Publications by authors named "Akihiko Kondo"

599 Publications

Advances in metabolic engineering of Corynebacterium glutamicum to produce high-value active ingredients for food, feed, human health, and well-being.

Essays Biochem 2021 Jun 7. Epub 2021 Jun 7.

Institute of Systems Biotechnology, Saarland University, Saarbrücken, Germany.

The soil microbe Corynebacterium glutamicum is a leading workhorse in industrial biotechnology and has become famous for its power to synthetise amino acids and a range of bulk chemicals at high titre and yield. The product portfolio of the microbe is continuously expanding. Moreover, metabolically engineered strains of C. glutamicum produce more than 30 high value active ingredients, including signature molecules of raspberry, savoury, and orange flavours, sun blockers, anti-ageing sugars, and polymers for regenerative medicine. Herein, we highlight recent advances in engineering of the microbe into novel cell factories that overproduce these precious molecules from pioneering proofs-of-concept up to industrial productivity.
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http://dx.doi.org/10.1042/EBC20200134DOI Listing
June 2021

Reprogramming Escherichia coli pyruvate-forming reaction towards chorismate derivatives production.

Metab Eng 2021 May 24;67:1-10. Epub 2021 May 24.

Center for Sustainable Resource Science, RIKEN, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan; Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan.

Microbial metabolic pathway engineering is a potent strategy used worldwide to produce aromatic compounds. We drastically rewired the primary metabolic pathway of Escherichia coli to produce aromatics and their derivatives. The metabolic pathway of E. coli was compartmentalized into the production and energy modules. We focused on the pyruvate-forming reaction in the biosynthesis pathway of some compounds as the reaction connecting those modules. E. coli strains were engineered to show no growth unless pyruvate was synthesized along with the compounds of interest production. Production of salicylate and maleate was demonstrated to confirm our strategy's versatility. In maleate production, the production, yield against the theoretical yield, and production rate reached 12.0 g L, 67%, and up to fourfold compared to that in previous reports, respectively; these are the highest values of maleate production in microbes to our knowledge. The results reveal that our strategy strongly promotes the production of aromatics and their derivatives.
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http://dx.doi.org/10.1016/j.ymben.2021.05.005DOI Listing
May 2021

Lutein production with Chlorella sorokiniana MB-1-M12 using novel two-stage cultivation strategies - metabolic analysis and process improvement.

Bioresour Technol 2021 Apr 24;334:125200. Epub 2021 Apr 24.

Department of Chemical Engineering, National Cheng Kung University, Tainan 701, Taiwan; Department of Chemical and Materials Engineering, College of Engineering, Tunghai University, Taichung 407, Taiwan; Research Center for Smart Sustainable Circular Economy, Tunghai University, Taichung 407, Taiwan. Electronic address:

Microalgae-derived carotenoids have increasingly been considered as feasible green alternatives for synthetic antioxidants. In this study, the lutein high-yielding strain (Chlorella sorokiniana MB-1; henceforth MB-1) and its mutant derivative (C. sorokiniana MB-1-M12; henceforth M12) were evaluated for their growth, biomass production, and lutein accumulation in three different cultivation modes - photoautotrophy, mixotrophy, and heterotrophy. M12 could grow effectively under heterotrophic conditions, but the lutein content was lower, indicating the necessity of photo-induction for lutein accumulation. Metabolic analysis of MB-1 and M12 in autotrophic growth in the presence of carbon dioxide indicated that carbon assimilation and channeling of the fixed metabolites towards carotenoid accumulation was elevated in M12 compared to MB-1. Novel two-stage alternative cultivation strategies (Autotrophic/Heterotrophic and Mixotrophic/Heterotrophic cultures) were applied for enhancing lutein production in M12. Maximum lutein quantity (6.17 mg/g) and production (33.64 mg/L) were obtained with the TSHM strategy that is considered the best two-stage operation.
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http://dx.doi.org/10.1016/j.biortech.2021.125200DOI Listing
April 2021

C-metabolic flux analysis in glycerol-assimilating strains of Saccharomyces cerevisiae.

J Gen Appl Microbiol 2021 May 8. Epub 2021 May 8.

School of Life Science and Technology, Tokyo Institute of Technology.

Glycerol is an attractive raw material for the production of useful chemicals using microbial cells. We previously identified metabolic engineering targets for the improvement of glycerol assimilation ability in Saccharomyces cerevisiae based on adaptive laboratory evolution (ALE) and transcriptome analysis of the evolved cells. We also successfully improved glycerol assimilation ability by the disruption of the RIM15 gene encoding a Greatwall protein kinase together with overexpression of the STL1 gene encoding the glycerol/H symporter. To understand glycerol assimilation metabolism in the evolved glycerol-assimilating strains and STL1-overexpressing RIM15 disruptant, we performed metabolic flux analysis using C-labeled glycerol. Significant differences in metabolic flux distributions between the strains obtained from the culture after 35 and 85 generations in ALE were not found, indicating that metabolic flux changes might occur in the early phase of ALE (i.e., before 35 generations at least). Similarly, metabolic flux distribution was not significantly changed by RIM15 gene disruption. However, fluxes for the lower part of glycolysis and the TCA cycle were larger and, as a result, flux for the pentose phosphate pathway was smaller in the STL1-overexpressing RIM15 disruptant than in the strain obtained from the culture after 85 generations in ALE. It could be effective to increase flux for the pentose phosphate pathway to improve the glycerol assimilation ability in S. cerevisiae.
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http://dx.doi.org/10.2323/jgam.2020.10.001DOI Listing
May 2021

Accelerated glucose metabolism in hyphae-dispersed Aspergillus oryzae is suitable for biological production.

J Biosci Bioeng 2021 Apr 22. Epub 2021 Apr 22.

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

Recently, a hyphae-dispersed type of filamentous fungus Aspergillus oryzae was constructed via genetic engineering, and industrial applications are expected due to the ease of handling and to the level of protein production properties. In this study, we constructed cellulase-expressing strains using wild-type and hyphae-dispersed strains to investigate the correlation between protein productivity and metabolism. Compared with the original strain, the hyphae-dispersed cellulase-expressing strain showed elevated cellulase activity, rapid glucose consumption, increased mycelial dry weight, an increased expression of cellulase genes, and activated respiration activity. Comparative metabolomic analysis showed fewer metabolites in the glycolysis and TCA cycles in the dispersed strains than in the original strains. These results indicate that the flux of carbohydrate metabolism in the hyphae-dispersed strains is smoother than that in the original strains. Such efficient metabolic flux would contribute to efficient energy conversion and to sufficient energy supply to anabolisms, such as mycelial growth and protein production. Our findings suggest that the hyphae-dispersed strains could be a useful host not only for protein production but also for the biological production of various chemicals such as organic acids.
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http://dx.doi.org/10.1016/j.jbiosc.2021.03.017DOI Listing
April 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

Direct 1,3-butadiene biosynthesis in Escherichia coli via a tailored ferulic acid decarboxylase mutant.

Nat Commun 2021 04 13;12(1):2195. Epub 2021 Apr 13.

Center for Sustainable Resource Science, RIKEN, Yokohama, Japan.

The C4 unsaturated compound 1,3-butadiene is an important monomer in synthetic rubber and engineering plastic production. However, microorganisms cannot directly produce 1,3-butadiene when glucose is used as a renewable carbon source via biological processes. In this study, we construct an artificial metabolic pathway for 1,3-butadiene production from glucose in Escherichia coli by combining the cis,cis-muconic acid (ccMA)-producing pathway together with tailored ferulic acid decarboxylase mutations. The rational design of the substrate-binding site of the enzyme by computational simulations improves ccMA decarboxylation and thus 1,3-butadiene production. We find that changing dissolved oxygen (DO) levels and controlling the pH are important factors for 1,3-butadiene production. Using DO-stat fed-batch fermentation, we produce 2.13 ± 0.17 g L 1,3-butadiene. The results indicate that we can produce unnatural/nonbiological compounds from glucose as a renewable carbon source via a rational enzyme design strategy.
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http://dx.doi.org/10.1038/s41467-021-22504-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8044207PMC
April 2021

Enhancing carbohydrate repartitioning into lipid and carotenoid by disruption of microalgae starch debranching enzyme.

Commun Biol 2021 Apr 9;4(1):450. Epub 2021 Apr 9.

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

Light/dark cycling is an inherent condition of outdoor microalgae cultivation, but is often unfavorable for lipid accumulation. This study aims to identify promising targets for metabolic engineering of improved lipid accumulation under outdoor conditions. Consequently, the lipid-rich mutant Chlamydomonas sp. KOR1 was developed through light/dark-conditioned screening. During dark periods with depressed CO fixation, KOR1 shows rapid carbohydrate degradation together with increased lipid and carotenoid contents. KOR1 was subsequently characterized with extensive mutation of the ISA1 gene encoding a starch debranching enzyme (DBE). Dynamic time-course profiling and metabolomics reveal dramatic changes in KOR1 metabolism throughout light/dark cycles. During light periods, increased flux from CO through glycolytic intermediates is directly observed to accompany enhanced formation of small starch-like particles, which are then efficiently repartitioned in the next dark cycle. This study demonstrates that disruption of DBE can improve biofuel production under light/dark conditions, through accelerated carbohydrate repartitioning into lipid and carotenoid.
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http://dx.doi.org/10.1038/s42003-021-01976-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8035404PMC
April 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

Four-carbon dicarboxylic acid production through the reductive branch of the open cyanobacterial tricarboxylic acid cycle in Synechocystis sp. PCC 6803.

Metab Eng 2021 05 17;65:88-98. Epub 2021 Mar 17.

School of Agriculture, Meiji University, 1-1-1, Higashimita, Tama-ku, Kawasaki, Kanagawa, 214-8571, Japan. Electronic address:

Succinate, fumarate, and malate are valuable four-carbon (C4) dicarboxylic acids used for producing plastics and food additives. C4 dicarboxylic acid is biologically produced by heterotrophic organisms. However, current biological production requires organic carbon sources that compete with food uses. Herein, we report C4 dicarboxylic acid production from CO using metabolically engineered Synechocystis sp. PCC 6803. Overexpression of citH, encoding malate dehydrogenase (MDH), resulted in the enhanced production of succinate, fumarate, and malate. citH overexpression increased the reductive branch of the open cyanobacterial tricarboxylic acid (TCA) cycle flux. Furthermore, product stripping by medium exchanges increased the C4 dicarboxylic acid levels; product inhibition and acidification of the media were the limiting factors for succinate production. Our results demonstrate that MDH is a key regulator that activates the reductive branch of the open cyanobacterial TCA cycle. The study findings suggest that cyanobacteria can act as a biocatalyst for converting CO to carboxylic acids.
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http://dx.doi.org/10.1016/j.ymben.2021.03.007DOI Listing
May 2021

Development of a Method for Fucoxanthin Production Using the Haptophyte Marine Microalga Pavlova sp. OPMS 30543.

Mar Biotechnol (NY) 2021 Apr 13;23(2):331-341. Epub 2021 Mar 13.

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

The natural pigment fucoxanthin has attracted global attention because of its superior antioxidant properties. The haptophyte marine microalgae Pavlova spp. are assumed to be promising industrial fucoxanthin producers as their lack of a cell wall could facilitate the commercialization of cultured cells as a whole food. This study screened promising Pavlova strains with high fucoxanthin content to develop an outdoor cultivation method for fucoxanthin production. Initial laboratory investigations of P. pinguis NBRC 102807, P. lutheri NBRC 102808, and Pavlova sp. OPMS 30543 identified OPMS 30543 as having the highest fucoxanthin content. The culture conditions were optimized for OPMS 30543. Compared with f/2 and Walne's media, the use of Daigo's IMK medium led to the highest biomass production and highest fucoxanthin accumulation. The presence of seawater elements in Daigo's IMK medium was necessary for the growth of OPMS 30543. OPMS 30543 was then cultured outdoors using acrylic pipe photobioreactors, a plastic bag, an open tank, and a raceway pond. Acrylic pipe photobioreactors with small diameters enabled the highest biomass production. Using an acrylic pipe photobioreactor with 60-mm diameter, a fucoxanthin productivity of 4.88 mg/L/day was achieved in outdoor cultivation. Thus, this study demonstrated the usefulness of Pavlova sp. OPMS 30543 for fucoxanthin production in outdoor cultivation.
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http://dx.doi.org/10.1007/s10126-021-10028-5DOI Listing
April 2021

Growth stimulation of Bifidobacterium from human colon using daikenchuto in an in vitro model of human intestinal microbiota.

Sci Rep 2021 Feb 25;11(1):4580. Epub 2021 Feb 25.

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

Daikenchuto (DKT) is a Japanese traditional herbal (Kampo) medicine containing ginseng, processed ginger, and Japanese or Chinese pepper. We aimed to determine how DKT affects human colonic microbiota. An in vitro microbiota model was established using fecal inocula collected from nine healthy volunteers, and each model was found to retain operational taxonomic units similar to the ones in the original human fecal samples. DKT was added to the in vitro microbiota model culture at a concentration of 0.5% by weight. Next-generation sequencing of bacterial 16S rRNA gene revealed a significant increase in the relative abundance of bacteria related to the Bifidobacterium genus in the model after incubation with DKT. In pure cultures, DKT significantly promoted the growth of Bifidobacterium adolescentis, but not that of Fusobacterium nucleatum or Escherichia coli. Additionally, in pure cultures, B. adolescentis transformed ginsenoside Rc to Rd, which was then probably utilized for its growth. Our study reveals the in vitro bifidogenic effect of DKT that likely contributes to its beneficial effects on the human colon.
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http://dx.doi.org/10.1038/s41598-021-84167-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7907203PMC
February 2021

Metabolic engineering of 1,2-propanediol production from cellobiose using beta-glucosidase-expressing E. coli.

Bioresour Technol 2021 Jun 16;329:124858. Epub 2021 Feb 16.

Center for Sustainable Resource Science, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan; Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan.

Microbial 1,2-propanediol production using renewable feedstock is a promising method for the sustainable production of value-added fuels and chemicals. We demonstrated the metabolically engineered Escherichia coli for improvement of 1,2-propanediol production using glucose and cellobiose. The deletion of competing pathways improved 1,2-propanediol production. To reduce carbon flux toward downstream glycolysis, the phosphotransferase system (PTS) was inactivated by ptsG gene deletion. The resultant strain, GL3/PD, produced 1.48 ± 0.01 g/L of 1,2-propanediol from 20 g/L of glucose. A sugar supply was engineered by coexpression of β-glucosidase (BGL). The strain expressing BGL produced 1,2-propanediol from cellobiose at a concentration of 0.90 ± 0.11 g/L with a yield of 0.15 ± 0.01 g/g glucose (cellobiose 1 g is equal to glucose 1.1 g). As cellobiose or cellooligosaccharides a carbon source, the feasibility of producing 1,2-propanediol using an E. coli strain engineered for β-glucosidase expression are demonstrated.
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http://dx.doi.org/10.1016/j.biortech.2021.124858DOI Listing
June 2021

Disruption of alpha-tubulin releases carbon catabolite repression and enhances enzyme production in Trichoderma reesei even in the presence of glucose.

Biotechnol Biofuels 2021 Feb 8;14(1):39. Epub 2021 Feb 8.

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

Background: Trichoderma reesei is a filamentous fungus that is important as an industrial producer of cellulases and hemicellulases due to its high secretion of these enzymes and outstanding performance in industrial fermenters. However, the reduction of enzyme production caused by carbon catabolite repression (CCR) has long been a problem. Disruption of a typical transcriptional regulator, Cre1, does not sufficiently suppress this reduction in the presence of glucose.

Results: We found that deletion of an α-tubulin (tubB) in T. reesei enhanced both the amount and rate of secretory protein production. Also, the tubulin-disrupted (ΔtubB) strain had high enzyme production and the same enzyme profile even if the strain was cultured in a glucose-containing medium. From transcriptome analysis, the ΔtubB strain exhibited upregulation of both cellulase and hemicellulase genes including some that were not originally induced by cellulose. Moreover, cellobiose transporter genes and the other sugar transporter genes were highly upregulated, and simultaneous uptake of glucose and cellobiose was also observed in the ΔtubB strain. These results suggested that the ΔtubB strain was released from CCR.

Conclusion: Trichoderma reesei α-tubulin is involved in the transcription of cellulase and hemicellulase genes, as well as in CCR. This is the first report of overcoming CCR by disrupting α-tubulin gene in T. reesei. The disruption of α-tubulin is a promising approach for creating next-generation enzyme-producing strains of T. reesei.
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http://dx.doi.org/10.1186/s13068-021-01887-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7869464PMC
February 2021

Efficient base editing in tomato using a highly expressed transient system.

Plant Cell Rep 2021 Apr 7;40(4):667-676. Epub 2021 Feb 7.

Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, 305-8572, Japan.

Key Message: Base editing in tomatoes was achieved by transient expression. The Solanaceae plants, particularly the tomato (Solanum lycopersicum), is of huge economic value worldwide. The tomato is a unique model plant for studying the functions of genes related to fruit ripening. Deeper understanding of tomatoes is of great importance for both plant research and the economy. Genome editing technology, such as CRISPR/Cas9, has been used for functional genetic research. However, some challenges, such as low transformation efficiency, remain with this technology. Moreover, the foreign Cas9 and gRNA expression cassettes must be removed to obtain null-segregants In this study, we used a high-level transient expression system to improve the base editing technology. A high-level transient expression system has been established previously using geminiviral replication and a double terminator. The pBYR2HS vector was used for this transient expression system. nCas9-CDA and sgRNA-SlHWS were introduced into this vector, and the protein and RNA were then transiently expressed in tomato tissues by agroinfiltration. The homozygous mutant produced by base editing was obtained in the next generation with an efficiency of about 18%. nCas9-free next-generation plants were 71%. All the homozygous base-edited plants in next generation are nCas9-free. These findings show that the high-level transient expression system is useful for base editing in tomatoes.
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http://dx.doi.org/10.1007/s00299-021-02662-zDOI Listing
April 2021

Semiological differences of focal onset bilateral motor (convulsive) seizure between mesial temporal lobe epilepsy and neocortical epilepsy.

Epilepsy Res 2021 Feb 7;170:106553. Epub 2021 Jan 7.

National Epilepsy Center, NHO Shizuoka Institute of Epilepsy and Neurological Disorders, Urushiyama 886, Aoi-ku, Shizuoka, Japan.

Objective: We investigated the semiology of focal onset bilateral motor (convulsive) seizure (FBMS) in patients with intractable focal epilepsy who underwent epilepsy surgery to understand its value in localizing the origin of the seizure.

Methods: The study included 20 patients who underwent resective surgery after intracranial video-EEG monitoring (iEEG) with a favorable seizure outcome (Engel class I), and had at least one FBMS during iEEG. The diagnosis was mesial temporal lobe epilepsy (MTLE) for 7 patients and neocortical epilepsy (NE) for 13 patients (lateral temporal lobe, 3; posterior cortex, 6; frontal lobe, 3; perirolandic, 1). Videotaped FBMSs were carefully analyzed.

Results: A generalized tonic phase appeared in all 7 patients with MTLE, but was absent in 6 of the 13 patients with NE (P = .044). Tonic cry was more frequently observed in MTLE than in NE (P = .012). Facial tonicity preceding limb tonicity was more frequently seen in patients with MTLE (P = .001).

Conclusion: Notably, patients with MTLE and those with NE showed semiological differences during bilateralization. FBMS includes not only focal to bilateral tonic-clonic seizure but also focal to bilateral clonic seizure.
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http://dx.doi.org/10.1016/j.eplepsyres.2021.106553DOI Listing
February 2021

The alga Euglena gracilis stimulates Faecalibacterium in the gut and contributes to increased defecation.

Sci Rep 2021 Jan 13;11(1):1074. Epub 2021 Jan 13.

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

The alga Euglena gracilis (E. gracilis) has recently gained attention as a health food, but its effects on human gut microbiota remain unknown. This study aimed to determine the effect of E. gracilis on gut microbiota and defecation due to modulation of microbiota composition in vitro and in vivo. The in vitro model simulating human colonic microbiota revealed that E. gracilis addition stimulated the growth of commensal Faecalibacterium. Further, E. gracilis addition enhanced butyrate production by Faecalibacterium prausnitzii. Paramylon, an insoluble dietary fibre that accumulates in E. gracilis and is the main component of E. gracilis, did not stimulate Faecalibacterium growth in vitro. Daily ingestion of 2 g of E. gracilis for 30 days increased bowel movement frequency as well as stool volume in 28 human participants. Collectively, these findings indicate that E. gracilis components other than paramylon, stimulate the growth of Faecalibacterium to improve digestive health as well as promote defecation by increasing butyrate production.
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http://dx.doi.org/10.1038/s41598-020-80306-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7806897PMC
January 2021

Enhanced methane production from cellulose using a two-stage process involving a bioelectrochemical system and a fixed film reactor.

Biotechnol Biofuels 2021 Jan 6;14(1). Epub 2021 Jan 6.

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

Background: It is desirable to improve the anaerobic digestion processes of recalcitrant materials, such as cellulose. Enhancement of methane (CH) production from organic molecules was previously accomplished through coupling a bioelectrochemical system (BES); however, scaling-up BES-based production is difficult. Here, we developed a two-stage process consisting of a BES using low-cost and low-reactive carbon sheets as the cathode and anode, and a fixed film reactor (FFR) containing conductive material, i.e., carbon fiber textiles (CFTs) (:BES → FFR). By controlling the cathodic current at 2.7 μA/cm without abiotic H production, the three-electrode BES system was operated to mimic a microbial electrolysis cell.

Results: The thermophilic BES (inlet pH: 6.1) and FFR (inlet pH: 7.5) were operated using hydraulic retention times (HRTs) of 2.5 and 4.2 days, respectively, corresponding to a cellulose load of 3555.6 mg-carbon (C)/(L day). The BES → FFR process achieved a higher CH yield (37.5%) with 52.8 vol% CH in the product gas compared to the non-bioelectrochemical system (NBES) → FFR process, which showed a CH yield of 22.1% with 46.8 vol% CH. The CH production rate (67.5 mM/day) obtained with the BER → FFR process was much higher than that obtained using electrochemical methanogenesis (0.27 mM/day). Application of the electrochemical system or CFTs improved the yields of CH with the NBES → FFR or BES → non-fixed film reactor process, respectively. Meta 16S rRNA sequencing revealed that putative cellulolytic bacteria (identified as Clostridium species) were present in the BES and NBES, and followed (BES→ and NBES→) FFR. Notably, H-consuming methanogens, Methanobacterium sp. and Methanosarcina sp., showed increased relative abundances in the suspended fraction and attached fraction of (BES→) FFR, respectively, compared to that of (NBES→) FFR, although these methanogens were observed at trace levels in the BES and NBES.

Conclusions: These results indicate that bioelectrochemical preprocessing at a low current effectively induces interspecies H transfer in the FFR with conductive material. Sufficient electrochemical preprocessing was observed using a relatively short HRT. This type of two-stage process, BES → FFR, is useful for stabilization and improvement of the biogas (CH) production from cellulosic material, and our results imply that the two-stage system developed here may be useful with other recalcitrant materials.
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http://dx.doi.org/10.1186/s13068-020-01866-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789537PMC
January 2021

CRISPR-derived genome editing technologies for metabolic engineering.

Metab Eng 2021 01 8;63:141-147. Epub 2020 Dec 8.

Engineering Biology Research Center, Kobe University, Japan; Graduate School of Science, Technology and Innovation, Kobe University, Japan. Electronic address:

In metabolic engineering, genome editing tools make it much easier to discover and evaluate relevant genes and pathways and construct strains. Clustered regularly interspaced palindromic repeats (CRISPR)-associated (Cas) systems now have become the first choice for genome engineering in many organisms includingindustrially relevant ones. Targeted DNA cleavage by CRISPR-Cas provides variousgenome engineering modes such as indels, replacements, large deletions, knock-in and chromosomal rearrangements, while host-dependent differences in repair pathways need to be considered. The versatility of the CRISPR system has given rise to derivative technologies that complement nuclease-based editing, which causes cytotoxicity especially in microorganisms. Deaminase-mediated base editing installs targeted point mutations with much less toxicity. CRISPRi and CRISPRa can temporarily control gene expression without changing the genomic sequence. Multiplex, combinatorial and large scale editing are made possible by streamlined design and construction of gRNA libraries to further accelerates comprehensive discovery, evaluation and building of metabolic pathways. This review summarizes the technical basis and recent advances in CRISPR-related genome editing tools applied for metabolic engineering purposes, with representative examples of industrially relevant eukaryotic and prokaryotic organisms.
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http://dx.doi.org/10.1016/j.ymben.2020.12.002DOI Listing
January 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

Multiple gene substitution by Target-AID base-editing technology in tomato.

Sci Rep 2020 11 24;10(1):20471. Epub 2020 Nov 24.

Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan.

The use of Target activation-induced cytidine deaminase (Target-AID) base-editing technology with the CRISPR-Cas 9 system fused with activation-induced cytidine deaminase (AID) resulted in the substitution of a cytidine with a thymine. In previous experiments focusing on a single target gene, this system has been reported to work in several plant species, including tomato (Solanum lycopersicum L.). In this research, we used Target-AID technology to target multiple genes related to carotenoid accumulation in tomato. We selected 3 genes, SlDDB1, SlDET1 and SlCYC-B, for their roles in carotenoid accumulation. Among 12 edited T lines, we obtained 10 independent T lines carrying nucleotide substitutions in the three targeted genes, with several allelic versions for each targeted gene. The two edited lines showed significant differences in carotenoid accumulation. These results demonstrate that Target-AID technology is a highly efficient tool for targeting multiple genes with several allelic versions.
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http://dx.doi.org/10.1038/s41598-020-77379-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7686336PMC
November 2020

Titanium oxide nano-radiosensitizers for hydrogen peroxide delivery into cancer cells.

Colloids Surf B Biointerfaces 2021 Feb 7;198:111451. Epub 2020 Nov 7.

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

Polyacrylic acid-modified titanium peroxide nanoparticles (PAA-TiO NPs) are promising radiosensitizers that enhance the therapeutic effect of X-ray irradiation after local injection into tumors. However, the mechanism for this reaction has remained unclear with the exception of the involvement of hydrogen peroxide (HO), which is released by PAA-TiO NPs to a liquid phase during dispersion. In the present study, a clonogenic assay was used to compare PAA-TiO NPs with free HO molecules to investigate the effect exerted on the radiosensitivity of cancer cells in vitro. A cell-free dialysis method revealed that a portion of the HO adsorbed onto the PAA-TiO NPs during synthesis could be released during a treatment regimen. The HO release lasted for 7 h, which was sufficient for one radiation treatment procedure. For in vitro experiments, cultured human pancreatic cancer cells took up PAA-TiO NPs in 10 min after administration. Interestingly, when the cells were washed with a buffer after treatment with either a PAA-TiO NP or HO solution, the intracellular HO levels remained higher with PAA-TiO NP treatment compared with the HO solution treatment. Furthermore, the effects of subsequent X-ray irradiation corresponded to the intracellular HO levels. These results indicate that PAA-TiO NPs are efficient carriers of HO into cancer cells and thus enhance the radiosensitivity.
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http://dx.doi.org/10.1016/j.colsurfb.2020.111451DOI Listing
February 2021

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

An ion-pair free LC-MS/MS method for quantitative metabolite profiling of microbial bioproduction systems.

Talanta 2021 Jan 8;222:121625. Epub 2020 Sep 8.

Graduate School of Science, Technology and Innovation, Kobe University, Japan; Engineering Biology Research Center, Kobe University, Japan.

Data-driven engineering of microbes has been demonstrated for the sustainable production of high-performance chemicals. Metabolic profiling analysis is essential to increase the productivity of target compounds. However, improvement of comprehensive analysis methodologies is required for the high demands of metabolic engineering. Therefore, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) based methodology was designed and applied to cover a wide target range with high precision. Ion-pair free separation of metabolites on a pentafluorophenyl propyl column enabled high-precision quantification of 113 metabolites. The method was further evaluated for high reproducibility and robustness. Target analytes consisted of primary metabolites and intermediate metabolites for microbial production of high-performance chemicals. 95 metabolites could be detected with high reproducibility of peak area (intraday data: CV<15%), and 53 metabolites could be sensitively determined within a wide dynamic linear range (3-4 orders of magnitude). The developed system was further applied to the metabolomic analysis of various prokaryotic and eukaryotic microorganisms. Differences due to culture media and metabolic phenotypes could be observed when comparing the metabolomes of conventional and non-conventional yeast. Furthermore, almost all Kluyveromyces marxianus metabolites could be detected with moderate reproducibility (CV<40%, among independent extractions), where 41 metabolites were detected with very high reproducibility (CV<15%). In addition, the accuracy was validated via a spike-and-recovery test,and 78 metabolites were detected with analyte recovery in the 80-120% range. Together these results establish ion-pair free metabolic profiling as a comprehensive and precise tool for data-driven bioengineering applications.
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http://dx.doi.org/10.1016/j.talanta.2020.121625DOI Listing
January 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

Prebiotic effects of yeast mannan, which selectively promotes Bacteroides thetaiotaomicron and Bacteroides ovatus in a human colonic microbiota model.

Sci Rep 2020 10 15;10(1):17351. Epub 2020 Oct 15.

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

Yeast mannan (YM) is an indigestible water-soluble polysaccharide of the yeast cell wall, with a notable prebiotic effect on the intestinal microbiota. We previously reported that YM increased Bacteroides thetaiotaomicron abundance in in vitro rat faeces fermentation, concluding that its effects on human colonic microbiota should be investigated. In this study, we show the effects of YM on human colonic microbiota and its metabolites using an in vitro human faeces fermentation system. Bacterial 16S rRNA gene sequence analysis showed that YM administration did not change the microbial diversity or composition. Quantitative real-time PCR analysis revealed that YM administration significantly increased the relative abundance of Bacteroides ovatus and B. thetaiotaomicron. Moreover, a positive correlation was observed between the relative ratio (with or without YM administration) of B. thetaiotaomicron and B. ovatus (r = 0.92), suggesting that these bacteria utilise YM in a coordinated manner. In addition, YM administration increased the production of acetate, propionate, and total short-chain fatty acids. These results demonstrate the potential of YM as a novel prebiotic that selectively increases B. thetaiotaomicron and B. ovatus and improves the intestinal environment. The findings also provide insights that might be useful for the development of novel functional foods.
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http://dx.doi.org/10.1038/s41598-020-74379-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7562712PMC
October 2020

Retained absolute pitch after selective amygdalohippocampectomy.

Epilepsy Behav Rep 2020 3;14:100378. Epub 2020 Jul 3.

National Epilepsy Center, NHO Shizuoka Institute of Epilepsy and Neurological Disorders, Urushiyama 886, Aoi-ku, Shizuoka 420-8688, Japan.

This study assessed the pre-operative chronic condition and effect of epilepsy surgery in a 21-year-old Japanese woman with drug-resistant right temporal lobe epilepsy (TLE). For this patient, it was crucially important to preserve language and her music capabilities, including absolute pitch (AP), which is found in the general population at less than 0.1%. The patient became seizure free, and her AP capability was preserved after selective amygdalohippocampectomy in the non-dominant right hemisphere. Most of the neuropsychological test (WAIS-III and WMS-R) scores remained in the normal range, except for low scores in verbal memory and markedly improved attention/concentration index. The patient's pre- and postoperative brain function related to language and music capabilities were investigated using functional magnetic resonance imaging (fMRI) based on two language tasks and a music task (listening to melodies). While task performance was similar in pre- and postoperative examinations, her brain activation patterns markedly differed. The most striking difference was during the music task: areas with significant activation existed in the bilateral frontal and temporal lobes before surgery, whereas postoperative activation was confined to a very limited region in the left angular gyrus. The authors speculate that the surgery triggered some change in functional organization in the brain, which contributed to preserving her capabilities.
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http://dx.doi.org/10.1016/j.ebr.2020.100378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494675PMC
July 2020

Optimization of 1,2,4-butanetriol production from xylose in Saccharomyces cerevisiae by metabolic engineering of NADH/NADPH balance.

Biotechnol Bioeng 2021 01 29;118(1):175-185. Epub 2020 Sep 29.

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

1,2,4-Butanetriol (BT) is used as a precursor for the synthesis of various pharmaceuticals and the energetic plasticizer 1,2,4-butanetriol trinitrate. In Saccharomyces cerevisiae, BT is biosynthesized from xylose via heterologous four enzymatic reactions catalyzed by xylose dehydrogenase, xylonate dehydratase, 2-ketoacid decarboxylase, and alcohol dehydrogenase. We here aimed to improve the BT yield in S. cerevisiae by genetic engineering. First, the amount of the key intermediate 2-keto-3-deoxy-xylonate as described previously was successfully reduced in 41% by multiple integrations of Lactococcus lactis 2-ketoacid decarboxylase gene kdcA into the yeast genome. Since the heterologous BT synthetic pathway is independent of yeast native metabolism, this manipulation has led to NADH/NADPH imbalance and deficiency during BT production. Overexpression of the NADH kinase POS5Δ17 lacking the mitochondrial targeting sequence to relieve NADH/NADPH imbalance resulted in the BT titer of 2.2 g/L (31% molar yield). Feeding low concentrations of glucose and xylose to support the supply of NADH resulted in BT titer of 6.6 g/L with (57% molar yield). Collectively, improving the NADH/NADPH ratio and supply from glucose are essential for the construction of a xylose pathway, such as the BT synthetic pathway, independent of native yeast metabolism.
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http://dx.doi.org/10.1002/bit.27560DOI Listing
January 2021

Consolidated bioprocessing of corn cob-derived hemicellulose: engineered industrial as efficient whole cell biocatalysts.

Biotechnol Biofuels 2020 8;13:138. Epub 2020 Aug 8.

CEB-Centre of Biological Engineering, University of Minho, Campus Gualtar, Braga, Portugal.

Background: Consolidated bioprocessing, which combines saccharolytic and fermentative abilities in a single microorganism, is receiving increased attention to decrease environmental and economic costs in lignocellulosic biorefineries. Nevertheless, the economic viability of lignocellulosic ethanol is also dependent of an efficient utilization of the hemicellulosic fraction, which contains xylose as a major component in concentrations that can reach up to 40% of the total biomass in hardwoods and agricultural residues. This major bottleneck is mainly due to the necessity of chemical/enzymatic treatments to hydrolyze hemicellulose into fermentable sugars and to the fact that xylose is not readily consumed by -the most used organism for large-scale ethanol production. In this work, industrial strains, presenting robust traits such as thermotolerance and improved resistance to inhibitors, were evaluated as hosts for the cell-surface display of hemicellulolytic enzymes and optimized xylose assimilation, aiming at the development of whole-cell biocatalysts for consolidated bioprocessing of corn cob-derived hemicellulose.

Results: These modifications allowed the direct production of ethanol from non-detoxified hemicellulosic liquor obtained by hydrothermal pretreatment of corn cob, reaching an ethanol titer of 11.1 g/L corresponding to a yield of 0.328 g/g of potential xylose and glucose, without the need for external hydrolytic catalysts. Also, consolidated bioprocessing of pretreated corn cob was found to be more efficient for hemicellulosic ethanol production than simultaneous saccharification and fermentation with addition of commercial hemicellulases.

Conclusions: These results show the potential of industrial strains for the design of whole-cell biocatalysts and paves the way for the development of more efficient consolidated bioprocesses for lignocellulosic biomass valorization, further decreasing environmental and economic costs.
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http://dx.doi.org/10.1186/s13068-020-01780-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414751PMC
August 2020