JBB : Journal of Bioscience and Bioengineering

JBB Vol. 125表紙  

JBB 新着20報



  • Enhanced production of lactate-based polyesters in Escherichia coli from a mixture of glucose and xylose by Mlc-mediated catabolite derepression
    Publication date: Available online 10 January 2018
    Source:Journal of Bioscience and Bioengineering

    Author(s): Ryosuke Kadoya, Ken'ichiro Matsumoto, Kenji Takisawa, Toshihiko Ooi, Seiichi Taguchi

    Lignocellulose-utilizing biorefinery is a promising strategy for the sustainable production of value-added products such as bio-based polymers. Simultaneous consumption of glucose and xylose in Escherichia coli was achieved by overexpression of the gene encoding Mlc, a multiple regulator of glucose and xylose uptake. This catabolite derepression gave the enhancement in the production of poly (15 mol% lactate-co-3-hydroxybutyrate), up to 65% from 50% (wild-type strain) in the cellular contents, of the Mlc-overexpressing strain of E. coli on a mixture of glucose and xylose as carbon sources. Microscopic analysis indicated that the Mlc-overexpressing strain showed the enlargement of cell volume in the presence and absence of polymer production, consequently making an expanded volumetric space available for enhanced polymer accumulation. The enhanced polymer production by the catabolite derepression was also reproducible using the biomass, Miscanthus × giganteus (hybrid Miscanthus), which was cultivated in the farm of Hokkaido University.





  • Characterization of newly isolated Pseudonocardia sp. N23 with high 1,4-dioxane-degrading ability
    Publication date: Available online 2 January 2018
    Source:Journal of Bioscience and Bioengineering

    Author(s): Norifumi Yamamoto, Yuji Saito, Daisuke Inoue, Kazunari Sei, Michihiko Ike

    This study was conducted to elucidate the 1,4-dioxane degradation characteristics of a newly isolated 1,4-dioxane-degrading bacterial strain and evaluate the applicability of the strain to biological 1,4-dioxane removal from wastewater. A bacterial strain (designated strain N23) capable of degrading 1,4-dioxane as the sole carbon and energy source was isolated from an enrichment culture prepared from 1,4-dioxane-contaminated groundwater. Strain N23 was phylogenetically identified as belonging to the genus Pseudonocardia, based on 16S rRNA gene sequencing. 1,4-Dioxane degradation experiments revealed that strain N23 is capable of constitutive 1,4-dioxane degradation. Further, this strain exhibited the highest specific 1,4-dioxane degradation rate of 0.230 mg-1,4-dioxane (mg-protein)−1 h−1 among 1,4-dioxane-degrading bacteria with constitutively expressed degrading enzymes reported to date. In addition, strain N23 was shown to degrade up to 1100 mg L−1 of 1,4-dioxane without significant inhibition, and to maintain a high level of 1,4-dioxane degradation activity under a wide pH (pH 3.8–8.2) and temperature (20–35 °C) range. In particular, the specific 1,4-dioxane degradation rate, even at pH 3.8, was 83% of the highest rate at pH 7.0. In addition, strain N23 was capable of utilizing ethylene glycol and diethylene glycol, which are both considered to be present in 1,4-dioxane-containing industrial wastewater, as the sole carbon source. The present results indicate that strain N23 exhibits the potential for 1,4-dioxane removal from industrial wastewater.





  • Comparative study of reactor performance and microbial community in psychrophilic and mesophilic biogas digesters under solid state condition
    Publication date: Available online 2 January 2018
    Source:Journal of Bioscience and Bioengineering

    Author(s): Suzhen Wei, Yanfei Guo

    Psychrophilic (15°C) and mesophilic (35°C) reactor performance and microbial community dynamics were compared when the biogas fermenters were performed at high altitude and solid state condition using animal manure and highland barley straw as substrate. Longer biogas fermentation time, higher peak methane content and lower volatile fatty acids (VFA) accumulation were found at psychrophilic condition compared to that of at mesophilic condition although the biogas production in both temperature conditions was similar. The cumulative biogas production at 35°C and 15°C were 246 (±5) and 225 (±7) ml/g volatile solids, respectively. The highest total VFA concentration under 35°C was 10,796 (±310) mg/kg total solid, while it only reached to 2346 (±87) mg/kg total solid at the condition of 15°C. Additionally, the variation of pH, soluble chemical oxygen demand and total ammonia nitrogen during the anaerobic digestion under psychrophilic condition were much smaller than that of under mesophilic condition. Polymerase chain reaction and denaturing gradient gel electrophoresis analysis followed by 16S rDNA sequencing showed that bacteria of genera Bacillus and Clostridium and archaea of genera Methanosarcina and Methanosaeta played a pivotal role during the biogas production.





  • Platform construction of molecular breeding for utilization of brown macroalgae
    Publication date: January 2018
    Source:Journal of Bioscience and Bioengineering, Volume 125, Issue 1

    Author(s): Toshiyuki Takagi, Kouichi Kuroda, Mitsuyoshi Ueda

    Brown macroalgae are characterized by a large size and high productivity without requiring arable land, fresh water, and fertilizer. Furthermore, since brown macroalgae contain little or no lignin, simple biorefinery processing can efficiently produce sugars from this material. Therefore, brown macroalgae have attracted attention as an alternative feedstock for bioethanol production. However, the utilization of biotechnologies previously developed for terrestrial biomass processing results in difficulties in the bioconversion of brown macroalgae. Recently, several studies have developed biotechnologies for using major carbohydrates of brown macroalgae, such as laminarin, mannitol, and alginate. This review focuses on these fermentation biotechnologies using natural or engineered microorganisms.





  • Type I collagen promotes primary cilia growth through down-regulating HDAC6-mediated autophagy in confluent mouse embryo fibroblast 3T3-L1 cells
    Publication date: January 2018
    Source:Journal of Bioscience and Bioengineering, Volume 125, Issue 1

    Author(s): Qian Xu, Weiwei Liu, Xiaoling Liu, Wuxiyar Otkur, Toshihiko Hayashi, Masayuki Yamato, Hitomi Fujisaki, Shunji Hattori, Shin-ichi Tashiro, Takashi Ikejima

    Primary cilia are microtubule-based organelles that extend from nearly all vertebrate cells. Abnormal ciliogenesis and cilia length are suggested to be associated with hypertension and obesity as well as diseases such as Meckel–Gruber syndrome. Extracellular matrix (ECM), comprising cellular microenvironment, influences cell shape and proliferation. However, influence of ECM on cilia biogenesis has not been well studied. In this study we examined the effects of type I collagen (col I), the major component of ECM, on primary cilia growth. When cultured on collagen-coated dishes, confluent 3T3-L1 cells were found to exhibit fibroblast-like morphology, which was different from the cobblestone-like shape on non-coated dishes. The level of autophagy in the cells cultured on col I-coated dishes was attenuated compared with the cells cultured on non-coated dishes. The cilia of the cells cultured on col I-coated dishes became longer, accompanying increased expression of essential proteins for cilia assembly. Transfection of the siRNA targeting microtubule-associated protein light chain 3 (LC3) further enhanced the length of primary cilia, suggesting that col I positively regulated cilia growth through inhibition of autophagy. Histone deacetylase 6 (HDAC6), which was suggested as a mediator of autophagy in our previous study on primary cilia, was down-regulated with col I. 3T3-L1 cells treated with the siRNA against HDAC6 reduced the autophagy level and enhanced collagen-induced cilia elongation, implying that HDAC6 was involved in mediating autophagy. In conclusion, col I promotes cilia growth through repressing the HDAC-autophagy pathway that can be involved in the interaction between primary cilia and col I.





  • Unique transcriptional profile of native persisters in Escherichia coli
    Publication date: January 2018
    Source:Journal of Bioscience and Bioengineering, Volume 125, Issue 1

    Author(s): Shinya Matsumoto, Yuto Kawai, Satoshi Miyagawa, Yuka Iwamoto, Shujiro Okuda, Alicia Sánchez-Gorostiaga, Miguel Vicente, Satoshi Tsuneda

    Non-dividing persisters, bacteria that can survive in the presence of antibiotics by pausing their metabolic activity, are among the many causes of the refractory nature of bacterial infections. Here we constructed a recombinant Escherichia coli strain that enables to distinguish non-dividing from dividing cell based on Z-ring during cell division. Then, non-dividing cells and dividing cells were successfully separated using a fluorescence activated cell sorter. The sorted non-dividing cells showed significantly higher tolerance toward ofloxacin than dividing cells, which indicates that persisters were concentrated with the methodology. Transcriptional analysis revealed that genes involved in guanosine tetraphosphate synthesis are upregulated in persisters, which represses transcription and DNA replication and leads to ofloxacin tolerance. Lactate dehydrogenase and several ATP-binding cassette transporters were upregulated in persisters to adapt to anaerobic metabolism. In addition, nitrite and dimethyl sulfoxide (DMSO) may be used as reducible substrates for alternative energy generation pathways. Our methodology revealed a unique transcriptional profile of E. coli persisters.





  • High-level expression and characterization of solvent-tolerant lipase
    Publication date: January 2018
    Source:Journal of Bioscience and Bioengineering, Volume 125, Issue 1

    Author(s): Yu Wang, Dan Luo, Yunshi Zhao, Shufang Tian, Wenpeng Deng, Chunhua Li, Lixin Ma

    In this study, the coding sequence of the lipase from Proteus sp. SW1 was optimized via codon optimization and subjected to expression in Pichia pastoris GS115. The maximum enzyme yield was 387 mg/L in the supernatants of the shake-flask culture. The purified recombinant lipase exhibited a specific activity of 130 U/mg toward p-nitrophenyl Laurate. Its optimum pH and temperature were 8.0 and 40°C, respectively. It was highly stable and even activated in water-miscible solvents, showing over 102% residual activity after 24 h incubation in ethanol, acetone, isopropanol and acetonitrile. In addition, the enzyme showed promoted activity with the increasing concentrations of methanol/ethanol and exhibited the maximum activity at 80%. In a solvent-free system for biodiesel synthesis with a one-step addition of methanol, the recombinant lipase displayed a 87% conversion rate toward palm oil at the high water content of 80%. The highly improved expression level and activity of the recombinant lipase may contribute to enable its commercial-scale production, and the unique properties would make it a particularly promising biocatalyst for biodiesel production in the future.





  • Molecular cloning and functional characterization of NADPH-dependent cytochrome P450 reductase from the green microalga Botryococcus braunii, B race
    Publication date: January 2018
    Source:Journal of Bioscience and Bioengineering, Volume 125, Issue 1

    Author(s): Chung-Yau Tsou, Shigeki Matsunaga, Shigeru Okada

    The green microalga Botryococcus braunii of the B race accumulates various lipophilic compounds containing a 10,11-oxidosqualene epoxide moiety in addition to large amounts of triterpene hydrocarbons. While 2,3-squalene epoxidases have already been isolated and characterized from the alga, the enzyme that catalyzes the 10,11-epoxidation of squalene has remained elusive. In order to obtain a molecular tool to explore a 10,11-squalene epoxidase, cDNA cloning of an NADPH-dependent cytochrome P450 reductase (CPR) that is required by both squalene epoxidases and cytochrome P450 enzymes was carried out. The isolated cDNA contained an open reading frame (1998 bp) that encoded for a protein with 665 amino acid residues with a predicted molecular weight of 71.46 kDa and a theoretical pI of 5.49. Analysis of the deduced amino acid sequence revealed the presence of conserved motifs, including FMN, FAD, and NADPH binding domains, which are typical of other CPRs and necessary for enzyme activity. By truncation of the N-terminal transmembrane anchor and addition of a 6× His-tag, BbCPR was heterologously produced in Escherichia coli and purified by Ni-NTA affinity chromatography. The purified recombinant enzyme showed optimal reducing activity of cytochrome c at around a neutral pH at a temperature range of 30–37°C. For steady state kinetic parameters, the recombinant enzyme had a k m for cytochrome c and NADPH of 11.7±1.6 and 9.4±1.4 μM, and a k cat for cytochrome c and NADPH of 2.78±0.09 and 3.66±0.11 μmol/min/mg protein, respectively. This is the first study to perform the functional characterization of a CPR from eukaryotic microalgae.





  • New nucleoside hydrolase with transribosylation activity from Agromyces sp. MM-1 and its application for enzymatic synthesis of 2′-O-methylribonucleosides
    Publication date: January 2018
    Source:Journal of Bioscience and Bioengineering, Volume 125, Issue 1

    Author(s): Yuuki Mitsukawa, Makoto Hibi, Narihiro Matsutani, Nobuyuki Horinouchi, Satomi Takahashi, Jun Ogawa

    Microorganisms were screened for transribosylation activity between 2′-O-methyluridine (2′-OMe-UR) and nucleobases, for the purpose of developing a biotransformation process to synthesize 2′-O-methylribonucleosides (2′-OMe-NRs), which are raw materials for nucleic acid drugs. An actinomycete, Agromyces sp. MM-1 was found to produce 2′-O-methyladenosine (2′-OMe-AR) when whole cells were used in a reaction mixture containing 2′-OMe-UR and adenine. The enzyme responsible for the transribosylation was partially purified from Agromyces sp. MM-1 cells through a six-step separation procedure, and identified as a nucleoside hydrolase family enzyme termed AgNH. AgNH was a bi-functional enzyme catalyzing both hydrolysis towards 2′-OMe-NRs and transribosylation between 2′-OMe-UR and various nucleobases as well as adenine. In the hydrolysis reaction, AgNH preferred guanosine analogues as its substrates. In the transribosylation reaction, AgNH showed strong activity towards 6-chloroguanine, with 25-fold relative activity when adenine was used as the acceptor substrate. The transribosylation reaction product from 2′-OMe-UR and 6-chloroguanine was determined to 2′-O-methyl-6-chloroguanosine (2′-OMe-6ClGR). Under the optimal conditions, the maximum molar yield of 2′-OMe-6ClGR reached 2.3% in a 293-h reaction, corresponding to 440 mg/L.





  • Purification and characterization of a cellulase-free, thermostable endo-xylanase from Streptomyces griseorubens LH-3 and its use in biobleaching on eucalyptus kraft pulp
    Publication date: January 2018
    Source:Journal of Bioscience and Bioengineering, Volume 125, Issue 1

    Author(s): Hao Wu, Xianbo Cheng, Yongfeng Zhu, Wei Zeng, Guiguang Chen, Zhiqun Liang

    Xylanase is an important enzyme involved in degrading xylan. In this study, an extracellular cellulase-free, thermostable endo-xylanase which was produced by Streptomyces griseorubens LH-3 with bagasse semi-cellulose as a carbon source was purified and characterized. The xylanase was purified 4-fold with a recovery yield of 21.6% by precipitation with 25–55% (NH4)2SO4, Mono Q ion exchange chromatography and sephacryl S-200 HR gel filtration chromatography. It appeared as a monomeric protein on SDS-PAGE gel and had an apparent molecular weight of 45.5 kDa with specific activity of 434 IU/mg. Using birchwood xylan as substrate, the maximum velocity (V max) and Michaelis–Menten constant (K m) were found to be 1.44 mg/ml and 2.05 μmol/min mg, respectively. The purified xylanase was active at pH 4.0–8.0 with an optimum pH of 5.0. It was stable at temperatures between 30°C and 50°C, exhibiting maximum activity at 60°C. Hg2+ and Al3+ inhibited the enzyme activity significantly. Enzymatic product analysis indicated that the enzyme was an endo-xylanase, whose hydrolysis products were mainly a series of short-chain xylooligosaccharides. Furthermore, it was used for biobleaching of eucalyptus kraft pulp, and results showed that this purified xylanase increased the brightness of the pulp by 14.5% and reduced the kappa number by 24.5%. All these industrially relevant characteristics made it had potential application in the pulp and paper industry as a biobleaching agent.





  • Inhibition of Saccharomyces cerevisiae growth by simultaneous uptake of glucose and maltose
    Publication date: January 2018
    Source:Journal of Bioscience and Bioengineering, Volume 125, Issue 1

    Author(s): Haruyo Hatanaka, Hitoshi Mitsunaga, Eiichiro Fukusaki

    Saccharomyces cerevisiae expresses α-glucoside transporters, such as MalX1p (X=1(Agt1p), 2, 3, 4, and 6), which are proton symporters. These transporters are regulated at transcriptional and posttranslational levels in the presence of glucose. Malt wort contains glucose, maltose, and maltotriose, and the assimilation of maltose is delayed as a function of glucose concentration. With the objective of increasing beer fermentation rates, we characterized α-glucoside transporters and bred laboratory yeasts that expressed various α-glucoside transporters for the simultaneous uptake of different sugars. Mal21p was found to be the most resistant transporter to glucose-induced degradation, and strain (HD17) expressing MAL21 grew on a medium containing glucose or maltose, but not on a medium containing both sugars (YPDM). This unexpected growth defect was observed on a medium containing glucose and >0.1% maltose but was not exhibited by a strain that constitutively expressed maltase. The defect depended on intracellular maltose concentration. Although maltose accumulation caused a surge in turgor pressure, addition of sorbitol to YPDM did not increase growth. When strain HD17 was cultivated in a medium containing only maltose, protein synthesis was inhibited at early times but subsequently resumed with reduction in accumulated maltose, but not if the medium was exchanged for YPDM. We conclude that protein synthesis was terminated under the accumulation of maltose, regardless of extracellular osmolarity, and HD17 could not resume growth, because the intracellular concentration of maltose did not decrease due to insufficient synthesis of maltase. Yeast should incorporate maltose after expressing adequate maltase in beer brewing.





  • Bioconversion of biodiesel-derived crude glycerol into lipids and carotenoids by an oleaginous red yeast Sporidiobolus pararoseus KM281507 in an airlift bioreactor
    Publication date: January 2018
    Source:Journal of Bioscience and Bioengineering, Volume 125, Issue 1

    Author(s): Atchara Manowattana, Charin Techapun, Masanori Watanabe, Thanongsak Chaiyaso

    Here we tested the bioconversion of biodiesel-derived crude glycerol by the oleaginous red yeast Sporidiobolus pararoseus KM281507 in two bioreactors types (stirred-tank and airlift). High production yields (biomass, 10.62 ± 0.21 g/L; lipids, 3.26 ± 0.13 g/L; β-carotene, 30.64 ± 0.05 mg/L; total carotenoids, 46.59 ± 0.07 mg/L) were achieved in a 3.0 L airlift bioreactor under uncontrolled pH regimes (initial pH 5.63). Under optimized conditions (6.0 vvm aeration rate; 60 ± 5% constant dissolved oxygen [DO] maintained by flushing pure oxygen [O2] into the vessel; 10,000 Lux light irradiation) volumetric production in the airlift bioreactor was further increased (biomass, 19.30 ± 1.07 g/L; lipids, 6.61 ± 0.04 g/L, β-carotene, 109.75 ± 0.21 mg/L; total carotenoids 151.00 ± 2.71 mg/L). Production was also recorded at a S. pararoseus KM281507 growth rate of 0.16 ± 0.00 h−1 (lipids, 0.94 ± 0.04 g/L/d; β-carotene, 15.68 ± 0.40 mg/L/d; total carotenoids, 21.56 ± 0.20 mg/L/d). Lipids from S. pararoseus KM281507 had a high unsaturated fatty acid content, with oleic acid (C18:1) accounting for 80% of all fatty acids. This high oleic acid content makes S. pararoseus KM281507 well-suited as a third generation biodiesel feedstock. Our findings show that airlift bioreactors are suitable for bioconversion of crude glycerol into lipids and carotenoids using S. pararoseus KM281507. This approach is advantageous because of its ease of operation, cost efficiency, and low energy consumption.





  • Leucine responsive regulatory protein is involved in methionine metabolism and polyamine homeostasis in acetic acid bacterium Komagataeibacter europaeus
    Publication date: January 2018
    Source:Journal of Bioscience and Bioengineering, Volume 125, Issue 1

    Author(s): Yuri Ishii, Naoki Akasaka, Hisao Sakoda, Ryota Hidese, Shinsuke Fujiwara

    The leucine responsive regulatory protein (Lrp) is a global transcription factor that regulates the expression of genes involved in amino acid metabolism. To identify metabolic pathways and related genes under the control of Lrp in the acetic acid bacterium Komagataeibacter europaeus, the Kelrp null mutant (KGMA7110), which requires supplementation of all 20 amino acids for normal growth, was cultivated in minimal media containing or lacking particular amino acids. The results confirmed that KGMA7110 was auxotrophic for methionine and its catabolites S-adenosylmethionine (SAM) and spermidine (SPD). Quantitative reverse-transcription PCR analysis revealed lower metK (SAM synthetase) and mdtI (SPD efflux pump) expression in KGMA7110 than in wild-type KGMA0119. By contrast, these genes were significantly up-regulated in the Kelrp mutant lacking the putative C-terminal ligand-sensing domain (KGMA7203), indicating abnormal regulation of target genes by the KeLrp variant in KGMA7203. KGMA7110 (0.69±0.27 μM) and KGMA7203 (4.90±0.61 μM) excreted lower and higher quantities of SPD, respectively, than KGMA0119 (2.28±0.26 μM). This was attributed to imbalanced carbon flow caused by Kelrp disruption that respectively attenuated and stimulated metK and mdtI expression. These findings indicate that KeLrp plays a key role in SAM biosynthesis and intracellular polyamine homeostasis in K. europaeus.





  • Selection of yeast Saccharomyces cerevisiae promoters available for xylose cultivation and fermentation
    Publication date: January 2018
    Source:Journal of Bioscience and Bioengineering, Volume 125, Issue 1

    Author(s): Yumiko Nambu-Nishida, Yuri Sakihama, Jun Ishii, Tomohisa Hasunuma, Akihiko Kondo

    To efficiently utilize xylose, a major sugar component of hemicelluloses, in Saccharomyces cerevisiae requires the proper expression of varied exogenous and endogenous genes. To expand the repertoire of promoters in engineered xylose-utilizing yeast strains, we selected promoters in S. cerevisiae during cultivation and fermentation using xylose as a carbon source. To select candidate promoters that function in the presence of xylose, we performed comprehensive gene expression analyses using xylose-utilizing yeast strains both during xylose and glucose fermentation. Based on microarray data, we chose 29 genes that showed strong, moderate, and weak expression in xylose rather than glucose fermentation. The activities of these promoters in a xylose-utilizing yeast strain were measured by lacZ reporter gene assays over time during aerobic cultivation and microaerobic fermentation, both in xylose and glucose media. In xylose media, P TDH3 , P FBA1 , and P TDH1 were favorable for high expression, and P SED1 , P HXT7 , P PDC1 , P TEF1 , P TPI1 , and P PGK1 were acceptable for medium–high expression in aerobic cultivation, and moderate expression in microaerobic fermentation. P TEF2 allowed moderate expression in aerobic culture and weak expression in microaerobic fermentation, although it showed medium–high expression in glucose media. P ZWF1 and P SOL4 allowed moderate expression in aerobic cultivation, while showing weak but clear expression in microaerobic fermentation. P ALD3 and P TKL2 showed moderate promoter activity in aerobic cultivation, but showed almost no activity in microaerobic fermentation. The knowledge of promoter activities in xylose cultivation obtained in this study will permit the control of gene expression in engineered xylose-utilizing yeast strains that are used for hemicellulose fermentation.





  • A search for microorganisms producing medium-chain alkanes from aldehydes
    Publication date: January 2018
    Source:Journal of Bioscience and Bioengineering, Volume 125, Issue 1

    Author(s): Masakazu Ito, Hiromi Kambe, Shigenobu Kishino, Masayoshi Muramatsu, Jun Ogawa

    Microorganisms with medium-chain alkane-producing activity are promising for the bio-production of drop-in fuel. In this study, we screened for microorganisms producing tridecane from tetradecanal. The activity of aldehyde decarbonylation was found in a wide range of microbes. In particular, the genus Klebsiella in the Enterobacteriaceae family was found to have a high ability to produce alkanes from aldehydes via enzyme catalyzed reaction.





  • Analysis of ambient pH stress response mediated by iron and copper intake in Schizosaccharomyces pombe
    Publication date: January 2018
    Source:Journal of Bioscience and Bioengineering, Volume 125, Issue 1

    Author(s): Yujiro Higuchi, Hikari Mori, Takeo Kubota, Kaoru Takegawa

    The molecular mechanism of tolerance to alkaline pH is well studied in model fungi Aspergillus nidulans and Saccharomyces cerevisiae. However, how fission yeast Schizosaccharomyces pombe survives under alkaline stress remains largely unknown, as the genes involved in the alkaline stress response pathways of A. nidulans and S. cerevisiae were not found in the genome of this organism. Since uptake of iron and copper into cells is important for alkaline tolerance in S. cerevisiae, here we examined whether iron and copper uptake processes were involved in conferring tolerance to alkaline stress in S. pombe. We first revealed that S. pombe wild-type strain could not grow at a pH higher than 6.7. We further found that the growths of mutants harboring disruption in the iron uptake-related gene frp1 + , fio1 + or fip1 + were severely inhibited under ambient pH stress condition. In contrast, derepression of these genes, by deletion of their repressor gene fep1 + , caused cells to acquire resistance to pH stress. Together, these results suggested that uptake of iron is essential for ambient pH tolerance in S. pombe. We also found that copper is required for the pH stress response because disruptants of ctr4 + , ctr5 + , ccc2 + and cuf1 + genes, all of which are needed for regulating intracellular Cu+, displayed ambient pH sensitivity. Furthermore, supplementing Fe2+ and Cu2+ ions to the culture media improved growth under ambient pH stress. Taken together, our results suggested that uptake of iron and copper is the crucial factor needed for the adaptation of S. pombe to ambient pH stress.





  • Microbial behavior and changes in food constituents during fermentation of Japanese sourdoughs with different rye and wheat starting materials
    Publication date: January 2018
    Source:Journal of Bioscience and Bioengineering, Volume 125, Issue 1

    Author(s): Akihito Fujimoto, Keisuke Ito, Madoka Itou, Noriko Narushima, Takayuki Ito, Akihisa Yamamoto, Satoru Hirayama, Soichi Furukawa, Yasushi Morinaga, Takahisa Miyamoto

    Sourdough is a food item made by kneading grain flour and water together and allowing fermentation through the action of lactic acid bacteria (Lactobacillales) and yeast. Typically, Japanese bakeries make sourdough with rye flour, wheat flour, malt extract, and water and allow spontaneous fermentation for 6 days. We compared the microbial behavior and food components, such as organic acids, sugars, and free amino acids, of sourdoughs made using two different rye and wheat flours during the 6-day fermentation period. Comparisons were made for two types of rye and wheat flours, using different production sites and different milling, distribution, and storage conditions. The microbial count was evaluated using different culture media. All sourdough types showed a significant increase in lactic acid levels on fermentation day 2 and a decrease in free amino acid levels on day 4. Low overall lactic acid production and little fluctuation in sugar levels occurred in sourdough made from French ingredients. For sourdough made from Japanese ingredients, sugar levels (chiefly glucose, sucrose, and maltose) declined on fermentation day 1, increased on day 2, and declined by day 5. With the French ingredients, no yeast cells were detected until day 3, and many acid precursors of sourdough flavor components were detected. Yet with the Japanese ingredients, 106/g yeast cells were detected on days 3–5, as well as sourdough-flavor esters and alcohols. Differences in raw material quality affected the microbial behavior and changes in food constituents during the fermentation process and, consequently, the sourdough flavor.





  • Selective production of deacetylated mannosylerythritol lipid, MEL-D, by acetyltransferase disruption mutant of Pseudozyma hubeiensis
    Publication date: January 2018
    Source:Journal of Bioscience and Bioengineering, Volume 125, Issue 1

    Author(s): Masaaki Konishi, Motoki Makino

    Mannosylerythritol lipids (MELs) are produced by several smut fungi of the Ustilaginaceae family; they are promising microbial biosurfactants and have excellent surface-active and self-assembling properties. Pseudozyma hubeiensis is a candidate for abundant MEL production and produces large amounts of 4-O-[(4′-mono-O-acetyl-2′,3′-di-O-alkanoyl)-β-d-mannopyranosyl]-meso-erythritol (MEL-C). An acetyltransferase disruption mutant of P. hubeiensis, SY62-MM36, was obtained to selectively produce deacetylated 4-O-[(2′,3′-di-O-alkanoyl)-β-d-mannopyranosyl]-meso-erythritol (MEL-D), and the structures of the products were determined. Lower mobility of major spots of the mutant on silica gel thin-layer chromatography verified its more hydrophilic nature than that of wild-type MEL-A, B, and C. Structural analyses confirmed the product to be MEL-D, which comprises acyl chains of caproic acid (C6:0), capric acid (C10:0), and lauric acid (C12:0). The critical micelle concentration (CMC) and the surface tension (γCMC) of the MEL-D were 2.0 × 10−5 M and 29.7 mN/m, respectively. SY62-MM36 also produced a minor product that was estimated as triacylated MEL-D. The triacylated MEL-D had a CMC of 3.5 × 10−5 M and a γCMC of 29.6 mN/m. In water, MEL-D formed a lamella liquid crystal phase over a broad range of concentrations. By fed-batch cultivation, the mutant produced 91.6 ± 6.3 g/L of MEL-D for 7 days.





  • Effects of glucose, lactate and basic FGF as limiting factors on the expansion of human induced pluripotent stem cells
    Publication date: January 2018
    Source:Journal of Bioscience and Bioengineering, Volume 125, Issue 1

    Author(s): Ikki Horiguchi, Yusuke Urabe, Keiichi Kimura, Yasuyuki Sakai

    Pluripotent stem cells (PSCs) are one of the promising cell sources for tissue engineering and drug screening. However, mass production of induced pluripotent stem cells (iPSCs) is still developing. Especially, a huge amount of culture medium usage causes expensive cost in the mass production process. In this report, we reduced culture medium usage by extending interval of changing culture medium. In parallel, we also increased glucose concentration and supplied heparan sulfate to avoid depletion of glucose and bFGF, respectively. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses showed that reducing medium change frequency increased differentiation marker expressions but high glucose concentration downregulated these expressions. In contrast, heparan sulfate did not prevent differentiation marker expressions. According to analyses of growth rate, cell growth with extended medium change interval was decreased in later stage of log growth phase despite the existence of high glucose concentration and heparan sulfate. This result and culturing iPSCs with lactate showed that the accumulation of excreted lactate decreased the growth rate regardless of pH control. Conclusively, these experiments show that adding glucose and removing lactate are important to expand iPSCs with reduced culture medium usage. This knowledge should be useful to design economical iPSC mass production and differentiation system.





  • Optimized transitory ectopic expression of promastigote surface antigen protein in Nicotiana benthamiana, a potential anti-leishmaniasis vaccine candidate
    Publication date: January 2018
    Source:Journal of Bioscience and Bioengineering, Volume 125, Issue 1

    Author(s): Séverine Lacombe, Martine Bangratz, Jean-Paul Brizard, Elodie Petitdidier, Julie Pagniez, Drissa Sérémé, Jean-Loup Lemesre, Christophe Brugidou

    In recent years, plants have been shown to be an efficient alternative expression system for high-value pharmaceuticals such as vaccines. However, constitutive expression of recombinant protein remains uncertain on their level of production and biological activity. To overcome these problems, transitory expression systems have been developed. Here, a series of experiments were performed to determine the most effective conditions to enhance vaccine antigen transient accumulation in Nicotiana benthamiana leaves using the promastigote surface antigen (PSA) from the parasitic protozoan Leishmania infantum. This protein has been previously identified as the major antigen of a licensed canine anti-leishmaniasis vaccine. The classical prokaryote Escherichia coli biosystem failed in accumulating PSA. Consequently, the standard plant system based on N. benthamiana has been optimized for the production of putatively active PSA. First, the RNA silencing defense mechanism set up by the plant against PSA ectopic expression was abolished by using three viral suppressors acting at different steps of the RNA silencing pathway. Then, we demonstrated that the signal peptide at the N-terminal side of the PSA is required for its accumulation. The PSA ER signaling and retention with the PSA signal peptide and the KDEL motif, respectively were optimized to significantly increase its accumulation. Finally, we demonstrate that the production of recombinant PSA in N. benthamiana leaves allows the conservation of its immunogenic property. These approaches demonstrate that based on these optimizations, plant based systems can be used to effectively produce the biological active PSA protein.