JBB : Journal of Bioscience and Bioengineering

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  • Genome-wide screening of transcription factor deletion targets in Escherichia coli for enhanced production of lactate-based polyesters
    Publication date: May 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 5

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

    Engineered Escherichia coli is a useful platform for production of lactate (LA)-based polyester poly[LA-co-3-hydroxybutyrate (3HB)] from renewable sugars. Here we screened all non-lethal transcription factor deletions of E. coli for efficient production of the polymer. This approach aimed at drawing out the latent potential of the host for efficient polymer production via indirect positive effects. Among 252 mutants from Keio Collection tested, eight mutants (ΔpdhR, ΔcspG, ΔyneJ, ΔchbR, ΔyiaU, ΔcreB, ΔygfI and ΔnanK) accumulated greater amount of polymer (6.2–10.1 g/L) compared to the parent strain E. coli BW25113 (5.1 g/L). The mutants increased polymer production per cell (1.1–1.5-fold) without significant change in cell density. The yield of the polymer from glucose was also higher for the selected mutants (0.34–0.38 g/g) than the parent strain (0.27 g/g). Therefore, the deletions of transcription factors should channel the carbon flux towards polymer production. It should be noted that the screening employed in this study identified beneficial mutants without analyzing causal relationship between the mutation and the enhanced polymer production. This approach, therefore, should be applicable to broad range of fermentation productions.





  • N-terminal SKIK peptide tag markedly improves expression of difficult-to-express proteins in Escherichia coli and Saccharomyces cerevisiae
    Publication date: May 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 5

    Author(s): Teruyo Ojima-Kato, Satomi Nagai, Hideo Nakano

    Despite advances in microbial protein expression systems, low production of proteins remains a great concern for some genes. Here we report that the insertion of a short peptide tag, consisting of Ser-Lys-Ile-Lys (SKIK), adjacent to the start codon of genes encoding difficult-to-express proteins can increase protein expression in Escherichia coli and Saccharomyces cerevisiae. Protein expression levels of a mouse monoclonal antibody (mAb), rabbit mAbs obtained from clonal B cells, and an artificially designed peptide were significantly increased simply by the addition of the SKIK tag in E. coli systems. In particular, a ∼30-fold increase in protein production was observed for the mouse mAb, and the artificially designed peptide band became detectable in sodium dodecyl sulfate-poly acrylamide gel electrophoresis after coomassie brilliant blue staining or western blotting on adding the SKIK tag. The tag also increased the expression of tagged proteins in S. cerevisiae and an E. coli cell-free protein synthesis system. Although the mechanism of high protein expression on addition of the tag is unclear, our findings offer great benefits to biotechnology research and industry.





  • Effect of acetate as a co-feedstock on the production of poly(lactate-co-3-hydroxyalkanoate) by pflA-deficient Escherichia coli RSC10
    Publication date: May 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 5

    Author(s): Lucia Salamanca-Cardona, Ryan A. Scheel, Kouhei Mizuno, N. Scott Bergey, Arthur J. Stipanovic, Ken'ichiro Matsumoto, Seiichi Taguchi, Christopher T. Nomura

    Developing Escherichia coli strains that are tolerant to acetate toxicity is important in light of an increased interest in the efficient utilization of lignocellulosic biomass feedstocks for the biosynthesis of value-added products. In this study, four strains known to produce polyhydroxyalkanoates (PHAs) from the typical hemicellulosic sugar xylose were tested for their tolerance to acetate. E. coli RSC10 was found to be tolerant of acetate, both in growth and fermentation studies. In the presence of acetate the strain showed a >2-fold increase in overall yields compared to using xylose alone as the feedstock. More importantly, the strain was found to be able to utilize acetate as a feedstock for biosynthesis of PHAs, with complete depletion of acetate (25 mM) at 9 h when acetate was the sole feedstock. Higher concentrations of acetate showed greater inhibition of fermentation than growth with a reduction of 90% in PHA yields at 100 mM. Additionally, the present work provides data to support the potential of acetate as a modulator for the control of composition of PHAs that incorporate lactate (LA) monomers into the copolymer from hemicellulose derived sugars.





  • Medium optimization based on yeast's elemental composition for glutathione production in Saccharomyces cerevisiae
    Publication date: May 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 5

    Author(s): Maximilian Schmacht, Eric Lorenz, Ulf Stahl, Martin Senz

    The production of glutathione (GSH) or GSH enriched yeast is still in the focus of research driven by a high industrial interest. In this study, an optimal growth rate for GSH production via Saccharomyces cerevisiae Sa-07346 was investigated. To further improve the fermentation process in a way that it is independent of lots, the influence of different WMIX medium compositions on biomass and GSH production was studied. Thereby, the fermentation medium was adjusted based on yeast's elemental composition. The resulting chemically defined fermentation medium led to high cell densities in fed-batches. Therefore, it has the potential to be applied successfully for other high cell density yeast fermentation processes. As cysteine is the key component for GSH production, different cysteine addition strategies were studied and finally, a continuous cysteine feeding was applied in the late stage of fermentation. Thereby, a GSH concentration of 1459 ± 57 mg/l was reached by continuously feeding cysteine, which meant an increase to 253% compared to the control without cysteine addition (577 mg/l GSH).





  • Butyrate production under aerobic growth conditions by engineered Escherichia coli
    Publication date: May 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 5

    Author(s): Naoya Kataoka, Alisa S. Vangnai, Thunyarat Pongtharangkul, Toshiharu Yakushi, Kazunobu Matsushita

    Butyrate is an important industrial platform chemical. Although several groups have reported butyrate production under oxygen-limited conditions by a native producer, Clostridium tyrobutylicum, and by a metabolically engineered Escherichia coli, efforts to produce butyrate under aerobic growth conditions have met limited success. Here, we constructed a novel butyrate synthetic pathway that functions under aerobic growth conditions in E. coli, by modifying the 1-butanol synthetic pathway reported previously. The pathway consists of phaA (acetyltransferase) and phaB (NADPH-dependent acetoacetyl-CoA reductase) from Ralstonia eutropha, phaJ ((R)-specific enoyl-CoA hydratase) from Aeromonas caviae, ter (trans-enoyl-CoA reductase) from Treponema denticola, and endogenous thioesterase(s) of E. coli. To evaluate the potential of this pathway for butyrate production, culture conditions, including pH, oxygen supply, and concentration of inorganic nitrogen sources, were optimized in a mini-jar fermentor. Under the optimal conditions, butyrate was produced at a concentration of up to 140 mM (12.3 g/L in terms of butyric acid) after 54 h of fed-batch culture.





  • Identification and characterization of a novel N-acyl-homoserine lactonase gene in Sphingomonas ursincola isolated from industrial cooling water systems
    Publication date: May 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 5

    Author(s): Tomohiro Morohoshi, Niina Sato, Taro Iizumi, Airi Tanaka, Tsukasa Ikeda

    Biofilm formation by bacteria is one of the main causes of fouling in industrial cooling water systems. In many gram-negative bacteria, biofilm formation is regulated by N-acyl-homoserine lactone (AHL)-mediated quorum sensing. In this study, we isolated three AHL-degrading bacteria from cooling water systems and identified them as Sphingomonas ursincola. The draft genome sequence of S. ursincola A1 revealed the presence of an AHL-degrading gene homolog, designated qsdS. The qsdS region was also amplified by PCR from the genomes of the other two S. ursincola strains, SF1 and SF8. Escherichia coli DH5α harboring a QsdS-expressing plasmid showed high degradative activity against AHLs with short and 3-oxo-substituted acyl chains. High-performance liquid chromatography analysis revealed that QsdS is an AHL lactonase, an enzyme that catalyzes AHL ring opening. Furthermore, heterologous expression of QsdS in Pseudomonas aeruginosa PAO1 resulted in degradation of endogenous AHLs and interfered with the quorum-sensing-regulated phenotype.





  • Acetylation and deacetylation for sucralose preparation by a newly isolated Bacillus amyloliquefaciens WZS01
    Publication date: May 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 5

    Author(s): Jie Sun, Liang Chen, Bo Lou, Yanbing Bai, Xinjun Yu, Man Zhao, Zhao Wang

    Sucralose is a non-nutritive artificial sweetener used in a broad range of foods and beverages. In the present study, Bacillus amyloliquefaciens WZS01 was isolated, identified, and used as a catalyst both in regioselective acylation and deacetylation for sucralose preparation. Bacterial cells were immobilized on polyurethane foam and utilized to synthesize sucrose-6-acetate regioselectively. The yield of sucrose-6-acetate was >95% with 60 mM sucrose after 22 h of reaction. Free cells could hydrolyze 75 mM sucralose-6-acetate to produce sucralose with >99% yield after 24 h of reaction. B. amyloliquefaciens WZS01 could be considered a potential biocatalyst for sucralose preparation.





  • Nitrite oxidation kinetics of two Nitrospira strains: The quest for competition and ecological niche differentiation
    Publication date: May 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 5

    Author(s): Norisuke Ushiki, Masaru Jinno, Hirotsugu Fujitani, Toshikazu Suenaga, Akihiko Terada, Satoshi Tsuneda

    Nitrite oxidation is an aerobic process of the nitrogen cycle in natural ecosystems, and is performed by nitrite-oxidizing bacteria (NOB). Also, nitrite oxidation is a rate-limiting step of nitrogen removal in wastewater treatment plants (WWTPs). Although Nitrospira is known as dominant NOB in WWTPs, information on their physiological properties and kinetic parameters is limited. Here, we report the kinetic parameters and inhibition of nitrite oxidation by free ammonia in pure cultures of Nitrospira sp. strain ND1 and Nitrospira japonica strain NJ1, which were previously isolated from activated sludge in a WWTP. The maximum nitrite uptake rate ( V max _ NO 2 ) and the half-saturation constant for nitrite uptake ( K m _ NO 2 ) of strains ND1 and NJ1 were 45 ± 7 and 31 ± 5 (μmol NO2 /mg protein/h), and 6 ± 1 and 10 ± 2 (μM NO2 ), respectively. The V max _ NO 2 and K m _ NO 2 of two strains indicated that they adapt to low-nitrite-concentration environments like activated sludge. The half-saturation constants for oxygen uptake ( K m _ O 2 ) of the two strains were 4.0±2.5 and 2.6±1.1 (μM O2), respectively. The K m _ O 2 values of the two strains were lower than those of other NOB, suggesting that Nitrospira in activated sludge could oxidize nitrite in the hypoxic environments often found in the interiors of biofilms and flocs. The inhibition thresholds of the two strains by free ammonia were 0.85 and 4.3 (mg-NH3 l−1), respectively. Comparing the physiological properties of the two strains, we suggest that tolerance for free ammonia determines competition and partitioning into ecological niches among Nitrospira populations.





  • Overexpression of endogenous 1-deoxy-d-xylulose 5-phosphate synthase (DXS) in cyanobacterium Synechocystis sp. PCC6803 accelerates protein aggregation
    Publication date: May 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 5

    Author(s): Kai Kudoh, Shingo Hotta, Midori Sekine, Rintaro Fujii, Arisu Uchida, Genma Kubota, Yusuke Kawano, Masaki Ihara

    1-Deoxy-d-xylulose 5-phosphate synthase (DXS) is a rate-limiting enzyme in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, which is responsible for the production of precursors of all isoprenoids. In a previous study, we had examined the overexpression of an endogenous DXS in a Synechocystis sp. PCC6803 mutant (DXS_ox), and found that the dxs mRNA level was 4-fold higher than that in the wild-type (WT) strain. However, the DXS protein level was only 1.5-fold higher, leading to the assumption that the level might be regulated by post-transcriptional events. In this study, we have additionally introduced an exogenous isoprene synthase (IspS; which can release MEP pathway products from the cell as gaseous isoprene) into the WT and DXS_ox strains (WT-isP and DXSox-isP strains, respectively), and their detailed DXS expression profiles were investigated from the induction phase through to the late-logarithmic phase. In the induction phase, the isoprene productivity of the DXSox-isP strain was slightly but significantly (1.4- to 1.8-fold) higher than that of the WT-isP strain, whereas the levels were comparable in the other phases. Interestingly, the ratios of soluble:insoluble DXS protein were remarkably low in the DXSox-isP strain during the induction phase to the early-logarithmic phase, resulting in a moderate level of soluble DXS. All our results suggested that the high translation rate of DXS disturbs the refolding process of DXS. To enhance the concentration of the active DXS in cyanobacteria, the enhancement of the DXS maturation system or the introduction of exogenous and robust DXS proteins might be necessary.





  • Importance of sulfide interaction with iron as regulator of the microbial community in biogas reactors and its effect on methanogenesis, volatile fatty acids turnover, and syntrophic long-chain fatty acids degradation
    Publication date: May 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 5

    Author(s): Sepehr Shakeri Yekta, Ryan M. Ziels, Annika Björn, Ulf Skyllberg, Jörgen Ejlertsson, Anna Karlsson, Matilda Svedlund, Magnus Willén, Bo H. Svensson

    The inhibitory effects of sulfide on microbial processes during anaerobic digestion have been widely addressed. However, other effects of sulfide are less explored, given that sulfide is a potential sulfur source for microorganisms and its high reactivity triggers a suit of abiotic reactions. We demonstrated that sulfide interaction with Fe regulates the dynamics and activities of microbial community during anaerobic digestion. This was manifested by the S:Fe molar ratio, whose increase adversely influenced the acetoclastic methanogens, Methanosaeta, and turnover of acetate. Dynamics of hydrogenotrophic methanogens, Methanoculleus and Methanobrevibacter, were presumably influenced by sulfide-induced changes in the partial pressure of hydrogen. Interestingly, conversion of the long-chain fatty acid (LCFA), oleate, to methane was enhanced together with the abundance of LCFA-degrading, β-oxidizing Syntrophomonas at an elevated S:Fe molar ratio. The results suggested that sulfur chemical speciation is a controlling factor for microbial community functions in anaerobic digestion processes.





  • Bacterial community dynamics in a biodenitrification reactor packed with polylactic acid/poly (3-hydroxybutyrate-co-3-hydroxyvalerate) blend as the carbon source and biofilm carrier
    Publication date: May 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 5

    Author(s): Tianlei Qiu, Ying Xu, Min Gao, Meilin Han, Xuming Wang

    While heterotrophic denitrification has been widely used for treating such nitrogen-rich wastewater, it requires the use of additional carbon sources. With fluctuations in the nitrate concentration in the influent, controlling the C/N ratio to avoid carbon breakthrough becomes difficult. To overcome this obstacle, solid-phase denitrification (SPD) using biodegradable polymers has been used, where denitrification and carbon source biodegradation depend on microorganisms growing within the reactor. However, the microbial community dynamics in continuous-flow SPD reactors have not been fully elucidated yet. Here, we aimed to study bacterial community dynamics in a biodenitrification reactor packed with a polylactic acid/poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PLA/PHBV) blend as the carbon source and biofilm carrier. A lab-scale denitrifying reactor filled with a PLA/PHBV blend was used. With 85 mg/L of influent NO3–N concentration and a hydraulic retention time (HRT) of 2.5 h, more than 92% of the nitrate was removed. The bacterial community of inoculated activated sludge had the highest species richness in all samples. Bacterial species diversity in the reactor first decreased and then increased to a stable level. Diaphorobacter species were predominant in the reactor after day 24. In total, 178 clones were retrieved from the 16S rRNA gene clone library constructed from the biofilm samples in the reactor at 62 days of operation, and 80.9% of the clones were affiliated with Betaproteobacteria. Of these, 97.2% were classified into phylotypes corresponding to Diaphorobacter nitroreducens strain NA10B with 99% sequence similarity. Diaphorobacter, Rhizobium, Acidovorax, Rubrivivax, Azospira, Thermomonas, and Acidaminobacter constituted the biofilm microflora in the stably running reactor.





  • Analysis of metabolisms and transports of xylitol using xylose- and xylitol-assimilating Saccharomyces cerevisiae
    Publication date: May 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 5

    Author(s): Tatsunori Tani, Hisataka Taguchi, Takashi Akamatsu

    To clarify the relationship between NAD(P)+/NAD(P)H redox balances and the metabolisms of xylose or xylitol as carbon sources, we analyzed aerobic and anaerobic batch cultures of recombinant Saccharomyces cerevisiae in a complex medium containing 20 g/L xylose or 20 g/L xylitol at pH 5.0 and 30°C. The TDH3p–GAL2 or gal80Δ strain completely consumed the xylose within 24 h and aerobically consumed 92–100% of the xylitol within 96 h, but anaerobically consumed only 20% of the xylitol within 96 h. Cells of both strains grew well in aerobic culture. The addition of acetaldehyde (an effective oxidizer of NADH) increased the xylitol consumption by the anaerobically cultured strain. These results indicate that in anaerobic culture, NAD+ generated in the NAD(P)H-dependent xylose reductase reaction was likely needed in the NAD+-dependent xylitol dehydrogenase reaction, whereas in aerobic culture, the NAD+ generated by oxidation of NADH in the mitochondria is required in the xylitol dehydrogenase reaction. The role of Gal2 and Fps1 in importing xylitol into the cytosol and exporting it from the cells was analyzed by examining the xylitol consumption in aerobic culture and the export of xylitol metabolized from xylose in anaerobic culture, respectively. The xylitol consumptions of gal80Δ gal2Δ and gal80Δ gal2Δ fps1Δ strains were reduced by 81% and 88% respectively, relative to the gal80Δ strain. The maximum xylitol concentration accumulated by the gal80Δ, gal80Δ gal2Δ, and gal80Δ gal2Δ fps1Δ strains was 7.25 g/L, 5.30 g/L, and 4.27 g/L respectively, indicating that Gal2 and Fps1 transport xylitol both inward and outward.





  • Characterization of particulate matter binding peptides screened from phage display
    Publication date: May 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 5

    Author(s): Aw Wei Liang Alvin, Masayoshi Tanaka, Mina Okochi

    Particulate matter (PM), especially particulates with diameters of less than 2.5 μm, can penetrate the alveolar region and increase the risk of respiratory diseases. This has stimulated research efforts to develop detection methods so that counter measures can be taken. In this study, four PM binding peptides were obtained by phage display and binding characteristics of these peptides were investigated using the peptide array. The strongest binding peptide, WQDFGAVRSTRS, displayed a binding property, measured in terms of spot intensity, 11.4 times higher than that of the negative control, AAAAA. Inductively coupled plasma mass spectrometry (ICPMS) analysis of the transition metal compounds in the PM bound to the peptide spots was performed, and two peptides showed higher binding towards Cu and Zn compounds in PM. These results suggest that the screened peptides could serve as an indicator of transition metal compounds, which are related to adverse health effects, contained in PM.





  • Synthetic metabolic bypass for a metabolic toggle switch enhances acetyl-CoA supply for isopropanol production by Escherichia coli
    Publication date: May 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 5

    Author(s): Yuki Soma, Taiki Yamaji, Fumio Matsuda, Taizo Hanai

    Almost all synthetic pathways for biofuel production are designed to require endogenous metabolites in glycolysis, such as phosphoenolpyruvate, pyruvate, and acetyl-CoA. However, such metabolites are also required for bacterial cell growth. To reduce the metabolic imbalance between cell growth and target chemical production, we previously constructed a metabolic toggle switch (MTS) as a conditional flux redirection tool controlling metabolic flux of TCA cycle toward isopropanol production. This approach succeeded to improve the isopropanol production titer and yield while ensuring sufficient cell growth. However, excess accumulation of pyruvate, the precursor for acetyl-CoA synthesis, was also observed. In this study, for efficient conversation of pyruvate to acetyl-CoA (pyruvate oxidation), we designed a synthetic metabolic bypass composed of poxB and acs with the MTS for acetyl-CoA supply from the excess pyruvate. When this designed bypass was expressed at the appropriate expression level associated with the conditional metabolic flux redirection, pyruvate accumulation was prevented, and the isopropanol production titer and yield were improved. Final isopropanol production titer of strain harboring MTS with the synthetic metabolic bypass improved 4.4-fold compared with strain without metabolic flux regulation, and it was 1.3-fold higher than that of strain harboring the conventional MTS alone. Additionally, glucose consumption was also improved 1.7-fold compared with strain without metabolic flux regulation. On the other hand, introduction of the synthetic metabolic bypass alone showed no improvement in isopropanol production and glucose consumption. These results showed that the improvement in bio-production process caused by synergy between the MTS and the synthetic metabolic bypass.





  • Acidic pH with coordinated reduction of basic fibroblast growth factor maintains the glioblastoma stem cell-like phenotype in vitro
    Publication date: May 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 5

    Author(s): Elizabeth M. Haley, Samantha G. Tilson, Ursula L. Triantafillu, Justin W. Magrath, Yonghyun Kim

    Glioblastoma stem cells (GSCs) are a unique subpopulation of cells within glioblastoma multiforme (GBM) brain tumors that possess the ability to self-renew and differentiate into bulk tumor cells. GSCs are resistant to currently available treatments and are the likely culprit behind tumor relapse in GBM patients. However, GSCs are currently inaccessible to the larger scientific community because obtaining a sufficient number of GSCs remains technically challenging and cost-prohibitive. Thus, the objective of this study was to develop a more efficient GSC culture strategy that results in a higher cell yield of GSCs at a lower cost. We observed that the basic fibroblast growth factor (bFGF) is indispensable in allowing GSCs to retain an optimal stem cell-like phenotype in vitro, but little change was seen in their stemness when grown with lower concentrations of bFGF than the established protocol. Interestingly, a dynamic fluctuation of GSC protein marker expression was observed that corresponded to the changes in the bFGF concentration during the culture period. This suggested that bFGF alone did not control stem cell-like phenotype; rather, it was linked to the fluctuations of both bFGF and media pH. We demonstrated that a high level of stem cell-like phenotype could be retained even when lowering bFGF to 8 ng/mL when the media pH was simultaneously lowered to 6.8. These results provide the proof-of-concept that GSC expansion costs could be lowered to a more economical level and warrant the use of pH- and bFGF-controlled bioprocessing methodologies to more optimally expand GSCs in the future.





  • Image-based cell quality evaluation to detect irregularities under same culture process of human induced pluripotent stem cells
    Publication date: May 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 5

    Author(s): Risako Nagasaka, Yuto Gotou, Kei Yoshida, Kei Kanie, Kazunori Shimizu, Hiroyuki Honda, Ryuji Kato

    To meet the growing demand for human induced pluripotent stem cells (iPSCs) for various applications, technologies that enable the manufacturing of iPSCs on a large scale should be developed. There are several technological challenges in iPSC manufacturing technology. Image-based cell quality evaluation technology for monitoring iPSC quality in culture enables the manufacture of intact cells for further applications. Although several studies have reported the effectiveness of image-based evaluation of iPSCs, it remains challenging to detect irregularities that may arise using the same processing operations during quality evaluation of automated processing. In this study, we investigated the evaluation performance of image-based cell quality analysis in detecting small differences that can result from human measurement, even when the same protocol is followed. To imitate such culture conditions, by image-analysis guided colony pickup, we changed the proportions of morphologically different subpopulations: “good morphology, regular morphology correlated with undifferentiation marker expression” and “bad morphology, irregular morphology correlated with loss of undifferentiation marker expression”. In addition, comprehensive gene-expression and metabolomics analyses were carried out for the same samples to investigate performance differences. Our data shows an example of investigating the usefulness and sensitivity of quality evaluation methods for iPSC quality monitoring.





  • Evaluation of a newly developed mid-infrared sensor for real-time monitoring of yeast fermentations
    Publication date: May 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 5

    Author(s): Robert Schalk, Daniel Geoerg, Jens Staubach, Matthias Raedle, Frank-Juergen Methner, Thomas Beuermann

    A mid-infrared (MIR) sensor using the attenuated total reflection (ATR) technique has been developed for real-time monitoring in biotechnology. The MIR-ATR sensor consists of an IR emitter as light source, a zinc selenide ATR prism as boundary to the process, and four thermopile detectors, each equipped with an optical bandpass filter. The suitability of the sensor for practical application was tested during aerobic batch-fermentations of Saccharomyces cerevisiae by simultaneous monitoring of glucose and ethanol. The performance of the sensor was compared to a commercial Fourier transform mid-infrared (FT-MIR) spectrometer by on-line measurements in a bypass loop. Sensor and spectrometer were calibrated by multiple linear regression (MLR) in order to link the measured absorbance in the transmission ranges of the four optical sensor channels to the analyte concentrations. For reference analysis, high-performance liquid chromatography (HPLC) was applied. Process monitoring using the sensor yielded in standard errors of prediction (SEP) of 6.15 g/L and 1.36 g/L for glucose and ethanol. In the case of the FT-MIR spectrometer the corresponding SEP values were 4.34 g/L and 0.61 g/L, respectively. The advantages of optical multi-channel mid-infrared sensors in comparison to FT-MIR spectrometer setups are the compactness, easy process implementation and lower price.





  • Corrigendum to “Experimental evolution and gene knockout studies reveal AcrA-mediated isobutanol tolerance in Ralstonia eutropha” [J Biosci Bioeng 122 (2016) 64–69]
    Publication date: May 2017
    Source:Journal of Bioscience and Bioengineering, Volume 123, Issue 5

    Author(s): Amanda C. Bernardi, Claudia S. Gai, Jingnan Lu, Anthony J. Sinskey, Christopher J. Brigham







  • Establishment of MicroRNA delivery system by PP7 bacteriophage-like particles carrying cell-penetrating peptide
    Publication date: Available online 22 April 2017
    Source:Journal of Bioscience and Bioengineering

    Author(s): Yanli Sun, Yanhua Sun, Ronglan Zhao

    MicroRNAs have great therapeutic potential in cancer and other diseases. However, their instability and low in vivo delivery efficiency limits their application. Recombinant PP7 bacteriophage-based virus-like particles (VLPs) could protect microRNAs against rapid degradation by RNase by packaging specific exogenous pre-microRNAs using the pac site. Insertion of a cell-penetrating peptide (CPP) into the AB-loop of VLPs could significantly improve the delivery efficiency of microRNAs into mammalian cells. Unlike other microRNA delivery methods (viral or non-viral vectors), recombinant PP7 VLPs carrying a CPP and microRNA could be efficiently expressed in Escherichia coli using the one-plasmid double expression system. Here we showed that PP7 VLPs carrying a CPP penetrated hepatoma SK-HEP-1 cells and delivered the pre-microRNA-23b, which was processed into a mature product within 24 h; a concentration of 10 nM was sufficient for the inhibition of hepatoma cell migration via the downregulation of liver-intestine cadherin expression. Furthermore, PP7 VLPs carrying a CPP and a pre-microRNA were not infectious, replicative, or cytotoxic. Therefore, recombinant PP7 VLPs can be used for simultaneous and targeted delivery of both microRNAs and peptides because of their ability to package specific exogenous RNA using the pac site and to display peptides.





  • Microbial secretion of lactate-enriched oligomers for efficient conversion into lactide: A biological shortcut to polylactide
    Publication date: Available online 22 April 2017
    Source:Journal of Bioscience and Bioengineering

    Author(s): Camila Utsunomia, Ken'ichiro Matsumoto, Sakiko Date, Chiaki Hori, Seiichi Taguchi

    Recently, we have succeeded in establishing the microbial platform for the secretion of lactate (LA)-based oligomers (D-LAOs), which consist of D-LA and d-3-hydroxybutyrate (d-3HB). The secretory production of D-LAOs was substantially enhanced by the supplementation of diethylene glycol (DEG), which resulted in the generation of DEG-capped oligomers at the carboxyl terminal (referred as D-LAOs-DEG). The microbial D-LAOs should be key compounds for the synthesis of lactide, an important intermediate for polylactides (PLAs) production, eliminating the costly chemo-oligomerization step in the PLA production process. Therefore, in order to demonstrate a proof-of-concept, here, we attempted to convert the D-LAOs-DEG into lactide via metal-catalyzed thermal depolymerization. As a result, D-LAOs-DEG containing 68 mol% LA were successfully converted into lactide, revealing that the DEG bound to D-LAOs-DEG does not inhibit the conversion into lactide. However, the lactide yield (4%) was considerably lower than that of synthetic LA homo-oligomers (33%). We presumed that 3HB units in the polymer chain blocked the lactide formation, and therefore, we investigated the LA enrichment in the oligomers. As the results, the combination of an LA-overproducing Escherichia coli mutant (Δdld and ΔpflA) with the use of xylose as a carbon source exhibited synergistic effect to increase LA fraction in the oligomers up to 89 mol%. The LA-enriched D-LAOs-DEG were converted into lactide with greater yield (18%). These results demonstrated that a greener shortcut route for PLA production can be created by using the microbial D-LAOs secretion system.