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Applied Microbiology and Biotechnology (v.67, #6)
The remarkable Rhodococcus erythropolis by Carla C. C. R. de Carvalho; M. Manuela R. da Fonseca (pp. 715-726).
Rhodococcus erythropolis cells contain a large set of enzymes that allow them to carry out an enormous number of bioconversions and degradations. Oxidations, dehydrogenations, epoxidations, hydrolysis, hydroxylations, dehalogenations and desulfurisations have been reported to be performed by R. erythropolis cells or enzymes. This large array of enzymes fully justifies the prospective application of this bacterium in biotechnology.
Feasibility of acrylic acid production by fermentation by Adrie J. J. Straathof; Susana Sie; Telma T. Franco; Luuk A. M. van der Wielen (pp. 727-734).
Acrylic acid might become an important target for fermentative production from sugars on bulk industrial scale, as an alternative to its current production from petrochemicals. Metabolic engineering approaches will be required to develop a host microorganism that may enable such a fermentation process. Hypothetical metabolic pathways for insertion into a host organism are discussed. The pathway should have plausible mass and redox balances, plausible biochemistry, and plausible energetics, while giving the theoretically maximum yield of acrylate on glucose without the use of aeration or added electron acceptors. Candidate metabolic pathways that might lead to the theoretically maximum yield proceed via β-alanine, methylcitrate, or methylmalonate-CoA. The energetics and enzymology of these pathways, including product excretion, should be studied in more detail to confirm this. Expression of the selected pathway in a host organism will require extensive genetic engineering. A 100,000-tons/year fermentation process for acrylic acid production, including product recovery, was conceptually designed based on the supposition that an efficient host organism for acrylic acid production can indeed be developed. The designed process is economically competitive when compared to the current petrochemical process for acrylic acid. Although the designed process is highly speculative, it provides a clear incentive for development of the required microbial host, especially considering the environmental sustainability of the designed process.
Natural polysaccharides as electroactive polymers by Victoria L. Finkenstadt (pp. 735-745).
Electroactive polymers (EAPs), a new class of materials, have the potential to be used for applications like biosensors, environmentally sensitive membranes, artificial muscles, actuators, corrosion protection, electronic shielding, visual displays, solar materials, and components in high-energy batteries. The commercialization of synthetic EAPs, however, has so far been severely limited. Biological polymers offer a degree of functionality not available in most synthetic EAPs. Carbohydrate polymers are produced with great frequency in nature. Starch, cellulose, and chitin are some of the most abundant natural polymers on earth. Biopolymers are a renewable resource and have a wide range of uses in nature, functioning as energy storage, transport, signaling, and structural components. In general, electroactive materials with polysaccharide matrices reach conductance levels comparable with synthetic ion-conducting EAPs. This review gives a brief history of EAPs, including terminology, describes evaluation methods, and reports on the current progress of incorporating polysaccharides as matrices for doped, blended, and grafted electroactive materials.
Novel method of inactivation of human immunodeficiency virus type 1 by the freeze pressure generation method by T. Otake; T. Kawahata; H. Mori; Y. Kojima; K. Hayakawa (pp. 746-751).
It has been reported that high-pressure (over 600 MPa) treatment at room temperature inactivates human immunodeficiency virus type 1 (HIV-1), and it has recently been shown that the high pressure generated by the expansion of water due to freezing (freeze pressure generation method, or FPGM) has an inactivating effect on bacteria and fungi. In this study, we examined the effects of treatment by FPGM on HIV-1. A sturdy vessel filled with water and securely closed with a lid was kept at 0°C to −30°C. High pressures of 200 MPa and 250 MPa were generated at −20°C and −30°C, respectively. When T-cell-tropic and macrophage-tropic laboratory strains of HIV-1 were kept at −10°C, the virus infectivity decreased to approximately 1/100, and was completely lost at −20°C and −30°C. Four T-cell-tropic and four macrophage-tropic laboratory strains and clinical isolates of HIV-1 became completely inactivated at −30°C. Treatment by FPGM at −20°C to −30°C reduced HIV-1 reverse transcriptase activity to approximately one tenth. In addition, treatment by FPGM at −20°C was found to destroy the ability of HIV-1 to bind to CD4+ cells. In conclusion, this study showed that treatment by FPGM at −20°C to −30°C destroyed the infectivity of a wide range of HIV-1 strains, and suggested that the mechanisms of HIV-1 inactivation were the reduction in viral enzyme activity and the loss of the cell-binding ability of a viral envelope protein.
Enhancement of ginsenoside biosynthesis in high-density cultivation of Panax notoginseng cells by various strategies of methyl jasmonate elicitation by Wei Wang; Zhan-Ying Zhang; Jian-Jiang Zhong (pp. 752-758).
A single addition of 200 μM methyl jasmonate (MJA) to high-density cell cultures of Panax notoginseng enhanced ginsenoside production in both shake-flask (250 ml) and airlift bioreactor (ALR; 1 l working volume). Repeated elicitation with two additions of 200 μM MJA during cultivation further induced the ginsenoside biosynthesis in both cultivation vessels. The content of ginsenosides Rg1, Re, Rb1 and Rd in the ALR was increased from, respectively, 0.18±0.01, 0.21±0.01, 0.21±0.02 and 0 mg per100 mg dry cell weight (DW) in untreated cell cultures (control) to 0.32±0.02, 0.36±0.02, 0.72±0.06 and 0.08±0.01 mg per100 mg DW with a single addition of MJA and further increased to 0.43±0.02, 0.46±0.03, 1.09±0.07 and 0.14±0.02 mg per100 mg DW with two additions of MJA. Interestingly, the activity of the Rb1 biosynthetic enzyme (UDPG-ginsenoside Rd glucosyltransferase), was also increased with a single elicitation by MJA and increased again by a repeated elicitation, which coincided well with the trend in the increase in Rb1 content. In order to further improve the cell density and ginsenoside production, a strategy of MJA repeated elicitation combined with sucrose feeding was adopted. The final cell density and total ginsenoside content in the ALR reached 27.3±1.5 g/l and 2.02±0.06 mg per100 mg DW; and the maximum production of ginsenoside Rg1, Re, Rb1 and Rd was 111.8±4.7, 117.2±4.6, 290.2±5.1 and 32.7±8.1 mg/l, respectively. The strategies demonstrated and the information obtained in this work are useful for the efficient large-scale production of bioactive ginsenosides by plant cell cultures.
Optimization for the production of water-soluble polysaccharide from Pleurotus citrinopileatus in submerged culture and its antitumor effect by Jinn-Chyi Wang; Shu-Hui Hu; Zeng-Chin Liang; Chi-Jung Yeh (pp. 759-766).
In recent years, a number of studies have been done on the physiological effects of water-soluble polysaccharides (WSPS) and their antitumor and immuno-enhancing properties. Many edible mushrooms, in particular those rich in WSPS, not only taste good but also contain ingredients beneficial to the physiology of the human body. In this study, response surface methodology was used to determine the optimal conditions for the production of WSPS, including the C/N ratio, initial pH, and incubation temperature. The highest yield of WSPS was obtained by incubation with a C/N ratio of 40, initial pH 5.5, and an incubation temperature of 25°C. WSPS were extracted by alcohol precipitation from the fermented broth of edible Pleurotus citrinopileatus. These extracts, referred to as SPPC in this paper, had a molecular mass of more than 105 Da and were largely made up of glucose and mannose. SPPC was fed to mice which had artificial pulmonary metastatic tumors. Changes in the percentage of the numbers of tumor cells and immune cells were determined by flow cytometry. Daily feeding of SPPC at a dosage of 50 mg/kg to tumor-bearing mice for 12 days resulted in a significant increase in the number of T cells, CD4+ cells, CD8+ cells, and macrophages, compared with mice that were not fed any SPPC. The proliferation rate of the pulmonary sarcoma lesions slowed down.
Emulsan quantitation by Nile red quenching fluorescence assay by Guillermo R. Castro; Bridget K. Larson; Bruce Panilaitis; David L. Kaplan (pp. 767-770).
A Nile red fluorescent technique to quantify 20–200 μg ml−1 of emulsan was developed. Nile red dissolved in DMSO showed an adsorption peak at 552 nm, and emission peak at 636 nm, with molar extinction coefficient of 19,600 cm−1 M−1. Nile red fluorescence in DMSO was proportionally quenched by emulsan and the quenching was time-dependent. The assay was used to follow the production of emulsan by cultures of Acinetobacter venetianus RAG-1.
Biotransformations for the production of the chiral drug (S)-Duloxetine catalyzed by a novel isolate of Candida tropicalis by Pankaj Soni; U. C. Banerjee (pp. 771-777).
A yeast strain, Candida tropicalis PBR-2, isolated from soil, is capable of carrying out the enantioselective reduction of N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine to (S)-N,N-dimethyl-3-hydroxy-3-(2-thienyl)-1-propanamine, a key intermediate in the synthesis of the chiral drug (S)-Duloxetine. The organism produced the enantiopure (S)-alcohol with a good yield (>80%) and almost absolute enantioselectivity, with an enantiomeric excess (ee) >99%. Parameters of the bioreduction reaction were optimized and the optimal temperature and pH for the reduction were found to be 30°C and 7.0, respectively. The optimized substrate and the resting cell concentration were 1 g/l and 250 g/l, respectively. The preparative-scale reaction using resting cells of C. tropicalis yielded the (S)-alcohol at 84–88% conversion and ee >99%.
Purification and characterization of a biodegradable plastic-degrading enzyme from Aspergillus oryzae by Hiroshi Maeda; Youhei Yamagata; Keietsu Abe; Fumihiko Hasegawa; Masayuki Machida; Ryoji Ishioka; Katsuya Gomi; Tasuku Nakajima (pp. 778-788).
We used biodegradable plastics as fermentation substrates for the filamentous fungus Aspergillus oryzae. This fungus could grow under culture conditions that contained emulsified poly-(butylene succinate) (PBS) and emulsified poly-(butylene succinate-co-adipate) (PBSA) as the sole carbon source, and could digest PBS and PBSA, as indicated by clearing of the culture supernatant. We purified the PBS-degrading enzyme from the culture supernatant, and its molecular mass was determined as 21.6 kDa. The enzyme was identified as cutinase based on internal amino acid sequences. Specific activities against PBS, PBSA and poly-(lactic acid) (PLA) were determined as 0.42 U/mg, 11 U/mg and 0.067 U/mg, respectively. To obtain a better understanding of how the enzyme recognizes and hydrolyzes PBS/PBSA, we investigated the environment of the catalytic pocket, which is divided into carboxylic acid and alcohol recognition sites. The affinities for different substrates depended on the carbon chain length of the carboxylic acid in the substrate. Competitive inhibition modes were exhibited by carboxylic acids and alcohols that consisted of C4-C6 and C3-C8 chain lengths, respectively. Determination of the affinities for different chemicals indicated that the most preferred substrate for the enzyme would consist of butyric acid and n-hexanol.
Chromosomal integration of sfp gene in Bacillus subtilis to enhance bioavailability of hydrophobic liquids by Young-Ki Lee; Seong-Bin Kim; Chan-Sun Park; Jong-Guk Kim; Hee-Mock Oh; Byung-Dae Yoon; Hee-Sik Kim (pp. 789-794).
Bacillus subtilis C9 effectively degrades aliphatic hydrocarbons up to a chain length of C19 and produces a lipopeptide-type biosurfactant, surfactin, yet it has no genetic competency. Therefore, to obtain a transformable surfactin producer, the sfp gene cloned from B. subtilis C9 was integrated into the chromosome of B. subtilis 168, a non-surfactin producer, by homologous recombination. The transformants reduced the surface tension of the culture broth from 70.0 mN/m to 28.0 mN/m, plus the surface-active compound produced by the transformants exhibited the same Rf value as that from B. subtilis C9 and authentic surfactin in a thin-layer chromatographic analysis. The integration of the sfp gene into the chromosome of B. subtilis 168 was confirmed by Southern hybridization. Like B. subtilis C9, the transformants readily degraded n-hexadecane, although the original strain did not. It was also statistically confirmed that the hydrocarbon degradation of the transformants was highly correlated to their surfactin production by the determination of the correlation coefficient (r2=0.997, P<0.01). Therefore, these results indicate that the surfactin produced from B. subtilis enhances the bioavailability of hydrophobic liquids.
A novel actinomycete strain de-replication approach based on the diversity of polyketide synthase and nonribosomal peptide synthetase biosynthetic pathways by Angel Ayuso; Desmond Clark; Ignacio González; Oscar Salazar; Annaliesa Anderson; Olga Genilloud (pp. 795-806).
The actinomycetes traditionally represent one of the most important sources for the discovery of new metabolites with biological activity; and many of these are described as being produced by polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). We present a strain characterization system based on the metabolic potential of microbial strains by targeting these biosynthetic genes. After an initial evaluation of the existing bias derived from the PCR detection in a well defined biosynthetic systems, we developed a new fingerprinting approach based on the restriction analysis of these PKS and NRPS amplified sequences. This method was applied to study the distribution of PKS and NRPS biosynthetic systems in a collection of wild-type actinomycetes isolated from tropical soil samples that were evaluated for the production of antimicrobial activities. We discuss the application of this tool as an alternative characterization approach for actinomycetes and we comment on the relationship observed between the presence of PKS-I, PKS-II and NRPS sequences and the antimicrobial activities observed in some of the microbial groups tested.
Examination of fungal stress response genes using Saccharomyces cerevisiae as a model system: targeting genes affecting aflatoxin biosynthesis by Aspergillus flavus Link by Jong H. Kim; Bruce C. Campbell; Jiujiang Yu; Noreen Mahoney; Kathleen L. Chan; Russell J. Molyneux; Deepak Bhatnagar; Thomas E. Cleveland (pp. 807-815).
Saccharomyces cerevisiae served as a model fungal system to examine functional genomics of oxidative stress responses and reactions to test antioxidant compounds. Twenty-two strains of S. cerevisiae, including a broad spectrum of singular gene deletion mutants, were exposed to hydrogen peroxide (H2O2) to examine phenotypic response to oxidative stress. Responses of particular mutants treated with gallic, tannic or caffeic acids, or methyl gallate, during H2O2 exposure, indicated that these compounds alleviated oxidative stress. These compounds are also potent inhibitors of aflatoxin biosynthesis in Aspergillus flavus. To gain further insights into a potential link between oxidative stress and aflatoxin biosynthesis, 43 orthologs of S. cerevisiae genes involved in gene regulation, signal transduction (e.g., SHO1, HOG1, etc.) and antioxidation (e.g., CTT1, CTA1, etc.) were identified in an A. flavus expressed sequence tag library. A successful exemplary functional complementation of an antioxidative stress gene from A. flavus, mitochondrial superoxide dismutase (sodA), in a sod2Δ yeast mutant further supported the potential of S. cerevisiae deletion mutants to serve as a model system to study A. flavus. Use of this system to further examine functional genomics of oxidative stress in aflatoxigenesis and reduction of aflatoxin biosynthesis by antioxidants is discussed.
Homozygous diploid deletion strains of Saccharomyces cerevisiae that determine lag phase and dehydration tolerance by Riccardo D’Elia; Patricia L. Allen; Kelly Johanson; Cheryl A. Nickerson; Timothy G. Hammond (pp. 816-826).
This study identifies genes that determine length of lag phase, using the model eukaryotic organism, Saccharomyces cerevisiae. We report growth of a yeast deletion series following variations in the lag phase induced by variable storage times after drying-down yeast on filters. Using a homozygous diploid deletion pool, lag times ranging from 0 h to 90 h were associated with increased drop-out of mitochondrial genes and increased survival of nuclear genes. Simple linear regression (R2 analysis) shows that there are over 500 genes for which >70% of the variation can be explained by lag alone. In the genes with a positive correlation, such that the gene abundance increases with lag and hence the deletion strain is suitable for survival during prolonged storage, there is a strong predominance of nucleonic genes. In the genes with a negative correlation, such that the gene abundance decreases with lag and hence the strain may be critical for getting yeast out of the lag phase, there is a strong predominance of glycoproteins and transmembrane proteins. This study identifies yeast deletion strains with survival advantage on prolonged storage and amplifies our understanding of the genes critical for getting out of the lag phase.
Xylose chemostat isolates of Saccharomyces cerevisiae show altered metabolite and enzyme levels compared with xylose, glucose, and ethanol metabolism of the original strain by Juha-Pekka Pitkänen; Eija Rintala; Aristos Aristidou; Laura Ruohonen; Merja Penttilä (pp. 827-837).
The efficient conversion of xylose-containing biomass hydrolysate by the ethanologenic yeast Saccharomyces cerevisiae to useful chemicals such as ethanol still remains elusive, despite significant efforts in both strain and process development. This study focused on the recovery and characterization of xylose chemostat isolates of a S. cerevisiae strain that overexpresses xylose reductase- and xylitol dehydrogenase-encoding genes from Pichia stipitis and the gene encoding the endogenous xylulokinase. The isolates were recovered from aerobic chemostat cultivations on xylose as the sole or main carbon source. Under aerobic conditions, on minimal medium with 30 g l−1 xylose, the growth rate of the chemostat isolates was 3-fold higher than that of the original strain (0.15 h−1 vs 0.05 h−1). In a detailed characterization comparing the metabolism of the isolates with the metabolism of xylose, glucose, and ethanol in the original strain, the isolates showed improved properties in the assumed bottlenecks of xylose metabolism. The xylose uptake rate was increased almost 2-fold. Activities of the key enzymes in the pentose phosphate pathway (transketolase, transaldolase) increased 2-fold while the concentrations of their substrates (pentose 5-phosphates, sedoheptulose 7-phosphate) decreased correspondingly. Under anaerobic conditions, on minimal medium with 45 g l−1 xylose, the ethanol productivity (in terms of cell dry weight; CDW) of one of the isolates increased from 0.012 g g−1 CDW h−1 to 0.017 g g−1 CDW h−1 and the yield from 0.09 g g−1 xylose to 0.14 g g−1 xylose, respectively.
Efficient decontamination of zearalenone, the mycotoxin of cereal pathogen, by transgenic yeasts through the expression of a synthetic lactonohydrolase gene by Naoko Takahashi-Ando; Takeshi Tokai; Hiroshi Hamamoto; Isamu Yamaguchi; Makoto Kimura (pp. 838-844).
Zearalenone (ZEN), an estrogenic mycotoxin produced by several Fusarium species, is converted to a non-estrogenic product by a detoxifying enzyme of Clonostachys rosea. Previously, we investigated whether recombinant Saccharomyces cerevisiae carrying this detoxification gene, zhd101, can remove 2 μg ml−1 of ZEN in a liquid culture. Although the transgenic yeasts eliminated most of the ZEN, they also converted a significant amount to a poor substrate, β-zearalenol, which remained in the medium. In this study, we synthesized a codon-optimized zhd101 gene and investigated whether the transgenic yeast strain can overcome the problem of insufficient detoxification of ZEN. Importantly, within 48 h of incubation at 28°C or 8 h of incubation at 37°C, the transgenic yeasts completely eliminated 2 μg ml−1 of ZEN in the medium without accumulating even a trace amount of β-zearalenol. The result suggests that incomplete ZEN detoxification attributed to the action of an endogenous yeast β-reductase can be overcome by simply increasing the expression of the detoxifying gene.
Biodegradation of the organochlorine insecticide, endosulfan, and the toxic metabolite, endosulfan sulfate, by Klebsiella oxytoca KE-8 by Gi-Seok Kwon; Ho-Yong Sohn; Kee-Sun Shin; Eungbin Kim; Bu-Il Seo (pp. 845-850).
Biodegradation of endosulfan, a chlorinated cyclodiene insecticide, is generally accompanied by production of the more toxic and more persistent metabolite, endosulfan sulfate. Since our reported endosulfan degrader, Klebsiella pneumoniae KE-1, failed to degrade endosulfan sulfate, we tried to isolate an endosulfan sulfate degrader from endosulfan-polluted soils. Through repetitive enrichment and successive subculture using mineral salt medium containing endosulfan or endosulfan sulfate as the sole source of carbon and energy, we isolated a bacterium capable of degrading endosulfan sulfate as well as endosulfan. The bacterium KE-8 was identified as Klebsiella oxytoca from the results of 16S rDNA sequence analysis. In biodegradation assays with KE-8 using mineral salt medium containing endosulfan (150 mg l−1) or endosulfan sulfate (173 mg l−1), the biomass was rapidly increased to an optical density at 550 nm of 1.9 in 4 days and the degradation constants for α- and β-endosulfan, and endosulfan sulfate were 0.3084, 0.2983 and 0.2465 day−1, respectively. Analysis of the metabolites further suggested that K. oxytoca KE-8 has high potential as a biocatalyst for bioremediation of endosulfan and/or endosulfan sulfate.
Demineralization of red crab shell waste by lactic acid fermentation by W. J. Jung; J. H. Kuk; K. Y. Kim; R. D. Park (pp. 851-854).
Lactic acid fermentation was applied to demineralize red crab shell waste using Lactobacillus paracasei subsp. tolerans KCTC-3074. Various concentrations (0, 2.5, 5.0, 10.0%) of glucose were supplemented as an initial carbon source and various amounts (2.5, 5.0, 10.0%) of the bacterial culture were inoculated as a starter. Microbial growth was very dependent on glucose concentration but little dependent on inoculum level. The pH decreased rapidly from pH 8 to pH 6 during the first day, at all three inoculum levels. At day 5 of fermentation, the 2.5, 5.0, and 10.0% inoculum levels with 10% glucose supply gave pH 5.5, 5.1, and 4.6, respectively, i.e. a decrease in pH concomitant with an increase in the inoculum level. The total titratable acidities (TTA) at day 5 for 2.5, 5.0, and 10.0% inoculum levels with 10% glucose supplement were 3.1, 4.5, and 8.3%, and the relative residual ash contents were 26.6, 25.9, and 19.0%, respectively. A negative relationship was found between pH and demineralization level (r2=0.8571), but there was a positive relationship between TTA and demineralization level (r2=0.5532).