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Applied Microbiology and Biotechnology (v.66, #6)
Gibberellin biosynthesis in fungi: genes, enzymes, evolution, and impact on biotechnology by Bettina Tudzynski (pp. 597-611).
Gibberellins (GAs) constitute a large family of tetracyclic diterpenoid carboxylic acids, some members of which function as growth hormones in higher plants. As well as being phytohormones, GAs are also present in some fungi and bacteria. In recent years, GA biosynthetic genes from Fusarium fujikuroi and Arabidopsis thaliana have been cloned and well characterised. Although higher plants and the fungus both produce structurally identical GAs, there are important differences indicating that GA biosynthetic pathways have evolved independently in higher plants and fungi. The fact that horizontal gene transfer of GA genes from the plant to the fungus can be excluded, and that GA genes are obviously missing in closely related Fusarium species, raises the question of the origin of fungal GA biosynthetic genes. Besides characterisation of F. fujikuroi GA pathway genes, much progress has been made in the molecular analysis of regulatory mechanisms, especially the nitrogen metabolite repression controlling fungal GA biosynthesis. Basic research in this field has been shown to have an impact on biotechnology. Cloning of genes, construction of knock-out mutants, gene amplification, and regulation studies at the molecular level are powerful tools for improvement of production strains. Besides increased yields of the final product, GA3, it is now possible to produce intermediates of the GA biosynthetic pathway, such as ent-kaurene, ent-kaurenoic acid, and GA14, in high amounts using different knock-out mutants. This review concentrates mainly on the fungal biosynthetic pathway, the genes and enzymes involved, the regulation network, the biotechnological relevance of recent studies, and on evolutionary aspects of GA biosynthetic genes.
Inhibitors of virus replication: recent developments and prospects by Julia Magden; Leevi Kääriäinen; Tero Ahola (pp. 612-621).
The search for inhibitors of viral replication is dependent on understanding the events taking place at the molecular level during viral infection. All the essential steps during the viral life cycle are potential targets for antiviral drugs. Classical inhibitors of herpesvirus replication cause chain termination during viral DNA replication. Similarly, the HIV reverse transcriptase is the major target of anti-HIV compounds. The broad-spectrum antiviral agent ribavirin affects viral nucleic acid replication by multiple mechanisms. Another major enzyme encoded by many viruses is a protease responsible for the processing of virus-encoded polyproteins. The HIV protease has been very successfully targeted, and hepatitis C virus and rhinovirus protease inhibitors are being actively developed. The complex series of interactions during virus entry is a rapidly emerging and promising target for inhibitors of HIV and many other viruses. New anti-influenza drugs inhibit virus release from infected cells. Several stages of the viral life cycle remain incompletely characterized and are therefore poorly exploited in antiviral strategies. These include, among others, the RNA capping reactions catalyzed by many viruses, as well as the membrane association of replication complexes which is common to all positive-strand RNA viruses.
Strategies and perspectives for genetic improvement of wine yeasts by Paolo Giudici; Lisa Solieri; Andrea M. Pulvirenti; Stefano Cassanelli (pp. 622-628).
Recent developments in expression profile and proteomic techniques illustrated that the main oenological traits of wine yeasts are complex and influenced by several genes, each of them identified as absolutely essential. Only for monogenic properties the genetic improvement programmes of wine yeasts can be performed by alteration of individual genes. Ideally the most productive way of improving the whole-cell biocatalysts is by evolution of the entire cell genome. In this article we briefly review the main genetic improvement techniques applied in new and optimised wine strains construction, paying particular attention to blind and whole genome strategies, such as the sexual recombination and genome shuffling.
Enantioselective reduction of carbonyl compounds by whole-cell biotransformation, combining a formate dehydrogenase and a (R)-specific alcohol dehydrogenase by Marianne Ernst; Björn Kaup; Michael Müller; Stephanie Bringer-Meyer; Hermann Sahm (pp. 629-634).
A whole-cell biotransformation system for the reduction of prochiral carbonyl compounds, such as methyl acetoacetate, to chiral hydroxy acid derivatives [methyl (R)-3-hydroxy butanoate] was developed in Escherichia coli by construction of a recombinant oxidation/reduction cycle. Alcohol dehydrogenase from Lactobacillus brevis catalyzes a highly regioselective and enantioselective reduction of several ketones or keto acid derivatives to chiral alcohols or hydroxy acid esters. The adh gene encoding for the alcohol dehydrogenase of L. brevis was expressed in E. coli. As expected, whole cells of the recombinant strain produced only low quantities of methyl (R)-3-hydroxy butanoate from the substrate methyl acetoacetate. Therefore, the fdh gene from Mycobacterium vaccae N10, encoding NAD+-dependent formate dehydrogenase, was functionally coexpressed. The resulting two-fold recombinant strain exhibited an in vitro catalytic alcohol dehydrogenase activity of 6.5 units mg−1 protein in reducing methyl acetoacetate to methyl (R)-3-hydroxy butanoate with NADPH as the cofactor and 0.7 units mg−1 protein with NADH. The in vitro formate dehydrogenase activity was 1.3 units mg−1 protein. Whole resting cells of this strain catalyzed the formation of 40 mM methyl (R)-3-hydroxy butanoate from methyl acetoacetate. The product yield was 100 mol% at a productivity of 200 μmol g−1 (cell dry weight) min−1. In the presence of formate, the intracellular [NADH]/[NAD+] ratio of the cells increased seven-fold. Thus, the functional overexpression of alcohol dehydrogenase in the presence of formate dehydrogenase was sufficient to enable and sustain the desired reduction reaction via the relatively low specific activity of alcohol dehydrogenase with NADH, instead of NADPH, as a cofactor.
Wood adhesives prepared from lucerne fiber fermentation residues of Ruminococcus albus and Clostridium thermocellum by P. J. Weimer; R. G. Koegel; L. F. Lorenz; C. R. Frihart; W. R. Kenealy (pp. 635-640).
Fermentation residues (consisting of incompletely fermented fiber, adherent bacterial cells, and a glycocalyx material that enhanced bacterial adherence) were obtained by growing the anaerobic cellulolytic bacteria Ruminococcus albus 7 or Clostridium thermocellum ATCC 27405 on a fibrous fraction derived from lucerne (Medicago sativa L.). The dried residue was able to serve as an effective co-adhesive for phenol–formaldehyde (PF) bonding of aspen veneer sheets to one another. Testing of the resulting plywood panels revealed that the adhesive, formulated to contain 30% of its total dry weight as fermentation residue, displayed shear strength and wood failure values under both wet and dry conditions that were comparable with those of industry standards for PF that contained much smaller amounts of fillers or extenders. By contrast, PF adhesives prepared with 30% of dry weight as either unfermented lucerne fiber or conventional fillers or extenders rather than as fermentation residues, displayed poor performance, particularly under wet conditions.
Growth rates of Dekkera/Brettanomyces yeasts hinder their ability to compete with Saccharomyces cerevisiae in batch corn mash fermentations by D. A. Abbott; S. H. Hynes; W. M. Ingledew (pp. 641-647).
Growth rates determined by linear regression analysis revealed that Saccharomyces cerevisiae consistently grew more rapidly than Brettanomyces yeasts under a wide array of batch fermentative conditions, including acetic acid stress, in normal gravity (ca. 20°Plato) mashes made from ground corn. Brettanomyces yeasts only grew more rapidly than S. cerevisiae when acetic acid concentrations were elevated to industrially irrelevant levels (>0.45%, w/v). Furthermore, the three Brettanomyces isolates used in this study failed to produce significant quantities of acetic acid under pure culture fermentative conditions. In fact, the small amounts of acetic acid which accumulated in pure culture fermentations of whole corn mash were below the concentration required to inhibit the growth and metabolism of S. cerevisiae. Acetic acid concentrations in pure culture Brettanomyces fermentations exceeded 0.05% (w/v) only in media containing low levels of glucose (<4%, w/v) or when aeration rates were elevated to at least 0.03 vol. air vol.−1 mash min−1. Consequently, it was concluded that Brettanomyces yeasts would not be capable of competing with S. cerevisiae in industrial batch fermentations of whole corn mash based solely on growth rates, nor would they be capable of producing inhibitory concentrations of acetic acid in such fermentations.
A novel fungal ω3-desaturase with wide substrate specificity from arachidonic acid-producing Mortierella alpina 1S-4 by Eiji Sakuradani; Takahiro Abe; Keita Iguchi; Sakayu Shimizu (pp. 648-654).
A filamentous fungus, Mortierella alpina 1S-4, is capable of producing not only arachidonic acid (AA; 20:4n-6) but also eicosapentaenoic acid (EPA; 20:5n-3) below a cultural temperature of 20°C. Here, we describe the isolation and characterization of a gene (maw3) that encodes a novel ω3-desaturase from M. alpina 1S-4. Based on the conserved sequence information for M. alpina 1S-4 Δ12-desaturase and Saccharomyces kluyveri ω3-desaturase, the ω3-desaturase gene from M. alpina 1S-4 was cloned. Homology analysis of protein databases revealed that the amino acid sequence showed 51% identity, at the highest, with M. alpina 1S-4 Δ12-desaturase, whereas it exhibited 36% identity with Sac. kluyveri ω3-desaturase. The cloned cDNA was confirmed to encode the ω3-desaturase by its expression in the yeast Sac. cerevisiae. Analysis of the fatty acid composition of the yeast transformant demonstrated that 18-carbon and 20-carbon n-3 polyunsaturated fatty acids (PUFAs) were accumulated through conversion of exogenous 18-carbon and 20-carbon n-6 PUFAs. The substrate specificity of the M. alpina 1S-4 ω3-desaturase differs from those of the known fungal ω3-desaturases from Sac. kluyveri and Saprolegnia diclina. Plant, cyanobacterial and Sac. kluyveri ω3-desaturases desaturate 18-carbon n-6 PUFAs, Spr. diclina ω3-desaturase desaturates 20-carbon n-6 PUFAs and Caenorhabditis elegans ω3-desaturase prefers 18-carbon n-6 PUFAs as substrates rather than 20-carbon n-6 PUFAs. The substrate specificity of M. alpina 1S-4 ω3-desaturase is rather similar to that of C. elegans ω3-desaturase, but the M. alpina ω3-desaturase can more effectively convert AA into EPA when expressed in yeast. The M. alpina 1S-4 ω3-desaturase is the first known fungal desaturase that uses both 18-carbon and 20-carbon n-6 PUFAs as substrates.
Molecular and functional characterization of a levansucrase from the sourdough isolate Lactobacillus sanfranciscensis TMW 1.392 by Markus Tieking; Matthias A. Ehrmann; Rudi F. Vogel; Michael G. Gänzle (pp. 655-663).
Exopolysaccharides (EPS) produced in situ by sourdough lactobacilli affect rheological properties of dough as well as bread quality and may serve as prebiotics. The aim of this study was to characterize EPS-formation by Lactobacillus sanfranciscensis TMW 1.392 at the molecular level. A levansucrase gene from L. sanfranciscensis TMW 1.392 encompassing 2,300 bp was sequenced. This levansucrase is predicted to be a cell-wall associated protein of 879 amino acids with a relative molecular weight (MR) of 90,000. The levansucrase gene was heterologously expressed in Escherichia coli and purified to homogeneity. The recombinant enzyme exhibited transferase and hydrolase activities and produced glucose, fructose, 1-kestose and levan from sucrose; truncation of the N-terminal domain did not affect catalytic activity. Kestose formation was enhanced relative to fructose and levan formation by low temperature or high sucrose levels. During growth in wheat doughs, strain TMW 1.392 utilized sucrose to form fructose, 1-kestose, and fructan, whereas a levansucrase deletion mutant, L. sanfranciscensis TMW 1392Δlev, lost the ability to hydrolyze sucrose, and did not produce fructan or 1-kestose. These results indicate that, in L. sanfranciscensis TMW 1.392, sucrose metabolism and formation of fructan and 1-kestose is dependent on the activity of a single enzyme, levansucrase.
Characterisation of a secondary alcohol dehydrogenase from Xanthomonas campestris DSM 3586 by Tuomas Salusjärvi; Niels Hvorslev; Andrei N Miasnikov (pp. 664-667).
The chromosomal locus NP_636946 of Xanthomonas campestris DSM 3586 (ATCC 33913) which was earlier presumed to encode a quinoprotein glucose dehydrogenase has been cloned, expressed in Escherichia coli and the recombinant enzyme has been characterised. It was found to have no glucose dehydrogenase activity but to be active on many different polyols and diols, aliphatic alcohols, certain aldonic acids and amino-sugars. The product of d-gluconic acid oxidation was 5-keto-d-gluconic acid. The enzyme differs from polyol/gluconate dehydrogenases found in Gluconobacter by its single-chain architecture, different substrate specificity and much higher (20- to 30-fold) expression level in E.coli.
A Gluconobacter oxydans mutant converting glucose almost quantitatively to 5-keto-d-gluconic acid by Mustafa Elfari; Seung-Wook Ha; Christoph Bremus; Marcel Merfort; Viola Khodaverdi; Ute Herrmann; Hermann Sahm; Helmut Görisch (pp. 668-674).
Gluconobacter oxydans converts glucose to gluconic acid and subsequently to 2-keto-d-gluconic acid (2-KGA) and 5-keto-d-gluconic acid (5-KGA) by membrane-bound periplasmic pyrroloquinoline quinone-dependent and flavin-dependent dehydrogenases. The product pattern obtained with several strains differed significantly. To increase the production of 5-KGA, which can be converted to industrially important l-(+)-tartaric acid, growth parameters were optimized. Whereas resting cells of G. oxydans ATCC 621H converted about 11% of the available glucose to 2-KGA and 6% to 5-KGA, with growing cells and improved growth under defined conditions (pH 5, 10% pO2, 0.05% pCO2) a conversion yield of about 45% 5-KGA from the available glucose was achieved. As the accumulation of the by-product 2-KGA is highly disadvantageous for an industrial application of G. oxydans, a mutant was generated in which the membrane-bound gluconate-2-dehydrogenase complex was inactivated. This mutant, MF1, grew in a similar way to the wild type, but formation of the undesired 2-KGA was not observed. Under improved growth conditions, mutant MF1 converted the available glucose almost completely (84%) into 5-KGA. Therefore, this newly developed recombinant strain is suitable for the industrial production of 5-KGA.
Establishment of a gene transfer system for Rhodothermus marinus by S. H. Bjornsdottir; S. H. Thorbjarnardottir; G. Eggertsson (pp. 675-682).
Genetic manipulation of Rhodothermus marinus has been hampered by the lack of a selection system for gene transfer. We report construction of a Rhodothermus/Escherichia coli shuttle plasmid, containing the R. marinus trpB gene, based on pUC18 and the cryptic R. marinus plasmid pRM21. A plasmid-less R. marinus recipient strain was selected on the basis of growth characteristics and absence of restriction activity. The shuttle plasmid, pRM100, was successfully introduced into a TrpB− mutant of the recipient strain using electroporation and was found to transform it to prototrophy. No loss or rearrangement of pRM100 was observed after growth for 80 generations in non-selective medium. The relative copy numbers of pRM100 and of the parental plasmid, pRM21, were determined as 7±1 and 42±4, respectively. The shuttle plasmid was used to optimize an electroporation protocol, and the maximal number of transformants obtained was 4.3±0.7×106 cfu/μg DNA at 22.5 kV/cm, 200 Ω and 25 μF. Transformation failed, however, after chemical preparation of cells according to several protocols. This is the first report of genetic transformation in the genus Rhodothermus.
Improvement of pCOR plasmid copy number for pharmaceutical applications by F. Soubrier; B. Laborderie; B. Cameron (pp. 683-688).
Production of pharmaceutical-grade plasmid DNA is becoming important as the demand for clinical batches is steadily growing. pCOR plasmids have been specifically designed and used for gene delivery into humans, and have been produced by high cell-density fermentation with a yield of 100 mg/l. This yield could probably be increased as long as the release specifications of bulk plasmid remain the same, particularly in terms of plasmid sequence. We report here the use of genetic approaches in Escherichia coli to increase the copy number of pCOR. The bacterial gene encoding the π initiator-protein, which plays a pivotal role in pCOR replication, was mutagenized. A fluorescence-based screening methodology in E. coli was used to identify novel copy-up mutations. A particular combination of copy-up mutations translated into a 3–5-fold increase in monomer pCOR plasmid DNA per biomass unit.
Quorum-sensing antagonist (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone influences siderophore biosynthesis in Pseudomonas putida and Pseudomonas aeruginosa by Dacheng Ren; Rongjun Zuo; Thomas K. Wood (pp. 689-695).
Siderophore synthesis of Pseudomonas putida F1 was found to be regulated by quorum sensing since normalized siderophore production (per cell) increased 4.2-fold with cell density after the cells entered middle exponential phase; similarly, normalized siderophore concentrations in Pseudomonas aeruginosa JB2 increased 28-fold, and a 5.5-fold increase was seen for P. aeruginosa PAO1. Further evidence of the link between quorum sensing and siderophore synthesis of P. putida F1 was that the quorum-sensing-disrupter (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) from the marine red alga Delisea pulchra was found to inhibit the formation of the siderophore produced by P. putida F1 in a concentration-dependent manner, with 57% siderophore synthesis repressed by 100 μg/ml furanone. In contrast, this furanone did not affect the siderophore synthesis of Burkholderia cepacia G4 at 20–40 μg/ml, and stimulated siderophore synthesis of P. aeruginosa JB2 2.5- to 3.7-fold at 20–100 μg/ml. Similarly, 100 μg/ml furanone stimulated siderophore synthesis in P. aeruginosa PAO1 about 3.5-fold. The furanone appears to interact with the quorum-sensing machinery of P. aeruginosa PAO1 since it stimulates less siderophore synthesis in the P. aeruginosa qscR quorum-sensing mutant (QscR is a negative regulator of LasI, an acylated homoserine lactone synthase).
Chemotaxis of Pseudomonas stutzeri OX1 and Burkholderia cepacia G4 toward chlorinated ethenes by Gönül Vardar; Paola Barbieri; Thomas K. Wood (pp. 696-701).
The chemotactic responses of Pseudomonas putida F1, Burkholderia cepacia G4, and Pseudomonas stutzeri OX1 were investigated toward toluene, trichloroethylene (TCE), tetrachloroethylene (PCE), cis-1,2-dichloroethylene (cis-DCE), trans-1,2-dichloroethylene (trans-DCE), 1,1-dichloroethylene (1,1-DCE), and vinyl chloride (VC). P. stutzeri OX1 and P. putida F1 were chemotactic toward toluene, PCE, TCE, all DCEs, and VC. B. cepacia G4 was chemotactic toward toluene, PCE, TCE, cis-DCE, 1,1-DCE, and VC. Chemotaxis of P. stutzeri OX1 grown on o-xylene vapors was much stronger than when grown on o-cresol vapors toward some chlorinated ethenes. Expression of toluene-o-xylene monooxygenase (ToMO) from touABCDEF appears to be required for positive chemotaxis attraction, and the attraction is stronger with the touR (ToMO regulatory) gene.
Biodegradation of an endocrine-disrupting chemical, di-2-ethylhexyl phthalate, by Bacillus subtilis No. 66 by C. S. Quan; Q. Liu; W. J. Tian; J. Kikuchi; S. D. Fan (pp. 702-710).
A bacterial strain capable of rapidly degrading di-2-ethylhexyl phthalate (DEHP) was isolated from soil and identified as Bacillus subtilis. The organism also utilized di-butyl phthalate, di-ethyl phthalate, di-pentyl phthalate, di-propyl phthalate, and phthalic acid as sole carbon sources; and their biodegradation ratio was over 99%, when the incubation was performed for 5 days at 30°C. The microorganism degraded di-2-ethylhexyl phthalate and di-butyl phthalate through the intermediate formation of mono-2-ethylhexyl phthalate and mono-butyl phthalate, which were then metabolized to phthalic acid and further by a protocatechuate pathway, as evidenced by oxygen uptake studies and GC-MS analysis. The decontamination of soil polluted with di-2-ethylhexyl phthalate by B. subtilis was investigated. Experimental results showed that the strain could degrade about 80% of 5 mM DEHP simply by adding 8% culture medium to soil, indicating that the degradation can occur even when other organisms are present.
Treatment of dairy effluents in an aerobic granular sludge sequencing batch reactor by N. Schwarzenbeck; J. M. Borges; P. A. Wilderer (pp. 711-718).
Aerobic granular sludge can successfully be cultivated in a sequencing batch reactor (SBR) treating dairy wastewater. Attention has to be paid to the fact that suspended solids are always present in the effluent of aerobic granular sludge reactors, making a post-treatment step necessary. Sufficient post-treatment can be achieved through a sedimentation process with a hydraulic retention time of 15–30 min. After complete granulation and the separation of biomass from the effluent, removal efficiencies of 90% CODtotal, 80% Ntotal and 67% Ptotal can be achieved at a volumetric exchange ratio of 50% and a cycle duration of 8 h. Effluent values stabilize at around 125 mg l−1 CODdissolved. The maximum applicable loading rate is nevertheless limited, as the stability of aerobic granules very much depends on the presence of distinct feast and famine conditions and the degradation of real wastewaters shows slower kinetics compared with synthetic wastewaters. As loading rate and volumetric exchange ratio are coupled in an SBR system, the potential of granular sludge for improving process efficiency is also limited.
The ability of white-rot fungi to degrade the endocrine-disrupting compound nonylphenol by Ana Soares; Karin Jonasson; Enrique Terrazas; Benoit Guieysse; Bo Mattiasson (pp. 719-725).
Phanerochaete chrysosporium, Pleurotus ostreatus, Trametes versicolor and Bjerkandera sp. BOL13 were tested for their ability to degrade the endocrine-disrupting compound nonylphenol at an initial concentration of 100 mg l−1. The highest removals were achieved with T. versicolor and Bjerkandera sp. BOL13, which were able to degrade 97 mg l−1 and 99 mg l−1 of nonylphenol in 25 days of incubation, respectively. Nonylphenol removal was associated with the production of laccase by T. versicolor, but the levels of laccase, manganese peroxidase and lignin peroxidase produced by Bjerkandera sp. BOL13 were very low. At 14°C, T. versicolor and Bjerkandera sp. BOL13 sustained the removal of 88 mg l−1 and 79 mg l−1 of nonylphenol, respectively. No pollutant removal was recorded at 4°C, although both fungi could grow at this temperature in the absence of nonylphenol. A microtoxicity assay showed that the fungi produced compounds that were toxic to Vibrio fischerii; and thus a reduction in toxicity could not be correlated with nonylphenol metabolism. T. versicolor and Bjerkandera sp. BOL13 were capable of colonizing soil artificially contaminated with 430 mg kg−1 of nonylphenol. Only 1.3±0.1% of nonylphenol remained in the soil after 5 weeks of incubation.
Influence of the carbon/nitrogen/phosphorus ratio on polycyclic aromatic hydrocarbon degradation by Mycobacterium and Sphingomonas in soil by Natalie M. Leys; Leen Bastiaens; Willy Verstraete; Dirk Springael (pp. 726-736).
Biodegradation of polycyclic aromatic hydrocarbons (PAHs) in the environment is often limited due to unfavorable nutrient conditions for the bacteria that use these PAHs as sole source of carbon and energy. Mycobacterium and Sphingomonas are 2 PAH-degrading specialists commonly present in PAH-polluted soil, but not much is known about their specific nutrient requirements. By adding different inorganic supplements of nitrogen (N) and phosphorus (P), affecting the overall carbon/nitrogen/phosphorus ratio of soil in soil slurry degradation tests, we investigated the impact of soil inorganic N and P nutrient conditions on PAH degradation by PAH-degrading Sphingomonas and Mycobacterium strains. The general theoretically calculated C/N/P ratio of 100/10/1 (expressed in moles) allowed rapid PAH metabolization by Sphingomonas and Mycobacterium strains without limitation. In addition, PAH-degradation rate and extent was not affected when ca. ten times lower concentrations of N and P were provided, indicating that Sphingomonas and Mycobacterium strains are capable of metabolizing PAHs under low nutrient conditions. Nor does PAH-degradation seem to be affected by excesses of N and P creating an imbalanced C/N/P ratio. However, supplements of N and P salts increased the salinity of soil slurry solutions and seriously limited or even completely blocked biodegradation.
Expression and production of llama variable heavy-chain antibody fragments (VHHs) by Aspergillus awamori
by Vivi Joosten; Robin J. Gouka; Cees A. M. J. J. van den Hondel; C. Theo Verrips; B. Christien Lokman (pp. 737-737).