Check out our New Publishers' Select for Free Articles

Applied Microbiology and Biotechnology (v.75, #5)

Metagenomics, biotechnology with non-culturable microbes by Christel Schmeisser; Helen Steele; Wolfgang R. Streit (pp. 955-962).

Metagenomics as a new field of research has been developed over the past decade to elucidate the genomes of the non-cultured microbes with the goal to better understand global microbial ecology on the one side, and on the other side it has been driven by the increasing biotechnological demands for novel enzymes and biomolecules. Since it is well accepted that the majority of all microbes has not yet been cultured, the not-yet-cultivated microbes represent a shear unlimited and intriguing resource for the development of novel genes, enzymes and chemical compounds for use in biotechnology. However, with respect to biotechnology, metagenomics faces now two major challenges. Firstly, it has to identify truly novel biocatalysts to fulfil the needs of industrial processes and green chemistry. Secondly, the already available genes and enzymes need to be implemented in production processes to further prove the value of metagenome-derived sequences.

Keywords: Metagenomics; Biocatalysis; Non-cultivated microbes; Environmental genomics

Molecular tools for analysis of gene function in parasitic microorganisms by Markus Meissner; Carolina Agop-Nersesian; William J. Sullivan Jr. (pp. 963-975).

With the completion of several genome sequences for parasitic protozoa, research in molecular parasitology entered the “post-genomic” era. Accompanied by global transcriptome and proteome analysis, huge datasets have been generated that have added many novel candidates to the list of drug and vaccine targets. The challenge is now to validate these factors and to bring science back to the bench to perform a detailed characterization. In some parasites, like Trypanosoma brucei, high-throughput genetic screens have been established using RNA interference [for a detailed review, see Motyka and Englund (2004)]. In most protozoan parasites, however, more time-consuming approaches have to be employed to identify and characterize the function of promising candidates in detail. This review aims to summarize the status of molecular genetic tools available for a variety of protozoan pathogens and discuss how they can be implemented to advance our understanding of parasite biology.

Keywords: Protozoan parasites; Transfection; Recombinase; Transcription; Inducible system; RNA interference

Chemistry, physiological properties, and microbial production of hydroxycitric acid by Takashi Yamada; Hiroyuki Hida; Yasuhiro Yamada (pp. 977-982).

The tropical plants Garcinia cambogia and Hibiscus subdariffa produce hydroxycitric acid (HCA), of which the absolute configurations are (2S,3S) and (2S,3R), respectively. (2S,3S)-HCA is an inhibitor of ATP-citrate lyase, which is involved in fatty acid synthesis. (2S,3R)-HCA inhibits pancreatic α-amylase and intestinal α-glucosidase, leading to a reduction in carbohydrate metabolism. In this study, we review current knowledge on the structure, biological occurrence, and physiological properties of HCA. The availability of HCA is limited by the restricted habitat of its source plants and the difficulty of stereoselective organic synthesis. Hence, in our recent study, thousands of microbial strains were screened and finally two bacterial strains were, for the first time, found to produce trace amounts of HCA. The HCA variants produced were the Hibiscus-type (2S,3R) enantiomer. Subsequent genome shuffling rapidly generated a mutant population with improved HCA yield relative to the parent strain of bacteria. These bacteria are a potential alternative source of natural HCA.

Keywords: Hydroxycitric acid; Microbial production; Genome shuffling; Streptomyces

Fetal calf serum-free culture of Chinese hamster ovary cells employing fish serum by M. Fujiwara; R. Tsukada; Y. Tsujinaga; M. Takagi (pp. 983-987).

The effects of fish serum on cell growth and human granulocyte-macrophage colony-stimulating factor (hGM-CSF) production in an adhesion culture of Chinese hamster ovary (CHO) cells DR1000L4N were investigated and compared with those of fetal calf serum (FCS). Although fish serum did not stimulate the initial adhesion of CHO cells to culture dishes, it prompted cell growth after cell adhesion with FCS for 24 h. The cell density in the fish serum medium reached 75% that in the FCS medium. Fish serum promoted cell adhesion to and cell growth on collagen-coated dishes. The cell-specific production rate of hGM-CSF in the fish serum medium on collagen-coated dishes was almost the same as that in the FCS medium.

Keywords: Fish serum; Fetal calf serum; Chinese hamster ovary cells

The antibacterial and immunostimulative effect of chitosan-oligosaccharides against infection by Staphylococcus aureus isolated from bovine mastitis by Jin-San Moon; Hee-Kyung Kim; Hye Cheong Koo; Yi-Seok Joo; Hyang-mi Nam; Yong Ho Park; Mun-Il Kang (pp. 989-998).

Based on our previous study evaluating the in vivo cure efficacy of chitosan on bovine mastitis, a more water-soluble chitosan-oligosaccharide (OCHT) with a high degree of deacetylation and low molecular weight was prepared to obtain high antibiotic efficacy. The growth of Staphylococcus aureus isolated from bovine mastitis was inhibited within 10 min of treatment with OCHT in concentrations ranging from 0.0001 to 0.5%. Additionally, electron microscopic observation indicated that the surface of the OCHT-treated bacteria was expanded, distorted, and lysed compared to that of the control bacteria. In mice, the proportion of monocytes was elevated, and the levels of interleukin-6 and interferon-γ sharply increased l h after the peritoneal inoculation of the OCHT (0.5 to 1 mg per mouse). Mice challenged intraperitoneally with S. aureus (2.5 × 10⁸ colony forming units) after oral treatment with OCHT (0.5 to 2 mg per day) for 7 days showed a higher survival rate (70–100%) than that of the control (10%). We suggest that the OCHT prepared in this study is a potential agent for the prevention and treatment of bovine mastitis based on its strong antibacterial activity against S. aureus as well as the immunostimulative effect it exhibits on murine infection by S. aureus.

Keywords: Chitosan-oligosaccharides; Staphylococcus aureus ; Antibacterial activity; Immunostimulative effect

Unique butyric acid incorporation patterns for salinosporamides A and B reveal distinct biosynthetic origins by Ginger Tsueng; Katherine A. McArthur; Barbara C. M. Potts; Kin S. Lam (pp. 999-1005).

Feeding sodium butyrate (0.25–1 mg/ml) to cultures of Salinispora tropica NPS21184 enhanced the production of salinosporamide B (NPI-0047) by 319% while inhibiting the production of salinosporamide A (NPI-0052) by 26%. Liquid chromatography mass spectrometry analysis of the crude extract from the strain NPS21184 fed with 0.5 mg/ml sodium [U-¹³C₄]butyrate indicated that butyrate was incorporated as a contiguous four-carbon unit into NPI-0047 but not into NPI-0052. Nuclear magnetic resonance analysis of NPI-0047 and NPI-0052 purified from the sodium [U-¹³C₄]butyrate-supplemented culture extract confirmed this incorporation pattern. The above finding is the first direct evidence to demonstrate that the biosynthesis of NPI-0047 is different from NPI-0052, and NPI-0047 is not a precursor of NPI-0052.

Keywords: Salinispora tropica ; Salinosporamides; Biosynthesis; Butyric acid

Improvement in lactic acid production from starch using α-amylase-secreting Lactococcus lactis cells adapted to maltose or starch by Kenji Okano; Sakurako Kimura; Junya Narita; Hideki Fukuda; Akihiko Kondo (pp. 1007-1013).

To achieve direct and efficient lactic acid production from starch, a genetically modified Lactococcus lactis IL 1403 secreting α-amylase, which was obtained from Streptococcus bovis 148, was constructed. Using this strain, the fermentation of soluble starch was achieved, although its rate was far from efficient (0.09 g l⁻¹ h⁻¹ lactate). High-performance liquid chromatography revealed that maltose accumulated during fermentation, and this was thought to lead to inefficient fermentation. To accelerate maltose consumption, starch fermentation was examined using L. lactis cells adapted to maltose instead of glucose. This led to a decrease in the amount of maltose accumulation in the culture, and, as a result, a more rapid fermentation was accomplished (1.31 g l⁻¹ h⁻¹ lactate). Maximum volumetric lactate productivity was further increased (1.57 g l⁻¹ h⁻¹ lactate) using cells adapted to starch, and a high yield of lactate (0.89 g of lactate per gram of consumed sugar) of high optical purity (99.2% of l-lactate) was achieved. In this study, we propose a new approach to lactate production by α-amylase-secreting L. lactis that allows efficient fermentation from starch using cells adapted to maltose or starch before fermentation.

Keywords: Adaptation to maltose or starch; α-amylase; Lactic acid fermentation from starch; Lactococcus lactis

Optimization of medium composition for the production of alkaline β-mannanase by alkaliphilic Bacillus sp. N16-5 using response surface methodology by Shan-shan Lin; Wen-fang Dou; Hong-yu Xu; Hua-zhong Li; Zheng-Hong Xu; Yan-he Ma (pp. 1015-1022).

In this work, a 2² factorial design was employed combining with response surface methodology (RSM) to optimize the medium compositions for the production of alkaline β-mannanase by alkaliphilic Bacillus sp. N16-5 isolated previously from sediment of Wudunur Soda Lake in Inner Mongolia, China. The central composite design (CCD) used for the analysis of treatment combinations showed that a second-order polynomial regression model was in good agreement with experimental results, with R ² = 0.9829 (P < 0.05). The maximum activity was obtained at NaCl concentration (84.4 g l⁻¹) and sodium glutamate (3.11 g l⁻¹) and a high medium pH around 10.0. Under such conditions, the activity of alkaline β-mannanase achieved 310.1 U/ml in the scale of 5-l fermenter, which was increased nearly twice compared with the original. Through optimization, the substrates shifted from the expensive substrates, such as locust bean gum and peptone, to the inexpensive ones such as konjac powder, soymeal, and sodium glutamate. The experiment results also suggested that the environmental conditions of high salinity and high alkalinity, as well as the inducer substrates, play very important roles in the production of the alkaline β-mannanase by alkaliphilic Bacillus sp. N16-5.

Keywords: Alkaliphilic Bacillus sp.; Alkaline β-mannanase; Optimization; Response surface methodology

Microencapsulated bacterial cells can be used to produce the enzyme feruloyl esterase: preparation and in-vitro analysis by Jasmine Bhathena; Arun Kulamarva; Aleksandra Malgorzata Urbanska; Christopher Martoni; Satya Prakash (pp. 1023-1029).

Biotechnological production of ferulic acid, a precursor of vanillin, is an attractive alternative for various industries due to the high price and demand for natural ferulic acid. Feruloyl esterase has been identified as a key enzyme involved in microbial transformations of ferulic acid to vanillin. Several fungal feruloyl esterases have been purified and characterized for their use in the production of ferulic acid. This paper, for the first time, discusses the use of lactic acid bacteria for the production of ferulic acid. Specifically, we have used Lactobacillus cells and microencapsulation so that ferulic acid can be produced continuously using various types of fermentation systems. Bacteria were encapsulated in alginate-poly-l-lysine-alginate (APA) microcapsules, and the production of ferulic acid by lactobacilli was detected using a real-time high-performance liquid chromatography (HPLC)-based assay. Results show that ferulic acid can be produced using microencapsulated Lactobacillus fermentum (ATCC 11976) with significant levels of biological feruloyl esterase activity.

Cofactor regeneration in phototrophic cyanobacteria applied for asymmetric reduction of ketones by Jan Havel; Dirk Weuster-Botz (pp. 1031-1037).

The obligate photoautotrophic cyanobacterium Synechococcus PCC7942 and the photoheterotrophic heterocystous cyanobacterium Noctoc muscorum are able to reduce prochiral ketones asymmetrically to optical pure chiral alcohols without light. An example is the synthesis of S-pentafluoro(phenyl-)ethanol with an enantiomeric excess >99% if 2′-3′-4′-5′-6′-pentafluoroacetophenone is used as substrate. If no light is available for regeneration of the cofactor nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH), glucose is used as cosubstrate. Membrane disintegration during asymmetric reduction promotes cytosolic energy generating metabolic pathways. Observed regulatory effects depicted by an adenosine triphosphate (ATP) to nicotinamide adenine dinucleotide phosphate (oxidized form) (NADP⁺) ratio of 3:1 for efficient cofactor recycling indicate a metabolization via glycolisis. The stoichiometric formation of the by-product acetate (1 mol acetate/1 mol chiral alcohol) indicates homoacetic acid fermentation for cofactor regeneration including the obligate photoautotrophic cyanobacterium Synechococcus PCC7942.

High yield of mannosylglycerate production by upshock fermentation and bacterial milking of trehalose-deficient mutant Thermus thermophilus RQ-1 by K. Egorova; T. Grudieva; C. Morinez; J. Kube; H. Santos; M. S. da Costa; G. Antranikian (pp. 1039-1045).

A production process, using upshock fermentation and osmotic downshock, for the effective production/excretion of mannosylglycerate (MG) by the trehalose-deficient mutant of the strain Thermus thermophilus RQ-1 has been developed. In the first phase of fed-batch fermentation, the knockout mutant was grown at 70°C on a NaCl-free medium. After the culture reached the end of the exponential growth phase, upshift in temperature and NaCl concentration was applied. The temperature was increased to 77°C, and NaCl was added up to 3.0% and kept constant during the second phase of fermentation. Although this shift in cultivation parameters caused a dramatic drop of cell density, a significant improvement in accumulation of MG up to 0.64 μmol/mg protein compared to batch fermentations (0.31 μmol/mg protein) was achieved. A total yield of 4.6 g MG/l of fermentation broth was obtained in the dialysis bioreactor with a productivity of 0.29 g MG l⁻¹ h⁻¹. The solute was released from the harvested biomass by osmotic downshock using demineralized water at 70°C. More than 90% of the intracellularly accumulated solute was recovered from the water fraction. The process was very efficient, as hyperosmotic shock, release of the solute, and reiterative fed-batch fermentation could be repeated at least four times.

Keywords: Mannosylglycerate; Fermentation; Thermus

Production of medium-chain-length hydroxyalkanoic acids from Pseudomonas putida in pH stat by Lei Wang; Wolfgang Armbruster; Dieter Jendrossek (pp. 1047-1053).

Pseudomonas putida GP01 cells that had accumulated medium-chain-length polyhydroxyalkanoates (PHA_MCL) secreted 3-hydroxyoctanoate and 3-hydroxyhexanoate when incubated in alkaline buffers. The release of acids strongly decreased the pH resulting in less efficient secretion of 3HA_MCL at neutral pH. To increase the yield of secreted MCL-hydroxyalkanoates, experiments at constant pH in a pH stat apparatus were performed. High acid releasing rates were recorded for the wild type GP01 at pH 9.2 (0.60 mmol acid h⁻¹ g⁻¹ cellular dry weight [cdw]). At more alkaline constant pH values (pH 9.3–11), the initial acid secretion rates were even higher but rapidly decreased by time. When acid secretion of PHA depolymerase mutant GPo500 was tested (pH 9.2), considerably lower rates compared to wild type were recorded (0.18 mmol acid h⁻¹ g⁻¹ cdw). Determination of dissolved oxygen during acid release indicated different respiratoric activity in wild type (low) and mutant (high). Acid release of mutant, but not of the wild type, could be enhanced by aeration. Determination of PHA content of cells after alkaline incubation showed that the wild type had lost most of its accumulated PHA, whereas the PHA content of the depolymerase mutant was not significantly reduced. Considerable amounts of 3HA_MCL were secreted by the wild type, but only little 3HA_MCL were found for the depolymerase mutant. In summary, 3HA_MCL can be more efficiently produced at constant high pH than by incubation without pH control. High PHA depolymerase activity enabled the wild type to compensate for the high external pH by secretion of PHA hydrolysis products, whereas production of protons at aerobic conditions presumably was responsible for the major portion of the observed acid releasing rates in the depolymerase mutant.

Keywords: PHB; PHA; Intracellular PHA depolymerase; Medium-chain-length hydroxyalkanoic acids; 3-Hydroxyoctanoate; pH stat

Construction of a thermostable cytochrome P450 chimera derived from self-sufficient mesophilic parents by Sabine Eiben; Heike Bartelmäs; Vlada B. Urlacher (pp. 1055-1061).

The P450 monooxygenases CYP102A1 from Bacillus megaterium and CYP102A3 from Bacillus subtilis are fusion flavocytochromes comprising of a P450 heme domain and a FAD/FMN reductase domain. This protein organization is responsible for the extraordinary catalytic activities making both monooxygenases promising enzymes for biocatalysis. CYP102A1 and CYP102A3 are fatty acid hydroxylases that share 65% identity, and their mutants are able to oxidize a wide range of substrates. In an attempt to increase the process stability of CYP102A1, we exchanged the more unstable reductase domain of CYP102A1 with the more stable reductase domain of CYP102A3. Stability of the chimeric fusion protein was determined spectrophotometrically as well as by measuring the hydroxylation activity towards 12-para-nitrophenoxydodecanoic acid (12-pNCA) after incubation at elevated temperatures. In the reaction with 12-pNCA, the new chimeric protein exhibited 88 and 38% of the activity of CYP102A3 and CYP102A1, respectively, but was able to hydroxylate substrates within a wider temperature range compared with the parental enzymes. Maximum activity was obtained at 51°C, and the half-life at 50°C was with 100 min more than ten times longer than that of CYP102A1 (8 min).

Keywords: Cytochrome P450 monooxygenase; Thermostability; Chimera; Self-sufficient

Synthesis of highly hydroxylated aromatics by evolved biphenyl dioxygenase and subsequent dihydrodiol dehydrogenase by Kazutoshi Shindo; Yuko Shindo; Tomoko Hasegawa; Ayako Osawa; Osamu Kagami; Kensuke Furukawa; Norihiko Misawa (pp. 1063-1069).

The evolved bphA1 (2049) gene, in which nine amino acids from the Pseudomonas pseudoalcaligenes KF707 BphA1 were changed to those from the Burkholderia xenovorans LB400 BphA1 (M247I, H255Q, V258I, G268A, D303E, -313G, S324T, V325I, and T376N), was expressed in Escherichia coli along with the bphA2A3A4 and bphB genes derived from strain KF707. This recombinant E. coli cells converted biphenyl and several heterocyclic aromatic compounds into the highly hydroxylated products such as biphenyl-2,3,2′,3′-tetraol (from biphenyl), 2-(2,3-dihydroxyphenyl)benzoxazole-4,5-diol (from 2-phenylbenzoxazole), and 2-(2,5-dihydroxyphenyl)benzoxazole-4,5-diol [from 2-(2-hydroxyphenyl)benzoxazole]. The antioxidative activity of these generated compounds was markedly higher than that of the original substrate used.

Keywords: Hydroxylated aromatics; Biphenyl dioxygenase; Dihydrodiol dehydrogenase

A Paenibacillus sp. dextranase mutant pool with improved thermostability and activity by Erika Hild; Stevens M. Brumbley; Michael G. O’Shea; Helena Nevalainen; Peter L. Bergquist (pp. 1071-1078).

Random mutagenesis was used to create a library of chimeric dextranase (dex1) genes. A plate-screening protocol was developed with improved thermostability as a selection criterion. The mutant library was screened for active dextranase variants by observing clearing zones on dextran-blue agar plates at 50°C after exposure to 68°C for 2 h, a temperature regime at which wild-type activity was abolished. A number of potentially improved variants were identified by this strategy, five of which were further characterised. DNA sequencing revealed ten nucleotide substitutions, ranging from one to four per variant. Thermal inactivation studies showed reduced (2.9-fold) thermostability for one variant and similar thermostability for a second variant, but confirmed improved thermostability for three mutants with 2.3- (28.9 min) to 6.9-fold (86.6 min) increases in half-lives at 62°C compared to that of the wild-type enzyme (12.6 min). Using a 10-min assay, apparent temperature optima of the variants were similar to that of the wild type (T _opt 60°C). However, one of these variants had increased enzyme activity. Therefore, the first-generation dextranase mutant pool obtained in this study has sufficient molecular diversity for further improvements in both thermostability and activity through recombination (gene shuffling).

Keywords: Dextranase; Thermostability; Activity; Directed evolution; Paenibacillus sp.; Random mutagenesis

Construction of an S-layer protein exhibiting modified self-assembling properties and enhanced metal binding capacities by Katrin Pollmann; Sabine Matys (pp. 1079-1085).

The functional S-layer protein gene slfB of the uranium mining waste pile isolate Bacillus sphaericus JG-A12 was cloned as a polymerase chain reaction product into the expression vector pET Lic/Ek 30 and heterologously expressed in Escherichia coli Bl21(DE3). The addition of His tags to the N and C termini enabled the purification of the recombinant protein by Ni-chelating chromatography. The Ni binding capacity of the His-tagged recombinant S-layer protein was compared with that of the wild-type S layer. The inductively coupled plasma mass spectrometry analyses demonstrate a significantly enhanced Ni binding capability of the recombinant protein. In addition, the self-assembling properties of the purified modified S-layer proteins were studied by light microscopy and scanning electron microscopy. Whereas the wild-type S-layer proteins re-assembled into regular cylindric structures, the recombinant S-layer proteins reassembled into regular sheets that formed globular agglomerating structures. The nanoporous structure of the protein meshwork, together with its enhanced Ni binding capacity, makes the recombinant S-layer attractive as a novel self-assembling biological template for the fabrication of metal nanoclusters and construction of nanomaterials that are of technical interest.

Isolation and properties of a levo-lactonase from Fusarium proliferatum ECU2002: a robust biocatalyst for production of chiral lactones by Xian Zhang; Jian-He Xu; Yi Xu; Jiang Pan (pp. 1087-1094).

A fungus strain ECU2002, capable of enantioselectively hydrolyzing chiral lactones to optically pure hydroxy acids, was newly isolated from soil samples through two steps of screening and identified as Fusarium proliferatum (Matsushima) Nirenberg. From the crude extract of F. proliferatum ECU2002, a novel levo-lactonase was purified to homogeneity, with a purification factor of 460-folds and an overall yield of 9.7%, by ultrafiltration, acetone precipitation, and chromatographic separation through DEAE-Toyopearl, Butyl-Toyopearl, Hydroxyapatite, Toyoscreen-Super Q, and TSK-gel columns. The purified enzyme is a monomer; with a molecular mass of ca 68 kDa and a pI of 5.7 as determined by two-dimensional electrophoresis. The catalytic performance of the partially purified levo-lactonase was investigated, giving temperature and pH optima at 50°C and 7.5, respectively, for γ-butyrolactone hydrolysis. The substrate specificity of the partially purified lactonase was also examined using several useful lactones, among which α-hydroxy-γ-butyrolactone was the best substrate, with 448-fold higher lactonase activity as compared to γ-butyrolactone. The F. proliferatum lactonase preferentially hydrolyzed the levo enantiomer of butyrolactones, including β-butyrolactone, α-hydroxy-γ-butyrolactone, α-hydroxy-β,β-dimethyl-γ-butyrolactone (pantolactone), and β-hydroxy-γ-butyrolactone, affording (+)-hydroxy acids in high (94.8∼98.2%) enantiomeric excesses (ee) and good conversions (38.2∼44.2%). A simple immobilization of the crude lactonase with glutaraldehyde cross-linking led to a stable and easy-to-handle biocatalyst for catalytic resolution of chiral lactones. The immobilized lactonase also performed quite well in repeated batch resolution of dl-pantolactone at a concentration of 35% (w/v), retaining 67% of initial activity after ten cycles of reaction (corresponding to a half life of 20 cycles) and affording the product in 94∼97% ee, which can be easily enhanced to >99% ee after the d-hydroxy acid was chemically converted into l-lactone and crystallized.

Design of a secondary alcohol degradation pathway from Pseudomonas fluorescens DSM 50106 in an engineered Escherichia coli by Anett Kirschner; Josef Altenbuchner; Uwe T. Bornscheuer (pp. 1095-1101).

The genes encoding an alcohol dehydrogenase, Baeyer–Villiger monooxygenase and an esterase from P. fluorescens DSM 50106, which seemed to be metabolically connected based on the sequence of the corresponding open reading frames, were cloned into one vector (pABE) and functionally expressed in Escherichia coli. Overall expression levels were quite low, however, using whole cells of E. coli JM109 pABE expressing the three recombinant enzymes, conversion of secondary alcohols (C_n) to the corresponding primary alcohols (C_n−2) and acetic acid via ketone and ester was possible. In this way, 2-decanol was almost completely converted within 20 h at 30°C. Thus, it could be shown that the three enzymes are metabolically connected and that they are most probably involved in alkane degradation via sub-terminal oxidation of the acyclic aliphatic hydrocarbons.

Keywords: Baeyer–Villiger monooxygenase; Esterase; Alcohol dehydrogenase; Aliphatic alcohols; Pseudomonas fluorescens ; Degradation pathway

A novel-designed Escherichia coli for the production of various polyhydroxyalkanoates from inexpensive substrate mixture by Rui Li; Quan Chen; Peng George Wang; Qingsheng Qi (pp. 1103-1109).

To efficiently produce polyhydroxyalkanoates (PHAs) from substrate mixture containing various carbon sources, Escherichia coli phosphotransferase system (PTS) mutants were constructed, in which the carbon catabolite repression in the presence of glucose was released. When cultivated in the medium containing glucose and xylose, E. coli LR1010, harboring phaC Re and phaAB Re genes from Ralstronia eutropha, could simultaneously consume glucose and xylose and accumulate short-chain-length PHA, whereas E. coli LR1120 and LR1110, harboring phaC1 gene from Pseudomonas aeruginosa, could accumulate medium-chain-length PHA. At the same time, LR1110 could simultaneously utilize the mixture of glucose and fatty acids. Semiquantitative reverse transcription polymerase chain reaction analysis indicated that disrupting phosphoenolpyruvate: sugar PTS in this strain released the repression on fad genes.

Keywords: Escherichia coli ; Polyhydroxyalkanoates; PTS; Metabolic engineering

Cloning of the Arthrobacter sp. FG1 dehalogenase genes and construction of hybrid pathways in Pseudomonas putida strains by Francesca Radice; Viviana Orlandi; Valentina Massa; Vanessa Battini; Giovanni Bertoni; Walter Reineke; Paola Barbieri (pp. 1111-1118).

An Arthrobacter strain, able to utilize 4-chlorobenzoic acid as the sole carbon and energy source, was isolated and characterized. The first step of the catabolic pathway was found to proceed via a hydrolytic dehalogenation that leads to the formation of 4-hydroxybenzoic acid. The dehalogenase encoding genes (fcb) were sequenced and found highly homologous to and organized as those of other 4-chlorobenzoic acid degrading Arthrobacter strains. The fcb genes were cloned and successfully expressed in the heterologous host Pseudomonas putida PaW340 and P. putida KT2442 upper TOL, which acquired the ability to grow on 4-chlorobenzoic acid and 4-chlorotoluene, respectively. The cloned dehalogenase displayed a high specificity for para-substituted haloaromatics with affinity Cl > Br > I » F, in the order.

Keywords: Dehalogenase; fcb genes; 4-Chlorobenzoic acid; 4-Chlrotoluene

Genetic organization of the biosynthetic gene cluster for the indolocarbazole K-252a in Nonomuraea longicatena JCM 11136 by Seung-Young Kim; Jin-Soo Park; Choong-Sik Chae; Chang-Gu Hyun; Byoung Wook Choi; Jongheon Shin; Ki-Bong Oh (pp. 1119-1126).

Indolocarbazole metabolite K-252a is a natural product that was previously reported as a potent protein kinase C inhibitor with in vitro and in vivo potency. From a biosynthetic viewpoint, this compound possesses structurally interesting features such as an unusual furanosyl sugar moiety, which are absent in the well-studied staurosporine and rebeccamycin. A cosmid library from genomic DNA of Nonomuraea longicatena JCM 11136 was constructed and screened for the presence of genes to be involved in the biosynthesis of indolocarbazole K-252a. Using as a probe an internal fragment of vioB, a Chromobacterium violaceum gene encoding a multifunctional enzyme that catalyzes tryptophan decarboxylation and condensation reaction in violacein biosynthesis, we isolated a DNA region that directed the biosynthesis of K-252a when introduced into the heterologous expression host Streptomyces albus. Sequence analysis of 45 kb revealed genes for indolocarbazole core formation, glycosylation, and sugar methylation, as well as a regulatory gene and two resistance/secretion genes. The cloned genes should help to elucidate the molecular basis for indolocarbazole biosynthesis and generate new indolocarbazole analogues by genetic engineering.

Keywords: Nonomuraea longicatena ; Indolocarbazole; K-252a; Biosynthesis; Gene cluster; Heterologous expression

Identification of promoters of two dehydrogenase genes in Ketogulonicigenium vulgare DSM 4025 and their strength comparison in K. vulgare and Escherichia coli by Shulin Fu; Weicai Zhang; Aiguang Guo; Jianhua Wang (pp. 1127-1132).

Promoters of sorbose dehydrogenase gene sdh and sorbosone dehydrogenase gene sndh (Psdh and Psndh) in Ketogulonicigenium vulgare DSM 4025 were identified. The transcription initiation site (TIS) of Psdh was guanine 74 bp upstream of the start codon of sdh and the TIS of Psndh was adenine 113 bp upstream of the first codon of sndh. Comparing Psdh and Psndh, consensus sequences were found, which were TAVCVT (V=A, C or G) and THGAHC (H=A, C or T) for their putative −10 and −35 regions, respectively, and the spans between the 2 regions were 17 bp. Psdh and Psndh promoters may be constitutive in K. vulgare DSM 4025 when cultured in HJ medium. Semiquantitative RT-PCR analysis showed that the Psdh promoter was about 2.5 times stronger than Psndh in strength in K. vulgare DSM 4025. In Escherichia coli, Psdh and Psndh demonstrated strong activity with the former about two times stronger than the latter. DCIP decoloration method and reporter plasmids pSDH or pSNDH may be applied to discover promoters of genes in E. coli and to determine their strength in one step.

Keywords: Promoters; Determination; Strength comparison; Ketogulonicigenium vulgare DSM 4025; E. coli

Genomotyping of Pseudomonas putida strains using P. putida KT2440-based high-density DNA microarrays: implications for transcriptomics studies by Hendrik Ballerstedt; Rita J. M. Volkers; Astrid E. Mars; John E. Hallsworth; Vitor A. Martins dos Santos; Jaçek Puchalka; Joost van Duuren; Gerrit Eggink; Ken N. Timmis; Jan A. M. de Bont; Jan Wery (pp. 1133-1142).

Pseudomonas putida KT2440 is the only fully sequenced P. putida strain. Thus, for transcriptomics and proteomics studies with other P. putida strains, the P. putida KT2440 genomic database serves as standard reference. The utility of KT2440 whole-genome, high-density oligonucleotide microarrays for transcriptomics studies of other Pseudomonas strains was investigated. To this end, microarray hybridizations were performed with genomic DNAs of subcultures of P. putida KT2440 (DSM6125), the type strain (DSM291^T), plasmid pWW0-containing KT2440-derivative strain mt-2 (DSM3931), the solvent-tolerant P. putida S12, and several other Pseudomonas strains. Depending on the strain tested, 22 to 99% of all genetic elements were identified in the genomic DNAs. The efficacy of these microarrays to study cellular function was determined for all strains included in the study. The vast majority of DSM6125 genes encoding proteins of primary metabolism and genes involved in the catabolism of aromatic compounds were identified in the genomic DNA of strain S12: a prerequisite for reliable transcriptomics analyses. The genomotypic comparisons between Pseudomonas strains were used to construct highly discriminative phylogenetic relationships. DSM6125 and DSM3931 were indistinguishable and clustered together with strain S12 in a separate group, distinct from DSM291^T. Pseudomonas monteilii (DSM14164) clustered well with P. putida strains.

Keywords: Pseudomonas putida ; Transcriptomics; Genomotyping; Solvent-tolerant bacteria

Plant-growth-promoting compounds produced by two agronomically important strains of Azospirillum brasilense, and implications for inoculant formulation by D. Perrig; M. L. Boiero; O. A. Masciarelli; C. Penna; O. A. Ruiz; F. D. Cassán; M. V. Luna (pp. 1143-1150).

We evaluated phytohormone and polyamine biosynthesis, siderophore production, and phosphate solubilization in two strains (Cd and Az39) of Azospirillum brasilense used for inoculant formulation in Argentina during the last 20 years. Siderophore production and phosphate solubilization were evaluated in a chemically defined medium, with negative results. Indole 3-acetic acid (IAA), gibberellic acid (GA₃), and abscisic acid (ABA) production were analyzed by gas chromatography-mass spectrometry. Ethylene, polyamine, and zeatin (Z) biosynthesis were determined by gas chromatography-flame ionization detector and high performance liquid chromatography (HPLC-fluorescence and -UV), respectively. Phytohormones IAA, Z, GA₃, ABA, ethylene, and growth regulators putrescine, spermine, spermidine, and cadaverine (CAD) were found in culture supernatant of both strains. IAA, Z, and GA₃ were found in all two strains; however, their levels were significantly higher (p < 0.01) in Cd (10.8, 2.32, 0.66 μg ml⁻¹). ABA biosynthesis was significantly higher (p < 0.01) in Az39 (0.077 μg ml⁻¹). Ethylene and polyamine CAD were found in all two strains, with highest production in Cd cultured in NFb plus l-methionine (3.94 ng ml⁻¹ h⁻¹) and Az39 cultured in NFb plus l-lysine (36.55 ng ml⁻¹ h⁻¹). This is the first report on the evaluation of important bioactive molecules in strains of A. brasilense as potentially capable of direct plant growth promotion or agronomic yield increase. Az39 and Cd showed differential capability to produce the five major phytohormones and CAD in chemically defined medium. This fact has important technological implications for inoculant formulation as different concentrations of growth regulators are produced by different strains or culture conditions.

Keywords: Azospirillum ; Abscisic acid; Gibberellic acid; Indole 3-acetic acid; Ethylene; Cadaverine; Plant-growth-promoting rhizobacteria

Anaerobic co-metabolic oxidation of 4-alkylphenols with medium-length or long alkyl chains by Thauera sp., strain R5 by Atsushi Shibata; Arata Katayama (pp. 1151-1161).

A 4-alkylphenol-degrading facultative anaerobic bacterium, strain R5, was isolated from paddy soil after enrichment with 4-n-propylphenol, 4-n-butylphenol and 4-hydroxybenzoate (4-HBA) under nitrate-reducing conditions. Strain R5 is a Gram-negative rod bacillus grown on phenolic compounds with short alkyl chains (≤C2), organic acids and ethanol. The sequence of the 16S ribosomal RNA gene revealed that the strain is affiliated with Thauera sp. In the presence of 4-HBA as a carbon source, the strain transformed 4-n-alkylphenols with a medium or long-length alkyl chain (C3–C8) to the corresponding oxidised products as follows: 1-(4-hydroxyphenyl)-1-alkenes, -(4-hydroxyphenyl)-1-alkanones and/or 1-(4-hydroxyphenyl)-1-alcohols. The strain also transformed 4-i-propylphenol and 4-sec-butylphenol to (4-hydroxyphenyl)-i-propene and (4-hydroxyphenyl)-sec-butene but not 4-alkylphenols with tertiary alkyl chains (4-t-butylphenol or 4-t-octylphenol). The biotransformation did not proceed without another carbon source and was coupled with nitrate reduction. Biotransformation activity was high in the presence of p-cresol, 4-ethylphenol, 4′-hydroxyacetophenone and 4-HBA as carbon sources and low in the presence of organic acids and ethanol. We suggest that strain R5 co-metabolically transforms alkylphenols to the corresponding metabolites with oxidised alpha carbon in the alkyl chain during coupling with nitrate reduction.

Keywords: Anaerobic degradation; Nitrate reduction; Phenolic compound

Characterization of estrogen-degrading bacteria isolated from an artificial sandy aquifer with ultrafiltered secondary effluent as the medium by Jinxia Ke; Weiqin Zhuang; Karina Yew-Hoong Gin; Martin Reinhard; Lim Tok Hoon; Joo-Hwa Tay (pp. 1163-1171).

This study investigated the aerobic and anoxic biodegradation of four estrogens [estrone (E1), estradiol (E2), estriol (E3), and the synthetic 17α-ethinylestradiol (EE2)] in microcosms constructed with marine sand and ulftrafiltered (UF) secondary effluent. Three estrogen-degrading bacteria, LHJ1, LHJ3, and CYH, were isolated. Based on gram-stain morphology and 16S rRNA sequence homology, LHJ1 and LHJ3 belong to the genus Acinetobacter and Agromyces, respectively; CYH matched to 95% with the genus Sphingomonas. Aerobically LHJ3 degrades E3, CYH degrades E1, and all three isolates oxidize E2 to E1. Under anoxic conditions, CYH degrades E1 and LHJ3 degrades E2, whereas E3 and EE2 were not degraded by the three isolates; EE2 was transformed in microcosms incubated with site ground water. The degradation kinetics of E1 and E2 by CYH and E2 by LHJ3 under aerobic conditions was linearly correlated with the initial concentration, which ranged from 50 to 2,000 μg/l. The degradation of E1 by CYH under anoxic conditions followed Michaelis–Menten kinetics. 16α-Hydroxyestrone was found to be a transient transformation product of E3 under aerobic conditions.

Keywords: Estrogens; Biodegradation; Bacteria; Isolation; Tropical aquifer

Anaerobic growth and potential for amino acid production by nitrate respiration in Corynebacterium glutamicum by Seiki Takeno; Junko Ohnishi; Tomoha Komatsu; Tatsuya Masaki; Kikuo Sen; Masato Ikeda (pp. 1173-1182).

Oxygen limitation is a crucial problem in amino acid fermentation by Corynebacterium glutamicum. Toward this subject, our study was initiated by analysis of the oxygen-requiring properties of C. glutamicum, generally regarded as a strict aerobe. This organism formed colonies on agar plates up to relatively low oxygen concentrations (0.5% O₂), while no visible colonies were formed in the absence of O₂. However, in the presence of nitrate ( $${ ext{NO}}^{ - }_{{ ext{3}}} $$ ), the organism exhibited limited growth anaerobically with production of nitrite ( $${ ext{NO}}^{ - }_{{ ext{2}}} $$ ), indicating that C. glutamicum can use nitrate as a final electron acceptor. Assays of cell extracts from aerobic and hypoxic cultures yielded comparable nitrate reductase activities, irrespective of nitrate levels. Genome analysis revealed a narK2GHJI cluster potentially relevant to nitrate reductase and transport. Disruptions of narG and narJ abolished the nitrate-dependent anaerobic growth with the loss of nitrate reductase activity. Disruption of the putative nitrate/nitrite antiporter gene narK2 did not affect the enzyme activity but impaired the anaerobic growth. These indicate that this locus is responsible for nitrate respiration. Agar piece assays using l-lysine- and l-arginine-producing strains showed that production of both amino acids occurred anaerobically by nitrate respiration, indicating the potential of C. glutamicum for anaerobic amino acid production.

Keywords: Corynebacterium glutamicum ; Oxygen limitation; Nitrate reductase; Nitrate respiration; Amino acid production

Involvement of nitric oxide in cerebroside-induced defense responses and taxol production in Taxus yunnanensis suspension cells by Jian Wen Wang; Li Ping Zheng; Ren Xiang Tan (pp. 1183-1190).

This work was to characterize the generation of nitric oxide (NO) in Taxus yunnanensis cells induced by a fungal-derived cerebroside and the signal role of NO in the elicitation of plant defense responses and taxol production. (2S,2′R,3R,3′E,4E,8E)-1-O-β-d-glucopyranosyl-2-N-(2′-hydroxy-3′-octadecenoyl)-3-hydroxy-9-methyl-4,8-sphingadienine at 10 μg/ml induced a rapid and dose-dependent NO production in the Taxus cell culture, reaching a maximum within 5 h of the treatment. The NO donor sodium nitroprusside (SNP) potentiated cerebroside-induced H₂O₂ production and cell death. Inhibition of nitric oxide synthase activity by phenylene-1,3-bis(ethane-2-isothiourea) dihydrobromide or scavenging NO by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide partially blocked the cerebroside-induced H₂O₂ production and cell death. Moreover, NO enhanced cerebroside-induced activation of phenylalanine ammonium-lyase and accumulation of taxol in cell cultures. These results are suggestive of a role for NO as a new signal component for activating the cerebroside-induced defense responses and secondary metabolism activities of plant cells.

Keywords: Cerebroside; Nitric oxide; Oxidative responses; Phenylalanine ammonium-lyase; Taxol production; Taxus yunnanensis

Bioaugmentation and coexistence of two functionally similar bacterial strains in aerobic granules by He-Long Jiang; Abdul Majid Maszenan; Joo-Hwa Tay (pp. 1191-1200).

The survival of the inoculated microbial culture is critical for successful bioaugmentation but impossible to predict precisely. As an alternative strategy, bioaugmentation of a group of microorganisms may improve reliability of bioaugmentation. This study evaluated simultaneous bioaugmentation of two functionally similar bacterial strains in aerobic granules. The two strains, Pandoraea sp. PG-01 and Rhodococcus erythropolis PG-03, showed high phenol degradation and growth rates in phenol medium, but they were characterized as having a poor aggregation activity and weak bioflocculant-producing and biofilm-forming abilities. In the spatially homogeneous batch conditions, strain PG-01 with higher growth rates outcompeted strain PG-03. However, the two strains could stably coexist in the spatially heterogeneous conditions. Then the two strains were mixed and bioaugmented into activated sludge in two sequencing batch reactors, which were operated with the different settling times of 5 and 30 min, respectively. Aerobic granules were developed only in the reactor with a settling time of 5 min. Fluorescence in situ hybridization and denaturing gradient gel electrophoresis showed that the two strains could coexist in aerobic granules but not in activated sludge. These findings suggested that the compact structure of aerobic granules provided spatial isolation for coexistence of competitively superior and inferior strains with similar functions.

Keywords: Aerobic granules; Bioaugmentation; Coexistence; Competition; Phenol degradation; Spatial effect

Effects of ammonia on hydrolysis of proteins and lipids from fish residues by Fan Lü; Pin-Jing He; Li-Ming Shao; Duu-Jong Lee (pp. 1201-1208).

This study investigated the influence of ammonia on the hydrolysis rates of proteins and lipids in fish residues under mesophilic anaerobic incubation at a neutral pH. The hydrolysis kinetics of the fish residues, which contained primarily proteins and lipids, were examined at initial ammonia concentrations of 0–16 g N l⁻¹. Carbon hydrolysis was suppressed more by ammonium in the acidogenesis phase than in the acidogenesis/methanogenesis period of a single-stage anaerobic digestion. Conversely, hydrolysis of compounds containing nitrogen was similarly suppressed by ammonia during acidogenesis and acidogenesis/methanogenesis phases of a single-stage anaerobic digestion. Parameter uncertainty analysis demonstrated that the proteins fraction in the fish residues was entirely biodegradable. Model fitting demonstrated that two fractions of lipid substrates exist, namely, easy and hard to biodegrade with hydrolysis rates that were affected differently by ammonia content.

Keywords: Anaerobic digestion; Fermentation; Kinetic modeling; Biodegradability; Anaerobic digestion model no.1 (ADM1)

Preparing a highly specific inert immunomolecular-magnetic beads for rapid detection and separation of S. aureus and group G Streptococcus by Xiao Xiao; Xu Yang; Ting Liu; Zhang Chen; Lingli Chen; Huidong Li; Le Deng (pp. 1209-1216).

The rapid detection and separation of Staphylococcus aureus and group G Streptococcus was based on the affinity chromatography interactions between Fc fragment of human IgG and protein A/G (located on the cell wall of S. aureus and group G Streptococcus). In this case, immobilization of antibodies had to take place in a different and complementary way than in the case of conventional immunosensors. In this study, three different kinds of immunomolecular-magnetic beads (IMB) were prepared for rapid detection and separation of S. aureus and group G Streptococcus (GGS). The Fc regions of the immobilized antibodies were fully accessible to adsorb protein A or protein G. On the contrary, conventional immunosensors had to have fully accessible Fab regions to facilitate the antigen–antibody recognition. It was suggested that the worse method of immobilization of the antibodies for conventional use would yield the better results for this specific use. In this study, we also perfectly solved the nonspecific adsorptions and interaction problems, which were the most serious critical problems for all kinds of sensors. It was achieved by blocking the excess surface groups of aldehyde IMB and the Fab region of the immobilized antibodies with aldehyde-dextran.

Keywords: Detection and separation; Affinity chromatography interactions; Immunomolecular-magnetic beads (IMB); Aldehyde-dextran

An automated process to extract plasmid DNA by alkaline lysis by Xiaolin Li; Huali Jin; Zhifang Wu; Simon Rayner; Jiangmei Yin; Yang Yu; Wenjuan Zhang; Zhonghuai He; Chen Wang; Bin Wang (pp. 1217-1223).

With advances in the development of DNA vaccines and gene therapy, there is a growing need for plasmid DNA with high quality for fundamental research and clinical trials. In this report, a scalable automated process for large-scale preparation of plasmid is described. This process is based on alkaline lysis and can be easily scaled up to meet demands for larger quantities. In the process, harvested bacteria are passed through two mixing chambers at controlled speeds to affect lysis and control alkalinity. The resulting solution is passed through a series of filters to remove contaminants, and ethanol precipitated. System parameters are examined to maximize the quantity and quality of the prepared plasmid. Using this procedure, plasmid can be extracted and purified from 1 l of Escherichia coli cultures at an OD600 nm of 50 in less than 45 min. The plasmid yields are approximately 90 mg/l culture.

Keywords: Plasmid; Large-scale; Alkaline lysis; Automated process

Sections

Personal tools

Applied Microbiology and Biotechnology (v.75, #5)

Sections

Personal tools

Local resources

Applied Microbiology and Biotechnology (v.75, #5)