JBB : Journal of Bioscience and Bioengineering

            

Journal of Bioscience and Bioengineering vol.123 cover

 



  • Enzymatic synthesis of 2′-O-methylribonucleosides with a nucleoside hydrolase family enzyme from Lactobacillus buchneri LBK78
    Publication date: June 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 6

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

    2′-O-Methylribonucleosides (2′-OMe-NRs) are promising raw materials for the production of nucleic acid drugs. We previously reported that LbNH, a nucleoside hydrolase from Lactobacillus buchneri LBK78 (NITE P-01581), was the first enzyme found to act on 2′-OMe-NRs. In the present study, we determined that LbNH also has the transribosylation activity between 2′-OMe-NRs and nucleobases, in addition to the hydrolyzing activity towards 2′-OMe-NRs. When 2′-O-methyluridine (2′-OMe-UR) and adenine were reacted with LbNH, 2′-O-methyladenosine (2′-OMe-AR) was produced. LbNH preferred purine nucleobases as its acceptor substrates for the transribosylation with 2′-OMe-UR as a donor substrate. Kinetic analysis of LbNH revealed that adenine behaved as a mixed inhibitor of the hydrolysis of 2′-OMe-UR. Under the optimal reaction conditions, the maximum molar yield of enzymatic 2′-OMe-AR produced reached 0.97% towards 2′-OMe-UR, corresponding to 0.16 g/L.





  • Identification of an acidic endo-polygalacturonase from Penicillium oxalicum CZ1028 and its broad use in major tropical and subtropical fruit juices production
    Publication date: June 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 6

    Author(s): Zhong Cheng, Dong Chen, Qingyan Wang, Liang Xian, Bo Lu, Yutuo Wei, Hongchi Tang, Zhilong Lu, Qixia Zhu, Yunlai Chen, Ribo Huang

    Endo-polygalacturonases play an important role on depectinization in fruit juices industry. A putative endo-polygalacturonase gene PoxaEnPG28A was cloned from Penicillium oxalicum CZ1028. PoxaEnPG28A consisted of a putative signal peptide and a catalytic domain belonging to glycoside hydrolase family 28, and it shared 72% identity with that of a functionally characterized endo-polygalacturonase from Trichoderma harzianum. Gene PoxaEnPG28A was successfully expressed in Pichia pastoris with a high yield of 1828.7 U/mL. The purified recombinant enzyme PoxaEnPG28A hydrolyzed polygalacturonic acid in endo-manner releasing oligo-galacturonates. PoxaEnPG28A showed maximal activity at pH 5.5 and 55°C, and was stable between pH 3.0 to 10.0 and below 45°C. The kinetic constants K m and V max of PoxaEnPG28A were calculated as 1.57 g/L and 14,641.29 U/mg, respectively. PoxaEnPG28A significantly improved the yields of fruit juices from banana, plantain, papaya, pitaya and mango. The high production level of the recombinant enzyme PoxaEnPG28A by P. pastoris and remarkable catalytic activity of PoxaEnPG28A toward five kinds of fruit juices made the enzyme a potential application in agriculture and food industries.





  • Improvement of operational stability of Ogataea minuta carbonyl reductase for chiral alcohol production
    Publication date: June 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 6

    Author(s): Kohsuke Honda, Mizuha Inoue, Tomohiro Ono, Kenji Okano, Yasumasa Dekishima, Hiroshi Kawabata

    Directed evolution of enantio-selective carbonyl reductase from Ogataea minuta was conducted to improve the operational stability of the enzyme. A mutant library was constructed by an error-prone PCR and screened using a newly developed colorimetric assay. The stability of a mutant with two amino acid substitutions was significantly higher than that of the wild type at 50°C in the presence of dimethyl sulfoxide. Site-directed mutagenesis analysis showed that the improved stability of the enzyme can be attributed to the amino acid substitution of V166A. The half-lives of the V166A mutant were 11- and 6.1-times longer than those of the wild type at 50°C in the presence and absence, respectively, of 20% (v/v) dimethyl sulfoxide. No significant differences in the substrate specificity and enantio-selectivity of the enzyme were observed. The mutant enzyme converted 60 mM 2,2,2-trifluoroacetophenone to (R)-(−)-α-(trifluoromethyl)benzyl alcohol in a molar yield of 71% whereas the conversion yield with an equivalent concentration of the wild-type enzyme was 27%.





  • Evaluation of tyrosinase inhibitory and antioxidant activities of Angelica dahurica root extracts for four different probiotic bacteria fermentations
    Publication date: June 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 6

    Author(s): Guey-Horng Wang, Chih-Yu Chen, Teh-Hua Tsai, Ching-Kuo Chen, Chiu-Yu Cheng, Yi-Hsin Huang, Min-Chi Hsieh, Ying-Chien Chung

    Angelica dahurica root (ADR), which shows strong antioxidant activity, is used in Chinese medicine. This study evaluated the tyrosinase inhibitory and antioxidant activities of ADR extracts fermented by four different probiotic bacteria: Bifidobacterium bifidum, Bifidobacterium lactis, Lactobacillus acidophilus, and Lactobacillus brevis. The ADR was first extracted using distilled water, 70% ethanol, and ethyl acetate, and then fermented by probiotic bacteria. The physiological characteristics of these fermented extracts, namely the antityrosinase activity, antioxidant activity, phenolic composition, and phenolic content, were evaluated and compared with those of unfermented extracts. Results showed that the water extracts after fermentation by probiotic bacteria exhibited the most favorable physiological characteristics. Among the extracts fermented by these probiotic bacteria, L. acidophilus-fermented ADR extract showed the most favorable physiological characteristics. The optimal IC50 values for antityrosinase activity, DPPH radical scavenging activity, and reducing power for L. acidophilus-fermented ADR extract were 0.07 ± 0.03, 0.12 ± 0.01, and 0.68 ± 0.06 mg/mL, respectively. Furthermore, the physiological activities of fermented extracts were considerably higher than those of unfermented extracts. The tyrosinase inhibition and melanin content of B16F10 melanoma cells, and cytotoxicity effects of the fermented ADR extracts on B16F10 cells were also evaluated. We found that the L. acidophilus-fermented ADR extract at 1.5 mg/mL showed significant cellular antityrosinase activity with low melanin production in B16F10 cells and was noncytotoxic to B16F10 cells. Among all probiotic bacteria, water-extracted ADR fermented by L. acidophilus for 48 h was found to be the best skincare agent or antioxidant agent.





  • Reduction of FR900525 using an S-(2-aminoethyl) l-cysteine-resistant mutant
    Publication date: June 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 6

    Author(s): Shiho Shimizu, Ayako Futase, Tatsuya Yokoyama, Satoshi Ueda, Hiroyuki Honda

    FK506 (tacrolimus), a macrolide compound with immunosuppressant activity, has been proven to have clinical importance and has been manufactured industrially since 1993 by using mutants with high FK506-production ability; these mutants have been developed from the wild strain Streptomyces tsukubaensis No. 9993. FR900525 is one of the by-products of FK506 production. However, there was no effective industrial method to separate FR900525 from FK506 due to the structural similarity between the two compounds. Therefore, reducing the level of FR900525 was a serious problem in the industrial strain A. In this study, we aimed to reduce the FR900525 production. We first determined that pipecolic acid level was a critical parameter for controlling FR900525 production in strain A. S-(2-Aminoethyl) l-cysteine (AEC)-resistant mutants has been reported to increase lysine productivity successfully in a variety of lysine-producing microorganisms. Therefore, next, we applied a selection of AEC-resistant mutants to enhance pipecolic acid biosynthesis. Finally, four AEC-resistant mutants were obtained from strain A using ultraviolet irradiation, and three of them showed less FR900525 productivity compared to the parental strain A. Our findings indicated that AEC resistance was effective phenotype marker for increasing pipecolic acid productivity and for reducing FR900525 production in S. tsukubaensis. Thus, our study provides an efficient method for reducing FR90025 level during FK506 biosynthesis.





  • Biocatalyst-mediated production of 11,15-dihydroxy derivatives of androst-1,4-dien-3,17-dione
    Publication date: June 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 6

    Author(s): Yuqian Qiao, Yanbing Shen, Wei Huang, Yanping Wang, Jiajia Ren, Ting Xia, Min Wang

    Hydroxylation of steroids at various positions is a powerful tool for the production of valuable pharmaceutical ingredients and precursors. Our paper reported the synchronous dihydroxylation of an efficient strain, i.e., Colletotrichum lini AS3.4486, at two points. C. lini AS3.4486 was selected from 10 strains; this strain can catalyze the dihydroxylation of androst-1,4-dien-3,17-dione at C-11α and C-15α positions. Transformation of ADD(I) by C. lini AS3.4486 produced metabolites II–IV. The structures of these compounds were elucidated by liquid chromatography–mass spectrometry (LC–MS), Fourier Transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and X-ray as 15-hydroxyandrost-1,4-dien-3,17-dione (15α-OH-ADD; II), 11,15-dihydroxyandrost-1,4-dien-3,17-dione (11,15-diOH-ADD; III), and 15,17β-dihy-droxyandrost-1,4-dien-3-one (15-OH-BD; BD is the abbreviation of boldenone; IV). III, as a novel compound, was reported for the first time. The course of conversion and mechanism about dihydroxylation reaction was also investigated. On the basis of time course analysis of hydroxylation, I underwent regioselective hydroxylation at 15 position and was subsequently converted to III and IV. Enzyme inhibition analysis showed that 11- and 15-hydroxylations were catalyzed by different hydroxylases. The effect of substrate concentration on I transformation was also determined. Results showed that the optimum concentration of I was 20 g/L, and the yield of III was up to 18.8 g/L.





  • Injury and recovery of Escherichia coli ATCC25922 cells treated by high hydrostatic pressure at 400–600 MPa
    Publication date: June 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 6

    Author(s): Keitarou Kimura, Kazuya Morimatsu, Takashi Inaoka, Kazutaka Yamamoto

    Escherichia coli cells were inactivated by high hydrostatic pressure (HHP) at 400–600 MPa and their recovery under various conditions was evaluated by colony counting and flow cytometer (FCM) analyses. The lag time in colony formation and an improved recovery of cells under less oxidative conditions (pyruvate addition to the medium and incubation in anaerobic conditions) were observed for HHP treated cells, which indicated that a significant portion of cells were injured and recovered during incubation after HHP treatment. The lag time for colony formation varied, which suggested a wave of resuscitation and recovered cells may multiply before other injured cells complete resuscitation. The recovery process was monitored by FCM: The FCM profile of cells stained using propidium iodide and SYTO9 indicated that while the majority of cells died just after HHP treatment, the staining pattern of possibly injured cells displayed a specific spectrum that gradually became consistent with that of the dead cell population and a living cell population simultaneously appeared. Pyruvate addition to the medium not only enhanced viability of HHP treated cells, but also reduced the lethal effect of HHP. These observations suggested that the degree of damage by HHP may differ cell-by-cell, and oxidative stress may continue after HHP treatment. Pyruvate addition to the recovery medium enhanced viability of E. coli cells inactivated by HHP treatment in tomato juice as well.





  • Breeding of a sake yeast mutant with enhanced ethyl caproate productivity in sake brewing using rice milled at a high polishing ratio
    Publication date: June 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 6

    Author(s): Toshinari Takahashi, Yusuke Ohara, Kazuo Sueno

    Sake yeast produces a fruity flavor known as ginjo-ko—which is mainly attributable to ethyl caproate and isoamyl acetate—during fermentation in sake brewing. The production of these flavor components is inhibited by unsaturated fatty acids derived from the outer layer of rice as raw material. We isolated three mutants (hec2, hec3, and hec6) with enhanced ethyl caproate productivity in sake brewing using rice milled at a high polishing ratio from a cerulenin-resistant mutant derived from the hia1 strain, which shows enhanced isoamyl acetate productivity. The hec2 mutant had the homozygous FAS2 mutation Gly1250Ser, which is known to confer high ethyl caproate productivity. When the homozygous FAS2 mutation Gly1250Ser was introduced into strain hia1, ethyl caproate productivity was increased but neither this nor intracellular caproic acid content approached the levels observed in the hec2 mutant, indicating that a novel mutation was responsible for the high ethyl caproate productivity. We also found that the expression of EEB1 encoding acyl-coenzyme A:ethanol O-acyltransferase (AEATase) and enzymatic activity were increased in the hec2 mutant. These results suggest that the upregulation of EEB1 expression and AEATase activity may also have contributed to the enhancement of ethyl caproate synthesis from ethanol and caproyl-CoA. Our findings are useful for the brewing of sake with improved flavor due to high levels of isoamyl acetate and ethyl caproate.





  • Recycling of metals from pretreated waste printed circuit boards effectively in stirred tank reactor by a moderately thermophilic culture
    Publication date: June 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 6

    Author(s): Ming-Chen Xia, Ya-Ping Wang, Tang-Jian Peng, Li Shen, Run-Lan Yu, Yuan-Dong Liu, Miao Chen, Jiao-Kun Li, Xue-Ling Wu, Wei-Min Zeng

    To seek a feasible technique for processing waste printed circuit boards (PCBs), pretreatment of PCBs by table separation and further bioleached by moderate thermophiles in a stirred tank reactor were investigated. The shaking table separation, conducted after grinding and sieving of PCBs, produced two fractions: metal-rich parts (RPCBs), which is more suitable for pyrometallurgy process than untreated PCBs, and metal-poor parts (PPCBs) with only 8.83% metals was then bioleached by a mixed culture of moderate thermophiles effectively. After adaptation, the mixed culture could tolerate 80 g/L PPCBs. The bioleaching results showed that metals recovery was 85.23% Zn, 76.59% Cu and 70.16% Al in only 7 days. Trace Pb and Sn were detected in the leachate because of precipitating. The microorganism community structure was analyzed by amplified ribosomal DNA restriction analysis. Two moderately thermophilic bacteria species were identified as Leptospirillum ferriphilum and Acidithiobacillus caldus. Furthermore, uncultured Thermoplasmatales archaeon was also detected in the leaching system. It was also shown that moderate thermophiles revealed best bioleaching ability when compared with mesophiles and the mixture of mesophiles and moderate thermophiles. Finally, we designed a two-stage process model according to the present study to achieve semi-industrial waste PCBs recycling and economic feasibility analysis indicated that the process was profitable.





  • Polyphosphate metabolism by purple non-sulfur bacteria and its possible application on photo-microbial fuel cell
    Publication date: June 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 6

    Author(s): Yen-Chun Lai, Chih-Ming Liang, Shu-Chuan Hsu, Ping-Heng Hsieh, Chun-Hsiung Hung

    A purple non-sulfur bacterium, Rhodopseudomonas palustris G11, was isolated from an activated sludge plant that treats domestic wastewater. This isolation resulted in the effective accumulation of polyphosphate in cells upon reaching the stationary growth phase. However, when the carbon and/or energy source were/was removed, this bacterium released intracellular polyphosphate or poly-β-hydroxybutyrate to obtain energy to grow or maintain its growth. Furthermore, a novel photo-microbial fuel cell (PMFC) design was proposed. The unique capability of purple non-sulfur bacteria to capture light energy for polyphosphate accumulation was maximized. After R. palustris G11 accumulated considerable polyphosphate and was transferred to a fresh medium, the PMFC system exhibited a maximum voltage of approximately 0.03 V undt illumination. The chemical oxygen demand removal efficiency, Coulomb efficiency, and power density were 95.8%, 0.62%, and 0.15 mW/m2, respectively. The test microorganisms converted most of the light energy in growth and caused the low power production. The microorganisms grew slowly and produced less power under dark conditions than under light illumination. However, these microorganisms used the previously stored polyphosphate or poly-β-hydroxybutyrate for electricity production when they were incubated in a growth-insufficient condition. This novel concept can be improved and optimized in the future for new PMFC applications, such as rechargeable cells, to treat wastewater and restore energy simultaneously.





  • Influence of rice straw polyphenols on cellulase production by Trichoderma reesei
    Publication date: June 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 6

    Author(s): Wei Zheng, Qin Zheng, Yiyun Xue, Jiajun Hu, Min-Tian Gao

    In this study, we found that during cellulase production by Trichoderma reesei large amounts of polyphenols were released from rice straw when the latter was used as the carbon source. We identified and quantified the phenolic compounds in rice straw and investigated the effects of the phenolic compounds on cellulase production by T. reesei. The phenolic compounds of rice straw mainly consisted of phenolic acids and tannins. Coumaric acid (CA) and ferulic acid (FA) were the predominant phenolic acids, which inhibited cellulase production by T. reesei. When the concentrations of CA and FA in the broth increased to 0.06 g/L, cellulase activity decreased by 23% compared with that in the control culture. Even though the rice straw had a lower tannin than phenolic acid content, the tannins had a greater inhibitory effect than the phenolic acids on cellulase production by T. reesei. Tannin concentrations greater than 0.3 g/L completely inhibited cellulase production. Thus, phenolic compounds, especially tannins are the major inhibitors of cellulase production by T. reesei. Therefore, we studied the effects of pretreatments on the release of phenolic compounds. Ball milling played an important role in the release of FA and CA, and hot water extraction was highly efficient in removing tannins. By combining ball milling with extraction by water, the 2-fold higher cellulase activity than in the control culture was obtained.





  • Fabrication of a fiber-type hepatic tissue by bottom-up method using multilayer spheroids
    Publication date: June 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 6

    Author(s): Tatsuya Okudaira, Ryohei Yabuta, Hiroshi Mizumoto, Toshihisa Kajiwara

    Liver regenerative medicine, a therapy using cultured hepatocytes or hepatic tissues, has the potential to replace liver transplantation. However, this therapeutic strategy has challenges to overcome, including in construction of the hepatic tissues. As an approach to fabricating functional 3D hepatic tissues, we focused on hepatocyte spheroids, which have high cell density and maintain high liver-specific functions. We employed a bottom-up method using spheroids, arranging hepatocytes and endothelial cells regularly at the time of tissue construction. This enabled a vascular network to be formed within the three-dimensional hepatic tissue. We included NIH/3T3 cells, known to promote vasculature formation by endothelial cells. We fabricated hepatocyte spheroids covered with human umbilical vein endothelial cells (HUVECs) and NIH/3T3 cells (EC-3T3-covered hepatocyte spheroids) and constructed the hepatic tissues by stacking these cell types in hollow fibers. We then performed histological and functional analyses of the resulting hepatic tissues. The hepatic tissues constructed by stacking EC-3T3-covered hepatocyte spheroids showed high liver-specific functions; that is, ammonia removal and albumin secretion. The HUVECs formed endothelial networks. In addition, hypoxia-inducible factor-1α (HIF-1α) expression was suppressed in the hepatic tissue throughout the culture period and the hepatic tissue was sufficiently strong for use in certain analyses and applications. In summary, we fabricated a functional 3D hepatic tissue by the bottom-up method using hepatocyte spheroids covered with HUVECs and NIH/3T3 cells.





  • Degradation of endothelial network in disordered tumor-containing cell sheet
    Publication date: June 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 6

    Author(s): Menglu Li, Masahiro Kino-oka

    Tumor angiogenesis is an important event in tumor malignancy; and the vasculature formed in tumor region is typically dysfunctional. Multiple factors are associated with tumor vessel abnormalities, but the precise mechanism has not been fully understood. In the present study, a tumor-containing cell sheet was prepared by mixing a small population of human embryonal rhabdomyosarcoma (RMS) cells (RDs) with human skeletal muscle myoblasts (HSMMs) to mimic muscle tissue invaded by RMS cells. Sheet fluidity and the extracellular matrix (ECM) meshwork of the tumor-containing cell sheet were found to be elevated and disordered, demonstrating the disruptive effect of tumor cells on sheet structure. When green fluorescent protein expressing human umbilical vein endothelial cells (GFP-HUVECs) were co-cultured with the tumor-containing cell sheet, an endothelial network was formed, but degraded faster as a result of activated migration of endothelial cells in the tumor-containing cell sheet. This study suggested that disorganized tissue structure facilitate tumor angiogenesis by activation of endothelial cell migration.





  • Evaluation of a novel system for analyzing hydrophilic blood metabolites
    Publication date: June 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 6

    Author(s): Shin Nishiumi, Keisuke Shima, Takeshi Azuma, Masaru Yoshida

    Metabolomics has recently been developed, and there have been a considerable number of metabolomics-based biomarker studies in the medical research field. Therefore, as a first step toward the practical use of metabolite biomarkers, a simple and quick sample preparation method involving metabolite extraction, metabolite measurement, and data analysis needs to be developed. In this study, we evaluated whether the use of simpler metabolite extraction methods would facilitate the stable analysis of hydrophilic blood metabolites during liquid chromatography/triple quadrupole mass spectrometry (LC/QqQMS)-based metabolome analysis. As a result, the anion and cation metabolites in plasma were stably analyzed via a methanol-based extraction procedure followed by ultrafiltration, and it was also confirmed that a lyophilization step was not necessary. When extraction was performed without a lyophilization step, approximately >50% and >80% of the detected metabolites had relative standard deviation values of <20% during LC/QqQMS-based anion and cation analyses, respectively. In addition, the plasma levels of l-valine, l-leucine, l-isoleucine, l-tyrosine, and l-phenylalanine were quantitatively measured using the corresponding stable isotopes; the SCLAM-2000, a fully automatic pre-treatment system for LC/MS that can be connected online to an LC/MS device; and an extraction procedure based on the simple procedure that we developed. Our findings suggest that simpler pretreatment procedures can be employed during LC/QqQMS-based metabolomics and might aid searches for metabolite biomarker candidates, the validation of metabolite biomarker candidates, and the practical use of metabolite biomarkers.





  • Detection of antibodies against hepatitis B virus surface antigen and hepatitis C virus core antigen in plasma with a waveguide-mode sensor
    Publication date: June 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 6

    Author(s): Takenori Shimizu, Torahiko Tanaka, Shigeyuki Uno, Hiroki Ashiba, Makoto Fujimaki, Mutsuo Tanaka, Koichi Awazu, Makoto Makishima

    In large-scale disasters, such as huge significant earthquakes, on-site examination for blood typing and infectious disease screening will be very helpful to save lives of victims who need surgical treatment and/or blood transfusion. However, physical damage, such as building collapse, electric power failure and traffic blockage, disrupts the capacity of the medical system. Portable diagnostic devices are useful in such cases of emergency. In this study, we evaluated a waveguide-mode sensor for detection of anti-hepatitis virus antibodies. First, we examined whether we can detect antigen–antibody interaction on a sensor chip immobilized hepatitis B virus surface (HBs) antigen and hepatitis C virus (HCV) core antigen using monoclonal mouse antibodies for HBs antigen and HCV core antigen. We obtained significant changes in the reflectance spectra, which indicate specific antigen–antibody interaction for anti-HBs antibody and anti-HCV antibody. Next, we examined the effect of horseradish peroxidase-conjugated secondary antibody using aminoethyl carbazole as the peroxidase substrate and found that the colorimetric reaction increases detection sensitivity for anti-HBs antibody more than 300 times. Finally, we successfully detected anti-HBs antibody in human blood samples with an enhancing method using a peroxidase reaction. Thus, a portable device utilizing a waveguide-mode sensor may be applied to on-site blood testing in emergency settings.





  • Nanaomycin H: A new nanaomycin analog
    Publication date: June 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 6

    Author(s): Takuji Nakashima, Toru Kimura, Rei Miyano, Hirotaka Matsuo, Tomoyasu Hirose, Aoi Kimishima, Kenichi Nonaka, Masato Iwatsuki, Jun Nakanishi, Yōko Takahashi, Satoshi Ōmura

    Physicochemical screening identified a new nanaomycin analog, nanaomycin H, which was isolated from a culture broth of Streptomyces rosa subsp. notoensis OS-3966. This microorganism is already known to produce seven nanaomycin compounds, (nanaomycin A to G). Structural elucidation of nanaomycin H showed it to be a pyranonaphthoquinone with a mycothiol moiety. A N-acetylcysteine S-conjugate of nanaomycin H, without α-glucosamine linked to myo-inositol moiety, mercapturic acid derivative, was also detected in the same culture broth. Mercapturic acid derivatives of secondary metabolites are known to be produced for xenobiotic metabolism outside microbial cells. Mycothiol acts as a detoxifier to help prevent cell damage from factors such as oxidative stress. The production of O2 generated by reduction of nanaomycin A is correlated with antibacterial activity. Mycothiol-containing nanaomycin H proved to be markedly decreased in O2 and did not express any notable antimicrobial activity. It is suggested that nanaomycin H is produced in the detoxification process.





  • Investigation of storage time-dependent alterations of enantioselective amino acid profiles in kimchi using liquid chromatography-time of flight mass spectrometry
    Publication date: Available online 27 May 2017
    Source:Journal of Bioscience and Bioengineering

    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.





  • Enhancement of butanol production by sequential introduction of mutations conferring butanol tolerance and streptomycin resistance
    Publication date: Available online 26 May 2017
    Source:Journal of Bioscience and Bioengineering

    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.





  • In vivo evolutionary engineering for ethanol-tolerance of Saccharomyces cerevisiae haploid cells triggers diploidization
    Publication date: Available online 25 May 2017
    Source:Journal of Bioscience and Bioengineering

    Author(s): Burcu Turanlı-Yıldız, Laurent Benbadis, Ceren Alkım, Tuğba Sezgin, Arman Akşit, Abdülmecit Gökçe, Yavuz Öztürk, Ahmet Tarık Baykal, Zeynep Petek Çakar, Jean M. François

    Microbial ethanol production is an important alternative energy resource to replace fossil fuels, but at high level, this product is highly toxic, which hampers its efficient production. Towards increasing ethanol-tolerance of Saccharomyces cerevisiae, the so far best industrial ethanol-producer, we evaluated an in vivo evolutionary engineering strategy based on batch selection under both constant (5%, v v−1) and gradually increasing (5–11.4%, v v−1) ethanol concentrations. Selection under increasing ethanol levels yielded evolved clones that could tolerate up to 12% (v v−1) ethanol and had cross-resistance to other stresses. Quite surprisingly, diploidization of the yeast population took place already at 7% (v v−1) ethanol level during evolutionary engineering, and this event was abolished by the loss of MKT1, a gene previously identified as being implicated in ethanol tolerance (Swinnen et al. Genome Res., 22, 975–984 (2012)). Transcriptomic analysis confirmed diploidization of the evolved clones with strong down-regulation in mating process, and in several haploid-specific genes. We selected two clones exhibiting the highest viability on 12% ethanol, and found productivity and titer of ethanol significantly higher than those of the reference strain under aerated fed-batch cultivation conditions. This higher fermentation performance could be related with a higher abundance of glycolytic and ribosomal proteins and with a relatively lower respiratory capacity of the evolved strain, as revealed by a comparative transcriptomic and proteomic analysis between the evolved and the reference strains. Altogether, these results emphasize the efficiency of the in vivo evolutionary engineering strategy for improving ethanol tolerance, and the link between ethanol tolerance and diploidization.





  • Improved laccase production by Funalia trogii in absorbent fermentation with nutrient carrier
    Publication date: Available online 22 May 2017
    Source:Journal of Bioscience and Bioengineering

    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.