JBB : Journal of Bioscience and Bioengineering

JBB Vol. 124表紙  

JBB 新着20報



  • Identification and characterization of lbpA, an indigoidine biosynthetic gene in the γ-butyrolactone signaling system of Streptomyces lavendulae FRI-5
    Publication date: October 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 4

    Author(s): Ivy Grace Umadhay Pait, Shigeru Kitani, Yohanes Novi Kurniawan, Maeda Asa, Takashi Iwai, Haruo Ikeda, Takuya Nihira

    Streptomyces lavendulae FRI-5 produces the blue pigment indigoidine and other secondary metabolites (d-cycloserine and nucleoside antibiotics). The production of these useful compounds is controlled by a signaling cascade mediated by the γ-butyrolactone autoregulator IM-2. Previously we revealed that the far regulatory island includes the IM-2 receptor, the IM-2 biosynthetic enzyme, and several transcriptional regulators, and that it contributes to the regulation of indigoidine production in response to the signaling molecule. Here, we found that the vicinity of the far regulatory island includes the putative gene cluster for the biosynthesis of indigoidine and unidentified compounds, and demonstrated that the expression of the gene cluster is under the control of the IM-2 regulatory system. Heterologous expression of lbpA, encoding a plausible nonribosomal peptide synthetase, in the versatile model host Streptomyces avermitilis SUKA22 led to indigoidine production, which was enhanced dramatically by feeding of the indigoidine precursor l-glutamine. These results confirmed that LbpA is an indigoidine biosynthetic enzyme in the IM-2 signaling cascade.





  • Characterization of the cellulosomal scaffolding protein CbpC from Clostridium cellulovorans 743B
    Publication date: October 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 4

    Author(s): Daichi Nakajima, Toshiyuki Shibata, Reiji Tanaka, Kouichi Kuroda, Mitsuyoshi Ueda, Hideo Miyake

    Clostridium cellulovorans 743B, an anaerobic and mesophilic bacterium, produces an extracellular enzyme complex called the cellulosome on the cell surface. Recently, we have reported the whole genome sequence of C. cellulovorans, which revealed that a total of 4 cellulosomal scaffolding proteins: CbpA, HbpA, CbpB, and CbpC were encoded in the C. cellulovorans genome. In particular, cbpC encoded a 429-residue polypeptide that includes a carbohydrate-binding module (CBM), an S-layer homology module, and a cohesin. CbpC was also detected in the culture supernatant of C. cellulovorans. Genomic DNA coding for CbpC was subcloned into a pET-22b+ vector in order to express and produce the recombinant protein in Escherichia coli BL21(DE3). Measurement of CbpC adsorption to crystalline cellulose indicated a dissociation constant of 0.60 μM, which is a similar to that of CBM from CbpA. We also subcloned the region encoding xylanase B (XynB) with the dockerin from C. cellulovorans and analyzed the interaction between XynB and CbpC by GST pull-down assay. It was observed that GST-CbpC assembles with XynB to form a minimal cellulosome. The activity of XynB against rice straw tended to be increased in the presence of CbpC. These results showed a synergistic effect on rice straw as a representative cellulosic biomass through the formation of a minimal cellulosome containing XynB bound to CbpC. Thus, our findings provide a foundation for the development of cellulosic biomass saccharification using a minimal cellulosome.





  • Improved laccase production by Funalia trogii in absorbent fermentation with nutrient carrier
    Publication date: October 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 4

    Author(s): Guanhua Li, Xiaoxue Liu, Lin Yuan

    A novel strategy of enhancing laccase production by absorbent fermentation was investigated. Peanut shell was used as nutrient carrier for laccase production by Funalia trogii IFP0027 in the absorbent fermentation. The maximum laccase production was reached to 11,900 U/l, which was 4.97 times higher than that of the control group. The results indicated that carbohydrates and phenolic substances especially flavonoids contained in peanut shell stimulated laccase production by F. trogii. Meanwhile, the peanut shell nutrient carrier could not only alleviate the oxidative damage, owing to strong scavenging activity on hydroxyl, but also relieve the mechanical stresses to form small and regular microbial pellets. Therefore, the absorbent fermentation using peanut shell as nutrient carrier shows enormous potential in enhancing laccase production.





  • Metabolic pathway analysis of the xylose-metabolizing yeast protoplast fusant ZLYRHZ7
    Publication date: October 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 4

    Author(s): Jingping Ge, Renpeng Du, Gang Song, Yuhuan Zhang, Wenxiang Ping

    Xylose is the second major fermentable sugar present in hard woods and herbs (after d-glucose). Therefore, efficient conversion of xylose to ethanol is essential for the commercialization of lignocellulosic ethanol, which may provide an ideal alternative to fossil fuels in the future. ZLYRHZ7 is a fusant produced by protoplast fusion between two different yeast species, Saccharomyces cerevisiae W5 and Candida shehatae 20335, which is able to utilize xylose to produce ethanol. To improve ethanol production and to quantitatively analyze metabolic pathway in ZLYRHZ7, we used high performance liquid chromatography (HPLC) to assess the utilization rates of xylose, xylitol, and xylulose, and to measure ethanol yields using xylose, xylitol, and xylulose as sole carbon sources. The ethanol yields reached 0.549±0.003, 0.567±0.003 and 0.544±0.005 g/g in 72 h, which indicated that the metabolic pathways from xylose to xylitol, xylitol to xylulose, and xylulose to ethanol, respectively, were functional. In addition, enzyme activity and qRT-PCR analyses showed that the xylose metabolism-related enzymes xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulose kinase (XK) and their respective genes were expressed at significantly higher levels in ZLYRHZ7 than in both S. cerevisiae W5 and C. shehatae 20335 at 24, 48, and 72 h of fermentation. These results clearly show that the fusant ZLYRHZ7, obtained by protoplast fusion of two different yeast species, has the ability to ferment xylose to produce ethanol.





  • Production of 3-hydroxypropionic acid via the malonyl-CoA pathway using recombinant fission yeast strains
    Publication date: October 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 4

    Author(s): Akiko Suyama, Yujiro Higuchi, Masahiro Urushihara, Yuka Maeda, Kaoru Takegawa

    3-Hydroxypropionic acid (3-HP) can be converted into derivatives such as acrylic acid, a source for producing super absorbent polymers. Although Escherichia coli has often been used for 3-HP production, it exhibits low tolerance to 3-HP. To circumvent this problem, we selected the fission yeast Schizosaccharomyces pombe as this microorganism has higher tolerance to 3-HP than E. coli. Therefore, we constructed S. pombe transformants overexpressing two genes, one encoding the S. pombe acetyl-CoA carboxylase (Cut6p) and the other encoding the malonyl-CoA reductase derived from Chloroflexus aurantiacus (CaMCR). To prevent the degradation of these expressed proteins, we employed an S. pombe protease-deficient strain. Moreover, to increase the cytosolic concentration of acetyl-CoA, we supplemented acetate to the medium, which improved 3-HP production. To further produce 3-HP by overexpressing Cut6p and CaMCR, we exploited the highly expressing S. pombe hsp9 promoter. Finally, culturing in high-density reached 3-HP production to 7.6 g/L at 31 h.





  • Enhancement of butanol production by sequential introduction of mutations conferring butanol tolerance and streptomycin resistance
    Publication date: October 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 4

    Author(s): Yukinori Tanaka, Ken Kasahara, Yutaka Hirose, Yu Morimoto, Masumi Izawa, Kozo Ochi

    Ribosome engineering, originally applied to Streptomyces lividans, has been widely utilized for strain improvement, especially for the activation of bacterial secondary metabolism. This study assessed ribosome engineering technology to modulate primary metabolism, taking butanol production as a representative example. The introduction into Clostridium saccharoperbutylacetonicum of mutations conferring resistance to butanol (ButR) and of the str mutation (SmR; a mutation in the rpsL gene encoding ribosomal protein S12), conferring high-level resistance to streptomycin, increased butanol production 1.6-fold, to 16.5 g butanol/L. Real-time qPCR analysis demonstrated that the genes involved in butanol metabolism by C. saccharoperbutylacetonicum were activated at the transcriptional level in the drug-resistant mutants, providing a mechanism for the higher yields of butanol by the mutants. Moreover, the activity of enzymes butyraldehyde dehydrogenase (AdhE) and butanol dehydrogenases (BdhAB), the key enzymes involved in butanol synthesis, was both markedly increased in the ButR SmR mutant, reflecting the significant up-regulation of adhE and bdhA at transcriptional level in this mutant strain. These results demonstrate the efficacy of ribosome engineering for the production of not only secondary metabolites but of industrially important primary metabolites. The possible ways to overcome the reduced growth rate and/or fitness cost caused by the mutation were also discussed.





  • Effects of exogenous isoprenoid diphosphates on in vivo attachment to bacteriochlorophyllide c in the green sulfur photosynthetic bacterium Chlorobaculum tepidum
    Publication date: October 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 4

    Author(s): Yoshitaka Saga, Hayato Yamashita

    Metabolic substitution of the esterifying chain in bacteriochlorophyll (BChl) c in green photosynthetic bacteria grown by supplementation of exogenous alcohols has attracted attentions to study supramolecular structures and biogenesis of major antenna complexes chlorosomes in these bacteria as well as BChl pigment biosynthesis. Actual substrates in the enzymatic attachment of the esterifying moieties to the precursor of BChl c, namely bacteriochlorophyllide (BChlide) c, in these bacteria are believed to be diphosphate esters of alcoholic substrates, although only intact alcohols have so far been supplemented in the bacterial cultures. We report herein BChl c compositions in the green sulfur photosynthetic bacterium Chlorobaculum tepidum by supplementation with geranyl and geranylgeranyl diphosphates. The supplementation of these diphosphates hardly produced BChl c derivatives esterified with geraniol and geranylgeraniol in Cba. tepidum, whereas these BChl c derivatives were accumulated by supplementation of intact geraniol and geranylgeraniol. The sharp contrast of the incorporation efficiency of the supplemental isoprenoid moieties in BChl c using the isoprenoid diphosphates to that by the isoprenoid alcohols was mainly ascribable to less penetration abilities of the diphosphate substrates into Cba. tepidum cells because of their anionic and polar diphosphate moiety.





  • Investigation of storage time-dependent alterations of enantioselective amino acid profiles in kimchi using liquid chromatography-time of flight mass spectrometry
    Publication date: October 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 4

    Author(s): Moyu Taniguchi, Yutaka Konya, Yosuke Nakano, Eiichiro Fukusaki

    Although naturally abundant amino acids are represented mainly by l-enantiomers, fermented foods are known to contain various d-amino acids. Enantiospecific profiles of food products can vary due to fermentation by bacteria, and such alterations may contribute to changes in food properties that would not be dependent exclusively on l-amino acids. Therefore, more attention should be paid to the study of temporal alterations of d-amino acid profiles during fermentation process. However, there have been very few studies reporting time-dependent profiling of d-amino acids because enantioseparation of widely targeted d-amino acids is technically difficult. This study aimed to achieve high throughput profiling of amino acids enantiomers. Enantioselective profiling of amino acids using CROWNPAK CR-I(+) column, liquid chromatography, time of flight mass spectrometry, and principle component analysis was performed to investigate time-dependent alterations in concentrations of free d- and l-amino acids in kimchi stored at 4°C or 25°C. We demonstrated significant changes in d- and l-amino acid profiles in kimchi stored at 25°C. In particular, concentrations of the amino acids d-Ala, d-Ser, d-allo-Ile, d-Leu, d-Asp, d-Glu, and d-Met became higher in kimchi with storage time. This is the first report of time-dependent alterations of d- and l-amino acid contents in kimchi. This study showed that our analytical method of enantioselective detection of amino acids using liquid chromatography time-of-flight mass spectrometry (LC-TOFMS) with CROWNPAK CR-I(+) enables high throughput food screening and can be recommended for advanced studies of the relationship between d-amino acid content and food properties.





  • Search for compounds contributing to onion-like off-flavor in beer and investigation of the cause of the flavor
    Publication date: October 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 4

    Author(s): Shigekuni Noba, Nana Yako, Minoru Kobayashi, Susumu Masuda, Tetsuya Watanabe

    Onion-like off-flavor is a highly undesirable property in beer. Although several compounds that impart onion-like odors have been identified, the individual contribution of these compounds to the onion-like off-flavor in beer is not clear. In the present study, we searched for compounds that impart an onion-like odor by gas chromatography (GC)-olfactometry. The analysis of several types of beer revealed that 2-mercapto-3-methyl-1-butanol (2M3MB) and 3-mercapto-3-methyl-1-butanol (3M3MB) were possible causative compounds. Based on the difference threshold values in beer (0.13 ng/mL for 2M3MB and 17.5 ng/mL for 3M3MB) and the quantification values of these compounds in beer samples, only 2M3MB was considered to contribute to the onion-like off-flavor in beer. A further formation factor analysis of 2M3MB revealed that 2M3MB was formed in hopped wort after fermentation, and that the concentration of 2M3MB increased following the hot aeration treatment of wort. These results suggest that preventing the hot aeration of wort is a key factor for reducing 2M3MB levels in beer. In a previous report, 3-methyl-2-buten-1-ol (3MBol) was speculated to be the precursor of 2M3MB and 3M3MB; however, the results of the present quantification analysis and wort addition tests indicate that 3MBol did not contribute to the formation of 2M3MB in the brewing process and that unknown precursors of 2M3MB originated in wort. Identifying the precursor of 2M3MB may facilitate elucidation of the mechanism of 2M3MB formation.





  • Anaerobic hydrogen production from unhydrolyzed mushroom farm waste by indigenous microbiota
    Publication date: October 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 4

    Author(s): Chiu-Yue Lin, Chyi-How Lay, I-Yuan Sung, Biswarup Sen, Chin-Chao Chen

    The cultivation of mushrooms generates large amounts of waste polypropylene bags stuffed with wood flour and bacterial nutrients that makes the mushroom waste (MW) a potential feedstock for anaerobic bioH2 fermentation. MW indigenous bacteria were enriched using thermophilic temperature (55°C) for use as the seed inoculum without any external seeding. The peak hydrogen production rate (6.84 mmol H2/L-d) was obtained with cultivation pH 8 and substrate concentration of 60 g MW/L in batch fermentation. Hydrogen production yield (HY) is pH and substrate concentration dependent with an HY decline occurring at pH and substrate concentration increasing from pH 8 to 10 and 60 to 80 g MW/L, respectively. The fermentation bioH2 production from MW is in an acetate-type metabolic path.





  • Development of an in situ evaluation system for neural cells using extracellular matrix-modeled gel culture
    Publication date: October 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 4

    Author(s): Takayuki Nagai, Yasuhiro Ikegami, Hideyuki Mizumachi, Nana Shirakigawa, Hiroyuki Ijima

    Two-dimensional monolayer culture is the most popular cell culture method. However, the cells may not respond as they do in vivo because the culture conditions are different from in vivo conditions. However, hydrogel-embedding culture, which cultures cells in a biocompatible culture substrate, can produce in vivo-like cell responses, but in situ evaluation of cells in a gel is difficult. In this study, we realized an in vivo-like environment in vitro to produce cell responses similar to those in vivo and established an in situ evaluation system for hydrogel-embedded cell responses. The extracellular matrix (ECM)-modeled gel consisted of collagen and heparin (Hep-col) to mimic an in vivo-like environment. The Hep-col gel could immobilize growth factors, which is important for ECM functions. Neural stem/progenitor cells cultured in the Hep-col gel grew and differentiated more actively than in collagen, indicating an in vivo-like environment in the Hep-col gel. Second, a thin-layered gel culture system was developed to realize in situ evaluation of the gel-embedded cells. Cells in a 200-μm-thick gel could be evaluated clearly by a phase-contrast microscope and immunofluorescence staining through reduced optical and diffusional effects. Finally, we found that the neural cells cultured in this system had synaptic connections and neuronal action potentials by immunofluorescence staining and Ca2+ imaging. In conclusion, this culture method may be a valuable evaluation system for neurotoxicity testing.





  • Influence of pH and neutralizing agent on anaerobic succinic acid production by a Corynebacterium crenatum strain
    Publication date: October 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 4

    Author(s): Xiaoju Chen, Xuefeng Wu, Shaotong Jiang, Xingjiang Li

    Environmental conditions, particularly pH, have significant effects on the efficiency and final titers of bio-based products. Therefore, these factors need to be identified to ensure the fermentation process is economically attractive. In this study, strategies for controlling pH were optimized to enhance succinic acid production by Corynebacterium crenatum J-2. The results indicate that pH 6.8 is the optimal value for anaerobic succinic acid production by C. crenatum J-2 in terms of productivity and titer. The use of Mg(OH)2 as the neutralizing agent for pH control resulted in the highest levels of succinic acid concentration, yield, and productivity; superior to the levels obtained with Ca(OH)2, KOH, and NaOH. Under conditions of pH 6.8 and Mg(OH)2 as the neutralizing agent, 45.7 g/L succinic acid was produced within 12 h during the prophase of anaerobic fermentation, resulting in a succinic acid productivity of 3.8 g/(L·h). Succinic acid concentration reached 53.8 g/L at 22 h, with a productivity of 2.45 g/(L·h). The results of this study will be useful for the development of highly efficient succinic acid production processes utilizing industrial Corynebacterium spp. strains.





  • Recombinant and chemo-/bio-orthogonal synthesis of liposomal thrombomodulin and its antithrombotic activity
    Publication date: October 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 4

    Author(s): Lin Wang, Rui Jiang, Yang Liu, Maosheng Cheng, Qingyu Wu, Xue-Long Sun

    Thrombomodulin (TM) is an endothelial cell membrane protein that acts as a major cofactor in the protein C anticoagulant pathway. The EGF-like domains 4–6 of TM (TM456) are essential for PC activation. In this study, we proposed a liposomal recombinant TM conjugate to mimic the membrane TM structure and its anticoagulant activity. First, a DSPE-PEG2000-TM456 was successfully synthesized by site-specific conjugation of azido-TM456 with DSPE-PEG2000-DBCO via copper-free click chemistry quantitatively. Then, liposome-TM456 was fabricated via direct liposome formation with the DSPE-PEG2000-TM456 and other lipids. This liposomal formulation of TM456 retained protein C activation activity as that of TM456. Also, liposome-TM456 was much more stable and had a longer plasma half-life than TM456 and DSPE-PEG2000-TM456, respectively. Moreover, liposome-TM456 showed in vivo anticoagulant effect by decreasing the mortality from 80% to 20% in a thrombin-induced thromboembolism mouse model. The reported liposome-TM456 conjugate mimics the endothelial TM anticoagulation activity and may serve as an effective anticoagulant agent candidate for future development.





  • Enhanced production of natural yellow pigments from Monascus purpureus by liquid culture: The relationship between fermentation conditions and mycelial morphology
    Publication date: October 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 4

    Author(s): Jun Lv, Bo-Bo Zhang, Xiao-Dong Liu, Chan Zhang, Lei Chen, Gan-Rong Xu, Peter Chi Keung Cheung

    Natural yellow pigments produced by submerged fermentation of Monascus purpureus have potential economic value and application in the food industry. In the present study, the relationships among fermentation conditions (in terms of pH and shaking/agitation speed), mycelial morphology and the production of Monascus yellow pigments were investigated in both shake-flask and scale-up bioreactor experiments. In the shake-flask fermentation, the highest yield of the Monascus yellow pigments was obtained at pH 5.0 and a shaking speed of 180 rpm. Microscopic images revealed that these results were associated with the formation of freely dispersed small mycelial pellets with shorter, thicker and multi-branched hyphae. Further investigation indicated that the hyphal diameter was highly correlated with the biosynthesis of the Monascus yellow pigments. In a scaled-up fermentation experiment, the yield of yellow pigments (401 U) was obtained in a 200-L bioreactor, which is the highest yield to the best of our knowledge. The present findings can advance our knowledge on the conditions used for enhancing the production of Monascus yellow pigments in submerged fermentation and facilitate large-scale production of these natural pigments.





  • Activation of unfolded protein response pathway is important for valproic acid mediated increase in immunoglobulin G productivity in recombinant Chinese hamster ovary cells
    Publication date: October 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 4

    Author(s): Kamal Prashad Segar, Vikas Chandrawanshi, Sarika Mehra

    Process engineering to improve product quality and titers is gaining importance at late-stage cell culture process development. Valproic acid, a US Food and Drug Administration-approved histone deacetylase (HDAC) inhibitor, has been shown to improve cell culture performance with higher productivities and minimal effect on the product quality. However, the wider physiological impact of valproic acid on recombinant cells has not been investigated till date. In this study, we investigate the role of unfolded protein response pathway when immunoglobulin G (IgG)-secreting Chinese hamster ovary (CHO) cells are treated with valproic acid, resulting in a 3-fold increase in product titers and productivity. It is found that cells undergo an early transient endoplasmic reticulum (ER) stress on treatment with valproic acid, and subsequently adapt to perform as high producers. Induction of chaperones through enhanced XBP1 splicing activity and ATF6 activation suggests an increase in protein processing activity in these cells. We show that in addition to the enhanced recombinant mRNA expression of IgG heavy chain and light chain, the activation of unfolded protein response (UPR) pathway is critical to the increase in productivity of cells on valproic acid treatment. Further, upregulation of the UPR pathway is not through HDAC inhibition alone. To our knowledge, this is the first attempt to arrive at a phenotype-genotype mechanistic understanding of how valproic acid treatment enhances productivity in recombinant CHO cells.





  • Size- and time-dependent growth properties of human induced pluripotent stem cells in the culture of single aggregate
    Publication date: October 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 4

    Author(s): Suman C. Nath, Masanobu Horie, Eiji Nagamori, Masahiro Kino-oka

    Aggregate culture of human induced pluripotent stem cells (hiPSCs) is a promising method to obtain high number of cells for cell therapy applications. This study quantitatively evaluated the effects of initial cell number and culture time on the growth of hiPSCs in the culture of single aggregate. Small size aggregates ((1.1 ± 0.4) × 101–(2.8 ± 0.5) × 101 cells/aggregate) showed a lower growth rate in comparison to medium size aggregates ((8.8 ± 0.8) × 101–(6.8 ± 1.1) × 102 cells/aggregate) during early-stage of culture (24–72 h). However, when small size aggregates were cultured in conditioned medium, their growth rate increased significantly. On the other hand, large size aggregates ((1.1 ± 0.2) × 103–(3.5 ± 1.1) × 103 cells/aggregate) showed a lower growth rate and lower expression level of proliferation marker (ki-67) in the center region of aggregate in comparison to medium size aggregate during early-stage of culture. Medium size aggregates showed the highest growth rate during early-stage of culture. Furthermore, hiPSCs proliferation was dependent on culture time because the growth rate decreased significantly during late-stage of culture (72–120 h) at which point collagen type I accumulated on the periphery of aggregate, suggesting blockage of diffusive transport of nutrients, oxygen and metabolites into and out of the aggregates. Consideration of initial cell number and culture time are important to maintain balance between autocrine factors secretion and extracellular matrix accumulation on the aggregate periphery to achieve optimal growth of hiPSCs in the culture of single aggregate.





  • Evaluation of method bias for determining bacterial populations in bacterial community analyses
    Publication date: October 2017
    Source:Journal of Bioscience and Bioengineering, Volume 124, Issue 4

    Author(s): Masayuki Takahashi, Yasuko Kita, Akihiro Mizuno, Nami Goto-Yamamoto

    Various methods are used for analyzing a bacterial community. We recently developed a method for quantifying each bacterium constituting the microbiota by combining a next-generation sequencing (NGS) analysis with a quantitative polymerase chain reaction (NGS-qPCR) assay. Our NGS-qPCR method is useful for analyzing a comprehensive bacterial community because it is enables the easy calculation of the amounts of each bacterium constituting the microbiota. However, it has not been confirmed whether the estimated bacterial community obtained using this NGS-qPCR method corresponds to the results obtained using conventional methods. Accordingly, we prepared model bacterial community samples and analyzed them by several methods (NGS-qPCR, species-specific qPCR, flow cytometry, total direct counting by epifluorescent microscopy [TDC], and plate count). The total bacterial cell densities determined by the PCR-based methods were largely consistent with those determined by the TDC method. There was a difference between the amounts of each bacterium analyzed by NGS-qPCR and species-specific qPCR, although the same trend was shown by both species-specific qPCR and NGS-qPCR. Our findings also demonstrated that there is a strong positive correlation between the cell densities of a specific bacterial group in craft beer samples determined by group-specific qPCR and NGS-qPCR, and there were no significant differences among quantification methods (we tested two bacterial groups: lactic acid bacteria and acetic acid bacteria). Thus, the NGS-qPCR method is a practical method for analyzing a comprehensive bacterial community based on a bacterial cell density.





  • Evaluation of rice tetraticopeptide domain-containing thioredoxin as a novel solubility-enhancing fusion tag in Escherichia coli
    Publication date: Available online 18 September 2017
    Source:Journal of Bioscience and Bioengineering

    Author(s): Wenjun Xiao, Li Jiang, Weiyu Wang, Ruyue Wang, Jun Fan

    Fusion of solubility-enhancing tag is frequently used for improving soluble production of target protein in Escherichia coli. The Arabidopsis tetraticopeptide domain-containing thioredoxin (TDX) has been documented to exhibit functions of disulfide reductase, foldase chaperone, and holdase chaperone. Here, we identified that fusion of rice TDX with the smaller size increased soluble expression levels of three fluorescent proteins with different fluorophores in the E. coli strain BL21(DE3) or the Rosetta (DE3) strain with coexpression of six rare tRNAs, but decreased conformational quality of certain fluorescent proteins, as comparison with the His6-tagged ones. Among five maize proteins, the rice TDX fusion carrier displayed higher solubility-enhancing activity than the yeast SUMO3 tag toward three proteins in both E. coli strains. Five fusion constructs were cleaved with the co-expressed TEV protease variant, but the released target proteins were partly insolubly aggregated in vivo. Attachment of the His6-tag to the TDX tagged proteins had little impact on protein solubility. After Ni-NTA purification, five His6-TDX tagged proteins displayed different apparent purities. Taken together, our work presents that rice TDX tag is a novel solubility enhancer.





  • Microbial behavior and changes in food constituents during fermentation of Japanese sourdoughs with different rye and wheat starting materials
    Publication date: Available online 15 September 2017
    Source:Journal of Bioscience and Bioengineering

    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.





  • Inhibition of Saccharomyces cerevisiae growth by simultaneous uptake of glucose and maltose
    Publication date: Available online 15 September 2017
    Source:Journal of Bioscience and Bioengineering

    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.