Publications by authors named "Jun Ishii"

142 Publications

Intraoperative guidance using ICG fluorescence imaging system for safe and precise laparoscopic liver resection.

Minerva Surg 2021 Jun 23;76(3):211-219. Epub 2021 Apr 23.

Division of General and Gastroenterological Surgery (Omori), Department of Surgery, Toho University Faculty of Medicine, Tokyo, Japan.

Background: Laparoscopic liver resection (LLR) has been spread as minimally invasive surgery for liver disease. Advances in surgical technique and devices enabled us to perform various procedures of LLR. Indocyanine green (ICG) fluorescence imaging has been suggested as useful tool to identify liver tumors, anatomical territory of liver parenchyma, and cholangiography in open liver surgery. Due to recent development, this technology can be applied in LLR. we describe safe and effective using of the ICG fluorescence imaging during LLR.

Methods: From September 2013 to August 2019, 34 patients were performed LLR using a total of 46 procedures by ICG fluorescence imaging system for purposes including identification of anatomic domain of the liver in 12 LLRs, detection of liver tumors in 30 nodules, or intraoperative cholangiography in 4 LLRs.

Results: During the detection of liver tumors, 25 nodules in 30 malignant to benign tumors were positively detected (83.3%). Although there has been no publication regarding information on ICG fluorescence imaging of low grade malignant or benign tumors, we found positive emission in focal nodular hyperplasia, an angiomyolipoma, and an intraductal papillary neoplasm of the bile duct. The identification of anatomic domain in the liver was successful in all 12 LLRs with negative and positive staining techniques. In the intraoperative cholangiography, all 4 tests were successfully performed. One of 4 patients were found to have biliary leakage which was repaired intraoperatively.

Conclusions: The ICG fluorescence imaging could be useful in safe and precise performance of LLR.
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http://dx.doi.org/10.23736/S2724-5691.21.08597-7DOI Listing
June 2021

Synthetic production of prenylated naringenins in yeast using promiscuous microbial prenyltransferases.

Metab Eng Commun 2021 Jun 19;12:e00169. Epub 2021 Mar 19.

Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.

Reconstitution of prenylflavonoids using the flavonoid biosynthetic pathway and prenyltransferases (PTs) in microbes can be a promising attractive alternative to plant-based production or chemical synthesis. Here, we demonstrate that promiscuous microbial PTs can be a substitute for regiospecific but mostly unidentified botanical PTs. To test the prenylations of naringenin, we constructed a yeast strain capable of producing naringenin from l-phenylalanine by genomic integration of six exogenous genes encoding components of the naringenin biosynthetic pathway. Using this platform strain, various microbial PTs were tested for prenylnaringenin production. screening demonstrated that the fungal AnaPT (a member of the tryptophan dimethylallyltransferase family) specifically catalyzed C-3' prenylation of naringenin, whereas SfN8DT-1, a botanical PT, specifically catalyzed C-8 prenylation. , the naringenin-producing strain expressing the microbial AnaPT exhibited heterologous microbial production of 3'-prenylnaringenin (3'-PN), in contrast to the previously reported production of 8-prenylnaringenin (8-PN) using the botanical SfN8DT-1. These findings provide strategies towards expanding the production of a variety of prenylated compounds, including well-known prenylnaringenins and novel prenylflavonoids. These results also suggest the opportunity for substituting botanical PTs, both known and unidentified, that display relatively strict regiospecificity of the prenyl group transfer.
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http://dx.doi.org/10.1016/j.mec.2021.e00169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8040282PMC
June 2021

Oligoether/Zwitterion Diblock Copolymers: Synthesis and Application as Cathode-Coating Material for Li Batteries.

Polymers (Basel) 2021 Mar 5;13(5). Epub 2021 Mar 5.

Department of Materials and Life Sciences, Sophia University, 7-1 Kioi-cho, Chiyoda-ku, Tokyo 102-8554, Japan.

Poly (ethylene oxide) (PEO) has been investigated as an ion-conductive matrix for several decades due to its excellent properties. However, further improvements are needed to enable a PEO-based ion-conductive matrix for practical applications. In order to develop novel solid polymer electrolytes based on zwitterions, we synthesized diblock copolymers (PPEGMA--SPBs) with oligoether and zwitterionic side-chains and evaluated their physico-chemical properties. PPEGMA--SPBs with various unit ratios were synthesized by RAFT polymerization. PPEGMA--SPBs with/without LiTFSA exhibited two distinct glass transition temperatures regardless of the unit ratio of PEGMA and SPB. AFM observations clearly revealed phase-separated structures. The ionic conductivity of PPEGMA--SPBs increased even at a high salt concentrations such as [EO]:[Li] = 6:1 and was over 10 S cm at 25 °C. This tendency is unusual in a PEO matrix. The oxidation stability of PPEGMA--SPBs was about 5.0 V vs. Li/Li, which is a higher value than that of PEO. The improvement of the electrochemical properties is attributed to the introduction of the SPB block into the block copolymers. PPEGMA--SPBs were evaluated as cathode-coating materials for Li batteries. The discharge capacity and coulombic efficiency of the cells employing the cathode (LiNiMnCoO (NMC)) coated with the block copolymers were much higher than those of the cell employing the pristine cathode at the 50th cycle in the cut-off voltage range of 3.0-4.6 V.
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http://dx.doi.org/10.3390/polym13050800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961851PMC
March 2021

Robust and flexible platform for directed evolution of yeast genetic switches.

Nat Commun 2021 03 23;12(1):1846. Epub 2021 Mar 23.

Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan.

A wide repertoire of genetic switches has accelerated prokaryotic synthetic biology, while eukaryotic synthetic biology has lagged in the model organism Saccharomyces cerevisiae. Eukaryotic genetic switches are larger and more complex than prokaryotic ones, complicating the rational design and evolution of them. Here, we present a robust workflow for the creation and evolution of yeast genetic switches. The selector system was designed so that both ON- and OFF-state selection of genetic switches is completed solely by liquid handling, and it enabled parallel screen/selection of different motifs with different selection conditions. Because selection threshold of both ON- and OFF-state selection can be flexibly tuned, the desired selection conditions can be rapidly pinned down for individual directed evolution experiments without a prior knowledge either on the library population. The system's utility was demonstrated using 20 independent directed evolution experiments, yielding genetic switches with elevated inducer sensitivities, inverted switching behaviours, sensory functions, and improved signal-to-noise ratio (>100-fold induction). The resulting yeast genetic switches were readily integrated, in a plug-and-play manner, into an AND-gated carotenoid biosynthesis pathway.
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http://dx.doi.org/10.1038/s41467-021-22134-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7988172PMC
March 2021

Factors associated with knowledges and attitudes about measles and rubella immunization in a non-health care occupational setting in Japan.

J Infect Chemother 2021 May 26;27(5):684-689. Epub 2020 Dec 26.

Infectious Disease Surveillance Center, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan.

Introduction: Elimination of measles and rubella has been achieved in several countries and some regions. After verified measles elimination, some countries have reported outbreaks among adults in occupational settings such as health care institution and school setting. Studies have reported that knowledge and attitude for measles and/or rubella are significantly associated with immunization uptake in adults, but few studies have been conducted in settings other than health care facilities and schools.

Methods: We conducted a cross-sectional study among 134 office employees during a routine health checkup in June 17-20, 2014, to examine the association between willingness to receive immunization and knowledge and attitudes.

Results: Approximately 75% had a protective level of antibody for measles (PA≥1:256) and rubella (HI ≥ 32 IU/mL). After adjustment for sex, age and immune status, the attitudes that immunization prevents measles (adjusted odds ratio [aOR] = 7.8, 95% confidence interval [95%CI]: 2.5-24.7) and prevents infection and transmission to others (aOR = 4.0, 95%CI: 1.4-11.4). Knowing that males are the vulnerable group for rubella infection (aOR = 5.8, 95%CI: 2.4-13.9), attitude that immunization prevents rubella infection (aOR = 7.9, 95%CI: 2.4-26.5), and prevents infection and transmit to others (aOR = 6.7, 95%CI: 2.2-20.1) were significantly associated with willingness to receive immunization after adjustment for sex, age, and immune status.

Conclusions: Studies have shown that physicians and other health care workers are important source of information for promotion of immunization. Thus, we recommend that physicians educate and promote immunization for measles and/or rubella to adults working in offices during routine health checks.
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http://dx.doi.org/10.1016/j.jiac.2020.12.003DOI Listing
May 2021

Exchange of endogenous and heterogeneous yeast terminators in Pichia pastoris to tune mRNA stability and gene expression.

Nucleic Acids Res 2020 12;48(22):13000-13012

Graduate School of Science, Technology and Innovation, Kobe University, Kobe 657-8501, Japan.

In the yeast Saccharomyces cerevisiae, terminator sequences not only terminate transcription but also affect expression levels of the protein-encoded upstream of the terminator. The non-conventional yeast Pichia pastoris (syn. Komagataella phaffii) has frequently been used as a platform for metabolic engineering but knowledge regarding P. pastoris terminators is limited. To explore terminator sequences available to tune protein expression levels in P. pastoris, we created a 'terminator catalog' by testing 72 sequences, including terminators from S. cerevisiae or P. pastoris and synthetic terminators. Altogether, we found that the terminators have a tunable range of 17-fold. We also found that S. cerevisiae terminator sequences maintain function when transferred to P. pastoris. Successful tuning of protein expression levels was shown not only for the reporter gene used to define the catalog but also using betaxanthin production as an example application in pathway flux regulation. Moreover, we found experimental evidence that protein expression levels result from mRNA abundance and in silico evidence that levels reflect the stability of mRNA 3'-UTR secondary structure. In combination with promoter selection, the novel terminator catalog constitutes a basic toolbox for tuning protein expression levels in metabolic engineering and synthetic biology in P. pastoris.
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http://dx.doi.org/10.1093/nar/gkaa1066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7736810PMC
December 2020

Metabolic design for selective production of nicotinamide mononucleotide from glucose and nicotinamide.

Metab Eng 2021 05 18;65:167-177. Epub 2020 Nov 18.

Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan; Engineering Biology Research Center, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan; Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan; RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro, Tsurumi, Yokohama, 230-0045, Japan.

β-Nicotinamide mononucleotide (NMN) is, one of the nucleotide compounds, a precursor of NAD and has recently attracted attention as a nutraceutical. Here, we develop a whole-cell biocatalyst using Escherichia coli, which enabled selective and effective high production of NMN from the inexpensive feedstock substrates glucose and nicotinamide (Nam). Notably, we identify two actively functional transporters (NiaP and PnuC) and a high-activity key enzyme (Nampt), permitting intracellular Nam uptake, efficient conversion of phosphoribosyl pyrophosphate (PRPP; supplied from glucose) and Nam to NMN, and NMN excretion extracellularly. Further, enhancement of the PRPP biosynthetic pathway and optimization of individual gene expression enable drastically higher NMN production than reported thus far. The strain extracellularly produces 6.79 g l of NMN from glucose and Nam, and the reaction selectivity from Nam to NMN is 86%. Our approach will be promising for low-cost, high-quality industrial production of NMN and other nucleotide compounds using microorganisms.
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http://dx.doi.org/10.1016/j.ymben.2020.11.008DOI Listing
May 2021

Comparative analyses of site-directed mutagenesis of human melatonin MTNR1A and MTNR1B receptors using a yeast fluorescent biosensor.

Biotechnol Bioeng 2021 02 6;118(2):863-876. Epub 2020 Nov 6.

Engineering Biology Research Center, Kobe University, Kobe, Japan.

Melatonin is an indoleamine neurohormone made by the pineal gland. Its receptors, MTNR1A and MTNR1B, are members of the G-protein-coupled receptor (GPCR) family and are involved in sleep, circadian rhythm, and mood disorders, and in the inhibition of cancer growth. These receptors, therefore, represent significant molecular targets for insomnia, circadian sleep disorders, and cancer. The yeast Saccharomyces cerevisiae is an attractive host for assaying agonistic activity for human GPCR. We previously constructed a GPCR-based biosensor employing a high-sensitivity yeast strain that incorporated both a chimeric yeast-human Gα protein and a bright fluorescent reporter gene (ZsGreen). Similar approaches have been used for simple and convenient measurements of various GPCR activities. In the current study, we constructed a fluorescence-based yeast biosensor for monitoring the signaling activation of human melatonin receptors. We used this system to analyze point mutations, including previously unreported mutations of the consensus sequences of MTNR1A and MTNR1B melatonin receptors and compared their effects. Most mutations in the consensus sequences significantly affected the signaling capacities of both receptors, but several mutations showed differences between these subtype receptors. Thus, this yeast biosensor holds promise for revealing the functions of melatonin receptors.
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http://dx.doi.org/10.1002/bit.27609DOI Listing
February 2021

Histopathological evaluation of the effectiveness of oral Eppikajutsuto treatment for lymphatic malformation.

J Pediatr Surg 2020 Sep 17. Epub 2020 Sep 17.

Department of Pathology, Graduate School of Medicine, Dokkyo Medical University, Tochigi, Japan. Electronic address:

Background: Lymphatic malformation (LM) is a congenital disease caused by lymphatic vessel malformation. Although standard therapies for LMs are sclerotherapy and/or surgical excision, a new therapy using Japanese herbal medicine Eppikajutsuto (TJ-28) has been recently reported as clinically effective. We aimed to experimentally confirm the therapeutic effectiveness of TJ-28 for LMs.

Methods: LM lesions were generated in the mesentery and peritoneum of mice by intraperitoneal injection of Freund's incomplete adjuvant. Mice with LMs were treated by gavage or dietary administration of TJ-28 for 2 months. Formalin-fixed paraffin-embedded tissue sections of mesentery and peritoneum tissues were histologically and immunohistochemically examined by focusing on lymph nodes and perinodal lymph vessels.

Results: Multiple Freund's incomplete adjuvant-associated foreign-body granulomas were formed in the mesentery and peritoneum, resulting in congestion of lymph fluid and dilatation of lymph vessels. The numbers and sizes of lymph nodes were not significantly different between TJ-28-treated and control groups. However, the luminal areas of lymphatic vessels were reduced significantly in the TJ-28 treatment group by both gavage and dietary administrations.

Conclusion: TJ-28 conspicuously reduced congestion of lymph fluid. This is the first histopathological evaluation of LM model mice to study the effectiveness of oral TJ-28 treatment.
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http://dx.doi.org/10.1016/j.jpedsurg.2020.09.007DOI Listing
September 2020

Repression of mitochondrial metabolism for cytosolic pyruvate-derived chemical production in Saccharomyces cerevisiae.

Microb Cell Fact 2019 Oct 15;18(1):177. Epub 2019 Oct 15.

Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka, 565-0871, Japan.

Background: Saccharomyces cerevisiae is a suitable host for the industrial production of pyruvate-derived chemicals such as ethanol and 2,3-butanediol (23BD). For the improvement of the productivity of these chemicals, it is essential to suppress the unnecessary pyruvate consumption in S. cerevisiae to redirect the metabolic flux toward the target chemical production. In this study, mitochondrial pyruvate transporter gene (MPC1) or the essential gene for mitophagy (ATG32) was knocked-out to repress the mitochondrial metabolism and improve the production of pyruvate-derived chemical in S. cerevisiae.

Results: The growth rates of both aforementioned strains were 1.6-fold higher than that of the control strain. C-metabolic flux analysis revealed that both strains presented similar flux distributions and successfully decreased the tricarboxylic acid cycle fluxes by 50% compared to the control strain. Nevertheless, the intracellular metabolite pool sizes were completely different, suggesting distinct metabolic effects of gene knockouts in both strains. This difference was also observed in the test-tube culture for 23BD production. Knockout of ATG32 revealed a 23.6-fold increase in 23BD titer (557.0 ± 20.6 mg/L) compared to the control strain (23.5 ± 12.8 mg/L), whereas the knockout of MPC1 revealed only 14.3-fold increase (336.4 ± 113.5 mg/L). Further investigation using the anaerobic high-density fermentation test revealed that the MPC1 knockout was more effective for ethanol production than the 23BD production.

Conclusion: These results suggest that the engineering of the mitochondrial transporters and membrane dynamics were effective in controlling the mitochondrial metabolism to improve the productivities of chemicals in yeast cytosol.
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http://dx.doi.org/10.1186/s12934-019-1226-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6794801PMC
October 2019

Congenital goitrous hypothyroidism is caused by dysfunction of the iodide transporter SLC26A7.

Commun Biol 2019 24;2:270. Epub 2019 Jul 24.

1Department of Pathology, Kyorin University School of Medicine, Tokyo, Japan.

Iodide transport and storage in the thyroid follicles is crucial for thyroid hormone synthesis. Pendrin, the iodide exporter that transports iodide to thyroid follicles, is responsible for Pendred syndrome, a disorder characterized by congenital hypothyroidism and hearing loss. However, thyroid hormone levels are basically normal in patients with Pendred syndrome, indicating the presence of another unknown iodide transporter. Here, we show that SLC26A7 is a novel iodide transporter in the thyroid. We observe that SLC26A7 is specifically expressed in normal thyroid tissues and demonstrate its function in iodide transport. Using whole-exome sequencing, we also find a homozygous nonsense mutation in (c.1498 C > T; p.Gln500Ter) in two siblings with congenital goitrous hypothyroidism. The mutated SLC26A7 protein shows an abnormal cytoplasmic localisation and lacks the iodide transport function. These results reveal that SLC26A7 functions as a novel iodide transporter in the thyroid and its dysfunction affects thyroid hormonogenesis in humans and causes congenital goitrous hypothyroidism.
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http://dx.doi.org/10.1038/s42003-019-0503-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6656751PMC
May 2020

Author Correction: Mechanism-based tuning of insect 3,4-dihydroxyphenylacetaldehyde synthase for synthetic bioproduction of benzylisoquinoline alkaloids.

Nat Commun 2019 May 22;10(1):2336. Epub 2019 May 22.

Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, 657-8501, Japan.

In the original version of this Article, the abbreviation of 3,4-dihydroxyphenylacetaldehyde synthase presented in the first paragraph of the Discussion section was given incorrectly as DYPAA. The correct abbreviation for this enzyme is DHPAAS. This error has been corrected in both the PDF and HTML versions of the Article.
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http://dx.doi.org/10.1038/s41467-019-10312-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6531545PMC
May 2019

Mechanism-based tuning of insect 3,4-dihydroxyphenylacetaldehyde synthase for synthetic bioproduction of benzylisoquinoline alkaloids.

Nat Commun 2019 05 1;10(1):2015. Epub 2019 May 1.

Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, 657-8501, Japan.

Previous studies have utilized monoamine oxidase (MAO) and L-3,4-dihydroxyphenylalanine decarboxylase (DDC) for microbe-based production of tetrahydropapaveroline (THP), a benzylisoquinoline alkaloid (BIA) precursor to opioid analgesics. In the current study, a phylogenetically distinct Bombyx mori 3,4-dihydroxyphenylacetaldehyde synthase (DHPAAS) is identified to bypass MAO and DDC for direct production of 3,4-dihydroxyphenylacetaldehyde (DHPAA) from L-3,4-dihydroxyphenylalanine (L-DOPA). Structure-based enzyme engineering of DHPAAS results in bifunctional switching between aldehyde synthase and decarboxylase activities. Output of dopamine and DHPAA products is fine-tuned by engineered DHPAAS variants with Phe79Tyr, Tyr80Phe and Asn192His catalytic substitutions. Balance of dopamine and DHPAA products enables improved THP biosynthesis via a symmetrical pathway in Escherichia coli. Rationally engineered insect DHPAAS produces (R,S)-THP in a single enzyme system directly from L-DOPA both in vitro and in vivo, at higher yields than that of the wild-type enzyme. However, DHPAAS-mediated downstream BIA production requires further improvement.
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http://dx.doi.org/10.1038/s41467-019-09610-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6494836PMC
May 2019

Biosynthesis of Novel Statins by Combining Heterologous Genes from Xylaria and Aspergillus.

ACS Synth Biol 2018 12 13;7(12):2783-2789. Epub 2018 Nov 13.

Technology Research Association of Highly Efficient Gene Design (TRAHED) , Tsukisamu-Higashi 2-17-2-1 , Toyohira, Sapporo , Hokkaido 062-8517 , Japan.

For many secondary metabolites, heterologous synthesis is the definitive step to determine their required biosynthetic genes. Using a multivector expression system in Saccharomyces cerevisiae, we reconstituted not only two natural statins from two fungal species, i.e., lovastatin from Aspergillus terreus and FR901512 from Xylaria grammica, but also new statin structures by mixing their genes. Combinatorial gene exchange experiments revealed the functional promiscuity of two polyketide synthases in A. terreus, lovB, and lovF; they could synthesize FR901512 with Xylaria genes. Key structure determinants of statins are essential accessory genes that are irreplaceable across species.
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http://dx.doi.org/10.1021/acssynbio.8b00392DOI Listing
December 2018

Heterologous production of free dihomo-γ-linolenic acid by Aspergillus oryzae and its extracellular release via surfactant supplementation.

J Biosci Bioeng 2019 Apr 13;127(4):451-457. Epub 2018 Oct 13.

Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo, Hokkaido 062-8517, Japan.

Free dihomo-γ-linolenic acid (DGLA) and its desaturated form, free arachidonic acid (ARA) are polyunsaturated free fatty acids (FFAs). They are useful raw materials to produce eicosanoid pharmaceuticals. In this study, we aimed at their production by the oleaginous filamentous fungus Aspergillus oryzae via metabolic engineering. Three genes encoding enzymes involved in the synthesis of DGLA and ARA, were isolated from the filamentous fungus Mortierella alpina that produces ARA in a triacylglycerol form. These genes were concatenated to promoters and terminators of highly expressed genes of A. oryzae, and the concatenated DNA fragments were further concatenated with each other to generate a single DNA fragment in the form of a biosynthetic gene cluster. By homologous recombination, the resulting DNA fragment was integrated to the chromosome of the A. oryzae acyl-CoA synthetase gene disruptant whose FFA productivity was enhanced at 9.2-fold more than the wild-type strain. The DNA-integrated disruptant produced free DGLA but did not produce free ARA. Thus, focusing on free DGLA, after removal of the gene for converting DGLA to ARA, the constructed strain produced free DGLA at 145 mg/l for 5 d. Also, by supplementing Triton X-100 surfactant at 1% to the culture, over 80% of free DGLA was released from cells without inhibiting the growth. Consequently, the constructed strain will be useful for attempting production of free DGLA-derived eicosanoids because it bypasses excision of free DGLA from triacylglycerols by lipase. To our knowledge, this is the first report on microbial production of free DGLA and its extracellular release.
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http://dx.doi.org/10.1016/j.jbiosc.2018.09.013DOI Listing
April 2019

Deletion of DNA ligase IV homolog confers higher gene targeting efficiency on homologous recombination in Komagataella phaffii.

FEMS Yeast Res 2018 11;18(7)

Engineering Biology Research Center, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan.

The non-conventional yeast Komagataella phaffii, formerly Pichia pastoris, is a popular host for recombinant protein production. The relatively lower gene targeting efficiency observed in this species occurs due to high levels of non-homologous recombination activity. In the current study, we explored the function of the K. phaffii homolog of DNA ligase IV (Dnl4p) by creating a DNL4-disrupted strain. To assess the roles of non-homologous end joining (NHEJ)-related proteins in this species, strains deleted for either or both genes encoding Dnl4p or the telomeric Ku complex subunit (Ku70p) were generated. These deletions were constructed by either of two distinct marker-recycling methods (yielding either a seamless gene deletion or a Cre-loxP-mediated gene deletion). The resulting dnl4- and/or ku70-deleted K. phaffii strains were used to evaluate gene targeting efficiency in gene knock-out and gene knock-in experiments. The Dnl4p-defective strain showed improved gene targeting efficiency for homologous recombination compared to the wild-type and Ku70p-deffective strains. The dnl4 ku70 double knock-out strain exhibited a further improvement in gene targeting efficiency. Thus, the K. phaffii dnl4 and dnl4 ku70 deletion strains are expected to serve as useful platforms for functional analysis and strain development in this species.
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http://dx.doi.org/10.1093/femsyr/foy074DOI Listing
November 2018

A pyruvate carbon flux tugging strategy for increasing 2,3-butanediol production and reducing ethanol subgeneration in the yeast .

Biotechnol Biofuels 2018 26;11:180. Epub 2018 Jun 26.

2Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871 Japan.

Background: The yeast is a promising host cell for producing a wide range of chemicals. However, attempts to metabolically engineer Crabtree-positive invariably face a common issue: how to reduce dominant ethanol production. Here, we propose a yeast metabolic engineering strategy for decreasing ethanol subgeneration involving tugging the carbon flux at an important hub branching point (e.g., pyruvate). Tugging flux at a central glycolytic overflow metabolism point arising from high glycolytic activity may substantially increase higher alcohol production in . We validated this possibility by testing 2,3-butanediol (2,3-BDO) production, which is routed via pyruvate as the important hub compound.

Results: By searching for high-activity acetolactate synthase (ALS) enzymes that catalyze the important first-step reaction in 2,3-BDO biosynthesis, and tuning several fermentation conditions, we demonstrated that a stronger pyruvate pulling effect (tugging of pyruvate carbon flux) is very effective for increasing 2,3-BDO production and reducing ethanol subgeneration by . To further confirm the validity of the pyruvate carbon flux tugging strategy, we constructed an evolved pyruvate decarboxylase (PDC)-deficient yeast (PDCΔ) strain that lacked three isozymes of PDC. In parallel with re-sequencing to identify genomic mutations, liquid chromatography-tandem mass spectrometry analysis of intermediate metabolites revealed significant accumulation of pyruvate and NADH in the evolved PDCΔ strain. Harnessing the high-activity ALS and additional downstream enzymes in the evolved PDCΔ strain resulted in a high yield of 2,3-BDO (a maximum of 0.41 g g glucose consumed) and no ethanol subgeneration, thereby confirming the utility of our strategy. Using this engineered strain, we demonstrated a high 2,3-BDO titer (81.0 g L) in a fed-batch fermentation using a high concentration of glucose as the sole carbon source.

Conclusions: We demonstrated that the pyruvate carbon flux tugging strategy is very effective for increasing 2,3-BDO production and decreasing ethanol subgeneration in Crabtree-positive . High activity of the common first-step enzyme for the conversion of pyruvate, which links to both the TCA cycle and amino acid biosynthesis, is likely important for the production of various chemicals by .
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http://dx.doi.org/10.1186/s13068-018-1176-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6020211PMC
June 2018

Short Oligopeptides for Biocompatible and Biodegradable Supramolecular Hydrogels.

Langmuir 2018 07 26;34(27):8065-8074. Epub 2018 Jun 26.

Department of Chemical Science and Engineering, Graduate School of Engineering , Kobe University , 1-1 Rokkodaicho , Nada-ku, Kobe 657-8501 , Japan.

Short Phe-rich oligopeptides, consisting of only four and five amino acids, worked as effective supramolecular hydrogelators for buffer solutions at low gelator concentrations (0.5-1.5 wt %). Among 10 different oligopeptides synthesized, peptide P1 (Ac-Phe-Phe-Phe-Gly-Lys) showed high gelation ability. Transmission electron microscopy observations suggested that the peptide molecules self-assembled into nanofibrous networks, which turned into gels. The hydrogel of peptide P1 showed reversible thermal gel-sol transition and viscoelastic properties typical of a gel. Circular dichroism spectra revealed that peptide P1 formed a β-sheetlike structure, which decreased with increasing temperature. The self-assembly of peptide P1 occurred even in the presence of nutrients in culture media and common surfactants. Escherichia coli and yeast successfully grew on the hydrogel. The hydrogel exhibited low cytotoxicity to animal cells. Finally, we demonstrated that functional compounds can be released from the hydrogel in different manners based on the interaction between the compounds and the hydrogel.
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http://dx.doi.org/10.1021/acs.langmuir.8b00362DOI Listing
July 2018

A Stable, Autonomously Replicating Plasmid Vector Containing Pichia pastoris Centromeric DNA.

Appl Environ Microbiol 2018 08 17;84(15). Epub 2018 Jul 17.

Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan

The methylotrophic yeast is widely used to produce recombinant proteins, taking advantage of this species' high-density cell growth and strong ability to secrete proteins. Circular plasmids containing the -specific autonomously replicating sequence () permit transformation of with higher efficiency than obtained following chromosomal integration by linearized DNA. Unfortunately, however, existing autonomously replicating plasmids are known to be inherently unstable. In this study, we used transcriptome sequencing (RNA-seq) data and genome sequence information to independently identify, on each of the four chromosomes, centromeric DNA sequences consisting of long inverted repeat sequences. By examining the chromosome 2 centromeric DNA sequence () in detail, we demonstrate that an ∼111-bp region located at one end of the putative centromeric sequence had autonomous replication activity. In addition, the full-length sequence, which contains two long inverted repeat sequences and a nonrepetitive central core region, is needed for the accurate replication and distribution of plasmids in Thus, we constructed a new, stable, autonomously replicating plasmid vector that harbors the entire sequence; this episome facilitates genetic manipulation in , providing high transformation efficiency and plasmid stability. Secretory production of recombinant proteins is the most important application of the methylotrophic yeast , a species that permits mass production of heterologous proteins. To date, the genetic engineering of has relied largely on integrative vectors due to the lack of user-friendly tools. Autonomously replicating plasmids are expected to facilitate genetic manipulation; however, the existing systems, which use autonomously replicating sequences (ARSs) such as the -specific ARS (), are known to be inherently unstable for plasmid replication and distribution. Recently, the centromeric DNA sequences of were identified in back-to-back studies published by several groups; therefore, a new episomal plasmid vector with centromere DNA as a tool for genetic manipulation of is ready to be developed.
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http://dx.doi.org/10.1128/AEM.02882-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6052274PMC
August 2018

Metabolic engineering of the 2-ketobutyrate biosynthetic pathway for 1-propanol production in Saccharomyces cerevisiae.

Microb Cell Fact 2018 Mar 9;17(1):38. Epub 2018 Mar 9.

Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe, Japan.

Background: To produce 1-propanol as a potential biofuel, metabolic engineering of microorganisms, such as E. coli, has been studied. However, 1-propanol production using metabolically engineered Saccharomyces cerevisiae, which has an amazing ability to produce ethanol and is thus alcohol-tolerant, has infrequently been reported. Therefore, in this study, we aimed to engineer S. cerevisiae strains capable of producing 1-propanol at high levels.

Results: We found that the activity of endogenous 2-keto acid decarboxylase and alcohol/aldehyde dehydrogenase is sufficient to convert 2-ketobutyrate (2 KB) to 500 mg/L 1-propanol in yeast. Production of 1-propanol could be increased by: (i) the construction of an artificial 2 KB biosynthetic pathway from pyruvate via citramalate (cimA); (ii) overexpression of threonine dehydratase (tdcB); (iii) enhancement of threonine biosynthesis from aspartate (thrA, thrB and thrC); and (iv) deletion of the GLY1 gene that regulates a competing pathway converting threonine to glycine. With high-density anaerobic fermentation of the engineered S. cerevisiae strain YG5C4231, we succeeded in producing 180 mg/L 1-propanol from glucose.

Conclusion: These results indicate that the engineering of a citramalate-mediated pathway as a production method for 1-propanol in S. cerevisiae is effective. Although optimization of the carbon flux in S. cerevisiae is necessary to harness this pathway, it is a promising candidate for the large-scale production of 1-propanol.
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http://dx.doi.org/10.1186/s12934-018-0883-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5844117PMC
March 2018

Combination Gemcitabine and WT1 Peptide Vaccination Improves Progression-Free Survival in Advanced Pancreatic Ductal Adenocarcinoma: A Phase II Randomized Study.

Cancer Immunol Res 2018 03 22;6(3):320-331. Epub 2018 Jan 22.

Department of Cancer Immunology, Osaka University Graduate School of Medicine, Osaka, Japan.

We investigated the efficacy of a Wilms' tumor gene 1 (WT1) vaccine combined with gemcitabine (GEMWT1) and compared it with gemcitabine (GEM) monotherapy for advanced pancreatic ductal adenocarcinoma (PDAC) in a randomized phase II study. We randomly assigned HLA-A*02:01- or HLA-A*24:02-positive patients with advanced PDAC to receive GEMWT1 or GEM. We assessed WT1-specific immune responses via delayed-type hypersensitivity (DTH) to the WT1 peptide and a tetramer assay to detect WT1-specific cytotoxic T lymphocytes (WT1-CTL). Of 91 patients enrolled, 85 were evaluable (GEMWT1: = 42; GEM: = 43). GEMWT1 prolonged progression-free survival [PFS; hazard ratio (HR), 0.66; = 0.084] and improved overall survival rate at 1 year (1-year OS%; GEMWT1: 35.7%; GEM: 20.9%). However, the difference in OS was not significant (HR: 0.82; = 0.363). These effects were particularly evident in metastatic PDAC (PFS: HR 0.51, = 0.0017; 1-year OS%: GEMWT1 27.3%; GEM 11.8%). The combination was well tolerated, with no unexpected serious adverse events. In patients with metastatic PDAC, PFS in the DTH-positive GEMWT1 group was significantly prolonged, with a better HR of 0.27 compared with the GEM group, whereas PFS in the DTH-negative GEMWT1 group was similar to that in the GEM group (HR 0.86; = 0.001). DTH positivity was associated with an increase in WT1-CTLs induced by the WT1 vaccine. GEM plus the WT1 vaccine prolonged PFS and may improve 1-year OS% in advanced PDAC. These clinical effects were associated with the induction of WT1-specific immune responses. .
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http://dx.doi.org/10.1158/2326-6066.CIR-17-0386DOI Listing
March 2018

Modifying Expression Modes of Human Neurotensin Receptor Type 1 Alters Sensing Capabilities for Agonists in Yeast Signaling Biosensor.

Biotechnol J 2018 Apr 30;13(4):e1700522. Epub 2017 Nov 30.

Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan.

Neurotensin receptor type 1 (NTSR1), a member of the G-protein-coupled receptor (GPCR) family, is naturally activated by binding of a neurotensin peptide, leading to a variety of physiological effects. The budding yeast Saccharomyces cerevisiae is a proven host organism for assaying the agonistic activation of human GPCRs. Previous studies showed that yeast cells can functionally express human NTSR1 receptor, permitting the detection of neurotensin-promoted signaling using a ZsGreen fluorescent reporter gene. However, the fluorescence intensity (sensitivity) of NTSR1-expressing yeast cells is low compared to that of yeast cells expressing other human GPCRs (e.g., human somatostatin receptors). The present study sought to increase the sensitivity of the NTSR1-expressing yeast for use as a fluorescent biosensor, including modification of the expression of human NTSR1 in yeast. Changes in the transcription, translation, and transport of the receptor are attempted by altering the promoter, consensus Kozak-like sequence, and secretion signal sequences of the NTSR1-encoding gene. The resulting yeast cells exhibited increased sensitivity to exogenously added peptide. The cells are further engineered by using cell-surface display technology to ensure that the agonistic peptides are secreted and tethered to the yeast cell wall, yielding cells with enhanced NTSR1 activation. This yeast biosensor holds promise for the identification of agonists to treat NTSR1-related diseases.
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http://dx.doi.org/10.1002/biot.201700522DOI Listing
April 2018

Sporadic minute medullary thyroid carcinoma with a double RET mutation: A case report.

Pathol Int 2017 Nov 27;67(11):580-584. Epub 2017 Sep 27.

Department of Pathology, Kyorin University School of Medicine, 6-20-2, Shinkawa, Mitaka, Tokyo, 181-8611.

We describe a 74-year-old man with a nodular goiter accompanied by an incidental sporadic minute medullary thyroid carcinoma (MTC). Histopathologically, the MTC was a well-defined 1.7 mm tumor in the upper one-third right lobe, with solid cell nests (SCNs) adjacent to the MTC. C-cells were scattered mainly around the SCNs, but C-cell hyperplasia was not evident in the background thyroid. The MTC cell phenotype was immunohistochemically identical to background C-cells, but was completely different from the SCN main cells. Direct DNA analyses of isolated MTC paraffin-embedded specimens revealed two RET proto-oncogene missense point mutations in exon 11 (i.e., C630R and C634W). The non-tumor thyroid tissue did not reveal any mutations. This study reports the smallest case of sporadic MTC with a double RET somatic mutation, substantiating that RET mutations can occur during a very early stage of carcinogenesis. The combined presence of C630R and C634W represent a novel somatic mutation in sporadic MTC. The present case indicates that the sporadic MTC originated from the surrounding C-cells of the SCNs without C-cell hyperplasia and that the SCN main cells may not be able to develop into an MTC.
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http://dx.doi.org/10.1111/pin.12588DOI Listing
November 2017

Transporter engineering in biomass utilization by yeast.

FEMS Yeast Res 2017 11;17(7)

Graduate School of Science, Technology, and Innovation, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan.

Biomass resources are attractive carbon sources for bioproduction because of their sustainability. Many studies have been performed using biomass resources to produce sugars as carbon sources for cell factories. Expression of biomass hydrolyzing enzymes in cell factories is an important approach for constructing biomass-utilizing bioprocesses because external addition of these enzymes is expensive. In particular, yeasts have been extensively engineered to be cell factories that directly utilize biomass because of their manageable responses to many genetic engineering tools, such as gene expression, deletion and editing. Biomass utilizing bioprocesses have also been developed using these genetic engineering tools to construct metabolic pathways. However, sugar input and product output from these cells are critical factors for improving bioproduction along with biomass utilization and metabolic pathways. Transporters are key components for efficient input and output activities. In this review, we focus on transporter engineering in yeast to enhance bioproduction from biomass resources.
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http://dx.doi.org/10.1093/femsyr/fox061DOI Listing
November 2017

Selection of yeast Saccharomyces cerevisiae promoters available for xylose cultivation and fermentation.

J Biosci Bioeng 2018 Jan 30;125(1):76-86. Epub 2017 Aug 30.

Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan; Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan; Biomass Engineering Program, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan. Electronic address:

To efficiently utilize xylose, a major sugar component of hemicelluloses, in Saccharomyces cerevisiae requires the proper expression of varied exogenous and endogenous genes. To expand the repertoire of promoters in engineered xylose-utilizing yeast strains, we selected promoters in S. cerevisiae during cultivation and fermentation using xylose as a carbon source. To select candidate promoters that function in the presence of xylose, we performed comprehensive gene expression analyses using xylose-utilizing yeast strains both during xylose and glucose fermentation. Based on microarray data, we chose 29 genes that showed strong, moderate, and weak expression in xylose rather than glucose fermentation. The activities of these promoters in a xylose-utilizing yeast strain were measured by lacZ reporter gene assays over time during aerobic cultivation and microaerobic fermentation, both in xylose and glucose media. In xylose media, P, P, and P were favorable for high expression, and P, P, P, P, P, and P were acceptable for medium-high expression in aerobic cultivation, and moderate expression in microaerobic fermentation. P allowed moderate expression in aerobic culture and weak expression in microaerobic fermentation, although it showed medium-high expression in glucose media. P and P allowed moderate expression in aerobic cultivation, while showing weak but clear expression in microaerobic fermentation. P and P showed moderate promoter activity in aerobic cultivation, but showed almost no activity in microaerobic fermentation. The knowledge of promoter activities in xylose cultivation obtained in this study will permit the control of gene expression in engineered xylose-utilizing yeast strains that are used for hemicellulose fermentation.
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http://dx.doi.org/10.1016/j.jbiosc.2017.08.001DOI Listing
January 2018

Effects of sorafenib and an adenylyl cyclase activator on in vitro growth of well-differentiated thyroid cancer cells.

Endocr J 2017 Nov 31;64(11):1115-1123. Epub 2017 Aug 31.

Department of Pathology, School of Medicine, Kyorin University, Mitaka-shi, 181-8611, Japan.

Well-differentiated thyroid carcinomas have driver mutations involving growth factor receptor-tyrosine kinases (RTKs) or their intracellular signaling pathway, that is, the mitogen-activated protein kinase (MAPK) pathway. Sorafenib is a multikinase inhibitor of RTKs and the MAPK pathway and has recently been used for the treatment of unresectable well-differentiated thyroid carcinoma. In normal thyroid follicular cells, stimulation of the thyroid-stimulating hormone (TSH) receptor activates the cyclic adenosine monophosphate (cAMP) pathway and promotes cell growth as well as hormonal secretion. However, an adenylyl cyclase (AC) activator, forskolin, has been reported to suppress the growth of thyroid carcinoma cells. To clarify the roles of the MAPK and cAMP pathways in proliferation of well-differentiated thyroid carcinoma cells, we compared the effects of sorafenib and forskolin in in vitro models. Sorafenib inhibited constitutive activation of the MAPK pathway, cyclin-dependent kinase 4 (CDK4), and phosphorylated retinoblastoma protein (RB) in 3 well-differentiated carcinoma cell lines, but it did not show sufficiently effective suppression of cell growth. Forskolin significantly suppressed the growth of all 3 cell lines and also activated the cAMP pathway and inhibited expression of cyclin D1. Our results suggest that activation of the cAMP pathway could be more potent than activation of the MAPK pathway in suppressing proliferation of well-differentiated thyroid cancer cells. We postulate that the AC activator suppresses growth of thyroid carcinoma cells through undetermined mechanisms.
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http://dx.doi.org/10.1507/endocrj.EJ16-0525DOI Listing
November 2017

Altered expression of cytokeratin 7 and CD117 in transitional mucosa adjacent to human colorectal cancer.

Oncol Lett 2017 Jul 11;14(1):119-126. Epub 2017 May 11.

Department of Pathology, Kyorin University School of Medicine, Mitaka, Tokyo 181-8611, Japan.

The multi-step progression of colorectal cancer through precancerous lesions (adenoma and dysplasia) is associated with cumulative molecular alterations, a number of which have also been demonstrated to be present in morphologically normal transitional mucosa adjacent to colorectal cancer. The cytoskeletal protein cytokeratin 7 (CK7) and the receptor tyrosine kinase, KIT proto-oncogene receptor tyrosine kinase (CD117), encoded by the proto-oncogene c-Kit, are lacking in normal colorectal crypt epithelium and are aberrantly expressed in a subset of colorectal cancer. The aim of the present study was to evaluate the expression of CK7 and CD117 in morphologically normal transitional mucosa adjacent to colorectal cancer. Immunohistochemical staining for CK7 and CD117 was performed in the mucosa adjacent to five groups of surgically resected colorectal tumors [low-grade adenoma, high-grade adenoma, mucosal adenocarcinoma, small-sized invasive adenocarcinoma (≤2 cm) and large-sized invasive adenocarcinoma (>2 cm)]. CK7 was expressed in the mucosa adjacent to a subset of colorectal tumors, and the positivity ratio increased according to tumor grade from low-grade adenoma up to small-sized invasive adenocarcinoma (61.2%). However, the positivity ratio of CK7 in the mucosa adjacent to the large-sized invasive adenocarcinoma (25.0%) was significantly lower compared with that of the next lower grade. CD117 was also expressed in the mucosa adjacent to a subset of colorectal tumors. In contrast to CK7, the positivity ratio of CD117 increased according to tumor grade from low-grade adenoma all the way through to the large-sized invasive adenocarcinoma (45.0%). Based on these results, the mechanism of CK7 and CD117 expression in the transitional mucosa adjacent to colorectal cancer may be different, and analysis of their individual expression may provide novel insights into the development and progression of colorectal cancer.
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http://dx.doi.org/10.3892/ol.2017.6156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5494860PMC
July 2017

Difference in chemical bonding between lithium and sodium salts: influence of covalency on their solubility.

Phys Chem Chem Phys 2017 Jul 26;19(26):17366-17372. Epub 2017 Jun 26.

School of Chemistry, Monash University, 17 Rainforest Walk, Clayton, Victoria 3800, Australia.

Recent thermal runaways in lithium-ion batteries have reinforced the focus on the research of safer electrolytes based on ionic liquids. A simple switch from organic solvents to ionic liquids has been proven difficult due to the decreased efficiency of batteries caused by decreased conductivity and increased viscosity of ionic liquids upon addition of lithium salts. The new trend in replacing lithium salts with a cheaper alternative, sodium salts, has resulted in rather poor solubility of sodium salts in commonly used ionic liquids. This phenomenon has been left largely unexplained. Herein, we present a high-level quantum chemical study of the chemical bonding of lithium and sodium salts coupled with ionic liquid anions. Due to their proximity to the anion, the 1s electrons on the lithium cation are found to become strongly polarized by the presence of the anion such that they start participating in the bonding, making it more covalent than originally thought. In sodium salts the 2s orbitals are rather removed from the anion, making its influence weaker. This polarization results in 90 kJ mol of difference in the interaction magnitude between lithium and sodium salts. Theoretical results have confirmed that increasing covalency in lithium salts results in their excellent solubility since these dissolve as ion-paired complexes. The downside of this ability is decreased conductivity as lithium salts are unlikely to easily dissociate in ionic liquids. Sodium salts are shown to maintain a high degree of ionicity, thus decreasing their chances of being solvated by ionic liquids as a result of their low concentration of ions per unit volume. The theoretical results are further underpinned by solubility studies of MX salts, where M = Li or Na and X = bis(trifluoromethylsulfonyl)imide (NTf), BF or PF, conducted in six different ionic liquids. Lithium salts consisting of BF or PF exhibited significantly better solubility than their sodium analogues by at least an order of magnitude. The findings of this work will have implications on the future direction of the development of safe electrolytes for lithium and sodium-ion secondary batteries.
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http://dx.doi.org/10.1039/c7cp03009fDOI Listing
July 2017

Glutathione production from mannan-based bioresource by mannanase/mannosidase expressing Saccharomyces cerevisiae.

Bioresour Technol 2017 Dec 1;245(Pt B):1400-1406. Epub 2017 Jun 1.

Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodaicho, Nada-ku, Kobe 657-8501, Japan. Electronic address:

This work aims to produce glutathione directly from mannan-based bioresources using engineered Saccharomyces cerevisiae. Mannan proved to be a valuable carbon source for glutathione production by this organism. Mannan-hydrolyzing S. cerevisiae was developed by heterologous expression of mannanase/mannosidase on its cell surface. This strain efficiently produced glutathione from mannose polysaccharide, β-1,4-mannan. Furthermore, it produced glutathione from locust bean gum (LBG), a highly dense and inexpensive mannan-based bioresource, as sole carbon source. Glutathione productivity from LBG was enhanced by engineering the glutathione metabolism of mannan-hydrolyzing S. cerevisiae. Expression of extracellular mannanase/mannosidase protein combined with intracellular metabolic engineering is potentially applicable to the efficient, environmentally friendly bioproduction of targeted products from mannan-based bioresources.
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http://dx.doi.org/10.1016/j.biortech.2017.05.190DOI Listing
December 2017

Heterologous expression of bacterial phosphoenol pyruvate carboxylase and Entner-Doudoroff pathway in Saccharomyces cerevisiae for improvement of isobutanol production.

J Biosci Bioeng 2017 Sep 20;124(3):263-270. Epub 2017 May 20.

Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address:

Bacterial phosphoenol pyruvate carboxylase (PPC) and enzymes in the Entner-Doudoroff (ED) pathway were heterologously expressed in Saccharomyces cerevisiae to improve the NADPH supply required for the bio-production of chemicals such as isobutanol. The heterologous expression of PPC from Synechocystis sp. PCC6803 increased in the isobutabol titer 1.45-fold (93.2±1.6 mg/L) in metabolically engineered S. cerevisiae strains producing isobutanol. This result suggested that the pyruvate and NADPH supply for isobutanol biosynthesis was activated by PPC overexpression. On the other hand, the expression of two enzymes organizing the ED pathway (6-phosphogluconate dehydratase [6PGD] and 2-dehydro-3-deoxy-phosphogluconate aldolase [KDPGA]) had no effect to isobutabol bio-production. Further analysis, however, revealed that additional expression of 6PGD and KDPGA improved the growth rate of S. cerevisiae strain BY4742 gnd1Δ. A C-labeling experiment using [1-C] glucose also suggested that metabolic flow levels in the ED pathway increased slightly with the additional expression. These results showed that the ED pathway was successfully constructed in S. cerevisiae, even though activity of the pathway was too weak to improve isobutanol biosynthesis.
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http://dx.doi.org/10.1016/j.jbiosc.2017.04.005DOI Listing
September 2017