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Applied Microbiology and Biotechnology (v.75, #4)

Biotechnological production of gluconic acid: future implications by Om V. Singh; Raj Kumar (pp. 713-722).

Gluconic acid (GA) is a multifunctional carbonic acid regarded as a bulk chemical in the food, feed, beverage, textile, pharmaceutical, and construction industries. The favored production process is submerged fermentation by Aspergillus niger utilizing glucose as a major carbohydrate source, which accompanied product yield of 98%. However, use of GA and its derivatives is currently restricted because of high prices: about US$ 1.20–8.50/kg. Advancements in biotechnology such as screening of microorganisms, immobilization techniques, and modifications in fermentation process for continuous fermentation, including genetic engineering programmes, could lead to cost-effective production of GA. Among alternative carbohydrate sources, sugarcane molasses, grape must show highest GA yield of 95.8%, and banana must may assist reducing the overall cost of GA production. These methodologies would open new markets and increase applications of GA.

Keywords: Gluconic acid; Microbial fermentation; Glucose oxidase; Alternative carbohydrate sources; Biotechnology; System biology

HIV-1 reverse transcriptase inhibitors by Yazan El Safadi; Valérie Vivet-Boudou; Roland Marquet (pp. 723-737).

Reverse transcriptase (RT) is one of the three enzymes encoded by the human immunodeficiency virus type 1 (HIV-1), the etiological agent of AIDS. Together with protease inhibitors, drugs inhibiting the RNA- and DNA-dependant DNA polymerase activity of RT are the major components of highly active antiretroviral therapy (HAART), which has dramatically reduced mortality and morbidity of people living with HIV-1/AIDS in developed countries. In this study, we focus on RT inhibitors approved by the US Food and Drugs Administration (FDA) or in phases II and III clinical trials. RT inhibitors belong to two main classes acting by distinct mechanisms. Nucleoside RT inhibitors (NRTIs) lack a 3′ hydroxyl group on their ribose or ribose mimic moiety and thus act as chain terminators. Non-NRTIs bind into a hydrophobic pocket close to the polymerase active site and inhibit the chemical step of the polymerization reaction. For each class of inhibitors, we review the mechanism of action, the resistance mechanisms selected by the virus, and the side effects of the drugs. We also discuss the main perspectives for the development of new RT inhibitors.

From scratch to value: engineering Escherichia coli wild type cells to the production of l-phenylalanine and other fine chemicals derived from chorismate by Georg A. Sprenger (pp. 739-749).

Recombinant strains of Escherichia coli K-12 for the production of the three aromatic amino acids (l-phenylalanine, l-tryptophan, l-tyrosine) have been constructed. The largest demand is for l-phenylalanine (l-Phe), as it can be used as a building block for the low-calorie sweetener, aspartame. Besides l-Phe, an increasing number of shikimic acid pathway intermediates can be produced from appropriate E. coli mutants with blocks in this pathway. The last common intermediate, chorismate, in E. coli not only serves for production of aromatic amino acids but can also be used for high-titer production of non-aromatic compounds, e.g., cyclohexadiene-transdiols. In an approach to diversity-oriented metabolic engineering (metabolic grafting), platform strains with increased flux through the general aromatic pathway were created by suitable gene deletions, additions, or rearrangements. Examples for rational strain constructions for l-phenylalanine and chorismate derivatives are given with emphasis on genetic engineering. As a result, l-phenylalanine producers are available, which were derived through several defined steps from E. coli K-12 wild type. These mutant strains showed l-phenylalanine titers of up to 38 g/l of l-phenylalanine (and up to 45.5 g/l using in situ product recovery). Likewise, two cyclohexadiene-transdiols could be recovered.

Biomass in the manufacture of industrial products—the use of proteins and amino acids by Elinor Scott; Francisc Peter; Johan Sanders (pp. 751-762).

The depletion in fossil feedstocks, increasing oil prices, and the ecological problems associated with CO₂ emissions are forcing the development of alternative resources for energy, transport fuels, and chemicals: the replacement of fossil resources with CO₂ neutral biomass. Allied with this, the conversion of crude oil products utilizes primary products (ethylene, etc.) and their conversion to either materials or (functional) chemicals with the aid of co-reagents such as ammonia and various process steps to introduce functionalities such as -NH₂ into the simple structures of the primary products. Conversely, many products found in biomass often contain functionalities. Therefore, it is attractive to exploit this to bypass the use, and preparation of, co-reagents as well as eliminating various process steps by utilizing suitable biomass-based precursors for the production of chemicals. It is the aim of this mini-review to describe the scope of the possibilities to generate current functionalized chemical materials using amino acids from biomass instead of fossil resources, thereby taking advantage of the biomass structure in a more efficient way than solely utilizing biomass for the production of fuels or electricity.

Biotransformation of piceid in Polygonum cuspidatum to resveratrol by Aspergillus oryzae by H. Wang; L. Liu; Y.-X. Guo; Y.-S. Dong; D.-J. Zhang; Z.-L. Xiu (pp. 763-768).

Biotransformation of piceid in Polygonum cuspidatum to resveratrol by Aspergillus oryzae was investigated in this study. Resveratrol is widely used in medicine, food, and cosmetic because of its pharmacological properties. However, it has a much lower content in plants compared with its glucoside piceid, which has a much lower bioavailability. Traditionally, the aglycone is acquired by acid or enzymatic hydrolysis of its glucoside, but the violent condition and the acid pollution in hydrolytic reaction and the high cost of the enzyme limit their industrial development. In this paper, fermentation of P. cuspidatum by A. oryzae was successfully performed, during which, piceid was converted to resveratrol with the highest yield of trans-resveratrol 1.35%, 3.6 times higher than that obtained from raw herb by microwave-assisted extraction. Scale-up production was also performed and the yield of trans-resveratrol was 3.1 times higher after 24 h incubation. Therefore, biotransformation is a better method to increase the yield of resveratrol because of its high yield and mild conditions.

Keywords: Biotransformation; Resveratrol; Piceid; Aspergillus oryzae ; Polygonum cuspidatum

Immunomodulatory effect of exo-polysaccharides from submerged cultured Cordyceps sinensis: enhancement of cytokine synthesis, CD11b expression, and phagocytosis by Mei-Chun Kuo; Chien-Yu Chang; Tso-Lin Cheng; Ming-Jiuan Wu (pp. 769-775).

Cordyceps sinensis is widely used as a traditional medicine for treatment of a wide variety of diseases or to maintain health. The immunomodulatory activity of polysaccharides prepared from submerged cultured C. sinensis BCRC36421 was investigated in human peripheral blood. Results demonstrated that Fr. A (exo-polysaccharides, 0.025 ∼ 0.1 mg/ml) induced the production of tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, and IL-10 dose-dependently. Fr. A, as low as 0.025 mg/ml, could significantly augment surface expression of CD11b in monocytes and polymorphonuclear neutrophils. Functional assay revealed that Fr. A (0.05 mg/ml) also elevated phagocytosis in monocytes and PMN. On the other hand, Fr. B (intracellular polysaccharides) only moderately induced TNF-α release, CD11b expression, and phagocytosis at the same concentrations. Our results indicate that the immunomodulatory components of submerged cultured C. sinensis mainly reside in the culture filtrate.

Keywords: Cordyceps sinensis ; Immunomodulatory effect; Phagocytosis; Cytokine; CD11b

5-Aminolevulinate production with recombinant Escherichia coli using a rare codon optimizer host strain by Weiqi Fu; Jianping Lin; Peilin Cen (pp. 777-782).

The 5-aminolevulinate (ALA) synthase gene (hemA) containing several codons rarely used by Escherichia coli was cloned from the genome of Rhodobacter sphaeroides and optimized in two strains of Escherichia coli: BL21(DE3) and Rosetta(DE3), which is a rare codon optimizer strain. The effects of initial isopropyl-β-d-thiogalactopyranoside (IPTG) concentration, induction time, and temperature on enzyme activity were studied and compared for two strains. The results indicated that the ALA synthase expressed by Rosetta(DE3)/pET-28a(+)-hemA was higher than that by BL21(DE3)/pET-28a(+)-hemA. The initial precursors, glycine and succinate, and initial glucose, which is an inhibitor for both ALA synthase and dehydratase, were observed to be the key factors affecting ALA production. ALA synthase activity was generally higher with Rosetta(DE3) than with BL21(DE3), so was ALA biosynthesis. Based on the optimal culture system using Rosetta(DE3), the yield of ALA achieved 3.8 g/l (29 mM) under the appropriate conditions in fermenter.

Astaxanthin production by Phaffia rhodozyma and Haematococcus pluvialis: a comparative study by A. R. Domínguez-Bocanegra; T. Ponce-Noyola; J. A. Torres-Muñoz (pp. 783-791).

Phaffia rhodozyma (now Xanthophyllomyces dendrorhous) and Haematococcus pluvialis are known as the major prominent microorganisms able to synthesize astaxanthin natural pigment. Important research efforts have been made to determine optimal conditions for astaxanthin synthesis. When the focus is on astaxanthin production, the maximal reported value of 9.2 mg/g cell is obtained within H. pluvialis grown on BAR medium, under continuous illumination (345 μmol photon m⁻² s⁻¹) and without aeration. Whereas fermentation by mutated R1 yeast grown on coconut milk produced 1,850 μg/g yeast. However, when looking at astaxanthin productivity, the picture is slightly different. The figures obtained with P. rhodozyma are rather similar to those of H. pluvialis. Maximal reported values are 170 μg/g yeast per day with a wild yeast strain and 370 μg/g yeast per day with mutated R1 yeast. In the case of H. pluvialis, maximal values ranged from 290 to 428 μg/g cell per day depending on the media (BG-11 or BAR), light intensity (177 μmol photon m⁻² s⁻¹), aeration, etc. The main aim of this work was to examine how astaxanthin synthesis, by P. rhodozyma and H. pluvialis, could be compared. The study is based on previous works by the authors where pigment productions have been reported.

Keywords: Haematococcus pluvialis ; Phaffia rhodozyma ; Astaxanthin synthesis; Coconut milk

Optimization of cellulase complex formulation for peashrub biomass hydrolysis by Debing Jing; Peijun Li; Xian-Zhe Xiong; Lihua Wang (pp. 793-800).

To improve efficiency and reduce cost, solid state simultaneous saccharification and fermentation of peashrub woody biomass was investigated under anaerobic conditions at 50°C, with a cellulase–inoculant mixture consisting of Trichoderma koningii cellulase, Aspergillus niger cellulase, and Lactobacillus. Experimental formulations were prepared according to uniform prescription design principles. By crude protein, crude fiber models constructed using multivariate regression in SPSS and solutions analysis through unconstrained mathematical optimization in Microsoft Excel, it was clearly revealed that low pH value (3.8) from lactic acid accumulation produced by Lactobacillus would ultimately limit enzymatic hydrolysis during long-term fermentation (30 days). It was shown that a cellulase complex with filter paper cellulase/carboxymethyl cellulase/cotton lyase/β-glucosidase/pectinase of activity ratios of 0.6:1:0.3:1:2.6 could effectively break peashrub cell wall structure by biodegradation of easily digested components and, then, release cellular contents to improve crude protein content. Thus, the enzymatic hydrolysis of peashrub biomass by the optimized cellulase complex could improve crude protein content by 45.3% (from 8.45 to 12.28%), although it only biodegraded about 10.90% of the crude fiber (from 44.45 to 40.08%).

Keywords: Simultaneous saccharification and fermentation; Cellulase–inoculant mixture; Peashrub woody biomass; Crude protein; Crude fiber; Lactobacillus spp.

A novel thermostable nitrilase superfamily amidase from Geobacillus pallidus showing acyl transfer activity by H. S. Makhongela; A. E. Glowacka; V. B. Agarkar; B. T. Sewell; B. Weber; R. A. Cameron; D. A. Cowan; S. G. Burton (pp. 801-811).

An amidase (EC 3.5.1.4) in branch 2 of the nitrilase superfamily, from the thermophilic strain Geobacillus pallidus RAPc8, was produced at high expression levels (20 U/mg) in small-scale fermentations of Escherichia coli. The enzyme was purified to 90% homogeneity with specific activity of 1,800 U/mg in just two steps, namely, heat-treatment and gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electron microscopic (EM) analysis of the homogenous enzyme showed the native enzyme to be a homohexamer of 38 kDa subunits. Analysis of the biochemical properties of the amidase showed that the optimal temperature and pH for activity were 50 and 7.0°C, respectively. The amidase exhibited high thermal stability at 50 and 60°C, with half-lives greater than 5 h at both temperatures. At 70 and 80°C, the half-life values were 43 and 10 min, respectively. The amidase catalyzed the hydrolysis of low molecular weight aliphatic amides, with d-selectivity towards lactamide. Inhibition studies showed activation/inhibition data consistent with the presence of a catalytically active thiol group. Acyl transfer reactions were demonstrated with acetamide, propionamide, isobutyramide, and acrylamide as substrates and hydroxylamine as the acyl acceptor; the highest reaction rate being with isobutyramide. Immobilization by entrapment in polyacrylamide gels, covalent binding on Eupergit C beads at 4°C and on Amberlite-XAD57 resulted in low protein binding and low activity, but immobilization on Eupergit C beads at 25°C with cross-linking resulted in high protein binding yield and high immobilized specific activity (80% of non-immobilized activity). Characterization of Eupergit C-immobilized preparations showed that the optimum reaction temperature was unchanged, the pH range was somewhat broadened, and stability was enhanced giving half-lives of 52 min at 70°C and 30 min at 80°C. The amidase has potential for application under high temperature conditions as a biocatalyst for d-selective amide hydrolysis producing enantiomerically pure carboxylic acids and for production of novel amides by acyl transfer.

Keywords: Amidase; Enantioselectivity; Substrate specificity; Thermostable; Characterization; Immobilization

Purification and characterization of a highly thermostable α-l-Arabinofuranosidase from Geobacillus caldoxylolyticus TK4 by Sabriye Canakci; Ali Osman Belduz; Badal C. Saha; Ahmet Yasar; Faik Ahmet Ayaz; Nurettin Yayli (pp. 813-820).

The gene encoding an α-l-arabinofuranosidase from Geobacillus caldoxylolyticus TK4, AbfATK4, was isolated, cloned, and sequenced. The deduced protein had a molecular mass of about 58 kDa, and analysis of its amino acid sequence revealed significant homology and conservation of different catalytic residues with α-l-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. A histidine tag was introduced at the N-terminal end of AbfATK4, and the recombinant protein was expressed in Escherichia coli BL21, under control of isopropyl-β-D-thiogalactopyranoside-inducible T7 promoter. The enzyme was purified by nickel affinity chromatography. The molecular mass of the native protein, as determined by gel filtration, was about 236 kDa, suggesting a homotetrameric structure. AbfATK4 was active at a broad pH range (pH 5.0–10.0) and at a broad temperature range (40–85°C), and it had an optimum pH of 6.0 and an optimum temperature of 75–80°C. The enzyme was more thermostable than previously described arabinofuranosidases and did not lose any activity after 48 h incubation at 70°C. The protein exhibited a high level of activity with p-nitrophenyl-α-l-arabinofuranoside, with apparent K _m and V _max values of 0.17 mM and 588.2 U/mg, respectively. AbfATK4 also exhibited a low level of activity with p-nitrophenyl-β-d-xylopyranoside, with apparent K _m and V _max values of 1.57 mM and 151.5 U/mg, respectively. AbfATK4 released l-arabinose only from arabinan and arabinooligosaccharides. No endoarabinanase activity was detected. These findings suggest that AbfATK4 is an exo-acting enzyme.

Keywords: α-l-Arabinofuranosidase; Geobacillus caldoxylolyticus ; Exo-acting; Thermostable; Xylosidase

Construction of a novel synergistic system for production and recovery of secreted recombinant proteins by the cell surface engineering by Seiji Shibasaki; Ai Kawabata; Jun Ishii; Shunsuke Yagi; Tetsuya Kadonosono; Michiko Kato; Nobuo Fukuda; Akihiko Kondo; Mitsuyoshi Ueda (pp. 821-828).

We determined whether the cocultivation of yeast cells displaying a ZZ-domain and secreting an Fc fusion protein can be a novel tool for the recovery of secreted recombinant proteins. The ZZ-domain from Staphylococcus aureus protein A was displayed on the cell surface of Saccharomyces cerevisiae under the control of the GAL1 promoter. Strain S. cerevisiae BY4742 cells displaying the ZZ-domain on their surface were used for cocultivation with cells that produce a target protein fused to the Fc fragment as an affinity tag. The enhanced green fluorescent protein or Rhizopus oryzae lipase was genetically fused to the N and C termini of the Fc fragment of human immunoglobulin G, respectively. Through analysis by fluorescence-activated cell sorting and enzymatic assay, it was demonstrated that these fusion proteins are successfully produced in the medium and recovered by affinity binding with the cell surface displaying the ZZ-domain. These results suggest that the ZZ-domain-displaying cell and Fc fusion protein-secreting cell can be applied to use in synergistic process of production and recovery of secreted recombinant proteins.

Keywords: ZZ-domain; Fc fragment; EGFP; Cell surface display; Secreted proteins

Influence of solubility-enhancing fusion proteins and organic solvents on the in vitro biocatalytic performance of the carotenoid cleavage dioxygenase AtCCD1 in a micellar reaction system by Martin Schilling; Frauke Patett; Wilfried Schwab; Jens Schrader (pp. 829-836).

The influence of the solubility-enhancing fusion proteins glutathione-S-transferase (GST) and NusA on the heterologous expression and in vitro biocatalytic performance of the carotenoid cleavage dioxygenase AtCCD1 from Arabidopsis thaliana was investigated. A micellar dispersion of the water-insoluble model substrate β-apo-8′-carotenal in combination with Triton X-100 was used for the spectrophotometric in vitro assays. Specific activity in the cellular extract was twofold increased by the use of GST as a carrier protein, whereas it was decreased by 70% when fused with NusA. Reduced molar activity of the purified fusion proteins was observed, which could not be regained by proteolytic removal of the carrier protein. The addition of organic solvents in the form of short-chain aliphatic alcohols positively influenced the enzyme activity. Optimization of the reaction medium led to an 18-fold activation, and a clear correlation could be found between the organic solvent concentration required for maximum activation and the log P of the solvent. The results provide a foundation for the development towards the application of carotenoid cleavage dioxygenases as in vitro biocatalysts for the production of norisoprenoids and apocarotenals from carotenoids.

Keywords: Carotenoid cleavage dioxygenase; Micellar reaction system; Fusion protein; Natural flavours; Hydrophobic substrate

Improved production of erythromycin A by expression of a heterologous gene encoding S-adenosylmethionine synthetase by Yong Wang; YiGuang Wang; Ju Chu; Yingping Zhuang; Lixin Zhang; Siliang Zhang (pp. 837-842).

An S-adenosylmethionine synthetase (SAM-s) gene from Streptomyces spectabilis was integrated along with vector DNA into the chromosome of a Saccharopolyspora erythraea E2. Elevated production of SAM was observed in the recombinant strain Saccharopolyspora erythraea E1. The results from the bioassay showed that the titer of erythromycin was increased from 920 IU ml⁻¹ by E₂ to approximately 2,000 IU ml⁻¹ by E₁. High performance liquid chromatography (HPLC) analysis revealed that there was a 132% increase in erythromycin A compared with the original strain, while the erythromycin B, the main impurity component in erythromycin, was decreased by 30%. The sporulation process was inhibited, while the SAM-s gene was expressed. The addition of the exogenous SAM also inhibited sporulation and promoted an increase in erythromycin titers.

Keywords: S-adenosylmethionine synthetase; Erythromycin A; Precision engineering; Saccharopolyspora erythraea

Characterization of the dszABC genes of Gordonia amicalis F.5.25.8 and identification of conserved protein and DNA sequences by John J. Kilbane II; John Robbins (pp. 843-851).

Gordonia amicalis F.5.25.8 has the unique ability to desulfurize dibenzothiophene and to metabolize carbazole [Santos et al., Appl Microbiol Biotechnol 71:355–362, 2006]. Efforts to amplify the dsz genes from G. amicalis F.5.25.8 based on polymerase chain reaction (PCR) primers designed using the dsz gene sequences of Rhodococcus erythropolis IGTS8 were mostly unsuccessful. A comparison of the protein sequences of dissimilar desulfurization enzymes (DszABC, BdsABC, and TdsABC) revealed multiple conserved regions. PCR primers targeting some of the most highly conserved regions of the desulfurization genes allowed us to amplify dsz genes from G. amicalis F.5.25.8. DNA sequence data that include nearly the entirety of the desulfurization operon as well as the promoter region were obtained. The most closely related dsz genes are those of G. alkinovorans strain 1B at 85% identity. The PCR primers reported here should be useful in microbial ecology studies and the amplification of desulfurization genes from previously uncharacterized microbial cultures.

Keywords: Gordonia ; Desulfurization; Biodesulfurization; dsz ; tds ; bds

Generation, annotation, and analysis of ESTs from four different Trichoderma strains grown under conditions related to biocontrol by Juan Antonio Vizcaíno; José Redondo; M. Belén Suárez; Rosa Elena Cardoza; Rosa Hermosa; Francisco Javier González; Manuel Rey; Enrique Monte (pp. 853-862).

The functional genomics project “TrichoEST” was developed focused on different taxonomic groups of Trichoderma with biocontrol potential. Four cDNA libraries were constructed, using similar growth conditions, from four different Trichoderma strains: Trichoderma longibrachiatum T52, Trichoderma asperellum T53, Trichoderma virens T59, and Trichoderma sp. T78. In this study, we present the analysis of the 8,160 expressed sequence tags (ESTs) generated. Each EST library was independently assembled and 1,000–1,300 unique sequences were identified in each strain. First, we queried our collection of ESTs against the NCBI nonredundant database using the BLASTX algorithm. Moreover, using the Gene Ontology hierarchy, we performed the annotation of 40.9% of the unique sequences. Later, based on the EST abundance, we examined the highly expressed genes in the four strains. A hydrophobin was found as the gene expressed at the highest level in two of the strains, but we also found that other unique sequences similar to the HEX1, QID3, and NMT1 proteins were highly represented in at least two of the Trichoderma strains.

Keywords: Functional genomics; Biological control; Mycoparasitism

A polysaccharide–peptide complex from abalone mushroom (Pleurotus abalonus) fruiting bodies increases activities and gene expression of antioxidant enzymes and reduces lipid peroxidation in senescence-accelerated mice by L. Li; T. B. Ng; M. Song; F. Yuan; Z. K. Liu; C. L. Wang; Y. Jiang; M. Fu; F. Liu (pp. 863-869).

The antioxidant effects of a polysaccharide–peptide complex (F22) from mushroom (Pleurotus abalonus)-fruiting bodies were studied. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) in the liver, kidney, and brain of senescence-accelerated mice showed a marked increase after treatment with the polysaccharide–peptide complex. Concurrently, the gene expression levels of SOD, CAT, and GPx, as determined with real-time polymerase chain reaction, were up-regulated in the liver, kidney, and brain, whereas the MDA content in these organs declined. The maximal lifespan of the mice was prolonged.

Keywords: Superoxide dismutase; Catalase; Glutathione peroxidase; Lipid peroxidation; Real-time PCR

Biosynthesis of polyhydroxybutyrate (PHB) and extracellular polymeric substances (EPS) by Ralstonia eutropha ATCC 17699 in batch cultures by Jin Wang; Han-Qing Yu (pp. 871-878).

The production of polyhydroxybutyrate (PHB) and extracellular polymeric substances (EPS) by Ralstonia eutropha ATCC 17699 at various glucose and (NH₄)₂SO₄ concentrations in batch cultures were investigated. The biosynthesis of EPS by R. eutropha closely coupled with cell growth, while PHB was synthesized only under nitrogen-deficient and cell-growth-limited conditions. Experimental results show that the specific PHB production rate had an exponential correlation with both specific cell growth rate and EPS production rate. Furthermore, PHB was observed as the main storage of carbon and energy source by R. eutropha under nitrogen-limited conditions. In addition, experiments were conducted based on central composite design to optimize the batch culture for a high PHB yield. The PHB yield on glucose reached a maximum value of 0.34 g/g at glucose concentrations of 38.2 g/l and (NH₄)₂SO₄ of 3.2 g/l.

Differential expression of carotenogenic genes and associated changes in pigment profile during regeneration of Haematococcus pluvialis cysts by Raman Vidhyavathi; Lakshmanan Venkatachalam; Burde Sandesh Kamath; Ravi Sarada; Gokare Aswathanarayana Ravishankar (pp. 879-887).

Haematococcus pluvialis is a green alga known to accumulate astaxanthin in extra-plastidic lipid vesicles under stress conditions. The present study revealed the influence of few cultural parameters and temperature treatments on regeneration efficiency of red cysts along with changes in pigment profile and expression of carotenogenic genes during regeneration. Regeneration efficiency has been improved by incubating less aged cyst cells in a medium containing ammonium carbonate, 16:8 light–dark cycle with a light intensity of 30 μmol m⁻² s⁻¹. During regeneration, there was a decrease in total astaxanthin, total carotenoids, and carotenoid to chlorophyll ratio, and increase in β-carotene, lutein, total chlorophyll, and chlorophyll a to b ratio. Expression analysis revealed the presence of transcripts of carotenogenic genes, phytoene synthase (PSY), phytoene desaturase (PDS), lycopene cyclase (LCY), β-carotene ketolase (BKT), and β-carotene hydroxylase (CHY) in cyst cells, and these transcripts were up regulated transiently upon transfer to favorable conditions. As the culture growth progressed, carotenogenic gene expressions were decreased and reached basal expression levels of green motile vegetative cells. In addition, this is the first report of detection of carotenogenic gene transcripts in red cysts, and their differential expression during regeneration. The present study suggests the use of red cysts as alternate inoculum for mass cultivation to combat protozoan predation.

Keywords: Astaxanthin; Carotenoid biosynthesis genes; Carotenoids; Extractability; Haematococcus pluvialis

Anaerobic growth of Corynebacterium glutamicum using nitrate as a terminal electron acceptor by Taku Nishimura; Alain A. Vertès; Yoshifumi Shinoda; Masayuki Inui; Hideaki Yukawa (pp. 889-897).

Corynebacterium glutamicum, a gram-positive soil bacterium, has been regarded as an aerobe because its growth by fermentative catabolism or by anaerobic respiration has, to this date, not been demonstrated. In this study, we report on the anaerobic growth of C. glutamicum in the presence of nitrate as a terminal electron acceptor. C. glutamicum strains R and ATCC13032 consumed nitrate and excreted nitrite during growth under anaerobic, but not aerobic, conditions. This was attributed to the presence of a narKGHJI gene cluster with high similarity to the Escherichia coli narK gene and narGHJI operon. The gene encodes a nitrate/nitrite transporter, whereas the operon encodes a respiratory nitrate reductase. Transposonal inactivation of C. glutamicum narG or narH resulted in mutants with impaired anaerobic growth on nitrate because of their inability to convert nitrate to nitrite. Further analysis revealed that in C. glutamicum, narK and narGHJI are cotranscribed as a single narKGHJI operon, the expression of which is activated under anaerobic conditions in the presence of nitrate. C. glutamicum is therefore a facultative anaerobe.

Keywords: Corynebacterium glutamicum ; Anaerobic growth; Nitrate/nitrite; Respiratory nitrate reductase; narKGHJI operon; Facultative anaerobe

Isolation and characterization of a nitrile hydrolysing acidotolerant black yeast—Exophiala oligosperma R1 by Sven Rustler; Andreas Stolz (pp. 899-908).

Different nitriles were used as sole sources of nitrogen in a series of enrichments under acidic conditions to isolate acidotolerant nitriles hydrolysing microorganisms. From an enrichment in Na–citrate–phosphate buffer at pH 4 with glucose as carbon source and phenylacetonitrile as sole source of nitrogen, a black yeast (strain R1) was obtained which was identified by subsequent 18S rRNA gene sequencing as Exophiala oligosperma. The growth conditions of the organism were optimized for the production of cell material and the induction of the nitrile converting activity. Resting cell experiments demonstrated that phenylacetonitrile was converted via phenylacetic acid and 2-hydroxyphenylacetic acid. The organism could grow at pH 4 with phenylacetonitrile as sole source of carbon, nitrogen, and energy. The nitriles hydrolysing activity was also detected in cell-free extracts and indications for a nitrilase activity were found. The cell-free extracts converted, in addition to phenylacetonitrile, also different substituted phenylacetonitriles. Whole cells of E. oligosperma R1 converted phenylacetonitrile with almost the same reaction rates in the pH range from pH 1.5–pH 9.

Keywords: Nitrilase; Acidotolerance; Black yeasts; Exophiala

n-Alkane assimilation and tert-butyl alcohol (TBA) oxidation capacity in Mycobacterium austroafricanum strains by Nicolas Lopes Ferreira; Hugues Mathis; Diane Labbé; Frédéric Monot; Charles W. Greer; Françoise Fayolle-Guichard (pp. 909-919).

Mycobacterium austroafricanum IFP 2012, which grows on methyl tert-butyl ether (MTBE) and on tert-butyl alcohol (TBA), the main intermediate of MTBE degradation, also grows on a broad range of n-alkanes (C₂ to C₁₆). A single alkB gene copy, encoding a non-heme alkane monooxygenase, was partially amplified from the genome of this bacterium. Its expression was induced after growth on n-propane, n-hexane, n-hexadecane and on TBA but not after growth on LB. The capacity of other fast-growing mycobacteria to grow on n-alkanes (C₁ to C₁₆) and to degrade TBA after growth on n-alkanes was compared to that of M. austroafricanum IFP 2012. We studied M. austroafricanum IFP 2012 and IFP 2015 able to grow on MTBE, M. austroafricanum IFP 2173 able to grow on isooctane, Mycobacterium sp. IFP 2009 able to grow on ethyl tert-butyl ether (ETBE), M. vaccae JOB5 (M. austroaafricanum ATCC 29678) able to degrade MTBE and TBA and M. smegmatis mc2 155 with no known degradation capacity towards fuel oxygenates. The M. austroafricanum strains grew on a broad range of n-alkanes and three were able to degrade TBA after growth on propane, hexane and hexadecane. An alkB gene was partially amplified from the genome of all mycobacteria and a sequence comparison demonstrated a close relationship among the M. austroafricanum strains. This is the first report suggesting the involvement of an alkane hydroxylase in TBA oxidation, a key step during MTBE metabolism.

Keywords: Mycobacterium austroafricanum ; Alkane hydroxylase; tert-Butyl alcohol (TBA) Oxidation; n-Alkanes; alkB Gene expression; MTBE; Metabolism

Isolation and characterization of novel atrazine-degrading microorganisms from an agricultural soil by Laurel L. Vibber; Michael J. Pressler; Gregory M. Colores (pp. 921-928).

Six previously undescribed microorganisms capable of atrazine degradation were isolated from an agricultural soil that received repeated exposures of the commonly used herbicides atrazine and acetochlor. These isolates are all Gram-positive and group with microorganisms in the genera Nocardioides and Arthrobacter, both of which contain previously described atrazine degraders. All six isolates were capable of utilizing atrazine as a sole nitrogen source when provided with glucose as a separate carbon source. Under the culture conditions used, none of the isolates could utilize atrazine as the sole carbon and nitrogen source. We used several polymerase-chain-reaction-based assays to screen for the presence of a number of atrazine-degrading genes and verified their identity through sequencing. All six isolates contain trzN and atzC, two well-characterized genes involved in the conversion of atrazine to cyanuric acid. An additional atrazine-degrading gene, atzB, was detected in one of the isolates as well, yet none appeared to contain atzA, a commonly encountered gene in atrazine impacted soils and atrazine-degrading isolates. Interestingly, the deoxyribonucleic acid sequences of trzN and atzC were all identical, implying that their presence may be the result of horizontal gene transfer among these isolates.

Keywords: Atrazine; Nocardioides; Arthrobacter; TrzN; AtzA; AtzC

Development of a 60-mer oligonucleotide microarray on the basis of benzene monooxygenase gene diversity by Shoko Iwai; Futoshi Kurisu; Hidetoshi Urakawa; Osami Yagi; Hiroaki Furumai (pp. 929-939).

We constructed a 60-mer oligonucleotide microarray on the basis of benzene monooxygenase gene diversity to develop a new technology for simultaneous detection of the functional gene diversity in environmental samples. The diversity of the monooxygenase genes associated with benzene degradation was characterized. A new polymerase chain reaction (PCR) primer set was designed using conserved regions of benzene monooxygenase gene (BO12 primer) and used for PCR-clone library analysis along with a previously designed RDEG primer which targeted the different types of benzene monooxygenase gene. We obtained 20 types of amino acid sequences with the BO12 primer and 40 with the RDEG primer. Phylogenetic analysis of the sequences obtained suggested the large diversity of the benzene monooxygenase genes. A total of 87 60-mer probes specific for each operational taxonomical unit were designed and spotted on a microarray. When genomic DNAs of single strains were used in microarray hybridization assays, corresponding sequences were successfully detected by the microarray without any false-negative signals. Hybridization with soil DNA samples showed that the microarray was able to detect sequences that were not detected in clone libraries. Constructed microarray can be a useful tool for characterizing monooxygenase gene diversity in benzene degradation.

Microbial population in the biomass adhering to supporting material in a packed-bed reactor degrading organic solid waste by Kengo Sasaki; Shin Haruta; Yoshiyuki Ueno; Masaharu Ishii; Yasuo Igarashi (pp. 941-952).

An anaerobic packed-bed reactor using carbon fiber textiles (CFT) as the supporting material was continuously operated using an artificial garbage slurry. 16S rRNA gene analysis showed that many bacteria in the biomass adhering to CFT were closely related to those observed from other anaerobic environments, although a wide variety of unidentified bacteria were also found. Dot blot hybridization results clarified that 16S rRNA levels of methanogens in the adhering biomass were higher than those in the effluent. Based on microscopic observation, the adhering biomass consisted of microorganisms, organic material, and void areas. Bacteria and Archaea detected by fluorescence in situ hybridization were distributed from the surface to the inner regions of the adhering biomass. Methanosarcina sp. tended to be more abundant in the inner part of the adhering biomass than at the surface. This is the first report to elucidate the structure of the microbial community on CFT in a packed-bed reactor.

Keywords: Packed-bed reactor; Microbial community; Organic solid waste

Fabrication and application of carbohydrate microarray for analyzing human serum antibody–carbohydrate interaction by Gang-Liang Huang; Xin-Ya Mei; Han-Xing Zhang; Peng-George Wang (pp. 953-953).

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Applied Microbiology and Biotechnology (v.75, #4)

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Applied Microbiology and Biotechnology (v.75, #4)