, for low glucose–high ethanol concentrations, under aerobic conditions, S26 is the most efficient acid-degrading strain, while under limited-aerobic conditions, all the S. cerevisiae strains studied display acetic acid degradation efficiencies identical to Z. bailii. Moreover, S26 strain also reveals capacity to decrease volatile acidity of wines. Together, the S. cerevisiae strains characterized herein appear promising for the oenological removal of volatile acidity of acidic wines.

Keywords: Volatile acidity; Deacidification; Acidic wines; Acidic grape musts; Yeast

Microalgae-based processes for the biodegradation of pretreated piggery wastewaters by Cristina González; Judith Marciniak; Santiago Villaverde; Pedro A. García-Encina; Raúl Muñoz (pp. 891-898).

The potential and limitations of photosynthetic oxygenation on carbon and nitrogen removal from swine slurry were investigated in batch experiments using Chlorella sorokiniana and an acclimated activated sludge as model microorganisms. While algal–bacterial systems exhibited similar performance than aerated activated sludge in tests supplied with four and eight times diluted slurry, a severe inhibition of the biodegradation process was recorded in undiluted and two times diluted wastewater. Daily pH adjustment to 7 in enclosed algal–bacterial tests at several swine slurry dilutions allowed the treatment of up to two times diluted slurries (containing up to 1,180 mg N-NH₄ ⁺ l⁻¹). The combination of high pH levels and high NH₄ ⁺ concentrations was thus identified as the main inhibitory factor governing the efficiency of photosynthetically oxygenated processes treating swine slurry. Measurements of soluble total organic carbon (TOC) and volatile fatty acids (VFA) present in the slurry suggested that VFA degradation (mainly acetic and propionic acid) accounted for most of the soluble TOC removal, especially during the initial stages of the biodegradation process. On the other hand, assimilation into biomass and nitrification to NO₂ ⁻ constituted the main NH₄ ⁺ removal processes in enclosed algal–bacterial systems.

Keywords: Algal–bacterial systems; Ammonia inhibition; Biodegradation; Chlorella sorokiniana ; Photosynthetic oxygenation; Swine slurry

Biodegradation of dimethyl phthalate by Sphingomonas sp. isolated from phthalic-acid-degrading aerobic granules by Ping Zeng; Benjamin Yan-Pui Moy; Yong-Hui Song; Joo-Hwa Tay (pp. 899-905).

Phthalic acid esters (PAEs) contamination in water, air, and soil is one of the major environmental concerns in many countries. Besides the PAE biodegradation process, the PAE degrading bacteria have become one of the focuses of study. This study reports the successful isolation of one kind of indigenous bacterium PA-02 from phthalic acid (PA)-degrading aerobic granules. Based on its 16S ribosomal DNA sequence, isolate PA-02 was identified as Sphingomonas genus with 100% similarity to Sphingomonas sp. strain D84532. Strain PA-02 was a Gram-negative, rod-shaped bacterium with strong auto-aggregation ability. In particular, the strain PA-02 possessed PAE-degrading ability without acclimation. Results of growth tests showed that strain PA-02 could degrade dimethyl phthalate (DMP), dibutyl phthalate, and diethylhexyl phthalate. The specific degradation rates of DMP and PA were concentration-dependent with maximum values of 0.4 g-DMP g⁻¹ biomass h⁻¹ and 1.3 g-PA g⁻¹ biomass h⁻¹, respectively. Kinetic studies also revealed that PA-02 was robust under high concentrations of DMP and PA. Even when the PA concentration was increased to 1,000.0 mg l⁻¹, the specific PA degradation rate was about 0.25 g-PA g⁻¹ biomass h⁻¹. The corresponding value for DMP was 0.067 g-DMP g⁻¹ biomass h⁻¹ at 1,000 mg l⁻¹.

Keywords: PAEs degradation; Dimethyl phthalate; 16S rDNA; Sphingomonas ; Kinetics

Autocatalysis in Reactive Black 5 biodecolorization by Rhodopseudomonas palustris W1 by Xingzu Wang; Xiang Cheng; Dezhi Sun (pp. 907-915).

Autocatalysis in biological decolorization of Reactive Black 5 (RB5) by Rhodopseudomonas palustris W1 was investigated in batch assays. An improvement of 1.5-fold in decolorization rate of RB5 was obtained by supplementing decolorization metabolites from 200 mg l⁻¹ RB5. Liquid chromatography-mass spectrometry and cyclic voltammetric analysis revealed that the constituent of dye precursors, from azo bonds breakage, with quinone-like structure and reversible oxidation–reduction activity can be used as redox mediators and was responsible for the catalytic reduction of RB5. The required amount of metabolites for catalytic decolorization was quite small, indicating its possible application in real textile wastewater treatment. Furthermore, decolorization metabolites of RB5 were shown as effective in catalyzing anaerobic decolorization of Direct Yellow 11, an azo dye without autocatalyic activity.

Keywords: Catalytic decolorization; Reactive Black 5; Redox mediator; Rhodopseudomonas palustris

Efficient cloning system for construction of gene silencing vectors in Aspergillus niger by José Miguel Oliveira; Douwe van der Veen; Leo H. de Graaff; Ling Qin (pp. 917-924).

An approach based on Gateway recombination technology to efficiently construct silencing vectors was developed for use in the biotechnologically important fungus Aspergillus niger. The transcription activator of xylanolytic and cellulolytic genes XlnR of A. niger was chosen as target for gene silencing. Silencing was based on the expression vector pXLNRir that was constructed and used in co-transformation. From all the strains isolated (N = 77), nine showed poor xylan-degrading activities in two semi-quantitative plate assays testing different activities for xylan degradation. Upon induction on d-xylose, transcript levels of xlnR were decreased in the xlnR-silenced strains, compared to a wild-type background. Under these conditions, the transcript levels of xyrA and xynB (two genes regulated by XlnR) were also decreased for these xlnR-silenced strains. These results indicate that the newly developed system for rapid generation of silencing vectors is an effective tool for A. niger, and this can be used to generate strains with a tailored spectrum of enzyme activities or product formation by silencing specific genes encoding, e.g., regulators such as XlnR.

Keywords: pFIRD1; Gateway cloning; Aspergillus niger ; Post-transcriptional gene silencing; XlnR

Construction and characterization of a 9-mer phage display pVIII-library with regulated peptide density by Annette Fagerlund; Astrid Hilde Myrset; Mari Ann Kulseth (pp. 925-936).

The construction of a new phagemid vector for display of peptides on the pVIII major coat protein of filamentous bacteriophage is described, in which expression of pVIII-peptide fusions was placed under the control of the arabinose-inducible P_BAD promoter. The new phagemid showed excellent capacity for the regulation of peptide expression, as judged by enzyme-linked immunosorbent assay (ELISA) and electron microscopy of immunogold-labeled FLAG peptides displayed on phages. Regulation of the density of peptide fusions displayed on phages may offer advantages in the search for new peptide ligands due to the possibility of regulating the stringency of binding, reducing selection based on avidity effects during biopanning. Furthermore, the peptide expression in the absence of inducer was effectively shut off, minimizing growth bias of individual clones. A 9-mer phage display library prepared using the constructed phagemid was generated by insertion of randomly synthesized oligonucleotides close to the N-terminal of the pVIII protein. The library comprised a total of 9.4 × 10⁹ unique transformants, and was confirmed to show high diversity. The functional utility of the library was confirmed by the successful affinity selection of peptides binding to matrix metalloproteinase-9 (MMP-9). The majority of selected peptides shared the consensus motif R(D/N)XXG(M/L)(V/I)XQ, not previously selected during biopanning against MMP-9.

Keywords: Phage display; MMP-9; Biopanning; Phagemid; P_BAD promoter

Tn5 transposase-assisted high-efficiency transformation of filamentous fungus Phoma herbarum YS4108 by Mingqian Feng; Tian Zhou; Wenjie Wei; Yongchun Song; Renxiang Tan (pp. 937-944).

Conventionally, filamentous fungi are transformed by using conidia or protoplasts as recipients. However, induction of sporulation is difficult in some fungi, and protoplasting is an awing, frequently frustrating, and batch-dependent work. In this study, we established a simple and convenient method to prepare single cells from mycelia without enzymatic protoplasting. As a case study on the pathogenic fungus Phoma herbarum YS4108, the single cells could be directly and highly efficiently transformed with the aid of Tn5 transposase. The optimal electric pulse delivery parameters were 25 μF in capacitance, 0.75 kV (0.2-cm cuvette) in voltage, and 400 Ω in resistance, under which the efficiency of transposase-assisted transformation (TNAT) was enhanced to two to threefold compared to that of non-TNAT method, resulting in >230 transformants/cuvette (10⁶ recipients). Further cell wall weakening of the single cells by lytic enzymes and linearization of the plasmid were found to have no effects on transformation efficiency, but vector linearization apparently lowered the background growth. The present study for the first time explained that Tn5 transposase could be used to increase transformation efficiency in filamentous fungi, and the method presented here may be of wide applicability in different studies and may be the first choice when transformation efficiency and convenience are priorities and mycelia have to be used as transformation recipients.

Keywords: Phoma herbarum YS4108; Transformation; Electroporation; Transposase; Fungi

Sections

Personal tools

Applied Microbiology and Biotechnology (v.80, #5)

Sections

Personal tools

Local resources

Applied Microbiology and Biotechnology (v.80, #5)