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Applied Microbiology and Biotechnology (v.66, #2)
Environmental genomics: exploring the unmined richness of microbes to degrade xenobiotics by L. Eyers; I. George; L. Schuler; B. Stenuit; S. N. Agathos; Said El Fantroussi (pp. 123-130).
Increasing pollution of water and soils by xenobiotic compounds has led in the last few decades to an acute need for understanding the impact of toxic compounds on microbial populations, the catabolic degradation pathways of xenobiotics and the set-up and improvement of bioremediation processes. Recent advances in molecular techniques, including high-throughput approaches such as microarrays and metagenomics, have opened up new perspectives and pointed towards new opportunities in pollution abatement and environmental management. Compared with traditional molecular techniques dependent on the isolation of pure cultures in the laboratory, microarrays and metagenomics allow specific environmental questions to be answered by exploring and using the phenomenal resources of uncultivable and uncharacterized micro-organisms. This paper reviews the current potential of microarrays and metagenomics to investigate the genetic diversity of environmentally relevant micro-organisms and identify new functional genes involved in the catabolism of xenobiotics.
Industrial bioconversion of renewable resources as an alternative to conventional chemistry by Th. Willke; K.-D. Vorlop (pp. 131-142).
There are numerous possibilities for replacing chemical techniques with biotechnological methods based on renewable resources. The potential of biotechnology (products, technologies, metabolic pathways) is for the most part well known. Often the costs are still the problem. Biotechnological advances have the best chances for replacing some fine chemicals. While the raw material costs are less of a consideration here, the environmental benefit is huge, as chemical-technical processes often produce a wide range of undesirable/harmful by-products or waste. In the case of bulk chemicals (
Regulation and degradation of HMGCo-A reductase by T. Panda; V. Amutha Devi (pp. 143-152).
The enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) controls the biosynthesis of cholesterol. Hypercholesterolemia and atherosclerosis are critical health risk factors. One way of controlling these risk factors is to manipulate regulation as well as degradation of HMGR. At present, a class of compounds called statins, which are HMGR inhibitors, are used for the treatment of hypercholesterolemia. However, statins suffer major setbacks as their use produces more adverse reactions than the desirable one of inhibiting the enzyme. Genetically engineered forms of HMGR are also studied in primitive life forms like bacteria, but detailed investigation of this enzyme in human systems is certainly required. Extensive studies have been made on the regulatory aspects of this enzyme, but no breakthrough is conspicuous in the clinical background to find an alternative treatment for hypercholesterolemia. The immediate need is to find an alternate way of regulating degradation of the enzyme. This review presents the importance of regulation and degradation of the HMGR enzyme in different systems to gain possible insight into alternative schemes for regulating this enzyme and, if these exist, the feasibility of extending them same to studies in mammalian systems. A high degree of similarity exists between mammalian and yeast HMGR. Detailed studies reported on the regulation and degradation of the yeast enzyme also throw more light on the mammalian system, leading to a better understanding of ways of controlling hypercholesterolemia.
Biotechnological lycopene production by mated fermentation of Blakeslea trispora by M. J. López-Nieto; J. Costa; E. Peiro; E. Méndez; M. Rodríguez-Sáiz; J. L. de la Fuente; W. Cabri; J. L. Barredo (pp. 153-159).
A semi-industrial process (800-l fermentor) for lycopene production by mated fermentation of Blakeslea trispora plus (+) and minus (−) strains has been developed. The culture medium was designed at the flask scale, using a program based on a genetic algorithm; and a fermentation process by means of this medium was developed. Fermentation involves separate vegetative phases for (+) and (−) strains and inoculation of the production medium with a mix of both together. Feeding with imidazole or pyridine, molecules known to inhibit lycopene cyclase enzymatic activity, enhanced lycopene accumulation. Different raw materials and physical parameters, including dissolved oxygen, stirring speed, air flow rate, temperature, and pH, were checked in the fermentor to get maximum lycopene production. Typical data for the fermentation process are presented and discussed. This technology can be easily scaled-up to an industrial application for the production of this carotenoid nowadays widely in demand.
Production of l-lactic acid from a mixture of xylose and glucose by co-cultivation of lactic acid bacteria by M. Taniguchi; T. Tokunaga; K. Horiuchi; K. Hoshino; K. Sakai; T. Tanaka (pp. 160-165).
The production of optically pure lactic acid in a high yield from xylose or a mixture of xylose and glucose, which is a model hydrolysate of lignocellulose, is described. In a single cultivation, Enterococcus casseliflavus produced 38 g/l of lactic acid with an optical purity of 96% enantiomeric excess (ee) and 6.4 g/l of acetic acid from 50 g/l of xylose when MRS medium was used. When a mixture of 50 g/l of xylose and 100 g/l of glucose was used as the carbon source in a cultivation of E. casseliflavus alone, glucose was converted to lactic acid in the early phase of the cultivation but xylose was hardly consumed. In a co-cultivation where E. casseliflavus and Lactobacillus casei specific for glucose were simultaneously inoculated, little or no lactic acid was produced after the glucose was almost consumed. A co-cultivation with two-stage inoculation (in which E. casseliflavus was added at a cultivation time of 40 h after L. casei cells were inoculated) resulted in complete consumption of 50 g/l of xylose and 100 g/l of glucose. In the co-cultivation, 95 g/l of lactic acid with a high optical purity of 96% ee was obtained at 192 h. Such a co-cultivation using two microorganisms specific for each sugar is considered to be one promising cultivation technique for the efficient production of lactic acid from a sugar mixture derived from lignocellulose.
H2-Producing bacterial communities from a heat-treated soil inoculum by Prabha Iyer; Mary Ann Bruns; Husen Zhang; Steve Van Ginkel; Bruce E. Logan (pp. 166-173).
Hydrogen gas (∼60% H2) was produced in a continuous flow bioreactor inoculated with heat-treated soil, and fed synthetic wastewater containing glucose (9.5 g l−1). The pH in the bioreactor was maintained at 5.5 to inhibit consumption of H2 by methanogens. The objective of this study was to characterize bacterial communities in the reactor operated under two different hydraulic retention times (HRTs of 30-h and 10-h) and temperatures (30°C and 37°C). At 30-h HRT, the H2 production rate was 80 ml h−1 and yield was 0.91 mol H2/mol glucose. At 10-h HRT, the H2 production rate was more than 5 times higher at 436 ml h−1, and yield was 1.61 mol H2/mol glucose. Samples were removed from the reactor under steady-state conditions for PCR-based detection of bacterial populations by ribosomal intergenic spacer analysis (RISA). Populations detected at 30-h HRT were more diverse than at 10-h HRT and included representatives of Bacillaceae, Clostridiaceae, and Enterobacteriaceae. At 10-h HRT, only Clostridiaceae were detected. When the temperature of the 10-h HRT reactor was increased from 30°C to 37°C, the steady-state H2 production rate increased slightly to 463 ml h−1 and yield was 1.8 mol H2/mol glucose. Compared to 30°C, RISA fingerprints at 37°C from the 10-h HRT bioreactor exhibited a clear shift from populations related to Clostridium acidisoli (subcluster Ic) to populations related to Clostridium acetobutylicum (subcluster Ib).
A new ether bond-splitting enzyme found in Gram-positive polyethylene glycol 6000-utilizing bacterium, Pseudonocardia sp. strain K1 by Manabu Yamashita; Akio Tani; Fusako Kawai (pp. 174-179).
Pseudonocardia sp. strain K1 is the only Gram-positive bacterium among the bacteria aerobically metabolizing polyethylene glycol (PEG). Generally, PEG is metabolized by an oxidative pathway in which a terminal alcohol group of PEG is oxidized to aldehyde and to carboxylic acid and then an ether bond is oxidatively cleaved. As the cell-free extract of Pseudonocardia sp. strain K1 has PEG dehydrogenase, PEG aldehyde dehydrogenase and diglycolic acid (DGA) dehydrogenase (DGADH) activities, all of which are constitutively formed, the strain has a metabolic pathway similar to that so far known. We purified an ether bond-splitting enzyme as DGADH. The molecular mass of the enzyme was estimated to be 55 kDa; and it consisted of two identical subunits. The enzyme oxidatively cleaved both an ether bond of PEG 3000 dicarboxylic acid and DGA. The N-terminal amino acid sequence of the purified enzyme had high homology with various superoxide dismutases and the enzyme had also superoxide dismutase activity. The atomic absorption spectrum showed that approximately one atom of Fe was included in each subunit of the enzyme. DGADH activity increased in the cells grown in a PEG medium supplemented with FeCl3. Thus, we concluded that the enzyme purified from Pseudonocardia sp. strain K1 is a new ether bond-splitting enzyme.
Cloning, expression and characterisation of CYP102A2, a self-sufficient P450 monooxygenase from Bacillus subtilis by M. Budde; S. C. Maurer; R. D. Schmid; V. B. Urlacher (pp. 180-186).
The gene encoding CYP102A2, a novel P450 monooxygenase from Bacillus subtilis, was cloned and expressed in Escherichia coli. The recombinant enzyme formed was purified by immobilised metal chelate affinity chromatography (IMAC) and characterised. CYP102A2 is a 119-kDa self-sufficient monooxygenase, consisting of an FMN/FAD-containing reductase domain and a heme domain. The deduced amino acid sequence of CYP102A2 exhibits a high level of identity with the amino acid sequences of CYP102A1 from B. megaterium (59%) and CYP102A3 from B. subtilis (60%). In reduced, CO-bound form, the enzyme shows a typical Soret band at 449 nm. It catalyses the oxidation of even- and odd-chain saturated and unsaturated fatty acids. In all reactions investigated, the products were the respective ω-3, ω-2 and ω-1 hydroxylated fatty acids. Activity was highest towards oleic acid (KM=17.36±1.4 μM, kcat=2,244±72 min−1) and linoleic acid (KM=12.25±1.8 μM, kcat=1,950±84 min−1). Comparison of a CYP102A2 homology model with the CYP102A1 crystal structure revealed significant differences in the substrate access channels, which might explain the differences in the catalytic properties of these two enzymes.
Effect of NADH dehydrogenase-disruption and over-expression on respiration-related metabolism in Corynebacterium glutamicum KY9714 by Nawarat Nantapong; Youko Kugimiya; Hirohide Toyama; Osao Adachi; Kazunobu Matsushita (pp. 187-193).
The function of type II NADH dehydrogenase (NDH-2) in Gram-positive Corynebacterium glutamicum was investigated by preparing strains with ndh, the NDH-2 gene, disrupted and over-expressed. Although disruption showed no growth defects on glucose minimum medium, the growth rate of the over-expressed strain was lower compared with its parent, C. glutamicum KY9714. Ndh-disruption and over-expression did not lead to a large change in the respiratory chain and energetics, including the cytochrome components and the H+/O ratio. However, in the strain that lacked NDH-2, membrane l-lactate oxidase activity increased, while NDH-2 over-expression led to decreased l-lactate and malate oxidase activities. In addition, relatively high cytoplasmic lactate dehydrogenase (LDH) activity was always present as was malate dehydrogenase, irrespective of NDH-2 level. Furthermore, l-lactate or malate-dependent NADH oxidase activity could be reproduced by reconstitution with the membranes and the cytoplasmic fraction isolated from the disruptant. These results suggest that coupling of LDH and the membrane l-lactate oxidase system, together with the malate-dependent NADH oxidase system, operates to oxidize NADH when the NDH-2 function is defective in C. glutamicum.
Overproduction of recombinant laccase using a homologous expression system in Coriolus versicolor by Shinya Kajita; Shinsuke Sugawara; Yasumasa Miyazaki; Masaya Nakamura; Yoshihiro Katayama; Kazuo Shishido; Yosuke Iimura (pp. 194-199).
One of the major extracellular enzymes of the white-rot fungus Coriolus versicolor is laccase, which is involved in the degradation of lignin. We constructed a homologous system for the expression of a gene for laccase III (cvl3) in C. versicolor, using a chimeric laccase gene driven by the promoter of a gene for glyceraldehyde-3-phosphate dehydrogenase (gpd) from this fungus. We transformed C. versicolor successfully by introducing both a gene for hygromycin B phosphotransferase (hph) and the chimeric laccase gene. In three independent experiments, we recovered 47 hygromycin-resistant transformants at a transformation frequency of 13 transformants μg−1 of plasmid DNA. We confirmed the introduction of the chimeric laccase gene into the mycelia of transformants by a polymerase chain reaction in nine randomly selected transformants. Overproduction of extracellular laccase by the transformants was revealed by a colorimetric assay for laccase activity. We examined the transformant (T2) that had the highest laccase activity and found that its activity was significantly higher than that of the wild type, particularly in the presence of copper (II). Our transformation system should contribute to the efficient production of the extracellular proteins of C. versicolor for the accelerated degradation of lignin and aromatic pollutants.
Process design for recombinant protein production based on the promoter, PmalK by Maria Boström; Gen Larsson (pp. 200-208).
PmalK is induced through activation of MalT, by the formation of maltotriose and cyclic adenosine monophosphate (cAMP). The possibility to influence endogenous inducer levels is used to vary the production rates in specifically designed production protocols. Induction based on a batch process protocol on maltose gives low production rates, as the result of a lack of cAMP, which is shown to be of major importance to fully induce this promoter. Two mechanisms are thus used to influence the levels of maltotriose and/or cAMP formation: (1) catabolite derepression achieved from low glucose concentration and (2) catabolite derepression/inducer exclusion from diauxic growth on glucose/maltose. Fed-batch processes based on limited amounts of glucose result in product accumulation of up to 10% of the total protein. Depending on the feed of limiting glucose, different production profiles are developed. The initial increase in the production rate is due to maltotriose formation from endogenous glycogen degradation while, later in the process, production can be further supported by elevated levels of cAMP, provided the feed rate is sufficiently low. The introduction of maltose after a preceding fed-batch process on glucose can be efficiently used to produce maltotriose in combination with cAMP formation in the event of catabolite derepression. This leads to higher production rates and a further increase in product accumulation of up to 30% of the total protein. The diauxic growth phase resulting from the shift in carbon source can be shortened and even avoided by the design of the preceding feed-rate of glucose. It is postulated that proper design of the inoculum and initial phases of production can reduce basal levels of product formation. With this promoter, the production rate can be as high as 65 units mg−1 h−1 and the time to reach a maximal production rate can be designed to take up to 8 h. Furthermore, the duration of the production rate can be as long as 7 h.
Characterization in Pseudomonas putida Cg1 of nahR and its role in bacterial survival in soil by W. Park; E. L. Madsen (pp. 209-216).
Sequencing, RFLP analyses and experiments utilizing a lacZ transcriptional reporter fused to the promoter regions of nahR and nahG in Pseudomonas putida Cg1 confirmed that regulation of naphthalene degradation in both P. putida Cg1 and the type strain, P. putida NCIB 9816-4, is consistent with that of NAH7 from P. putida G7. Two nahR knockout strains (RK1 and Cg1-NAHR from P. putida NCIB 9816-4 and Cg1, respectively) showed a growth defect in the presence of naphthalene as sole carbon and energy source. We hypothesized that nahR influences ecological fitness of bacteria in naphthalene-contaminated soil and tested this hypothesis using both parent and nahR-knockout strains introduced to soil microcosms with and without added naphthalene. After 21 days, loss of cell viability was pronounced in the presence of added naphthalene crystals for nahR mutants of both test bacteria, relative to the wild types. Diminished viable counts were attributed to toxicity. Thus, our data indicated that NahR in P. putida Cg1 is virtually identical to its homologues in other pseudomonads and that nahR is required for resistance to naphthalene toxicity, hence the persistence of bacterial cells in soil with high concentrations of naphthalene.
β-Glucan synthase induction in mushrooms grown on olive mill wastewaters by M. Reverberi; F. Di Mario; U. Tomati (pp. 217-225).
β-1-3-Glucan synthase activity and its induction by olive mill wastewaters (OMW) was studied in ten fungal strains (Auricularia auricula-judae, Lentinula edodes, Pleurotus eryngii, Stropharia aeruginosa, Agrocybe aegerita, P. pulmonarius, Armillaria mellea, P. ferulae, P. ostreatus, P. nebrodensis). A microtiter-based enzymatic assay on β-1-3-glucan synthase activity was carried out on all mycelia growth both on the control medium and on OMW. Among the fungi assayed, L. edodes β-1-3-glucan synthase was highly enhanced in OMW. The main components of OMW, i.e. phenols and lipids, were added separately to the control medium, to highlight the mechanism of L. edodes β-1-3-glucan synthase induction. A Southern blot analysis and PCR with degenerated primers were carried out to detect the presence of fks1-like genes in these Basidiomycetes. The sequences obtained from the ten Basidiomycota were remarkably similar to fks1 from Filobasidiella neoformans. Spectrofluorimetric and RT-PCR analyses of β-1-3-glucan synthase were performed on the mycelia of L. edodes. In this fungus, a strong stimulation of β-1-3-glucan synthase mRNA and protein was recorded in the presence of OMW and phenols.
Changes in the composition of polar and apolar crude oil fractions under the action of Microcoleus consortia by Tirso Garcia De Oteyza; Joan O. Grimalt; Elia Diestra; Antonio Solé; Isabel Esteve (pp. 226-232).
Cultures of Microcoleus consortia polluted with two different types of crude oil, one with high content in aliphatic hydrocarbons (Casablanca) and the other rich in sulphur and aromatic compounds (Maya), were grown for 50 days and studied for changes in oil composition. No toxic effects from these oils were observed on Microcoleus consortia growth. In fact, the interface layer between the oils and the water culture medium proved to be the ideal site for consortia development, leading to a wrapping effect of the oil layers by these organisms. Despite this affinity of cyanobacteria for the oil substrate, the changes in oil composition were small. Microcoleus consortia did not induce transformation in the aliphatic-rich oil, and the modifications in the sulphur and aromatic-rich oil were small. The latter essentially involved degradation of aliphatic heterocyclic organo-sulphur compounds such as alkylthiolanes and alkylthianes. Other groups of compounds, such as the alkylated monocyclic and polycyclic aromatic hydrocarbons, carbazoles, benzothiophenes and dibenzothiophenes, also underwent some degree of transformation, involving only the more volatile and less alkylated homologues.