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Applied Microbiology and Biotechnology (v.55, #5)
Directed evolution and the creation of enantioselective biocatalysts by K.-E. Jaeger; T. Eggert; A. Eipper; M. Reetz (pp. 519-530).
Directed evolution has emerged as a key technology to generate enzymes with new or improved properties that are of major importance to the biotechnology industry. A directed evolution approach starts with the identification of a target enzyme to be optimized and the cloning of the corresponding gene. An efficient expression system is needed before the target gene is subjected to random mutagenesis and/or in vitro recombination, thereby creating molecular diversity. Subsequently, improved enzyme variants are identified, preferably after being secreted into culture medium, by screening or selection for the desired property. The genes encoding the improved enzymes are then used to parent the next round of directed evolution. Enantioselectivity is a biocatalyst property of major biotechnological importance that is, however, difficult to deal with. We discuss recent examples of creating enantioselective biocatalysts by directed evolution.
Infrared-thermographic screening of the activity and enantioselectivity of enzymes by M. Reetz; M. Hermes; M. Becker (pp. 531-536).
The infrared radiation caused by the heat of reaction of an enantioselective enzyme-catalyzed transformation can be detected by modern photovoltaic infrared (IR)-thermographic cameras equipped with focal-plane array detectors. Specifically, in the lipase-catalyzed enantioselective acylation of racemic 1-phenylethanol, the (R)- and (S)-substrates are allowed to react separately in the wells of microtiter plates, the (R)-alcohol showing hot spots in the IR-thermographic images. Thus, highly enantioselective enzymes can be identified at kinetic resolution.
Two-step process for ketocarotenoid production by a green alga, Chlorococcum sp. strain MA-1 by D.-H. Zhang; Y.-K. Lee (pp. 537-540).
The production of ketocarotenoids (KCs) from Chlorococcum sp. strain MA-1was investigated by a two-step process. In the first step, 18 g biomass l–1 was achieved by feeding glucose to the heterotrophic cultures; in the second step, the high-density cultures were treated with light illumination or chemical stress in dark, respectively, to induce KC synthesis. Light-treated cultures could produce 103 mg total KCs l–1 and 32 mg astaxanthin l–1, three times higher than those from chemical-treated cultures, in the 10 days of induction. The percentages of individual KCs (hydroxyechinenone, canthaxanthin, adonirubin and astaxanthin) in the total KCs were not markedly influenced by the different stress conditions. The developed two-step process provides a feasible strategy for commercial production of ketocarotenoids by the green microalga, Chlorococcum sp. strain MA-1.
Biotransformation of limonene by Pseudomonas putida by T. Chatterjee; D. Bhattacharyya (pp. 541-546).
From a study of three fungal and 15 bacterial strains, it was observed that Pseudomonas putida MTCC 1072 oxidized limonene with the highest efficiency of. Fermentation of limonene by P. putida MTCC 1072 was conducted for 120 h at 30 °C at a fixed pH of 5.0. Major bioconversion products were isolated and characterized by Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopy, and by elemental analysis. The bioconversion products were identified as perillyl alcohol and p-menth-1-ene-6,8-diol, and under optimum conditions the yields were 36% and 44%, respectively (a rate kinetic model indicated corresponding limiting yields of 44% and 56%). No further degradation of the products was observed using this bacteria.
High cell density cultivation of Rhodococcus opacus for lipid production at a pilot-plant scale by I. Voss; A. Steinbüchel (pp. 547-555).
The triacylglycerol (TAG)-accumulating bacterium Rhodococcus opacus strain PD630 was investigated with respect to the fermentative production of TAGs consisting of an unusually high fraction of fatty acids with an odd-number of carbon atoms and unsaturated monoenic fatty acids from sugar beet molasses and sucrose. Fed-batch fermentations were optimized at the 30-l scale in a stirred tank bioreactor at 30°C using a mineral salts medium, which contained sugar beet molasses and sucrose as sole carbon sources. Approximately 37.5 g cell dry matter (CDM) per liter was the highest cell density that was obtained at that scale with a TAG content in the cells of 52%. This fermentative process was also applied to a 500-l pilot-plant scale. Cell densities as high as 18.4 g CDM per liter were obtained, and 42% of the sucrose present in the medium was converted into cell mass which consisted of 38.4% TAGs.
Growth and differentiation of permanent and secondary mouse myogenic cell lines on microcarriers by C. Bardouille; J. Lehmann; P. Heimann; H. Jockusch (pp. 556-562).
Myogenesis involves the determination of progenitor cells to myoblasts, their fusion to yield multinuclear myotubes, and the maturation of myotubes to muscle fibres. This development is reflected in a time pattern of gene expression, e.g. of genes coding for desmin, the myogenic factors myogenin and myoD, the acetylcholine receptor α-subunit and the muscular chloride channel ClC-1. We attempted to improve yields and myogenic differentiation in culture by using three-dimensional microcarrier systems. Out of a variety of carriers tested in stationary cultures, collagen-coated dextran Cytodex3 beads proved optimal for the proliferation and differentiation of the murine myogenic cell line C2C12. With C2C12 myoblasts in stationary and stirred systems (Spinner- and SuperSpinner flasks), surface adherence, differentiation into myotubes and expression of muscle-specific mRNAs on Cytodex3 beads were the same as in conventional cultures. Other carriers tested (DEAE cellulose, glass, plastic, cellulose, polyester) did not support growth and differentiation of C2C12 cells. The secondary mouse myogenic stem cells M12 and M2.7-MDX proliferated and differentiated well in stationary Cytodex3 cultures, but no differentiation occurred in Spinner flasks. As indicated by light and scanning electron microscopy, C2C12 myotubes formed not only on but also in between Cytodex beads. The secondary cell lines may succumb to shear forces under these conditions.
Efficient transformation of filamentous fungus Pleurotus ostreatus using single-strand carrier DNA by Toshikazu Irie; Yoichi Honda; Takashi Watanabe; Masaaki Kuwahara (pp. 563-565).
The effects of carrier DNAs on the transformation of the basidiomycete Pleurotus ostreatus were analyzed. When λ phage DNA was added to a transformation mixture containing protoplasts and Cbx R vector plasmid, an increased number of drug-resistant transformants was observed on a screening plate containing 2 µg carboxin/ml. The highest efficiency (about 200 transformants/µg vector plasmid) was obtained by the addition of heat-denatured λ DNA, which gave yields approximately 50-fold higher than the control experiment without a carrier DNA. To our knowledge, this is the first report on enhancement in transformation efficiency of fungal protoplasts by single strand carrier DNA.
Homologous expression of recombinant manganese peroxidase genes in ligninolytic fungus Pleurotus ostreatus by T. Irie; Y. Honda; T. Watanabe; M. Kuwahara (pp. 566-570).
Pleurotus ostreatus is a white-rot fungus known as an efficient degrader of lignin and also various organo-pollutants. Using a DNA-mediated transformation system, a molecular breeding approach to isolate overproducers of a manganese peroxidase isozyme, MnP3, was carried out. Recombinant mnp3 constructs under the control of P. ostreatus sdi1 expression signals were introduced into the wild-type P. ostreatus strain by co-transformation with a carboxin-resistant vector plasmid, pTM1. One of the recombinants obtained by a mating between two monokaryotic transformants, TMG9-C1, showed a several times higher level of MnP activity than the wild-type control in the early stage of liquid culture. Predominant transcription of the recombinant mnp3 in the strain was demonstrated by RT-PCR experiments. This is the first report of a genetically modified P. ostreatus strain with an expression system for recombinant genes.
High-rate 3-methylcatechol production in Pseudomonas putida strains by means of a novel expression system by L. Hüsken; R. Beeftink; J. de Bont; J. Wery (pp. 571-577).
The bioconversion of toluene into 3-methylcatechol was studied as a model system for the production of valuable 3-substituted catechols in general. For this purpose, an improved microbial system for the production of 3-methylcatechol was obtained. Pseudomonas putida strains containing the todC1C2BAD genes involved in the conversion of toluene into 3-methylcatechol were used as hosts for introducing extra copies of these genes by means of a novel integrative expression system. A construct was made containing an expression cassette with the todC1C2BAD genes cloned under the control of the inducible regulatory control region for naphthalene and phenanthrene degradation, nagR. Introducing this construct into wild-type P. putida F1, which degrades toluene via 3-methylcatechol, or into mutant P. putida F107, which accumulates 3-methylcatechol, yielded biocatalysts carrying multiple copies of the expression cassette. As a result, up to 14 mM (1.74 g l–1) of 3-methylcatechol was accumulated and the specific production rate reached a level of 105 µmol min–1 g–1cell dry weight, which is four times higher than other catechol production systems. It was shown that these properties were kept stable in the biocatalysts without the need for antibiotics in the production process. This is an important step for obtaining designer biocatalysts.
Deletion of a cytotoxic, N-terminal putative signal peptide results in a significant increase in production yields in Escherichia coli and improved specific activity of Cel12A from Rhodothermus marinus by K. Wicher; M. Abou-Hachem; S. Halldórsdóttir; S. Thorbjarnadóttir; G. Eggertsson; G. Hreggvidsson; E. Nordberg Karlsson; O. Holst (pp. 578-584).
The thermostable cellulase Cel12A from Rhodothermus marinus was produced at extremely low levels when expressed in Escherichia coli and was cytotoxic to the cells. In addition, severe aggregation occurred when moderately high concentrations of the enzyme were heat-treated at 65 °C, the growth optimum of R. marinus. Sequence analysis revealed that the catalytic module of this enzyme is preceded by a typical linker sequence and a highly hydrophobic putative signal peptide. Two deletion mutants lacking this hydrophobic region were cloned and successfully expressed in E. coli. These results indicated that the N-terminal putative signal peptide was responsible for the toxicity of the full-length enzyme in the host organism. This was further corroborated by cloning and expressing the hydrophobic N-terminal domain in E. coli, which resulted in extensive cell lysis. The deletion mutants, made up of either the catalytic module of Cel12A or the catalytic module and the putative linker sequence, were characterised and their properties compared to those of the full-length enzyme. The specific activity of the mutants was approximately three-fold higher than that of the full-length enzyme. Both mutant proteins were highly thermostable, with half-lives exceeding 2 h at 90 °C and unfolding temperatures up to 103 °C.
Pre-termination in aflR of Aspergillus sojae inhibits aflatoxin biosynthesis by K. Matsushima; P.-K. Chang; J. Yu; K. Abe; D. Bhatnagar; T. Cleveland (pp. 585-589).
The aflR gene product is the main transcriptional regulator of aflatoxin biosynthesis in Aspergillus parasiticus and Aspergillus flavus. Although A. sojae strains do not produce aflatoxins, they do have an aflR homologue. When compared with the aflR of A. parasiticus, the A. sojae gene contains two mutations: an HAHA motif and a premature stop codon. To investigate the functionality of the A. sojae aflR gene product, we used a GAL4 one-hybrid system in yeast. The transcription-activating activity of AflR from A. sojae was 15% of that from A. parasiticus. The introduction of an additional aflR from A. sojae into an A. parasiticus strain did not affect aflatoxin productivity. A hybrid aflR comprising the amino-terminal region of A. sojae aflR and the carboxy-terminal region of A. parasiticus aflR suppressed the effect associated with pre-termination of the A. sojae AflR. We conclude that the premature stop codon of the A. sojae aflR is the key to its functionality and leads to prevention of aflatoxin biosynthesis through loss of the transcription of aflatoxin biosynthesis-related genes.
Synthesis of optically pure ethyl (S)-4-chloro-3-hydroxybutanoate by Escherichia coli transformant cells coexpressing the carbonyl reductase and glucose dehydrogenase genes by N. Kizaki; Y. Yasohara; J. Hasegawa; M. Wada; M. Kataoka; S. Shimizu (pp. 590-595).
The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE) was investigated. Escherichia coli cells expressing both the carbonyl reductase (S1) gene from Candida magnoliae and the glucose dehydrogenase (GDH) gene from Bacillus megaterium were used as the catalyst. In an organic-solvent-water two-phase system, (S)-CHBE formed in the organic phase amounted to 2.58 M (430 g/l), the molar yield being 85%. E. coli transformant cells coproducing S1 and GDH accumulated 1.25 M (208 g/l) (S)-CHBE in an aqueous mono-phase system by continuously feeding on COBE, which is unstable in an aqueous solution. In this case, the calculated turnover of NADP+ (the oxidized form of nicotinamide adenine dinucleotide phosphate) to CHBE was 21,600 mol/mol. The optical purity of the (S)-CHBE formed was 100% enantiomeric excess in both systems. The aqueous system used for the reduction reaction involving E. coli HB101 cells carrying a plasmid containing the S1 and GDH genes as a catalyst is simple. Furthermore, the system does not require the addition of commercially available GDH or an organic solvent. Therefore this system is highly advantageous for the practical synthesis of optically pure (S)-CHBE.
Molecular cloning of a putative divalent-cation transporter gene as a new genetic marker for the identification of Lactobacillus brevis strains capable of growing in beer by Nobuyuki Hayashi; Mie Ito; Satoka Horiike; Hiroshi Taguchi (pp. 596-603).
Random amplified polymorphic DNA (RAPD) PCR analysis of Lactobacillus brevis isolates from breweries revealed that one of the random primers could distinguish beer-spoilage strains of L. brevis from non-spoilage strains. The 1.1-kb DNA fragment amplified from all beer-spoilers included one open reading frame, termed hitA (hop-inducible cation transporter), which encodes an integral membrane protein with 11 putative trans-membrane domains and a binding protein-dependent transport signature of a non-ATP binding membrane transporter common to several prokaryotic and eukaryotic transporters. The hitA polypeptide is homologous to the natural resistance-associated macrophage protein (Nramp) family characterized as divalent-cation transport proteins in many prokaryotic and eukaryotic organisms. Northern blot analysis indicated that the hitA transcripts are expressed in cells cultivated in MRS broth supplemented with hop bitter compounds, which act as mobile-carrier ionophores, dissipating the trans-membrane pH gradient in bacteria sensitive to the hop bitter compounds by exchanging H+ for cellular divalent cations such as Mn2+. This suggests that the hitA gene products may play an important role in making the bacteria resistant to hop bitter compounds in beer by transporting metal ions such as Mn2+ into cells that no longer maintain the proton gradient.
Improved antifungal activity of a mutant of Trichoderma harzianum CECT 2413 which produces more extracellular proteins by M. Rey; J. Delgado-Jarana; T. Benítez (pp. 604-608).
Trichoderma harzianum is a well-known biological control agent against fungal plant diseases. In order to select improved biocontrol strains from Trichoderma harzianum CECT 2413, a mutant has been isolated for its ability to produce wider haloes than the wild type, when hydrolysing pustulan, a polymer of β-1,6-glucan. The mutant possesses between two and four times more chitinase, β-1,3- and β-1,6-glucanase activities than the wild type, produces about three times more extracellular proteins and secretes higher amounts of a yellow pigment (α-pyrone). This mutant performed better than the wild type during in vitro experiments, overgrowing and sporulating on Rhizoctonia solani earlier, killing this pathogen faster and exerting better protection on grapes against Botrytis cinerea.
Insights into the genetic diversity of initial dioxygenases from PAH-degrading bacteria by Ralf Moser; Ulf Stahl (pp. 609-618).
Alpha subunit genes of initial polyaromatic hydrocarbon (PAH) dioxygenases were used as targets for the PCR detection of PAH-degrading strains of the genera Pseudomonas, Comamonas and Rhodococcus which were obtained from activated sludge or soil samples. Sequence analysis of PCR products from several Pseudomonas strains showed that alpha subunits (nahAc allele) of this genus are highly conserved. PCR primers for the specific detection of alpha subunit genes of initial PAH dioxygenases from Pseudomonas strains were not suitable for detecting the corresponding genes from the genera Comamonas and Rhodococcus. Southern analysis using a heterologous gene probe derived from the P. putida OUS82 PAH dioxygenase alpha subunit identified segments of the PAH-degradation gene cluster from C. testosteroni strain H. Parts of this gene cluster containing three subunits of the initial PAH dioxygenase were isolated. These three subunits [ferredoxin (pahAb), alpha (pahAc) and beta (pahAd) subunit] were amplified by PCR as one fragment and expressed in Escherichia coli DH5α, resulting in an active initial dioxygenase with the ability to transform indole and phenanthrene. The DNA sequence alignment of alpha subunits from C. testosteroni H and various PAH-degrading bacteria permitted the design of new primers and oligonucleotide probes which are useful for the detection of the initial PAH dioxygenases from strains of Pseudomonas, Comamonas and Rhodococcus.
Biodegradation of synthetic and naturally occurring mixtures of mono-cyclic aromatic compounds present in olive mill wastewaters by two aerobic bacteria by D. Di Gioia; F. Fava; L. Bertin; L. Marchetti (pp. 619-626).
Two bacterial strains, Ralstonia sp. LD35 and Pseudomonas putida DSM 1868, were assayed for their ability to degrade the monocyclic aromatic compounds commonly found in olive mill wastewaters (OMWs). The goal was to study the possibility of employing the two strains in the removal of these recalcitrant and toxic compounds from the effluents of anaerobic treatment plants fed with OMWs. At first, the two strains were separately assayed for their ability to degrade a synthetic mixture of nine aromatic acids present in OMWs, both in growing- and resting-cell conditions. Then, due to the complementary activity exhibited by the two strains, a co-culture of the two bacteria was tested under growing-cell conditions for degradation of the same synthetic mixture. Finally, the degradation activity of the co-culture on two fractions was studied. Both fractions one deriving from natural OMWs through reverse osmosis treatment and containing low-molecular weight organic molecules, and the other obtained from an anaerobic lab-scale treatment plant fed with OMWs, were rich in monocyclic aromatic compounds. The co-culture of the two strains was able to biodegrade seven of the nine components of the tested synthetic mix (2, 6-dihydroxybenzoic acid and 3, 4, 5-trimethoxybenzoic acid were the two undegraded compounds). In addition, an efficient biodegrading activity towards several aromatic molecules present in the two natural fractions was demonstrated.
Efficiency of naphthalene and salicylate degradation by a recombinant Pseudomonas putida mutant strain defective in glucose metabolism by S. Samanta; B. Bhushan; R. Jain (pp. 627-631).
Metabolically engineered microorganisms may have tremendous potential in removing toxic compounds from nature. In general, microorganisms prefer to utilize simpler carbon sources over toxic compounds when both are present in an environment and, therefore, the presence of simpler carbon sources may greatly reduce the efficiency of a microorganism towards toxic compounds. If a microorganism is prevented from utilizing simpler carbon sources, thereby making it totally dependent upon the toxic compounds, it should increase the specificity for and efficiency of degradation of the toxic compounds in the presence of other, simpler carbon sources. To test this hypothesis, the efficiency of naphthalene and salicylate degradation in the presence of glucose by a recombinant Pseudomonas putida strain mutated in glucose metabolism was determined and compared to the non-mutated strain. Results obtained indicate that the impairment of glucose metabolism leads to better degradation of naphthalene and salicylate in the presence of glucose.
Screening for microorganisms that produce only endo-inulinase by Regina M. Gern; Sandra A. Furlan; Jorge L. Ninow; Rainer Jonas (pp. 632-635).
Sixteen fungal strains reported in the literature as endo-inulinase producers and three bacterial strains, isolated from the dahlia rhizosphere, were analysed for endo-inulinase production. From four isolated strains (one fungus and three bacteria) the results were evaluated in terms of substrate consumption, cell growth and production of endo-inulinases. All three bacterial strains were sole endo-inulinase producers and, among these, strain Paenibacillus sp. CDB 003 was the most suitable for endo-inulinase production, as this enzyme produced inulobiose as the principal substrate as well as inulo-oligosaccharides with polymerisation degrees of 3–5.
Copper removal from aqueous solution using Aspergillus niger mycelia in free and polyurethane-bound form by K. Tsekova; S. Ilieva (pp. 636-637).
This study assesses the ability of mycelia of Aspergillus niger B-77 (both free and immobilized on polyurethane foam) to remove copper from single-ion solution. All experiments were conducted using 0.5 mM solutions of CuSO4·5H2 O. Mycelia immobilized on polyurethane foam cells showed a three-fold increase in uptake, compared with that of free cells. The efficiency of copper removal (mg Cu2+ removed/mg Cu2+ added) in a column system reached more than 99% before the break-through point was attained.
Sugar composition of biofilms produced by paper mill bacteria by L. Lindberg; B. Holmbom; O. Väisänen; A. Weber; M. Salkinoja-Salonen (pp. 638-643).
Biofilms of paper mill bacteria were cultivated in paper mill white water-simulating conditions on glass slides or stainless steel coupons in a laboratory culture system. The sugar content and composition of the biofilms were analysed and compared with the sugar composition of paper mill slimes. Acid methanolysis followed by gas chromatography revealed that Burkholderia was the major biofilm producer in pure culture, producing up to 50 µg of biofilm sugar cm–2 in 5 days in rich medium and 10 µg in paper mill simulating medium. A mixture of simulated paper mill water with a culture medium yielded more biofilm (100 µg cm–2) than either of the media alone, so the biofilm accumulation was not proportional to the available substrate. More biofilm accumulated on stainless steel coupons than on glass slides, and the steel-coupon biofilms contained slightly more uronic acids. The biofilm sugars contained mainly galactose, glucose, mannose, and rhamnose. In paper mill medium, the Burkholderia biofilm contained more galactose and glucose, and less rhamnose, than in rich laboratory medium. The sugar composition of paper mill slimes was quite similar to those of steel-cultured Burkholderia cepacia biofilms. This suggests that Burkholderia cepacia is responsible for much of the slime in the paper mill.
Enhancing the start-up of a UASB reactor treating domestic wastewater by adding a water extract of Moringa oleifera seeds by Y. Kalogo; A. M'Bassiguié Séka; W. Verstraete (pp. 644-651).
Water extract of Moringa oleifera seeds (WEMOS) was used to enhance the start-up of a self-inoculated upflow anaerobic sludge blanket (UASB) reactor treating raw domestic wastewater. Two reactors labelled control (RC) and WEMOS addition (RM) were started without special inoculum. Both reactors were fed continuously for 22 weeks with domestic wastewater containing an average total chemical oxygen demand (COD) of 320 mg O2/l and suspended solid (SS) of 165 mg/l. The reactors operated during the entire experimental period at 29°C and at a hydraulic retention time (HRT) of 4 h. The RM reactor received 2 ml WEMOS per litre of influent. WEMOS solution was prepared on the basis of 2.5% (w/v) ground M. oleifera seeds in water. The results of 22 weeks' operation showed an improvement in the performance of the RM compared to that of the RC. The dosage of WEMOS in the feed (1) shortened the biological start-up period by 20%, (2) increased acidogenic and methanogenic activity by a factor of 2.4 and 2.2 respectively, (3) increased the specific biogas production by a factor of 1.6, (4) favoured fast growth of the sludge bed, and (5) allowed the aggregation of coccoid bacteria and growth of microbial nuclei, which are precursors of anaerobic granulation.