JBB : Journal of Bioscience and Bioengineering

            

Journal of Bioscience and Bioengineering vol.123 cover

 



  • Enhanced transgene expression by plasmid-specific recruitment of histone acetyltransferase
    Publication date: March 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 3

    Author(s): Mika Nishihara, Genki N. Kanda, Tetsuya Suzuki, Shin'ichiro Yamakado, Hideyoshi Harashima, Hiroyuki Kamiya

    Histone acetylation is associated with the activation of genes on chromosomes. Transgene expression from plasmid DNA might be increased by the acetylation of histones bound to plasmid DNA. To examine this hypothesis, we employed a positive feedback system, using a fusion protein of the sequence-specific DNA binding domain of yeast GAL4 and the histone acetyltransferase (HAT) domain of mouse CREB-binding protein (GAL4-HAT), in which GAL4-HAT promotes its own expression as well as that of a reporter gene product (luciferase). The activator plasmid DNA carrying the gene encoding GAL4-HAT was introduced into mouse Hepa1-6 cells, together with the reporter plasmid DNA, by lipofection. Significantly increased luciferase expression was observed by the co-introduction of the activator plasmid DNA. Moreover, the acetylation of histones bound to the reporter plasmid DNA was enriched by the activator plasmid DNA. These results indicated that the GAL4-HAT system is useful for enhanced transgene expression.





  • Molecular changes in appearance of a cancer cell among normal HEK293T cells
    Publication date: March 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 3

    Author(s): Jihye Chung, Shunsuke Aburaya, Wataru Aoki, Mitsuyoshi Ueda

    In very early stages of cancer development, one or a few cells expressing cancer-associated genes appear among a much larger number of surrounding normal cells. To analyze the molecular changes induced by this co-existence, we artificially prepared transformed cells with complete loss of tumor suppressor gene, SCRIB, among normal human embryonic kidney (HEK293T) cells. A cell strain with SCRIB-knockout was successfully constructed by using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nuclease system and co-cultured with normal cells. By measuring the time-course changes in cell numbers when SCIRB-knockout cells (cancer model) or cells with normal level of SCRIB expression were respectively co-cultured with wild-type normal HEK293T cells, it was shown that the SCRIB-knockout strain was beneficial for proliferation when mixed together with normal cells. Moreover, as a result of proteome analysis on wild-type cells separated from co-culture with SCRIB-knockout cells, a total of 843 proteins were identified, among which 139 proteins were specific. Among the specifically identified proteins, 22 proteins were annotated to be involved in cytoskeletons including microtubule motor activity-associated proteins. It was implied that molecular changes in cytoskeletons occurred in normal cells when co-cultured with SCRIB knockout cells, but the SCRIB knockout might affect proliferation of the transformed cells with SCRIB knockout by defensive or offensive mechanism of surrounding normal cells.





  • Detailed analysis of targeted gene mutations caused by the Platinum-Fungal TALENs in Aspergillus oryzae RIB40 strain and a ligD disruptant
    Publication date: March 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 3

    Author(s): Osamu Mizutani, Takayuki Arazoe, Kenji Toshida, Risa Hayashi, Shuichi Ohsato, Tetsushi Sakuma, Takashi Yamamoto, Shigeru Kuwata, Osamu Yamada

    Transcription activator-like effector nucleases (TALENs), which can generate DNA double-strand breaks at specific sites in the desired genome locus, have been used in many organisms as a tool for genome editing. In Aspergilli, including Aspergillus oryzae, however, the use of TALENs has not been validated. In this study, we performed genome editing of A. oryzae wild-type strain via error of nonhomologous end-joining (NHEJ) repair by transient expression of high-efficiency Platinum-Fungal TALENs (PtFg TALENs). Targeted mutations were observed as various mutation patterns. In particular, approximately half of the PtFg TALEN-mediated deletion mutants had deletions larger than 1 kb in the TALEN-targeting region. We also conducted PtFg TALEN-based genome editing in A. oryzae ligD disruptant (ΔligD) lacking the ligD gene involved in the final step of the NHEJ repair and found that mutations were still obtained as well as wild-type. In this case, the ratio of the large deletions reduced compared to PtFg TALEN-based genome editing in the wild-type. In conclusion, we demonstrate that PtFg TALENs are sufficiently functional to cause genome editing via error of NHEJ in A. oryzae. In addition, we reveal that genome editing using TALENs in A. oryzae tends to cause large deletions at the target region, which were partly suppressed by deletion of ligD.





  • Engineering a family 27 carbohydrate-binding module into an Aspergillus usamii β-mannanase to perfect its enzymatic properties
    Publication date: March 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 3

    Author(s): Jianfang Li, Chunjuan Wang, Die Hu, Fengjiao Yuan, Xueqing Li, Shihan Tang, Minchen Wu

    A family 27 carbohydrate-binding module of a Thermotoga maritima β-mannanase (TmCBM27) was chosen from the carbohydrate-active enzyme database by computer-aided design, possessing the lowest binding free energy with mannopentaose. To improve the enzymatic properties of a glycoside hydrolase family 5 β-mannanase from Aspergillus usamii (AuMan5A), two fusion β-mannanases, AuMan5A-F-M and AuMan5A-R-M, were designed by fusing a TmCBM27 into its C-terminus linked with a flexible peptide F (GGGGS)3 and rigid peptide R (EAAAK)3. Two fusion enzyme genes, Auman5A-F-m and Auman5A-R-m, were constructed as designed theoretically by overlapping PCR. Then, Auman5A and two fusion genes were expressed in Pichia pastoris GS115. Three recombinant β-mannanases, reAuMan5A, reAuMan5A-F-M and reAuMan5A-R-M, were purified to homogeneity with specific activities of 230.6, 153.3 and 241.7 U/mg. The temperature optimum of reAuMan5A-R-M was 70°C, identical with that of reAuMan5A, while its thermostability and melting temperature (T m) reached 68°C and 74.9°C, being 8.0°C and 8.4°C higher than those of the latter, respectively. Additionally, the K m values of reAuMan5A-R-M, towards locust bean gum, konjac gum and guar gum, significantly decreased to 0.9, 1.9 and 2.5 mg/mL from 1.7, 3.8 and 4.2 mg/mL of reAuMan5A, while its k cat/K m (catalytic efficiency) values increased to 287.8, 163.7 and 84.4 mL/mg⋅s from 171.2, 97.6 and 56.0 mL/mg⋅s of the latter, respectively. These results verified that the fusion of a TmCBM27 into the C-terminus of AuMan5A mediated by (EAAAK)3 linker contributed to its improved thermostability and catalytic efficiency.





  • Exploration of the 1-deoxy-d-xylulose 5-phosphate synthases suitable for the creation of a robust isoprenoid biosynthesis system
    Publication date: March 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 3

    Author(s): Kai Kudoh, Genma Kubota, Rintaro Fujii, Yusuke Kawano, Masaki Ihara

    1-Deoxy-d-xylulose 5-phosphate synthase (DXS) is a rate-limiting enzyme in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, which is responsible for production of two precursors of all isoprenoids, isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP). Previously, we attempted the overexpression of endogenous DXS in Synechocystis sp. PCC6803, and revealed that although the mRNA level was 4-fold higher, the DXS protein level was only 1.5-fold higher compared with those of the original strain, suggesting the lability of endogenous DXS protein. Therefore, for the creation of a robust isoprenoid synthesis system, it is necessary to build a novel MEP pathway by combining stable enzymes. In this study, we expressed 11 dxs genes from 9 organisms in Escherichia coli and analyzed their protein solubility. Furthermore, we purified the recombinant DXSes and evaluated their specific activities and protease tolerance, thermostability, and feedback inhibition tolerance. Among DXSes we examined in this study, the highest protein solubility was observed in Paracoccus aminophilus DXS (PaDXS). The DXS with the highest activity was one from Rhodobacter capsulatus (RcDXSA). The highest protease tolerance, thermostability, and tolerance of feedback inhibition were found in Bacillus subtilis DXS (BsDXS), RcDXSA, PaDXS, BsDXS, respectively. These DXSes can be potentially used for the design of robust isoprenoid synthesis system.





  • Antifungal activities of selected essential oils against Fusarium oxysporum f. sp. lycopersici 1322, with emphasis on Syzygium aromaticum essential oil
    Publication date: March 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 3

    Author(s): Abhishek Sharma, Sasireka Rajendran, Ankit Srivastava, Satyawati Sharma, Bishwajit Kundu

    The antifungal effects of four essential oils viz., clove (Syzygium aromaticum), lemongrass (Cymbopogon citratus), mint (Mentha × piperita) and eucalyptus (Eucalyptus globulus) were evaluated against wilt causing fungus, Fusarium oxysporum f. sp. lycopersici 1322. The inhibitory effect of oils showed dose-dependent activity on the tested fungus. Most active being the clove oil, exhibiting complete inhibition of mycelial growth and spore germination at 125 ppm with IC50 value of 18.2 and 0.3 ppm, respectively. Essential oils of lemongrass, mint and eucalyptus were inhibitory at relatively higher concentrations. The Minimum inhibitory concentration (MIC) of clove oil was 31.25 ppm by broth microdilution method. Thirty one different compounds of clove oil, constituting approximately ≥99% of the oil, were identified by gas chromatography–mass spectroscopy analysis. The major components were eugenol (75.41%), E-caryophyllene (15.11%), α-humulene (3.78%) and caryophyllene oxide (1.13%). Effect of clove oil on surface morphology of F. oxysporum f. sp. lycopersici 1322 was studied by scanning electron microscopy (SEM) and atomic force microscopy (AFM). SEM observation revealed shrivelled hyphae while AFM observation showed shrunken and disrupted spores in clove oil treated samples. In pots, 5% aqueous emulsion of clove oil controlled F. oxysporum f. sp. lycopersici 1322 infection on tomato plants. This study demonstrated clove oil as potent antifungal agent that could be used as biofungicide for the control of F. oxysporum f. sp. lycopersici in both preventive and therapeutic manner.





  • Potential of water surface-floating microalgae for biodiesel production: Floating-biomass and lipid productivities
    Publication date: March 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 3

    Author(s): Masaki Muto, Daisuke Nojima, Liang Yue, Hideyuki Kanehara, Hideaki Naruse, Asuka Ujiro, Tomoko Yoshino, Tadashi Matsunaga, Tsuyoshi Tanaka

    Microalgae have been accepted as a promising feedstock for biodiesel production owing to their capability of converting solar energy into lipids through photosynthesis. However, the high capital and operating costs, and high energy consumption, are hampering commercialization of microalgal biodiesel. In this study, the surface-floating microalga, strain AVFF007 (tentatively identified as Botryosphaerella sudetica), which naturally forms a biofilm on surfaces, was characterized for use in biodiesel production. The biofilm could be conveniently harvested from the surface of the water by adsorbing onto a polyethylene film. The lipid productivity of strain AVFF007 was 46.3 mg/L/day, allowing direct comparison to lipid productivities of other microalgal species. The moisture content of the surface-floating biomass was 86.0 ± 1.2%, which was much lower than that of the biomass harvested using centrifugation. These results reveal the potential of this surface-floating microalgal species as a biodiesel producer, employing a novel biomass harvesting and dewatering strategy.





  • Development of intra-strain self-cloning procedure for breeding baker's yeast strains
    Publication date: March 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 3

    Author(s): Youji Nakagawa, Hiroyuki Ogihara, Chisato Mochizuki, Hideki Yamamura, Yuzuru Iimura, Masayuki Hayakawa

    Previously reported self-cloning procedures for breeding of industrial yeast strains require DNA from other strains, plasmid DNA, or mutagenesis. Therefore, we aimed to construct a self-cloning baker's yeast strain that exhibits freeze tolerance via an improved self-cloning procedure. We first disrupted the URA3 gene of a prototrophic baker's yeast strain without the use of any marker gene, resulting in a Δura3 homozygous disruptant. Then, the URA3 gene of the parental baker's yeast strain was used as a selection marker to introduce the constitutive TDH3 promoter upstream of the PDE2 gene encoding high-affinity cyclic AMP phosphodiesterase. This self-cloning procedure was performed without using DNA from other Saccharomyces cerevisiae strains, plasmid DNA, or mutagenesis and was therefore designated an intra-strain self-cloning procedure. Using this self-cloning procedure, we succeeded in producing self-cloning baker's yeast strains that harbor the TDH3p-PDE2 gene heterozygously and homozygously, designated TDH3p-PDE2 hetero and TDH3p-PDE2 homo strains, respectively. These self-cloning strains expressed much higher levels of PDE2 mRNA than the parental strain and exhibited higher viability after freeze stress, as well as higher fermentation ability in frozen dough, when compared with the parental strain. The TDH3p-PDE2 homo strain was genetically more stable than the TDH3p-PDE2 hetero strain. These results indicate that both heterozygous and homozygous strains of self-cloning PDE2-overexpressing freeze-tolerant strains of industrial baker's yeast can be prepared using the intra-strain self-cloning procedure, and, from a practical viewpoint, the TDH3p-PDE2 homo strain constructed in this study is preferable to the TDH3p-PDE2 hetero strain for frozen dough baking.





  • Characterization of fermented seaweed sauce prepared from nori (Pyropia yezoensis)
    Publication date: March 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 3

    Author(s): Motoharu Uchida, Hirotaka Kurushima, Kenji Ishihara, Yuko Murata, Ken Touhata, Noriko Ishida, Kentaro Niwa, Toshiyoshi Araki

    High-salt content seaweed sauces were prepared for the first time using nori (Pyropia yezoensis) by fermentation and characterized. Components and taste of the two nori sauces (NSs) prepared separately were compared with those of soy and fish sauces. The NSs were rich in total nitrogen compounds (1.5 g N/100 ml on average) and potassium (880 mg/100 g), and had a unique free amino acid composition (e.g., taurine 617 mg/100 g), explaining their unique taste as evaluated by a taste sensing system. As for their food function, inhibitory activity of angiotensin-converting enzyme was observed. As for their food safety, arsenic was detected at a 0.8 mg/100 g level in total, but inorganic arsenic was not detected (<0.05 mg/100 g) and not regarded as a problem. Allergy-causing substances contained in wheat, soy beans, and crustaceans were not detected (<0.1 mg/100 g) with NSs. These results suggest that the nori sauce has a high potential as a novel nutritional source for humans.





  • Identification and characterization of an enzyme involved in the biosynthesis of the 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone in yeast
    Publication date: March 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 3

    Author(s): Kenji Uehara, Jun Watanabe, Yoshinobu Mogi, Yuichiro Tsukioka

    4-Hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) is considered a key flavor compound in soy sauce. The compound has a caramel-like aroma and several important physiological activities, such as strong antioxidant activity. Here, we report the identification and characterization of an enzyme involved in the biosynthesis of HEMF in yeast. We fractionated yeast cell-free extract from Saccharomyces cerevisiae using column chromatography and partially purified a fraction with HEMF-forming activity. Peptide mass fingerprinting analysis showed that the partially purified fraction contains aldehyde reductase encoded by YNL134C. This reductase shares low sequence identity with enone oxidoreductase, which is responsible for the formation of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) and HEMF in plants. YNL134C was expressed heterologously in Escherichia coli, and the purified protein catalyzed the formation of HEMF from the mixture of Maillard reaction products, acetaldehydes, and NADPH. Multicopy expression in S. cerevisiae resulted in increased HEMF productivity, and gene knockout of YNL134C in S. cerevisiae resulted in decreased HEMF productivity. These data suggest that the translation product of YNL134C is the HEMF-producing enzyme in yeast. Detailed analyses of an intermediate in the enzymatic reaction mixture revealed that HEMF is synthesized from (2E)-2-ethylidene-4-hydroxy-5-methyl-3(2H)-furanone, which formed via Knoevenagel condensation between the acetaldehyde and 4-hydroxy-5-methyl-3(2H)-furanone derived from the Maillard reaction based on ribose and glycine, by YNL134Cp in an NADPH dependent manner. Overall, this study shed light on the molecular basis for the improvement of soy sauce flavor and the biotechnological production of HEMF.





  • Bioprospecting thermotolerant ethanologenic yeasts for simultaneous saccharification and fermentation from diverse environments
    Publication date: March 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 3

    Author(s): Jairam Choudhary, Surender Singh, Lata Nain

    Lignocellulosic biomass, a promising renewable energy source, can be used for the production of second generation bioethanol. Simultaneous saccharification and fermentation (SSF), the process which alleviates the problem of separate hydrolysis and fermentation (SHF), requires thermotolerant ethanologenic yeast for bioethanol production. Therefore, ten yeast strains isolated from diverse sources, belonging to various genera like Saccharomyces, Candida, Pichia and Wickerhamomyces were evaluated for their thermotolerance, sugar utilization pattern, inhibitor tolerance and ethanol production potential with glucose, xylose and alkali pretreated paddy straw. All the tested strains were found to be thermotolerant, capable of significant growth at 40°C. Candida tropicalis Y6 was capable of utilizing a wide range of sugars as compared with other yeast isolates. Strains of Candida showed better inhibitor tolerance as compared to Saccharomyces and Pichia strains and exhibited only 5.1–18.8% and 4.7–7.9% reduction in growth with furfural and 5-hydroxymethyl furfural, respectively. Saccharomyces cerevisiae JRC6, isolated from distillery waste, produced ethanol with 88.3% and 89.1% theoretical efficiency at 40°C and 42°C, respectively, from glucose. This strain also produced significantly higher amount of ethanol (3.8 g/L) with better fermentation efficiency (87.9%) from alkali pretreated paddy straw at 40°C, as compared with the other yeast strains. Therefore, S. cerevisiae JRC6, based on its ability to ferment sugars at a higher temperature, can be a promising candidate for production of ethanol from lignocellulosic biomass via SSF process.





  • Engineering Escherichia coli to bind to cyanobacteria
    Publication date: March 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 3

    Author(s): Zijian Zhang, Liuyi Meng, Congjian Ni, Lanqiu Yao, Fengyu Zhang, Yuji Jin, Xuelang Mu, Shiyu Zhu, Xiaoyu Lu, Shiyu Liu, Congyu Yu, Chenggong Wang, Pu Zheng, Jie Wu, Li Kang, Haoqian M. Zhang, Qi Ouyang

    We engineered Escherichia coli cells to bind to cyanobacteria by heterologously producing and displaying lectins of the target cyanobacteria on their surface. To prove the efficacy of our approach, we tested this design on Microcystis aeruginosa with microvirin (Mvn), the lectin endogenously produced by this cyanobacterium. The coding sequence of Mvn was C-terminally fused to the ice nucleation protein NC (INPNC) gene and expressed in E. coli. Results showed that E. coli cells expressing the INPNC::Mvn fusion protein were able to bind to M. aeruginosa and the average number of E. coli cells bound to each cyanobacterial cell was enhanced 8-fold. Finally, a computational model was developed to simulate the binding reaction and help reconstruct the binding parameters. To our best knowledge, this is the first report on the binding of two organisms in liquid culture mediated by the surface display of lectins and it may serve as a novel approach to mediate microbial adhesion.





  • Improvement of hydrogen fermentation of galactose by combined inoculation strategy
    Publication date: March 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 3

    Author(s): Periyasamy Sivagurunathan, Parthiban Anburajan, Gopalakrishnan Kumar, Pugazhendhi Arivalagan, Péter Bakonyi, Sang-Hyoun Kim

    This study evaluated the feasibility of anaerobic hydrogen fermentation of galactose, a red algal biomass sugar, using individual and combined mixed culture inocula. Heat-treated (90°C, 30 min) samples of granular sludge (GS) and suspended digester sludge (SDS) were used as inoculum sources. The type of mixed culture inoculum played an important role in hydrogen production from galactose. Between two inocula, granular sludge showed higher hydrogen production rate (HPR) and hydrogen yield (HY) of 2.2 L H2/L-d and 1.09 mol H2/mol galactoseadded, respectively. Combined inoculation (GS + SDS) led to an elevated HPR and HY of 3.1 L H2/L-d and 1.28 mol H2/mol galactoseadded, respectively. Acetic and butyric acids are the major organic acids during fermentation. Quantitative polymerase chain reaction (qPCR) revealed that the mixed culture generated using the combined inoculation contained a higher cluster I Clostridium abundance than the culture produced using the single inoculum.





  • Microbial fuel cells using Cellulomonas spp. with cellulose as fuel
    Publication date: March 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 3

    Author(s): Yuya Takeuchi, Wichean Khawdas, Yuji Aso, Hitomi Ohara

    Cellulomonas fimi, Cellulomonas biazotea, and Cellulomonas flavigena are cellulose-degrading microorganisms chosen to compare the degradation of cellulose. C. fimi degraded 2.5 g/L of cellulose within 4 days, which was the highest quantity among the three microorganisms. The electric current generation by the microbial fuel cell (MFC) using the cellulose-containing medium with C. fimi was measured over 7 days. The medium in the MFC was sampled every 24 h to quantify the degradation of cellulose, and the results showed that the electric current increased with the degradation of cellulose. The maximum electric power generated by the MFC was 38.7 mW/m2, and this numeric value was 63% of the electric power generated by an MFC with Shewanella oneidensis MR-1, a well-known current-generating microorganism. Our results showed that C. fimi was an excellent candidate to produce the electric current from cellulose via MFCs.





  • Phenol-degrading anode biofilm with high coulombic efficiency in graphite electrodes microbial fuel cell
    Publication date: March 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 3

    Author(s): Dongdong Zhang, Zhiling Li, Chunfang Zhang, Xue Zhou, Zhixing Xiao, Takanori Awata, Arata Katayama

    A microbial fuel cell (MFC), with graphite electrodes as both the anode and cathode, was operated with a soil-free anaerobic consortium for phenol degradation. This phenol-degrading MFC showed high efficiency with a current density of 120 mA/m2 and a coulombic efficiency of 22.7%, despite the lack of a platinum catalyst cathode and inoculation of sediment/soil. Removal of planktonic bacteria by renewing the anaerobic medium did not decrease the performance, suggesting that the phenol-degrading MFC was not maintained by the planktonic bacteria but by the microorganisms in the anode biofilm. Cyclic voltammetry analysis of the anode biofilm showed distinct oxidation and reduction peaks. Analysis of the microbial community structure of the anode biofilm and the planktonic bacteria based on 16S rRNA gene sequences suggested that Geobacter sp. was the phenol degrader in the anode biofilm and was responsible for current generation.





  • Cloud-point extraction of green-polymers from Cupriavidus necator lysate using thermoseparating-based aqueous two-phase extraction
    Publication date: March 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 3

    Author(s): Yoong Kit Leong, John Chi-Wei Lan, Hwei-San Loh, Tau Chuan Ling, Chien Wei Ooi, Pau Loke Show

    Polyhydroxyalkanoates (PHAs), a class of renewable and biodegradable green polymers, have gained attraction as a potential substitute for the conventional plastics due to the increasing concern towards environmental pollution as well as the rapidly depleting petroleum reserve. Nevertheless, the high cost of downstream processing of PHA has been a bottleneck for the wide adoption of PHAs. Among the options of PHAs recovery techniques, aqueous two-phase extraction (ATPE) outshines the others by having the advantages of providing a mild environment for bioseparation, being green and non-toxic, the capability to handle a large operating volume and easily scaled-up. Utilizing unique properties of thermo-responsive polymer which has decreasing solubility in its aqueous solution as the temperature rises, cloud point extraction (CPE) is an ATPE technique that allows its phase-forming component to be recycled and reused. A thorough literature review has shown that this is the first time isolation and recovery of PHAs from Cupriavidus necator H16 via CPE was reported. The optimum condition for PHAs extraction (recovery yield of 94.8% and purification factor of 1.42 fold) was achieved under the conditions of 20 wt/wt % ethylene oxide-propylene oxide (EOPO) with molecular weight of 3900 g/mol and 10 mM of sodium chloride addition at thermoseparating temperature of 60°C with crude feedstock limit of 37.5 wt/wt %. Recycling and reutilization of EOPO 3900 can be done at least twice with satisfying yield and PF. CPE has been demonstrated as an effective technique for the extraction of PHAs from microbial crude culture.





  • Metabolomic analysis of primary metabolites in citrus leaf during defense responses
    Publication date: March 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 3

    Author(s): Tomonori Asai, Tetsuya Matsukawa, Shin'ichiro Kajiyama

    Mechanical damage is one of the unavoidable environmental stresses to plant growth and development. Plants induce a variety of reactions which defend against natural enemies and/or heal the wounded sites. Jasmonic acid (JA) and salicylic acid (SA), defense-related plant hormones, are well known to be involved in induction of defense reactions and play important roles as signal molecules. However, defense related metabolites are so numerous and diverse that roles of individual compounds are still to be elucidated. In this report, we carried out a comprehensive analysis of metabolic changes during wound response in citrus plants which are one of the most commercially important fruit tree families. Changes in amino acid, sugar, and organic acid profiles in leaves were surveyed after wounding, JA and SA treatments using gas chromatography–mass spectrometry (GC/MS) in seven citrus species, Citrus sinensis, Citrus limon, Citrus paradisi, Citrus unshiu, Citrus kinokuni, Citrus grandis, and Citrus hassaku. GC/MS data were applied to multivariate analyses including hierarchical cluster analysis (HCA), primary component analysis (PCA), and orthogonal partial least squares-discriminant analysis (OPLS-DA) to extract stress-related compounds. HCA showed the amino acid cluster including phenylalanine and tryptophan, suggesting that amino acids in this cluster are concertedly regulated during responses against treatments. OPLS-DA exhibited that tryptophan was accumulated after wounding and JA treatments in all species tested, while serine was down regulated. Our results suggest that tryptophan and serine are common biomarker candidates in citrus plants for wound stress.





  • Lengthening of high-yield production levels of monoclonal antibody-producing Chinese hamster ovary cells by downregulation of breast cancer 1
    Publication date: March 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 3

    Author(s): Rima Matsuyama, Noriko Yamano, Namiko Kawamura, Takeshi Omasa

    The establishment process of high-producing Chinese hamster ovary (CHO) cells for therapeutic protein production is usually laborious and time consuming because of the low probability of obtaining stable, high-producing clones over a long term. Thus, development of an efficient approach is required to establish stable, high-producing cells. This study presents a novel method that can efficiently establish sustainably high-producing cell lines by acceleration of transgene amplification and suppression of transgene silencing. The effects of breast cancer 1 (BRCA1) downregulation on gene amplification efficiency and long-term productivity were investigated in CHO cells. Small interfering RNA expression vectors against BRCA1 were transfected into the CHO DG44-derived antibody-producing cell clone. Individual cell clones were obtained after induction of gene amplification in the presence of 400 nM methotrexate, which were cultured until passage 20. BRCA1-downregulated cell clones CHO B1Sa and B1Sb displayed 2.2- and 1.6-fold higher specific production rates than the S-Mock clone. Fluorescence in situ hybridization showed that transgene amplification occurred at a high frequency in B1Sa and B1Sb clones. Moreover, B1Sa and B1Sb clones at 20 passages had approximately 3.5- and 5.3-fold higher productivity than the S-Mock clone. Histone modification analysis revealed a decrease in an active mark for transcription, trimethylation of histone H3 at lysine 4 (H3K4), in the transgene locus of the S-Mock clone. However, H3K4 trimethylation levels were not decreased in B1Sa and B1Sb clones during long term culture. Our results suggest that high-producing cells, which maintain their productivity long-term, were efficiently established by BRCA1 downregulation.





  • Improved recombinant antibody production by CHO cells using a production enhancer DNA element with repeated transgene integration at a predetermined chromosomal site
    Publication date: March 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 3

    Author(s): Yoshinori Kawabe, Takanori Inao, Shodai Komatsu, Guan Huang, Akira Ito, Takeshi Omasa, Masamichi Kamihira

    Chinese hamster ovary (CHO) cells are one of the most useful host cell lines for the production of biopharmaceutical proteins. Although a series of production processes have been refined to improve protein productivity and cost performance, establishing producer cells is still time-consuming and labor-intensive. Recombinase-mediated site-specific gene integration into a predetermined chromosomal locus may enable predictable protein expression, reducing the laborious process of cell screening. We previously developed an accumulative site-specific gene integration system (AGIS) using Cre recombinase and mutated loxP sites for transgene integration and amplification in the CHO cell genome. Epigenetic modifier elements such as insulators are effective DNA cis-regulatory elements for stabilizing transgene expression. Here, we attempted to enhance transgene expression in recombinant CHO cells generated by AGIS using a production enhancer DNA element (PE) derived from the CHO genome. The PE was introduced into an expression unit for a recombinant scFv-Fc antibody. The effect on scFv-Fc productivity of PE position and orientation within the transgene was evaluated, while keeping the background chromosomal structure constant. For the optimal PE arrangement, scFv-Fc productivity was enhanced 2.6-fold compared with an expression unit without a PE. The enhancing effect of the PE on transgene expression was also observed when two or three PE-flanked expression units were inserted as tandem repeats. These results indicate that AGIS using the PE-flanked expression unit is a promising approach for establishing producer cell lines for biopharmaceutical protein production.





  • LiaRS reporter assay: A simple tool to identify lipid II binding moieties in lantibiotic nukacin ISK-1
    Publication date: March 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 3

    Author(s): Khaled M. Elsayed, Mohammad R. Islam, Abdullah-Al-Mahin, Jun-ichi Nagao, Takeshi Zendo, Kenji Sonomoto

    Binding to lipid II is an important step in the mode of action of most lantibiotics targeting the bacterial cell wall. We applied the Bacillus subtilis two-component system, LiaRS, that is known to respond to antibiotics interfering with lipid II cycle, in order to evaluate lipid II binding activity of known bacteriocins and also to identify lipid II binding moieties in lantibiotic nukacin ISK-1. Using this method, we confirmed that the methyllanthionine ring in nukacin ISK-1 is crucial for lipid II binding as previously indicated. In this study, we further identified that the three N-terminal lysine residues (K1, K2, and K3) and the glycine (G5) residue in nukacin ISK-1 are also important in lipid II binding.