JBB : Journal of Bioscience and Bioengineering

            

Journal of Bioscience and Bioengineering vol.123 cover

 



  • Self-excising Cre/mutant lox marker recycling system for multiple gene integrations and consecutive gene deletions in Aspergillus oryzae
    Publication date: April 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 4

    Author(s): Silai Zhang, Akihiko Ban, Naoki Ebara, Osamu Mizutani, Mizuki Tanaka, Takahiro Shintani, Katsuya Gomi

    In this study, we developed a self-excising Cre/loxP-mediated marker recycling system with mutated lox sequences to introduce a number of biosynthetic genes into Aspergillus oryzae. To construct the self-excising marker cassette, both the selectable marker, the Aspergillus nidulans adeA gene, and the Cre recombinase gene (cre), conditionally expressed by the xylanase-encoding gene promoter, were designed to be located between the mutant lox sequences, lox66 and lox71. However, construction of the plasmid failed, possibly owing to a slight expression of cre downstream of the fungal gene promoter in Escherichia coli. Hence, to avoid the excision of the cassette in E. coli, a 71-bp intron of the A. oryzae xynG2 gene was inserted into the cre gene. The A. oryzae adeA deletion mutant was transformed with the resulting plasmid in the presence of glucose, and the transformants were cultured in medium containing xylose as the sole carbon source. PCR analysis of genomic DNA from resultant colonies revealed the excision of both the marker and Cre expression construct, indicating that the self-excising marker cassette was efficient at removing the selectable marker. Using the marker recycling system, hyperproduction of kojic acid could be achieved in A. oryzae by the introduction of two genes that encode oxidoreductase and transporter. Furthermore, we also constructed an alternative marker recycling cassette bearing the A. nidulans pyrithiamine resistant gene (ptrA) as a dominant selectable marker.





  • Characterization and functional expression of a rubber degradation gene of a Nocardia degrader from a rubber-processing factory
    Publication date: April 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 4

    Author(s): Dao Viet Linh, Nguyen Lan Huong, Michiro Tabata, Shunsuke Imai, Sou Iijima, Daisuke Kasai, To Kim Anh, Masao Fukuda

    A rubber-degrading bacterial consortium named H2DA was obtained from an enrichment culture with natural rubber latex and rubber-processing factory waste in Vietnam. Gel permeation chromatography analysis revealed that only the strain NVL3 degraded synthetic poly(cis-1,4-isoprene) into low-molecular-weight intermediates among the three strains found in the H2DA. The 16S-rRNA gene sequence of NVL3 showed the highest identity with that of Nocardia farcinica DSM 43665T. NVL3 accumulated aldehyde intermediates from synthetic poly(cis-1,4-isoprene) on a rubber-overlay plate as indicated by Schiff's staining. NVL3 also degraded deproteinized natural rubber into low-molecular-weight aldehyde intermediates. A latex-clearing protein (lcp) gene ortholog was identified within the genome sequence of NVL3, and it showed a moderate amino-acid identity (54–75%) with the lcp genes from previously reported rubber degraders. The heterologous expression of the NVL3 lcp in Escherichia coli BL21(DE3) allowed us to purify the 46.8-kDa His-tagged lcp gene product (His-Lcp). His-Lcp degraded synthetic poly(cis-1,4-isoprene) and accumulated aldehyde intermediates from deproteinized natural rubber suggesting the functional expression of the lcp gene from a Nocardia degrader in E. coli. Quantitative reverse transcription PCR analysis indicated the strong transcriptional induction of the lcp gene in NVL3 in the presence of synthetic poly(cis-1,4-isoprene). These results suggest the involvement of the lcp gene in rubber degradation in NVL3.





  • Enhanced in-cell folding of reversibly cationized transcription factor using amphipathic peptide
    Publication date: April 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 4

    Author(s): Midori Futami, Tomoki Nakano, Mayu Yasunaga, Masahiro Makihara, Takashi Asama, Yoshihisa Hagihara, Yoshihiro Nakajima, Junichiro Futami

    The intracellular delivery of functionally active transcription factor proteins is emerging as a promising technique for artificial regulation of cellular functions. However, in addition to the cell membrane, which acts as a barrier to macromolecules, the aggregation-favored properties of structurally flexible transcription factor proteins limit the application of this method. In-cell folding technique can be used to overcome these issues. This technique solubilizes denatured protein by reversible alkyl-disulfide cationization (S-cationization), and simultaneously endows efficient intracellular delivery and folding to the biologically active conformation in the reducing environment of the cytosol. Because cationized protein is internalized into cells by adsorption-mediated endocytosis, endosomal escape is crucial for this technique. In this study, we utilized a sensitive luciferase reporter gene assay to quantitatively evaluate in-cell folding of the artificial transcription factor GAL4-VP16. Although the cationic moiety of S-cationized protein was slightly affected, co-transduction of amphipathic peptide Endo-PORTER dramatically improved in-cell folding efficiency. Live cell imaging of fluorescent-labeled GAL4-VP16 revealed that some of the proteins diffused into the cytosol and nucleus through co-transduction with Endo-PORTER. Real-time monitoring of light output of luciferase revealed the kinetics of in-cell folding, supporting that endosomal-release assisted by Endo-PORTER was stimulated by endosome acidification. Because this method can transduce proteins uniformly and repeatedly into living cells, S-cationized transcription factor proteins are widely applicable for the artificial regulation of cellular functions.





  • Characterization of a novel DyP-type peroxidase from Streptomyces avermitilis
    Publication date: April 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 4

    Author(s): Kanako Sugawara, Yuriko Nishihashi, Tomomi Narioka, Toru Yoshida, Mifumi Morita, Yasushi Sugano

    DyP-type peroxidases are a heme peroxidase family with unique properties whose members are widely distributed from prokaryotes to eukaryotes. DyP-type peroxidases are subdivided into class P, I and V based on structure-based sequence alignment. Class V enzymes possess degradation activities for anthraquinone dyes, and include extra sequences compared with class P and I. Class V enzymes are mainly found in fungi, with only two such proteins, AnaPX and DyP2, reported in bacteria. Here, we heterologously expressed, purified and biochemically characterized SaDyP2 protein, predicted to belong to class V. SaDyP2 was purified as a ∼50 kDa enzyme containing a heme cofactor and was found to oxidize the typical peroxidase substrates, ABTS and DMP. SaDyP2 was generally thermostable and exhibited a lower optimal pH, a feature typical of DyP-type peroxidases. It also degraded anthraquinone dyes, a specific substrate of DyP-type peroxidases, although the k cat for SaDyP2 was lower than that for other class V enzymes. The K m value of SaDyP2 for anthraquinone dye was similar to that of other enzymes of this class. Homology modeling revealed that the structure of SaDyP2 best fit that of class V enzymes.





  • pH-gradient ion-exchange microbial cell chromatography as a simple method for microbial separation
    Publication date: April 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 4

    Author(s): Yoshiteru Aoi, Yuji Kaneko, Satoshi Tsuneda

    Selective separation of specific microbial types from a heterogeneous microbial population, such as an environmental microbial community, is an important process for microbial research and biotechnological industries. In the present study, pH-gradient ion-exchange microbial cell chromatography (PIE-MCC) was developed as a new method for microbial separation. The proposed method enables target microorganisms to be separated from a microbial community based on differences in microbial surface characteristics, because these characteristics, such as the ζ (zeta)-potential, vary among microbial cells. PIE-MCC was conducted by controlling the adhesion and detachment of microbial cells to and from the carrier surface by manipulating the pH of the running buffer. As a proof of concept, microbial cell separation via PIE-MCC was demonstrated using pure-cultured strains, model mixtures of two different pure-cultured strains, and an environmental sample targeting uncultivated microorganisms; i.e., each pure-cultured strain showed unique chromatograms; specific single species were separated from the model mixture; and a specific, uncultivated target was separated from the environmental sample. The ζ-potential of several tested strains suggested that not only electrostatic interactions, but also other factors affected microbial adhesion to the carrier surface. The newly developed method has several potential advantages compared with other techniques, not only in terms of its microbial separation capability, but also in terms of its simplicity and ability to be scaled up. Thus, the method has the potential to be widely used for a variety of purposes in the microbiology and biotechnology fields.





  • Pyruvate dehydrogenase complex regulator (PdhR) gene deletion boosts glucose metabolism in Escherichia coli under oxygen-limited culture conditions
    Publication date: April 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 4

    Author(s): Soya Maeda, Kumiko Shimizu, Chie Kihira, Yuki Iwabu, Ryuichi Kato, Makoto Sugimoto, Satoru Fukiya, Masaru Wada, Atsushi Yokota

    Pyruvate dehydrogenase complex regulator (PdhR) is a transcriptional regulator that negatively regulates formation of pyruvate dehydrogenase complex (PDHc), NADH dehydrogenase (NDH)-2, and cytochrome bo 3 oxidase in Escherichia coli. To investigate the effects of a PdhR defect on glucose metabolism, a pdhR deletion mutant was derived from the wild-type E. coli W1485 strain by λ Red-mediated recombination. While no difference in the fermentation profiles was observed between the two strains under oxygen-sufficient conditions, under oxygen-limited conditions, the growth level of the wild-type strain was significantly decreased with retarded glucose consumption accompanied by by-production of substantial amounts of pyruvic acid and acetic acid. In contrast, the mutant grew and consumed glucose more efficiently than did the wild-type strain with enhanced respiration, little by-production of pyruvic acid, less production yield and rates of acetic acid, thus displaying robust metabolic activity. As expected, increased activities of PDHc and NDH-2 were observed in the mutant. The increased activity of PDHc may explain the loss of pyruvic acid by-production, probably leading to decreased acetic acid formation, and the increased activity of NDH-2 may explain the enhanced respiration. Measurement of the intracellular NAD+/NADH ratio in the mutant revealed more oxidative or more reductive intracellular environments than those in the wild-type strain under oxygen-sufficient and -limited conditions, respectively, suggesting another role of PdhR: maintaining redox balance in E. coli. The overall results demonstrate the biotechnological advantages of pdhR deletion in boosting glucose metabolism and also improve our understanding of the role of PdhR in bacterial physiology.





  • Expression of the alaE gene is positively regulated by the global regulator Lrp in response to intracellular accumulation of l-alanine in Escherichia coli
    Publication date: April 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 4

    Author(s): Kohei Ihara, Kazuki Sato, Hatsuhiro Hori, Yumiko Makino, Shuji Shigenobu, Tasuke Ando, Emiko Isogai, Hiroshi Yoneyama

    The alaE gene in Escherichia coli encodes an l-alanine exporter that catalyzes the active export of l-alanine using proton electrochemical potential. In our previous study, alaE expression was shown to increase in the presence of l-alanyl-l-alanine (Ala-Ala). In this study, the global regulator leucine-responsive regulatory protein (Lrp) was identified as an activator of the alaE gene. A promoter less β-galactosidase gene was fused to an alaE upstream region (240 nucleotides). Cells that were lacZ-deficient and harbored this reporter plasmid showed significant induction of β-galactosidase activity (approximately 17-fold) in the presence of 6 mM l-alanine, l-leucine, and Ala-Ala. However, a reporter plasmid possessing a smaller alaE upstream region (180 nucleotides) yielded transformants with strikingly low enzyme activity under the same conditions. In contrast, lrp-deficient cells showed almost no β-galactosidase induction, indicating that Lrp positively regulates alaE expression. We next performed an electrophoretic mobility shift assay (EMSA) and a DNase I footprinting assay using purified hexahistidine-tagged Lrp (Lrp-His). Consequently, we found that Lrp-His binds to the alaE upstream region spanning nucleotide −161 to −83 with a physiologically relevant affinity (apparent KD, 288.7 ± 83.8 nM). Furthermore, the binding affinity of Lrp-His toward its cis-element was increased by l-alanine and l-leucine, but not by Ala-Ala and d-alanine. Based on these results, we concluded that the gene expression of the alaE is regulated by Lrp in response to intracellular levels of l-alanine, which eventually leads to intracellular homeostasis of l-alanine concentrations.





  • Biosynthesis of eight-carbon volatiles from tomato and pepper pomaces by fungi: Trichoderma atroviride and Aspergillus sojae
    Publication date: April 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 4

    Author(s): Onur Güneşer, Yonca Karagül Yüceer

    The aim of this study was to investigate the possibility of using tomato and red pepper pomaces for the production eight-carbon volatiles by Trichoderma atroviride and Aspergillus sojae. The fermentation of tomato and pepper pomace-based media by both moulds was conducted in shake flasks and bioreactors. Microbial growth behaviours and fermentation abilities of T. atroviride and A. sojae under both fermentation conditions were followed by microbial counting. The production of flavours from tomato and pepper pomaces by fungal metabolism was determined by gas chromatography–olfactometry, gas chromatography–mass spectrometry and sensory analysis. The results showed that T. atroviride grew faster than A. sojae, and the survival of T. atroviride in the tomato pomace was longer than that of A. sojae. However, T. atroviride grew slower than A. sojae in the pepper pomace. Eight-carbon flavour compounds, including (Z)-1,5-octadien-3-ol, 1-octen-3-ol, (E)-2-octenal and (E)-2-octenol, were produced by T. atroviride and A. sojae from the tomato and pepper pomaces. The highest production levels (265.55 ± 2.79 and 187.47 ± 0.92 μg kg−1) were observed for 1-octen-3-ol in the tomato fermentation by T. atroviride and A. sojae, respectively. The relationships between volatile compounds and their flavour characteristics in tomato and pepper pomaces were analysed using principal component analysis.





  • Innovation Chinese rice wine brewing technology by bi-acidification to exclude rice soaking process
    Publication date: April 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 4

    Author(s): Xiao Lu Wei, Shuang Ping Liu, Jian Shen Yu, Yong Jian Yu, Sheng Hu Zhu, Zhi Lei Zhou, Jian Hu, Jian Mao

    As a traditional fermented alcoholic beverage of China, Chinese rice wine (CRW) had a long history of more than 5000 years. Rice soaking process was the most crucial step during CRW brewing process, because rice soaking quality directly determined the quality of CRW. However, rice soaking water would cause the eutrophication of water bodies and waste of water. The longer time of rice soaking, the higher the content of biogenic amine, and it would have a huge impact on human health. An innovation brewing technology was carried out to exclude the rice soaking process and the Lactobacillus was added to make up for the total acid. Compared to the traditional brewing technology, the new technology saved water resources and reduced environmental pollution. The concentration of biogenic amine was also decreased by 27.16%, which improving the security of the CRW. The esters increased led to more soft-tasted CRW and less aging time; the quality of CRW would be improved with less alcohol.





  • Ammonium conversion and its feedback effect on methane oxidation of Methylosinus sporium
    Publication date: April 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 4

    Author(s): Ruo He, Min Chen, Ruo-Chan Ma, Yao Su, Xuan Zhang

    Ammonium (NH4 +) is not only nitrogen source that can support methanotrophic growth, but also it can inhibit methane (CH4) oxidation by competing with CH4 for the active site of methane monooxygenase. NH4 + conversion and its feedback effect on the growth and activity of methanotrophs were evaluated with Methylosinus sporium used as a model methanotroph. Nitrogen sources could affect the CH4-derived carbon distribution, which varied with incubation time and nitrogen concentrations. More CH4-derived carbon was incorporated into biomass in the media with NH4 +–N, compared to nitrate–nitrogen (NO3 –N), as sole nitrogen source at the nitrogen concentrations of 10–18 mmol L−1. Although ammonia (NH3) oxidation activity of methanotrophs was considerably lower, only accounting for 0.01–0.06% of CH4 oxidation activity in the experimental cultures, NH4 + conversion could lead to the pH decrease and toxic intermediates accumulation in the their habits. Compared with NH4 +, nitrite (NO2 ) accumulation in the NH4 + conversion of methanotroph had stronger inhibition on its activity, especially the joint inhibition of NO2 accumulation and the pH decrease during the NH4 +–N conversion. These results suggested that more attention should be paid to the feedback effects of NH4 + conversion by methanotrophs to understand effects of NH4 + on CH4 oxidation in the environments.





  • Effect of freeze–thaw process on physical properties, microbial activities and population structures of anaerobic sludge
    Publication date: April 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 4

    Author(s): Chantaraporn Phalakornkule, Sasikarn Nuchdang, Maneerat Khemkhao, Wuttichai Mhuantong, Sarunyou Wongwilaiwalin, Sithichoke Tangphatsornruang, Verawat Champreda, Jiraprat Kitsuwan, Savitri Vatanyoopaisarn

    Effects of the freeze–thaw process on physical properties, cell viability, microbial activities and population structures of anaerobic sludge were investigated. It was found that the sludge volume index was greatly reduced from 16.4 mL/g in the original sludge to 4.0 mL/g in the solid fraction of the frozen–thawed sludge. Even though the freeze–thaw process decreased cell viability in the solid fraction of the frozen–thawed sludge, microbial activity tests showed that the freeze–thaw process enhanced acidogenic activity approximately 20%. The enhanced acidogenic activity of the solid fraction was in good agreement with the enrichment of Clostridiaceae, Porphyromonadaceae and Propionibacteriaceae found in the solid fraction. The relative abundances of Proteobacteria families Oxalobacteraceae, Moraxellaceae, and Pseudomonadaceae were found to be highest in the liquid fraction where they form a substantial proportion of the bacterial community (a total of 59%).





  • Inhibition kinetics of nitritation and half-nitritation of old landfill leachate in a membrane bioreactor
    Publication date: April 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 4

    Author(s): Yun Li, Zhaozhao Wang, Jun Li, Jia Wei, Yanzhuo Zhang, Baihang Zhao

    Nitritation can be used as a pretreatment for anaerobic ammonia oxidation (anammox). Various control strategies for nitritation and half-nitritation of old landfill leachate in a membrane bioreactor were investigated in this study and the inhibition kinetics of substrate, product and old landfill leachate on nitritation were analyzed via batch tests. The results demonstrated that old landfill leachate nitritation in the membrane bioreactor can be achieved by adjusting the influent loading and dissolved oxygen (DO). From days 105–126 of the observation period, the average effluent concentration was 871.3 mg/L and the accumulation rate of NO 2 N was 97.2%. Half-nitritation was realized quickly by adjusting hydraulic retention time and DO. A low-DO control strategy appeared to best facilitate long-term and stable operation. Nitritation inhibition kinetic experiments showed that the inhibition of old landfill leachate was stronger than that of the substrate ( NH 4 + N ) or product ( NO 2 N ) . The ammonia oxidation rate dropped by 22.2% when the concentration of old landfill leachate (calculated in chemical oxygen demand) was 1600.2 mg/L; further, when only free ammonia or free nitrous acid were used as a single inhibition factor, the ammonia oxidation rate dropped by 4.7–6.5% or 14.5–15.9%, respectively. Haldane, Aiba, and a revised inhibition kinetic model were adopted to separately fit the experimental data. The R 2 correlation coefficient values for these three models were 0.982, 0.996, and 0.992, respectively.





  • Pretreatment of lignocellulosic biomass by cattle rumen fluid for methane production: Bacterial flora and enzyme activity analysis
    Publication date: April 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 4

    Author(s): Yasunori Baba, Yu Matsuki, Yumi Mori, Yoshihisa Suyama, Chika Tada, Yasuhiro Fukuda, Masanori Saito, Yutaka Nakai

    We attempted to develop a pretreatment method for methane fermentation of lignocellulosic biomass using cattle rumen fluid, treated as slaughterhouse waste. When rapeseed (Brassica napus L.) was added to the methane fermentation after being solubilized with rumen fluid, 1.5 times more methane was produced compared with untreated rapeseed. Analysis of the bacterial flora during rumen fluid treatment using the MiSeq next-generation sequencer showed that the predominant phylum shifted from Bacteroidetes, composed of amylolytic Prevotella spp., to Firmicutes, composed of cellulolytic and xylanolytic Ruminococcus spp., in only 6 h. In total, 7 cellulolytic, 25 cello-oligosaccharolytic, and 11 xylanolytic bacteria were detected after investigating the most abundant sequences of detected taxa. The relative abundance of two Ruminococcus species (Ruminococcus albus and R. flavefaciens), known as cellulolytic, cello-oligosaccharolytic, and xylanolytic bacteria, increased with increasing cellulose and hemicellulose degradation rates, and, finally, comprised 48% of all operational taxonomic units. The chronological observation of enzyme activities showed that cellulolytic and xylanolytic activities increased 6 h later, and that oligosaccharolytic activity increased 24 h later. This study detected six bacteria that participate in the degradation of aromatics derived from lignin, which have rarely been reported in rumen fluid. The constitution of the detected bacteria suggests that the aromatics were converted into acetate via benzoate. The list of microbes that cover all lignocellulose-degrading candidates will provide fundamental knowledge for future studies focusing on rumen microbes.





  • Effect of carbon source on nitrogen removal in anaerobic ammonium oxidation (anammox) process
    Publication date: April 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 4

    Author(s): Weiqiang Zhu, Peiyu Zhang, Huiyu Dong, Jin Li

    Anaerobic ammonium oxidation (anammox) has been regarded as an efficient process to treat high-strength wastewater without organic carbon source. To investigate nitrogen removal performance of anammox in presence of organic carbon source can broaden its application on organic wastewater treatment. In this work, effect of carbon source on anammox process was explored. Operating temperature was set at 35 ± 1°C. Influent pH and hydraulic retention time were 7.5 and 6 h, respectively. Effluent NH 4 + N was affected little with COD no more than 480 mg/L. Independent of carbon source content, nitrite removal rate was around 99%. The variation of Δ NO 2 N / Δ NH 4 + N lagged behind Δ NO 3 N / Δ NH 4 + N at high COD content, and pH could be used as an indicator for NH 4 + N removal. Specific anammox activity dropped from 0.39 to 0.19  kg NH 4 + N / ( kgVSS·d ) at COD=720 mg/L. The remodified logistic model was quite appropriate for describing the nitrogen removal kinetics and predicting the performance of anammox process in presence of carbon source.





  • Temperature dependence of nitrogen removal activity by anammox bacteria enriched at low temperatures
    Publication date: April 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 4

    Author(s): Giri Park, Masashi Takekawa, Satoshi Soda, Michihiko Ike, Kenji Furukawa

    The anaerobic ammonium oxidation (anammox) process, which is applicable at ambient temperature, is necessary to develop more versatile nitrogen removal technologies. In this study, two anammox reactors, Low-R1 and Low-R2 inoculated with activated sludge respectively in Kumamoto and Hokkaido, Japan, achieved nitrogen removal rates (NRRs) of 1.5 kg-N/m3/day at 20°C. The specific anammox activity (SAA) of the Low-R1 and Low-R2 sludge samples had peaks, respectively, of 2.8±0.3 mg-N/g-VSS/h at 25°C and 4.2±0.3 mg-N/g-VSS/h at 30°C and dropped over the optimum temperature. Moreover, the SAA values of the Low-R1 and Low-R2 were higher at 10–25°C and 10–35°C, respectively, than that of an anammox reactor inoculated with activated sludge in Kumamoto operated at 35°C (Mod-R). The apparent activation energy for anammox of Low-R1, Low-R2, and Mod-R were 108 kJ/mol (10–25°C), 73 kJ/mol (10–30°C), and 89 kJ/mol (10–35°C), respectively. Candidatus Kuenenia stuttgartiensis dominated in the Mod-R sludge. The Low-R1 sludge was comprised of Ca. K. stuttgartiensis, Ca. Brocadia caroliniensis and Ca. B. fulgida and uncultured anammox-like or planctomycete-like bacteria. The Low-R2 sludge was comprised of various uncultured anammox-like or planctomycete-like bacteria. As Low-R2 was constructed, enrichment of freshwater anammox bacteria at low temperature with seed sludge collected from cold regions is expected to be an effective strategy for anammox applications under a wide temperature range.





  • Cell compaction influences the regenerative potential of passaged bovine articular chondrocytes in an ex vivo cartilage defect model
    Publication date: April 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 4

    Author(s): Michael Schmutzer, Attila Aszodi

    The loss and degradation of articular cartilage tissue matrix play central roles in the process of osteoarthritis (OA). New models for evaluating cartilage repair/regeneration are thus of great value for transferring various culture systems into clinically relevant situations. The repair process can be better monitored in ex vivo systems than in in vitro cell cultures. I have therefore established an ex vivo defect model prepared from bovine femoral condyles for evaluating cartilage repair by the implantation of cells cultured in various ways, e.g., monolayer-cultured cells or suspension or pellet cultures of articular bovine chondrocytes representing different cell compactions with variable densities of chondrocytes. I report that the integrin subunit α10 was significantly upregulated in suspension-cultured bovine chondrocytes at passage P2 compared with monolayer-cultured cells at P1 (p = 0.0083) and P2 (p < 0.05). Suspension-cultured cells did not promote cartilage repair when compared with implanted monolayer-cultured chondrocytes and pellets: 24.0 ± 0.66% for suspension cells, 46.4 ± 2.9% for monolayer cells, and 127.64 ± 0.90% for pellets (p < 0.0001) of the original defect volume (percentage of defect). Additional cultivation with chondrogenesis-promoting growth factors TGF-β1 and BMP-2 revealed an enhancing effect on cartilage repair in all settings. The advantage and innovation of this system over in vitro differentiation (e.g., micromass, pellet) assays is the possibility of examining and evaluating cartilage regeneration in an environment in which implanted cells are embedded within native surrounding tissue at the defect site. Such ex vivo explants might serve as a better model system to mimic clinical situations.





  • Rapid monitoring of RNA degradation activity in vivo for mammalian cells
    Publication date: April 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 4

    Author(s): Hidenori Tani, Hiroaki Sato, Masaki Torimura

    We have developed a rapid fluorescence assay based on fluorescence resonance energy transfer (FRET) for the monitoring of RNA degradation activity in mammalian cells. In this technique, double-stranded RNA (dsRNA) fluorescent probes are used. The dsRNA fluorescent probes consist of a 5′ fluorophore-labeled strand hybridized to a 3′ quencher-labeled strand, and the fluorescent dye is quenched by a quencher dye. When the dsRNA is degraded by nascent RNases in cells, the fluorescence emission of the fluorophore is induced following the degradation of the double strands. The degradation rates of the dsRNA are decelerated in response to chemical or environmental toxicity; therefore, in the case of cellular toxicity, the dsRNA is not degraded and remains intact, thus quenching the fluorescence. Unlike in conventional cell-counting assays, this new assay eliminates time-consuming steps, and can be used to simply evaluate the cellular toxicity via a single reaction. Our results demonstrate that this assay can rapidly quantify the RNA degradation rates in vivo within 4 h for three model chemicals. We propose that this assay will be useful for monitoring cellular toxicity in high-throughput applications.





  • Spectrophotometric assay for sensitive detection of glycerol dehydratase activity using aldehyde dehydrogenase
    Publication date: April 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 4

    Author(s): Eul-Soo Park, Sunghoon Park, Jong-Shik Shin

    Glycerol dehydratase (GDHt) is a pivotal enzyme for fermentative utilization of glycerol by catalyzing radical-mediated conversion of glycerol into 3-hydroxypropionaldehyde (3-HPA). Precise and sensitive monitoring of cellular GDHt activity during the fermentation process is a prerequisite for reliable metabolic analysis to afford efficient cellular engineering and process optimization. Here we report a new spectrophotometric assay for the sensitive measurement of the GDHt activity with a sub-nanomolar limit of detection (LOD). The assay method employs aldehyde dehydrogenase (ALDH) as a reporter enzyme, so the readout of the GDHt activity is recorded at 340 nm as an increase in UV absorbance which results from NADH generation accompanied by oxidation of 3-HPA to 3-hydroxypropionic acid (3-HP). The GDHt assay was performed under the reaction conditions where the ALDH activity overwhelms the GDHt activity (i.e., 50-fold higher activity of ALDH relative to GDHt activity), affording sensitive detection of GDHt with 360 pM LOD. The ALDH-coupled assay was used to determine kinetic parameters of GDHt for glycerol, leading to K M = 0.73 ± 0.09 mM and k cat = 400 ± 20 s−1 which are in reasonable agreements with the previous reports. Our assay method allowed measurement of even a 104-fold decrease in the cellular GDHt activity during fermentative production of 3-HP, which demonstrates the detection sensitivity much higher than the previous methods.





  • First characterization of an archaeal amino acid racemase with broad substrate specificity from the hyperthermophile Pyrococcus horikoshii OT-3
    Publication date: Available online 24 March 2017
    Source:Journal of Bioscience and Bioengineering

    Author(s): Ryushi Kawakami, Haruhiko Sakuraba, Taketo Ohmori, Toshihisa Ohshima

    A novel amino acid racemase with broad substrate specificity (BAR) was recently isolated from the hyperthermophilic archaeon Pyrococcus horikoshii OT-3. Characterization of this enzyme has been difficult, however, because the recombinant enzyme is produced mainly as an inclusion body in Escherichia coli. In this study, expression of the recombinant protein into the soluble fraction was markedly improved by co-expression with chaperone molecules. The purified enzyme retained its full activity after incubation at 80°C for at least 2 h in buffer (pH 7–10), making this enzyme the most thermostable amino acid racemase so far known. Besides the nine amino acids containing hydrophobic and aromatic amino acids previously reported (Kawakami et al., Amino Acids, 47, 1579–1587, 2015), the enzyme exhibited substantial activity toward Thr (about 42% of relative activity toward Phe) and showed no activity toward Arg, His, Gln, and Asn. The substrate specificity of this enzyme thus differs markedly from those of other known amino acid racemases. In particular, the high reaction rate with Trp and Tyr, in addition to Leu, Met and Phe as substrates is a noteworthy feature of this enzyme. The high reactivity toward Trp and Tyr, as well as extremely high thermostability, is likely a major advantage of using BAR for biochemical conversion of these aromatic amino acids.





  • Further enhancement of FR901469 productivity by co-overexpression of cpcA, a cross-pathway control gene, and frbF in fungal sp. No. 11243
    Publication date: Available online 22 March 2017
    Source:Journal of Bioscience and Bioengineering

    Author(s): Makoto Matsui, Tatsuya Yokoyama, Kaoru Nemoto, Toshitaka Kumagai, Koichi Tamano, Masayuki Machida, Takashi Shibata

    FR901469 is a secondary metabolite with antifungal activity, produced by fungal sp. No. 11243. In our previous study, we constructed the frbF overexpression mutant (TFH2-2) from the wild-type strain. FR901469 productivity of TFH2-2 was 3.4 times higher than that of the wild-type strain. To further enhance FR901469 productivity in TFH2-2, we attempted to find genes from the genome that limited the productivity as bottlenecks in this study. Based on both correlation analysis of gene expression level against FR901469 productivity and genome annotation information, the cross-pathway control gene A (cpcA) was most predicted as the bottleneck. The cpcA and frbF co-overexpression mutant named TFCH3 was then constructed from TFH2-2. As a result, FR901469 productivity of TFCH3 was enhanced at 1.8 times higher than that of TFH2-2. Transcriptome analysis revealed that many genes involved in amino acid biosynthesis and encoding tRNA ligases were significantly upregulated in TFCH3, which implied increase of amino acids as the substrates of FR901469 would be a reason of further productivity enhancement.