JBB : Journal of Bioscience and Bioengineering

JBB Vol. 126表紙  



  • Application of chromosomal gene insertion into Escherichia coli for expression of recombinant proteins

    Publication date: August 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 2

    Author(s): Tsutomu Nakamura, Daisuke Koma, Maki Oshima, Hideto Hoshino, Takashi Ohmoto, Koichi Uegaki

    Escherichia coli is the most popular organism used for producing recombinant proteins. However, the expression of recombinant proteins in E. coli sometimes results in the aggregation of proteins as an inclusion body in host cells. In such cases, it is necessary to optimize the refolding conditions to obtain the recombinant protein in its native form. Several techniques, such as reducing the concentration of the induction reagent during E. coli cultivation, have been developed to prevent the formation of inclusion bodies by controlling protein expression levels. In this study, we inserted one copy of a target gene under the control of T7 promoter into the E. coli chromosome using the Red-mediated recombination system. This system enabled soluble expression of the putative d-aminoacylase from Pyrococcus abyssi, which is expressed in an insoluble form following the use of conventional plasmid-based T7 promoter/polymerase systems. The relationship between the number of inserted gene copies and amount of soluble recombinant protein produced was evaluated by multiple insertions of the eGFP gene into the E. coli chromosome. The results revealed that the total expression from the insertion of one copy was around 1/5 that of the pET plasmid system and that expression increased as the inserted gene copy number increased up to five copies.

  • Comprehensive analysis of triacylglycerol lipases in the oleaginous diatom Fistulifera solaris JPCC DA0580 with transcriptomics under lipid degradation

    Publication date: August 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 2

    Author(s): Tatsuhiro Nomaguchi, Yoshiaki Maeda, Yue Liang, Tomoko Yoshino, Toru Asahi, Tsuyoshi Tanaka

    Microalgal biofuels are desirable alternatives for traditional liquid fuels, but further improvements of productivity are necessary for microalgal biofuels to be economically feasible. As strategies for improving lipid productivity, repression of lipid degradation has a great potential because the lipid accumulation level is determined by a balance between lipid synthesis and degradation. However, studies of lipid degradation and its primary functioning enzyme triacylglycerol (TAG) lipases in microalgae are currently limited. In this study, we report the comprehensive analysis of TAG lipases in the oleaginous diatom Fistulifera solaris JPCC DA0580. First, we confirmed that TAGs in the lipid accumulation organelles, oil bodies, were degraded when the cells were transferred from the nutrient depleted conditions to nutrient replete condition. Further analysis revealed that, after eicosapentaenoic acid (C20:5n-3)-containing TAGs are were degraded, its molecular skeletons were likely to be recycled to produce glycolipids and phospholipids for chloroplast regeneration and cell growth, respectively. Next, we searched putative TAG lipase genes from the draft genome sequence of F. solaris, and discovered 42 candidates based on the amino acid sequence homology analysis. Subsequent transcriptome analysis revealed that 16 of the 42 lipase genes were up-regulated during lipid degradation. Among the up-regulated lipases, a number of enzymes were predicted to localize in endoplasmic reticulum which is closely associated to the lipid accumulation organelles, oil bodies. Our study provided new insights of lipid degradation in oleaginous microalgae, and putative TAG lipases which could be candidates for metabolic engineering in future study to improve microalgal lipid productivity.

  • Expression of the human UDP-galactose transporter gene hUGT1 in tobacco plants' enhanced plant hardness

    Publication date: August 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 2

    Author(s): Tayebeh Abedi, Mohamed Farouk Mohamed Khalil, Kanae Koike, Yoshio Hagura, Yuma Tazoe, Nobuhiro Ishida, Kenji Kitamura, Nobukazu Tanaka

    We reported previously that tobacco plants transformed with the human UDP-galactose transporter 1 gene (hUGT1) had enhanced growth, displayed characteristic traits, and had an increased proportion of galactose (hyper-galactosylation) in the cell wall matrix polysaccharides. Here, we report that hUGT1-transgenic plants have an enhanced hardness. As determined by breaking and bending tests, the leaves and stems of hUGT1-transgenic plants were harder than those of control plants. Transmission electron microscopy revealed that the cell walls of palisade cells in leaves, and those of cortex cells and xylem fibers in stems of hUGT1-transgenic plants, were thicker than those of control plants. The increased amounts of total cell wall materials extracted from the leaves and stems of hUGT1-transgenic plants supported the increased cell wall thickness. In addition, the cell walls of the hUGT1-transgenic plants showed an increased lignin contents, which was supported by the up-regulation of lignin biosynthetic genes. Thus, the heterologous expression of hUGT1 enhanced the accumulation of cell wall materials, which was accompanied by the increased lignin content, resulting in the increased hardness of the leaves and stems of hUGT1-trangenic plants. The enhanced accumulation of cell wall materials might be related to the hyper-galactosylation of cell wall matrix polysaccharides, most notably arabinogalactan, because of the enhanced UDP-galactose transport from the cytosol to the Golgi apparatus by hUGT1, as suggested in our previous report.

  • Compatibility between weak gel and microorganisms in weak gel-assisted microbial enhanced oil recovery

    Publication date: August 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 2

    Author(s): Yi-Bin Qi, Cheng-Gang Zheng, Cheng-Yuan Lv, Zeng-Min Lun, Tao Ma

    To investigate weak gel-assisted microbial flooding in Block Wang Long Zhuang in the Jiangsu Oilfield, the compatibility of weak gel and microbe was evaluated using laboratory experiments. Bacillus sp. W5 was isolated from the formation water in Block Wang Long Zhuang. The rate of oil degradation reached 178 mg/day, and the rate of viscosity reduction reached 75.3%. Strain W5 could produce lipopeptide with a yield of 1254 mg/L. Emulsified crude oil was dispersed in the microbial degradation system, and the average diameter of the emulsified oil particles was 18.54 μm. Bacillus sp. W5 did not affect the rheological properties of the weak gel, and the presence of the weak gel did not significantly affect bacterial reproduction (as indicated by an unchanged microbial biomass), emulsification (surface tension is 35.56 mN/m and average oil particles size is 21.38 μm), oil degradation (162 mg/day) and oil viscosity reduction (72.7%). Core-flooding experiments indicated oil recovery of 23.6% when both weak gel and Bacillus sp. W5 were injected into the system, 14.76% when only the weak gel was injected, and 9.78% with strain W5 was injected without the weak gel. The results demonstrate good compatibility between strains W5 and the weak gel and highlight the application potential of weak gel-assisted microbial flooding.

  • Enhancement of fermentative hydrogen production from Spirogyra sp. by increased carbohydrate accumulation and selection of the biomass pretreatment under a biorefinery model

    Publication date: August 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 2

    Author(s): Tiago Pinto, Luísa Gouveia, Joana Ortigueira, Ganesh D. Saratale, Patrícia Moura

    In this work, hydrogen (H2) was produced through the fermentation of Spirogyra sp. biomass by Clostridium butyricum DSM 10702. Macronutrient stress was applied to increase the carbohydrate content in Spirogyra, and a 36% (w/w) accumulation of carbohydrates was reached by nitrogen depletion. The use of wet microalga as fermentable substrate was compared with physically and chemically treated biomass for increased carbohydrate solubilisation. The combination of drying, bead beating and mild acid hydrolysis produced a saccharification yield of 90.3% (w/w). The H2 production from Spirogyra hydrolysate was 3.9 L H2 L−1, equivalent to 146.3 mL H2 g−1 microalga dry weight. The presence of protein (23.2 ± 0.3% w/w) and valuable pigments, such as astaxanthin (38.8% of the total pigment content), makes this microalga suitable to be used simultaneously in both food and feed applications. In a Spirogyra based biorefinery, the potential energy production and food-grade protein and pigments revenue per cubic meter of microalga culture per year was estimated on 7.4 MJ, US $412 and US $15, respectively, thereby contributing to the cost efficiency and sustainability of the whole bioconversion process.

  • Sustainable approach in phlorotannin recovery from macroalgae

    Publication date: August 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 2

    Author(s): Shir Reen Chia, Pau Loke Show, Siew-Moi Phang, Tau Chuan Ling, Hwai Chyuan Ong

    In this present study, alcohol/salt liquid biphasic system was used to extract phlorotannin from brown macroalgae. Liquid biphasic system is a new green technology that integrated with various processes into one-step, by concentrating, separating and purifying the bioproduct in a unit operation. The solvent used is non-toxic and there is potential for solvent recovery which is beneficial to the environment. Phlorotannin is a bioactive compound that has gained much attention due to its health beneficial effect. Therefore, the isolation of phlorotannin is lucrative as it contains various biological activities that are capable to be utilised into food and pharmaceutical application. By using 2-propanol/ammonium sulphate system, the highest recovery of phlorotannin was 76.1% and 91.67% with purification factor of 2.49 and 1.59 from Padina australis and Sargassum binderi, respectively. A recycling study was performed and the salt phase of system was recycled where maximum salt recovery of 41.04% and 72.39% could be obtained from systems containing P. australis and S. binderi, respectively. Similar recovery of phlorotannin was observed after performing two cycles of the system, this concludes that the system has good recyclability and eco-friendly.

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  • Immobilization of Azospira sp. strain I13 by gel entrapment for mitigation of N2O from biological wastewater treatment plants: Biokinetic characterization and modeling

    Publication date: August 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 2

    Author(s): Toshikazu Suenaga, Ryo Aoyagi, Nozomi Sakamoto, Shohei Riya, Hidenori Ohashi, Masaaki Hosomi, Hideaki Tokuyama, Akihiko Terada

    Development of a strategy to mitigate nitrous oxide (N2O) emitted from biological sources is important in the nexus of wastewater treatment and greenhouse gas emission. To this end, immobilization of N2O-reducing bacteria as a biofilm has the potential to ameliorate oxygen (O2) inhibition of the metabolic activity of the bacteria. We demonstrated the effectiveness of calcium alginate gel entrapment of the nosZ clade II type N2O-reducing bacterium, Azospira sp. strain I13, in reducing levels of N2O, irrespective of the presence of O2. Azospira sp. strain I13 cells in the gel exhibited N2O reduction up to a maximum dissolved oxygen concentration of 100 μM in the bulk liquid. The maximum apparent N2O uptake rate, Vm,N2O, by gel immobilization did not appreciably decrease, retaining 72% of the N2O reduction rate of the cell suspension of Azospira sp. strain I13. Whereas gel immobilization increased the apparent half-saturation constant for N2O, Km,N2O, and the apparent O2 inhibition constant, KI,O2, representing the degree of O2 resistance, correspondingly increased. A mechanistic model introducing diffusion and the reactions of N2O consumption was used to describe the experimental observations. Incorporating Thieles modulus into the model determined an appropriate gel size to achieve N2O reduction even under aerobic conditions.

  • Predictive modelling of chromium removal using multiple linear and nonlinear regression with special emphasis on operating parameters of bioelectrochemical reactor

    Publication date: August 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 2

    Author(s): Anand Govind More, Sunil Kumar Gupta

    Bioelectrochemical system (BES) is a novel, self-sustaining metal removal technology functioning on the utilization of chemical energy of organic matter with the help of microorganisms. Experimental trials of two chambered BES reactor were conducted with varying substrate concentration using sodium acetate (500 mg/L to 2000 mg/L COD) and different initial chromium concentration (Cri) (10–100 mg/L) at different cathode pH (pH 1–7). In the current study mathematical models based on multiple linear regression (MLR) and non-linear regression (NLR) approach were developed using laboratory experimental data for determining chromium removal efficiency (CRE) in the cathode chamber of BES. Substrate concentration, rate of substrate consumption, Cri, pH, temperature and hydraulic retention time (HRT) were the operating process parameters of the reactor considered for development of the proposed models. MLR showed a better correlation coefficient (0.972) as compared to NLR (0.952). Validation of the models using t-test analysis revealed unbiasedness of both the models, with t critical value (2.04) greater than t-calculated values for MLR (−0.708) and NLR (−0.86). The root-mean-square error (RMSE) for MLR and NLR were 5.06 % and 7.45 %, respectively. Comparison between both models suggested MLR to be best suited model for predicting the chromium removal behavior using the BES technology to specify a set of operating conditions for BES. Modelling the behavior of CRE will be helpful for scale up of BES technology at industrial level.

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  • Dynamic bacterial community changes in the autothermal thermophilic aerobic digestion process with cell lysis activities, shaking and temperature increase

    Publication date: August 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 2

    Author(s): Huijun Cheng, Yuya Asakura, Kosuke Kanda, Ryo Fukui, Yoshihisa Kawano, Yuki Okugawa, Yukihiro Tashiro, Kenji Sakai

    Autothermal thermophilic aerobic digestion (ATAD) is conducted for stabilization of sludge waste and is driven by the action of various microorganisms under aerobic conditions. However, the mechanism controlling bacterial community changes during ATAD via three (initial, middle and final) phases is currently unclear. To investigate this mechanism, activity analysis and a microcosm assay with shaking were performed on a bacterial community during the initial, middle, and final phases of incubation. Cell lysis activities toward gram-negative bacteria, but not gram-positive bacteria, were detected in the ATAD samples in the middle and final phases. During shaking incubation in initial-phase samples at 30 °C, major operational taxonomic units (OTUs) related to Acinetobacter indicus and Arcobacter cibarius dramatically increased along with decreases in several major OTUs. In middle-phase samples at 45 °C, we observed a major alteration of OTUs related to Caldicellulosiruptor bescii and Aciditerrimonas ferrireducens, together with distinct decreases in several other OTUs. Final-phase samples maintained a stable bacterial community with major OTUs showing limited similarities to Heliorestis baculata, Caldicellulosiruptor bescii, and Ornatilinea apprima. In conclusion, the changes in the bacterial community observed during ATAD could be partially attributed to the cell lysis activity toward gram-negative bacteria in the middle and final phases. The microcosm assay suggested that certain physical factors, such as a high oxygen supply and shearing forces, also might contribute to bacterial community changes in the initial and middle phases, and to the stable bacterial community in the final phase of ATAD.

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  • Improving flavor metabolism of Saccharomyces cerevisiae by mixed culture with Wickerhamomyces anomalus for Chinese Baijiu making

    Publication date: August 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 2

    Author(s): Musu Zha, Baoguo Sun, Yiping Wu, Sheng Yin, Chengtao Wang

    Yeasts are the most important microorganisms in the fermentation of Chinese Baijiu and the interaction of these yeasts could impact the quality of Baijiu. In this study, we initially characterized the Baijiu yeasts and evaluated their fermentation potential. Wickerhamomyces anomalus GZ3 and Saccharomyces cerevisiae G20 were found to generate high yields of ethyl acetate (2.76 g/L) and 2-phenylethanol, respectively. Results also indicated that the use of W. anomalus along with S. cerevisiae increased volatile compounds production, the maximum ethyl acetate production was observed in S. cerevisiae and W. anomalus at 106:106 ratio and have increased by 33 % compared with single culture of W. anomalus. Besides, there was a significant increase of 2-phenylethanol (3.29 g/L) production in single culture of S. cerevisiae with the addition of l-phenylalanine. However, the conversion of l-phenylalanine in mixed culture had significant impact on the yeasts interaction and end flavor of Baijiu. Thus, the present study provided new insights into yeasts interactions in Baijiu fermentation and the effect of some primary metabolites on the end flavor and Baijiu quality.

  • Metabolomics approach to reduce the Crabtree effect in continuous culture of Saccharomyces cerevisiae

    Publication date: August 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 2

    Author(s): Makoto Imura, Ryo Iwakiri, Takeshi Bamba, Eiichiro Fukusaki

    The budding yeast Saccharomyces cerevisiae is an important microorganism for fermentation and the food industry. However, during production, S. cerevisiae commonly uses the ethanol fermentation pathway for glucose utilization if excess sugar is present, even in the presence of sufficient oxygen levels. This aerobic ethanol fermentation, referred to as the Crabtree effect, is one of the most significant reasons for low cell yield. To weaken the Crabtree effect in fed-batch and continuous culture, sugar flow should be limited. In addition, in continuous culture, the dilution rate must be reduced to avoid washing out cells. However, under such conditions, production speed might be sacrificed. It is difficult to solve this problem with the tradeoff between cell yield and production speed by using conventional tactics. However, a metabolomics approach may be an effective way to search for clues regarding metabolic modulation. Therefore, the purpose of this study was to reduce ethanol production in continuous culture of S. cerevisiae at a higher dilution rate through a metabolomics approach. We used a metabolomics analysis to identify metabolites that were drastically increased or decreased in continuous culture when the dilution rate shifted from biomass formation to ethanol fermentation. The individual addition of two of the selected metabolites, fumaric acid and malic acid, reduced ethanol production at a higher dilution rate. This result demonstrates the potential for using metabolomics approaches to identify metabolites that reduce ethanol production in continuous culture at high dilution rates.

  • Whole cell immobilization of refractory glucose isomerase using tris(hydroxymethyl)phosphine as crosslinker for preparation of high fructose corn syrup at elevated temperature

    Publication date: August 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 2

    Author(s): Dong-Xu Jia, Teng Wang, Zi-Jian Liu, Li-Qun Jin, Jia-Jia Li, Cheng-Jun Liao, De-Shui Chen, Yu-Guo Zheng

    Glucose isomerase (GI) responsible for catalyzing the isomerization from d-glucose to d-fructose, was an important enzyme for producing high fructose corn syrup (HFCS). In a quest to prepare HFCS at elevated temperature and facilitate enzymatic recovery, an effective procedure for whole cell immobilization of refractory Thermus oshimai glucose isomerase (ToGI) onto Celite 545 using tris(hydroxymethyl)phosphine (THP) as crosslinker was established. The immobilized biocatalyst showed an activity of approximate 127.3 U/(g·immobilized product) via optimization in terms of cells loading, crosslinker concentration and crosslinking time. The pH optimum of the immobilized biocatalyst was displaced from pH 8.0 of native enzyme to neutral pH 7.0. Compared with conventional glutaraldehyde (GLU)-immobilized cells, it possessed the enhanced thermostability with 70.1% residual activity retaining after incubation at 90°C for 72 h. Moreover, the THP-immobilized biocatalyst exhibited superior operational stability, in which it retained 85.8% of initial activity after 15 batches of bioconversion at 85°C. This study paved a way for reducing catalysis cost for upscale preparation of HFCS with higher d-fructose concentration.

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  • Enzymatic properties and the gene structure of a cold-adapted laminarinase from Pseudoalteromonas species LA

    Publication date: August 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 2

    Author(s): Daisuke Mitsuya, Takuya Sugiyama, Shuo Zhang, Yo Takeuchi, Masahiko Okai, Naoto Urano, Masami Ishida

    We isolated a laminarin-degrading cold-adapted bacterium strain LA from coastal seawater in Sagami Bay, Japan and identified it as a Pseudoalteromonas species. We named the extracellular laminarinase LA-Lam, and purified and characterized it. LA-Lam showed high degradation activity for Laminaria digitata laminarin in the ranges of 15–50°C and pH 5.0–9.0. The major terminal products degraded from L. digitata laminarin with LA-Lam were glucose, laminaribiose, and laminaritriose. The degradation profile of laminarioligosaccharides with LA-Lam suggested that the enzyme has a high substrate binding ability toward tetrameric or larger saccharides. Our results of the gene sequence and the SDS-PAGE analyses revealed that the major part of mature LA-Lam is a catalytic domain that belongs to the GH16 family, although its precursor is composed of a signal peptide, the catalytic domain, and three-repeated unknown regions.

  • Characterization and induction of phenolic acid decarboxylase from Aspergillus luchuensis

    Publication date: August 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 2

    Author(s): Mayumi Maeda, Masashi Tokashiki, Midori Tokashiki, Keiko Uechi, Susumu Ito, Toki Taira

    Awamori is a traditional distilled liquor in the Ryukyu Islands, made from steamed rice by the action of the black-koji mold Aspergillus luchuensis and awamori yeast Saccharomyces cerevisiae. One of the specific flavors in aged awamori kusu is vanillin, which is derived from ferulic acid (FA) in rice grains. FA is released from the cell wall material in the rice grain by ferulic acid esterase produced by A. luchuensis. Through decarboxylation of FA, 4-vinylguaiacol (4-VG) is produced, which is transferred to the distilled liquor, and converted to vanillin by natural oxidization during the aging process. However, the actual mechanism for conversion of FA to 4-VG in the awamori brewing process is unknown. A genetic sequence having homology to the phenolic acid decarboxylase (PAD)-encoding region from bacteria and the yeast Candida guilliermondii has been identified in A. luchuensis mut. kawachii. In the present study, recombinant PAD from A. luchuensis, designated as AlPAD, expressed as a homodimer, catalyzed the conversion of FA to 4-VG, displayed optimal catalytic activity at pH 5.7 and 40°C, and was stable up to 50°C. Both rice bran and FA could induce the bioconversion of FA to 4-VG and the expression of AlPAD in A. luchuensis. The amount of AlPAD determined using western blotting correlated with the level of FA decarboxylase activity during koji production. In awamori brewing process, AlPAD might be responsible for a part of the conversion of FA to 4-VG.

  • High-throughput cytotoxicity and antigen-binding assay for screening small bispecific antibodies without purification

    Publication date: August 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 2

    Author(s): Aruto Sugiyama, Mitsuo Umetsu, Hikaru Nakazawa, Teppei Niide, Ryutaro Asano, Takamitsu Hattori, Izumi Kumagai

    The cytotoxicity of T cell-recruiting antibodies with their potential to damage late-stage tumor masses is critically dependent on their structural and functional properties. Recently, we reported a semi-high-throughput process for screening highly cytotoxic small bispecific antibodies (i.e., diabodies). In the present study, we improved the high-throughput performance of this screening process by removing the protein purification stage and adding a stage for determining the concentrations of the diabodies in culture supernatant. The diabodies were constructed by using an Escherichia coli expression system, and each diabody contained tandemly arranged peptide tags at the C-terminus, which allowed the concentration of diabodies in the culture supernatant to be quantified by using a tag-sandwich enzyme-linked immunosorbent assay. When estimated diabody concentrations were used to determine the cytotoxicity of unpurified antibodies, results comparable to those of purified antibodies were obtained. In a surface plasmon resonance spectroscopy-based target-binding assay, contaminants in the culture supernatant prevented us from conducting a quantitative binding analysis; however, this approach did allow relative binding affinity to be determined, and the relative binding affinities of the unpurified diabodies were comparable to those of the purified antibodies. Thus, we present here an improved high-throughput process for the simultaneous screening and determination of the binding parameters of highly cytotoxic bispecific antibodies.

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  • Quinoprotein dehydrogenase functions at the final oxidation step of lankacidin biosynthesis in Streptomyces rochei 7434AN4

    Publication date: August 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 2

    Author(s): Yusuke Yamauchi, Yosi Nindita, Keisuke Hara, Asako Umeshiro, Yu Yabuuchi, Toshihiro Suzuki, Haruyasu Kinashi, Kenji Arakawa

    Reinvestigation of the metabolite profile in a disruptant of the quinoprotein dehydrogenase (orf23) gene revealed that the Orf23 protein catalyzes dehydrogenation of the C23-C25 lactate moiety to pyruvate during lankacidin biosynthesis in Streptomyces rochei 7434AN4. The dehydrogenase activity was expressed and detected in a soluble fraction of the Streptomyces lividans recombinant harboring orf23. The Orf23 protein preferentially converts lankacidinol to lankacidin C in the presence of pyrroloquinoline quinone (PQQ). Other lankacidinol derivatives, lankacidinol A and iso-lankacidinol, were also converted to the corresponding C-24 keto compounds, lankacidin A (=sedecamycin) and iso-lankacidin C. Addition of various divalent metal cations, especially Ca2+, enhanced the dehydrogenase activity, whereas EDTA completely inhibited. These findings confirmed that the quinoprotein dehydrogenase Orf23 functions at the final oxidation step of lankacidin biosynthesis.

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  • Genetic manipulation to overexpress rpaA altered photosynthetic electron transport in Synechocystis sp. PCC 6803

    Publication date: August 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 2

    Author(s): Satomi Arisaka, Haruna Sukigara, Takashi Osanai

    Cyanobacteria are a group of prokaryotic organisms that perform oxygenic photosynthesis using a similar photosynthetic apparatus as is used in higher plants and eukaryotic algae. Cyanobacteria are also known to have a circadian rhythm. Here, we evaluated the effects on photosynthesis caused by the genetic manipulation of RpaA, which is a response regulator of a two-component regulatory system responsible for the signal output from circadian clocks. Using the unicellular cyanobacterium Synechocystis sp. PCC 6803, photosynthetic activities and transcript levels of photosystem I and photosystems II in the rpaA-overexpressing strain were measured, and it was found that the parameters, such as Fv/Fm, Fv/Fm′, qP, and ϕII, obtained from chlorophyll fluorescence analysis were decreased by rpaA overexpression. These results suggest that rpaA overexpression modified photosynthetic electron transport under normal light conditions. Thus, we demonstrated that RpaA regulates photosynthesis in cyanobacteria and can be a potential target of photosynthetic engineering in this cyanobacterium.

  • Controlled cell morphology and liver-specific function of engineered primary hepatocytes by fibroblast layer cell densities

    Publication date: August 2018

    Source: Journal of Bioscience and Bioengineering, Volume 126, Issue 2

    Author(s): Yusuke Sakai, Makiko Koike, Daisuke Kawahara, Hideko Hasegawa, Tomomi Murai, Kosho Yamanouchi, Akihiko Soyama, Masaaki Hidaka, Mitsuhisa Takatsuki, Fumihiko Fujita, Tamotsu Kuroki, Susumu Eguchi

    Engineered primary hepatocytes, including co-cultured hepatocyte sheets, are an attractive to basic scientific and clinical researchers because they maintain liver-specific functions, have reconstructed cell polarity, and have high transplantation efficiency. However, co-culture conditions regarding engineered primary hepatocytes were suboptimal in promoting these advantages. Here we report that the hepatocyte morphology and liver-specific function levels are controlled by the normal human diploid fibroblast (TIG-118 cell) layer cell density. Primary rat hepatocytes were plated onto TIG-118 cells, previously plated 3 days before at 1.04, 5.21, and 26.1 × 103 cells/cm2. Hepatocytes plated onto lower TIG-118 cell densities expanded better during the early culture period. The hepatocytes gathered as colonies and only exhibited small adhesion areas because of the pushing force from proliferating TIG-118 cells. The smaller areas of each hepatocyte result in the development of bile canaliculi. The highest density of TIG-118 cells downregulated albumin synthesis activity of hepatocytes. The hepatocytes may have undergone apoptosis associated with high TGF-β1 concentration and necrosis due to a lack of oxygen. These occurrences were supported by apoptotic chromatin condensation and high expression of both proteins HIF-1a and HIF-1b. Three types of engineered hepatocyte/fibroblast sheets comprising different TIG-118 cell densities were harvested after 4 days of hepatocyte culture and showed a complete cell sheet format without any holes. Hepatocyte morphology and liver-specific function levels are controlled by TIG-118 cell density, which helps to design better engineered hepatocytes for future applications such as in vitro cell-based assays and transplantable hepatocyte tissues.

  • Novel biobutanol fermentation at a large extractant volume ratio using immobilized Clostridium saccharoperbutylacetonicum N1-4

    Publication date: Available online 14 July 2018

    Source: Journal of Bioscience and Bioengineering

    Author(s): Rizki Fitria Darmayanti, Yukihiro Tashiro, Takuya Noguchi, Ming Gao, Kenji Sakai, Kenji Sonomoto

    Product inhibition by butanol and acetone is a known drawback in acetone-butanol-ethanol (ABE) fermentation. Extractive fermentation improves butanol production by several ABE-producing Clostridium spp., but only low volume ratios (<4) of extractant to broth (Ve/Vb) have been studied. Here, a novel extractive fermentation process was developed using Clostridium saccharoperbutylacetonicum N1-4 and a large Ve/Vb ratio. A mixture of oleyl alcohol-tributyrin (1:1 (v/v)) yielded high distribution coefficients for both butanol (3.14) and acetone (0.660). Although a fed-batch culture using free cells and the oleyl alcohol-tributyrin mixture at a Ve/Vb ratio of 5 had a lag phase of >24 h, it produced a higher concentration of total butanol (i.e., butanol produced in all the phases per broth volume used) of 24.2 g/L-broth after 96 h compared with 14.4 g/L-broth at a Ve/Vb ratio of 1, resulting in a low butanol concentration in the aqueous phase. The use of cells immobilized with calcium alginate beads shortened the lag phase to <12 h. Butanol production was achieved not only in a 3-phase mode (extractant, beads, and tryptone-yeast extract-acetate (TYA) medium) but also in a 2-phase mode (extractant and beads containing TYA medium, without an aqueous phase) at a Ve/Vb ratio of 5, resulting butanol concentrations of 30.9 g/L-broth and 27.7 g/L-broth, respectively. The 3-phases fed-batch extractive fermentation at a Ve/Vb ratio of 10 showed a better performance compared with published reports: a total butanol concentration of 64.6 g/L-broth and a butanol yield to consumed sugar of 0.378 C-mol/C-mol.

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  • Down-regulation of pyruvate decarboxylase gene of white-rot fungus Phlebia sp. MG-60 modify the metabolism of sugars and productivity of extracellular peroxidase activity

    Publication date: Available online 12 July 2018

    Source: Journal of Bioscience and Bioengineering

    Author(s): Taichi Motoda, Megumi Yamaguchi, Taku Tsuyama, Ichiro Kamei

    Ethanologenic white-rot fungus Phlebia sp. MG-60-P2 produces ethanol directly from several lignocelluloses. Efficient gene silencing methods are needed for metabolic engineering of this fungus for biorefinery use. In this study, we evaluated the effectiveness of RNAi-mediated silencing of the pyruvate decarboxylase gene of Phlebia sp. MG-60-P2 (MGpdc1). The RNAi lines generated showed a variety of suppression levels of ethanol production and MGpdc1 expression, and two selected strains led to different metabolic fluxes, resulting in rapid accumulation of xylitol from xylose. Knockdown lines KD2 and KD10 showed different strength of silencing. The moderate-inhibition line (KD10) showed faster xylitol accumulation from xylose than the severe-inhibition line (KD2). KD2, KD10 and knockout line KO77 showed higher extracellular peroxidase activity than the wild-type. Gene silencing using RNAi for MGpdc1 in the ethanologenic white-rot fungus Phlebia sp. MG-60-P2 is an effective first step in metabolic engineering to produce other chemicals besides ethanol. This high efficiency of transformation and silencing effect will makes it possible to cotransform with multiple expression vectors which enhance the minor metabolic pathway or introduce exogenous metabolic reaction in Phlebia sp. MG-60-P2.