The Experts below are selected from a list of 18921 Experts worldwide ranked by ideXlab platform
Ken-ichi Yoshida - One of the best experts on this subject based on the ideXlab platform.
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A bacterial Cell Factory converting glucose into scyllo-inositol, a therapeutic agent for Alzheimer's disease.
Communications biology, 2020Co-Authors: Christophe Michon, Choong-min Kang, Sophia Karpenko, Kosei Tanaka, Shu Ishikawa, Ken-ichi YoshidaAbstract:A rare stereoisomer of inositol, scyllo-inositol, is a therapeutic agent that has shown potential efficacy in preventing Alzheimer’s disease. Mycobacterium tuberculosis ino1 encoding myo-inositol-1-phosphate (MI1P) synthase (MI1PS) was introduced into Bacillus subtilis to convert glucose-6-phosphate (G6P) into MI1P. We found that inactivation of pbuE elevated intraCellular concentrations of NAD+·NADH as an essential cofactor of MI1PS and was required to activate MI1PS. MI1P thus produced was dephosphorylated into myo-inositol by an intrinsic inositol monophosphatase, YktC, which was subsequently isomerized into scyllo-inositol via a previously established artificial pathway involving two inositol dehydrogenases, IolG and IolW. In addition, both glcP and glcK were overexpressed to feed more G6P and accelerate scyllo-inositol production. Consequently, a B. subtilis Cell Factory was demonstrated to produce 2 g L−1 scyllo-inositol from 20 g L−1 glucose. This Cell Factory provides an inexpensive way to produce scyllo-inositol, which will help us to challenge the growing problem of Alzheimer’s disease in our aging society. Michon et al. describe the use of a recombinant Bacillus subtilis as a Cell Factory capable of producing scyllo-inositol, a therapeutic compound for Alzheimer’s disease, from inexpensive glucose. They demonstrate that it could produce 2 g L−1 of scyllo-inositol from 20 g L−1 glucose.
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A bacterial Cell Factory converting glucose into scyllo-inositol, a therapeutic agent for Alzheimer's disease
Communications Biology, 2020Co-Authors: Christophe Michon, Choong-min Kang, Sophia Karpenko, Kosei Tanaka, Shu Ishikawa, Ken-ichi YoshidaAbstract:A rare stereoisomer of inositol, scyllo-inositol, is a therapeutic agent that has shown potential efficacy in preventing Alzheimer's disease. Mycobacterium tuberculosis ino1 encoding myo-inositol-1-phosphate (MI1P) synthase (MI1PS) was introduced into Bacillus subtilis to convert glucose-6-phosphate (G6P) into MI1P. We found that inactivation of pbuE elevated intra-Cellular concentrations of NAD + ·NADH as an essential cofactor of MI1PS and was required to activate MI1PS. MI1P thus produced was dephosphorylated into myo-inositol by an intrinsic inositol monophosphatase, YktC, which was subsequently isomerized into scyllo-inositol via a previously established artificial pathway involving two inositol dehydrogenases, IolG and IolW. In addition, both glcP and glcK were overexpressed to feed more G6P and accelerate scyllo-inositol production. Consequently, a B. subtilis Cell Factory was demonstrated to produce 2 g L −1 scyllo-inositol from 20 g L −1 glucose. This Cell Factory provides an inexpensive way to produce scyllo-inositol, which will help us to challenge the growing problem of Alzheimer's disease in our aging society.
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A new-generation of Bacillus subtilis Cell Factory for further elevated scyllo -inositol production
Microbial cell factories, 2017Co-Authors: Kosei Tanaka, Shu Ishikawa, Shinji Takenaka, Ayane Natsume, Ken-ichi YoshidaAbstract:A stereoisomer of inositol, scyllo-inositol (SI), has been regarded as a promising therapeutic agent for Alzheimer’s disease. However, this compound is relatively rare, whereas another stereoisomer of inositol, myo-inositol (MI) is abundant in nature. Bacillus subtilis 168 has the ability to metabolize inositol stereoisomers, including MI and SI. Previously, we reported a B. subtilis Cell Factory with modified inositol metabolism that converts MI into SI in the culture medium. The strain was constructed by deleting all genes related to inositol metabolism and overexpressing key enzymes, IolG and IolW. By using this strain, 10 g/l of MI initially included in the medium was completely converted into SI within 48 h of cultivation in a rich medium containing 2% (w/v) Bacto soytone. When the initial concentration of MI was increased to 50 g/l, conversion was limited to 15.1 g/l of SI. Therefore, overexpression systems of IolT and PntAB, the main transporter of MI in B. subtilis and the membrane-integral nicotinamide nucleotide transhydrogenase in Escherichia coli respectively, were additionally introduced into the B. subtilis Cell Factory, but the conversion efficiency hardly improved. We systematically determined the amount of Bacto soytone necessary for ultimate conversion, which was 4% (w/v). As a result, the conversion of SI reached to 27.6 g/l within 48 h of cultivation. The B. subtilis Cell Factory was improved to yield a SI production rate of 27.6 g/l/48 h by simultaneous overexpression of IolT and PntAB, and by addition of 4% (w/v) Bacto soytone in the conversion medium. The concentration of SI was increased even in the stationary phase perhaps due to nutrients in the Bacto soytone that contribute to the conversion process. Thus, MI conversion to SI may be further optimized via identification and control of these unknown nutrients.
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A second-generation Bacillus Cell Factory for rare inositol production.
Bioengineered, 2014Co-Authors: Kosei Tanaka, Shinji Takanaka, Ken-ichi YoshidaAbstract:Some rare inositol stereoisomers are known to exert specific health-promoting effects, including scyllo-inositol (SI), which is a promising therapeutic agent for Alzheimer disease. We recently reported a Bacillus subtilis Cell Factory that performed the efficient production of SI from the cheapest and most abundant isomer myo-inositol (MI). In the Cell Factory all “useless” genes involved in MI and SI metabolism were deleted and overexpression of the key enzymes, IolG and IolW, was appended. It converted 10 g/L MI into the same amount of SI in 48 h of cultivation. In this addendum, we discuss further improvement in the Cell Factory and its possible applications.
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An improved Bacillus subtilis Cell Factory for producing scyllo-inositol, a promising therapeutic agent for Alzheimer's disease.
Microbial cell factories, 2013Co-Authors: Kosei Tanaka, Shinji Takenaka, Shintaro Tajima, Ken-ichi YoshidaAbstract:Bacillus subtilis 168 possesses an efficient pathway to metabolize some of the stereoisomers of inositol, including myo-inositol (MI) and scyllo-inositol (SI). Previously we reported a prototype of a B. subtilis Cell Factory with modified inositol metabolism that converts MI into SI in the culture medium. However, it wasted half of initial 1.0% (w/v) MI, and the conversion was limited to produce only 0.4% (w/v) SI. To achieve a more efficient SI production, we attempted additional modifications. All “useless” genes involved in MI and SI metabolism were deleted. Although no elevation in SI production was observed in the deletion strain, it did result in no wastage of MI anymore. Thus additionally, overexpression of the key enzymes, IolG and IolW, was appended to demonstrate that simultaneous overexpression of them enabled complete conversion of all MI into SI. The B. subtilis Cell Factory was improved to yield an SI production rate of 10 g/L/48 h at least. The improved conversion was achieved only in the presence of enriched nutrition in the form of 2% (w/v) Bacto soytone in the medium, which may be due to the increasing demand for regeneration of cofactors.
Jens Nielsen - One of the best experts on this subject based on the ideXlab platform.
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Yeast Systems Biology: Model Organism and Cell Factory.
Biotechnology journal, 2019Co-Authors: Jens NielsenAbstract:For thousands of years, the yeast Saccharomyces cerevisiae (S. cerevisiae) has served as a Cell Factory for the production of bread, beer, and wine. In more recent years, this yeast has also served as a Cell Factory for producing many different fuels, chemicals, food ingredients, and pharmaceuticals. S. cerevisiae, however, has also served as a very important model organism for studying eukaryal biology, and even today many new discoveries, important for the treatment of human diseases, are made using this yeast as a model organism. Here a brief review of the use of S. cerevisiae as a model organism for studying eukaryal biology, its use as a Cell Factory, and how advances in systems biology underpin developments in both these areas, is provided.
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Production of fatty acid-derived oleochemicals and biofuels by synthetic yeast Cell factories
Nature communications, 2016Co-Authors: Yongjin J. Zhou, Jiufu Qin, Zhiwei Zhu, Nicolaas A. Buijs, Verena Siewers, Jens NielsenAbstract:Sustainable production of oleochemicals requires establishment of Cell Factory platform strains. The yeast Saccharomyces cerevisiae is an attractive Cell Factory as new strains can be rapidly implemented into existing infrastructures such as bioethanol production plants. Here we show high-level production of free fatty acids (FFAs) in a yeast Cell Factory, and the production of alkanes and fatty alcohols from its descendants. The engineered strain produces up to 10.4 g l(-1) of FFAs, which is the highest reported titre to date. Furthermore, through screening of specific pathway enzymes, endogenous alcohol dehydrogenases and aldehyde reductases, we reconstruct efficient pathways for conversion of fatty acids to alkanes (0.8 mg l(-1)) and fatty alcohols (1.5 g l(-1)), to our knowledge the highest titres reported in S. cerevisiae. This should facilitate the construction of yeast Cell factories for production of fatty acids derived products and even aldehyde-derived chemicals of high value.
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Kinetic models in industrial biotechnology - Improving Cell Factory performance.
Metabolic engineering, 2014Co-Authors: Joachim Almquist, Jens Nielsen, Marija Cvijovic, Vassily Hatzimanikatis, Mats JirstrandAbstract:An increasing number of industrial bioprocesses capitalize on living Cells by using them as Cell factories that convert sugars into chemicals. These processes range from the production of bulk chemicals in yeasts and bacteria to the synthesis of therapeutic proteins in mammalian Cell lines. One of the tools in the continuous search for improved performance of such production systems is the development and application of mathematical models. To be of value for industrial biotechnology, mathematical models should be able to assist in the rational design of Cell Factory properties or in the production processes in which they are utilized. Kinetic models are particularly suitable towards this end because they are capable of representing the complex biochemistry of Cells in a more complete way compared to most other types of models. They can, at least in principle, be used to in detail understand, predict, and evaluate the effects of adding, removing, or modifying molecular components of a Cell Factory and for supporting the design of the bioreactor or fermentation process. However, several challenges still remain before kinetic modeling will reach the degree of maturity required for routine application in industry. Here we review the current status of kinetic Cell Factory modeling. Emphasis is on modeling methodology concepts, including model network structure, kinetic rate expressions, parameter estimation, optimization methods, identifiability analysis, model reduction, and model validation, but several applications of kinetic models for the improvement of Cell factories are also discussed.
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industrial systems biology of saccharomyces cerevisiae enables novel succinic acid Cell Factory
PLOS ONE, 2013Co-Authors: Jens Nielsen, Jose Manuel Otero, Donatella Cimini, Kiran Raosaheb Patil, Simon Guldberg Poulsen, Lisbeth OlssonAbstract:Saccharomyces cerevisiae is the most well characterized eukaryote, the preferred microbial Cell Factory for the largest industrial biotechnology product (bioethanol), and a robust commerically compatible scaffold to be exploitted for diverse chemical production. Succinic acid is a highly sought after added-value chemical for which there is no native pre-disposition for production and accmulation in S. cerevisiae. The genome-scale metabolic network reconstruction of S. cerevisiae enabled in silico gene deletion predictions using an evolutionary programming method to couple biomass and succinate production. Glycine and serine, both essential amino acids required for biomass formation, are formed from both glycolytic and TCA cycle intermediates. Succinate formation results from the isocitrate lyase catalyzed conversion of isocitrate, and from the α-keto-glutarate dehydrogenase catalyzed conversion of α-keto-glutarate. Succinate is subsequently depleted by the succinate dehydrogenase complex. The metabolic engineering strategy identified included deletion of the primary succinate consuming reaction, Sdh3p, and interruption of glycolysis derived serine by deletion of 3-phosphoglycerate dehydrogenase, Ser3p/Ser33p. Pursuing these targets, a multi-gene deletion strain was constructed, and directed evolution with selection used to identify a succinate producing mutant. Physiological characterization coupled with integrated data analysis of transcriptome data in the metabolically engineered strain were used to identify 2nd-round metabolic engineering targets. The resulting strain represents a 30-fold improvement in succinate titer, and a 43-fold improvement in succinate yield on biomass, with only a 2.8-fold decrease in the specific growth rate compared to the reference strain. Intuitive genetic targets for either over-expression or interruption of succinate producing or consuming pathways, respectively, do not lead to increased succinate. Rather, we demonstrate how systems biology tools coupled with directed evolution and selection allows non-intuitive, rapid and substantial re-direction of carbon fluxes in S. cerevisiae, and hence show proof of concept that this is a potentially attractive Cell Factory for over-producing different platform chemicals.
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establishing a platform Cell Factory through engineering of yeast acetyl coa metabolism
Metabolic Engineering, 2013Co-Authors: Yun Chen, Verena Siewers, Laurent Daviet, Michel Schalk, Jens NielsenAbstract:Production of fuels and chemicals by industrial biotechnology requires efficient, safe and flexible Cell Factory platforms that can be used for production of a wide range of compounds. Here we developed a platform yeast Cell Factory for efficient provision of acetyl-CoA that serves as precursor metabolite for a wide range of industrially interesting products. We demonstrate that the platform Cell Factory can be used to improve the production of alpha-santalene, a plant sesquiterpene that can be used as a perfume by four-fold. This strain would be a useful tool to produce a wide range of acetyl-CoA-derived products.
Kenneth N Timmis - One of the best experts on this subject based on the ideXlab platform.
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efficient production of soluble recombinant single chain fv fragments by a pseudomonas putida strain kt2440 Cell Factory
Microbial Cell Factories, 2011Co-Authors: Thorben Dammeyer, Miriam Steinwand, Sarahc Kruger, Stefan Dubel, Michael Hust, Kenneth N TimmisAbstract:Recombinant antibody fragments have a wide range of applications in research, diagnostics and therapy. For many of these, small fragments like single chain fragment variables (scFv) function well and can be produced inexpensively in bacterial expression systems. Although Escherichia coli K-12 production systems are convenient, yields of different fragments, even those produced from codon-optimized expression systems, vary significantly. Where yields are inadequate, alternative production systems are needed. Pseudomonas putida strain KT2440 is a versatile biosafety strain known for good expression of heterologous genes, so we have explored its utility as a Cell Factory for production of scFvs. We have generated new broad host range scFv expression constructs and assessed their production in the Pseudomonas putida KT2440 host. Two scFvs bind either to human C-reactive protein or to mucin1, proteins of significant medical diagnostic and therapeutic interest, whereas a third is a model anti-lysozyme scFv. The KT2440 antibody expression systems produce scFvs targeted to the periplasmic space that were processed precisely and were easily recovered and purified by single-step or tandem affinity chromatography. The influence of promoter system, codon optimization for P. putida, and medium on scFv yield was examined. Yields of up to 3.5 mg/l of pure, soluble, active scFv fragments were obtained from shake flask cultures of constructs based on the original codon usage and expressed from the Ptac expression system, yields that were 2.5-4 times higher than those from equivalent cultures of an E. coli K-12 expression host. Pseudomonas putida KT2440 is a good Cell Factory for the production of scFvs, and the broad host range constructs we have produced allow yield assessment in a number of different expression hosts when yields in one initially selected are insufficient. High Cell density cultivation and further optimization and refinement of the KT2440 Cell Factory will achieve additional increases in the yields of scFvs.
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Efficient production of soluble recombinant single chain Fv fragments by a Pseudomonas putida strain KT2440 Cell Factory
Microbial Cell Factories, 2011Co-Authors: Thorben Dammeyer, Miriam Steinwand, Sarahc Kruger, Stefan Dubel, Michael Hust, Kenneth N TimmisAbstract:Background Recombinant antibody fragments have a wide range of applications in research, diagnostics and therapy. For many of these, small fragments like single chain fragment variables (scFv) function well and can be produced inexpensively in bacterial expression systems. Although Escherichia coli K-12 production systems are convenient, yields of different fragments, even those produced from codon-optimized expression systems, vary significantly. Where yields are inadequate, alternative production systems are needed. Pseudomonas putida strain KT2440 is a versatile biosafety strain known for good expression of heterologous genes, so we have explored its utility as a Cell Factory for production of scFvs. Results We have generated new broad host range scFv expression constructs and assessed their production in the Pseudomonas putida KT2440 host. Two scFvs bind either to human C-reactive protein or to mucin1, proteins of significant medical diagnostic and therapeutic interest, whereas a third is a model anti-lysozyme scFv. The KT2440 antibody expression systems produce scFvs targeted to the periplasmic space that were processed precisely and were easily recovered and purified by single-step or tandem affinity chromatography. The influence of promoter system, codon optimization for P. putida , and medium on scFv yield was examined. Yields of up to 3.5 mg/l of pure, soluble, active scFv fragments were obtained from shake flask cultures of constructs based on the original codon usage and expressed from the Ptac expression system, yields that were 2.5-4 times higher than those from equivalent cultures of an E. coli K-12 expression host. Conclusions Pseudomonas putida KT2440 is a good Cell Factory for the production of scFvs, and the broad host range constructs we have produced allow yield assessment in a number of different expression hosts when yields in one initially selected are insufficient. High Cell density cultivation and further optimization and refinement of the KT2440 Cell Factory will achieve additional increases in the yields of scFvs.
Thorben Dammeyer - One of the best experts on this subject based on the ideXlab platform.
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efficient production of soluble recombinant single chain fv fragments by a pseudomonas putida strain kt2440 Cell Factory
Microbial Cell Factories, 2011Co-Authors: Thorben Dammeyer, Miriam Steinwand, Sarahc Kruger, Stefan Dubel, Michael Hust, Kenneth N TimmisAbstract:Recombinant antibody fragments have a wide range of applications in research, diagnostics and therapy. For many of these, small fragments like single chain fragment variables (scFv) function well and can be produced inexpensively in bacterial expression systems. Although Escherichia coli K-12 production systems are convenient, yields of different fragments, even those produced from codon-optimized expression systems, vary significantly. Where yields are inadequate, alternative production systems are needed. Pseudomonas putida strain KT2440 is a versatile biosafety strain known for good expression of heterologous genes, so we have explored its utility as a Cell Factory for production of scFvs. We have generated new broad host range scFv expression constructs and assessed their production in the Pseudomonas putida KT2440 host. Two scFvs bind either to human C-reactive protein or to mucin1, proteins of significant medical diagnostic and therapeutic interest, whereas a third is a model anti-lysozyme scFv. The KT2440 antibody expression systems produce scFvs targeted to the periplasmic space that were processed precisely and were easily recovered and purified by single-step or tandem affinity chromatography. The influence of promoter system, codon optimization for P. putida, and medium on scFv yield was examined. Yields of up to 3.5 mg/l of pure, soluble, active scFv fragments were obtained from shake flask cultures of constructs based on the original codon usage and expressed from the Ptac expression system, yields that were 2.5-4 times higher than those from equivalent cultures of an E. coli K-12 expression host. Pseudomonas putida KT2440 is a good Cell Factory for the production of scFvs, and the broad host range constructs we have produced allow yield assessment in a number of different expression hosts when yields in one initially selected are insufficient. High Cell density cultivation and further optimization and refinement of the KT2440 Cell Factory will achieve additional increases in the yields of scFvs.
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Efficient production of soluble recombinant single chain Fv fragments by a Pseudomonas putida strain KT2440 Cell Factory
Microbial Cell Factories, 2011Co-Authors: Thorben Dammeyer, Miriam Steinwand, Sarahc Kruger, Stefan Dubel, Michael Hust, Kenneth N TimmisAbstract:Background Recombinant antibody fragments have a wide range of applications in research, diagnostics and therapy. For many of these, small fragments like single chain fragment variables (scFv) function well and can be produced inexpensively in bacterial expression systems. Although Escherichia coli K-12 production systems are convenient, yields of different fragments, even those produced from codon-optimized expression systems, vary significantly. Where yields are inadequate, alternative production systems are needed. Pseudomonas putida strain KT2440 is a versatile biosafety strain known for good expression of heterologous genes, so we have explored its utility as a Cell Factory for production of scFvs. Results We have generated new broad host range scFv expression constructs and assessed their production in the Pseudomonas putida KT2440 host. Two scFvs bind either to human C-reactive protein or to mucin1, proteins of significant medical diagnostic and therapeutic interest, whereas a third is a model anti-lysozyme scFv. The KT2440 antibody expression systems produce scFvs targeted to the periplasmic space that were processed precisely and were easily recovered and purified by single-step or tandem affinity chromatography. The influence of promoter system, codon optimization for P. putida , and medium on scFv yield was examined. Yields of up to 3.5 mg/l of pure, soluble, active scFv fragments were obtained from shake flask cultures of constructs based on the original codon usage and expressed from the Ptac expression system, yields that were 2.5-4 times higher than those from equivalent cultures of an E. coli K-12 expression host. Conclusions Pseudomonas putida KT2440 is a good Cell Factory for the production of scFvs, and the broad host range constructs we have produced allow yield assessment in a number of different expression hosts when yields in one initially selected are insufficient. High Cell density cultivation and further optimization and refinement of the KT2440 Cell Factory will achieve additional increases in the yields of scFvs.
Kosei Tanaka - One of the best experts on this subject based on the ideXlab platform.
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A bacterial Cell Factory converting glucose into scyllo-inositol, a therapeutic agent for Alzheimer's disease.
Communications biology, 2020Co-Authors: Christophe Michon, Choong-min Kang, Sophia Karpenko, Kosei Tanaka, Shu Ishikawa, Ken-ichi YoshidaAbstract:A rare stereoisomer of inositol, scyllo-inositol, is a therapeutic agent that has shown potential efficacy in preventing Alzheimer’s disease. Mycobacterium tuberculosis ino1 encoding myo-inositol-1-phosphate (MI1P) synthase (MI1PS) was introduced into Bacillus subtilis to convert glucose-6-phosphate (G6P) into MI1P. We found that inactivation of pbuE elevated intraCellular concentrations of NAD+·NADH as an essential cofactor of MI1PS and was required to activate MI1PS. MI1P thus produced was dephosphorylated into myo-inositol by an intrinsic inositol monophosphatase, YktC, which was subsequently isomerized into scyllo-inositol via a previously established artificial pathway involving two inositol dehydrogenases, IolG and IolW. In addition, both glcP and glcK were overexpressed to feed more G6P and accelerate scyllo-inositol production. Consequently, a B. subtilis Cell Factory was demonstrated to produce 2 g L−1 scyllo-inositol from 20 g L−1 glucose. This Cell Factory provides an inexpensive way to produce scyllo-inositol, which will help us to challenge the growing problem of Alzheimer’s disease in our aging society. Michon et al. describe the use of a recombinant Bacillus subtilis as a Cell Factory capable of producing scyllo-inositol, a therapeutic compound for Alzheimer’s disease, from inexpensive glucose. They demonstrate that it could produce 2 g L−1 of scyllo-inositol from 20 g L−1 glucose.
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A bacterial Cell Factory converting glucose into scyllo-inositol, a therapeutic agent for Alzheimer's disease
Communications Biology, 2020Co-Authors: Christophe Michon, Choong-min Kang, Sophia Karpenko, Kosei Tanaka, Shu Ishikawa, Ken-ichi YoshidaAbstract:A rare stereoisomer of inositol, scyllo-inositol, is a therapeutic agent that has shown potential efficacy in preventing Alzheimer's disease. Mycobacterium tuberculosis ino1 encoding myo-inositol-1-phosphate (MI1P) synthase (MI1PS) was introduced into Bacillus subtilis to convert glucose-6-phosphate (G6P) into MI1P. We found that inactivation of pbuE elevated intra-Cellular concentrations of NAD + ·NADH as an essential cofactor of MI1PS and was required to activate MI1PS. MI1P thus produced was dephosphorylated into myo-inositol by an intrinsic inositol monophosphatase, YktC, which was subsequently isomerized into scyllo-inositol via a previously established artificial pathway involving two inositol dehydrogenases, IolG and IolW. In addition, both glcP and glcK were overexpressed to feed more G6P and accelerate scyllo-inositol production. Consequently, a B. subtilis Cell Factory was demonstrated to produce 2 g L −1 scyllo-inositol from 20 g L −1 glucose. This Cell Factory provides an inexpensive way to produce scyllo-inositol, which will help us to challenge the growing problem of Alzheimer's disease in our aging society.
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A new-generation of Bacillus subtilis Cell Factory for further elevated scyllo -inositol production
Microbial cell factories, 2017Co-Authors: Kosei Tanaka, Shu Ishikawa, Shinji Takenaka, Ayane Natsume, Ken-ichi YoshidaAbstract:A stereoisomer of inositol, scyllo-inositol (SI), has been regarded as a promising therapeutic agent for Alzheimer’s disease. However, this compound is relatively rare, whereas another stereoisomer of inositol, myo-inositol (MI) is abundant in nature. Bacillus subtilis 168 has the ability to metabolize inositol stereoisomers, including MI and SI. Previously, we reported a B. subtilis Cell Factory with modified inositol metabolism that converts MI into SI in the culture medium. The strain was constructed by deleting all genes related to inositol metabolism and overexpressing key enzymes, IolG and IolW. By using this strain, 10 g/l of MI initially included in the medium was completely converted into SI within 48 h of cultivation in a rich medium containing 2% (w/v) Bacto soytone. When the initial concentration of MI was increased to 50 g/l, conversion was limited to 15.1 g/l of SI. Therefore, overexpression systems of IolT and PntAB, the main transporter of MI in B. subtilis and the membrane-integral nicotinamide nucleotide transhydrogenase in Escherichia coli respectively, were additionally introduced into the B. subtilis Cell Factory, but the conversion efficiency hardly improved. We systematically determined the amount of Bacto soytone necessary for ultimate conversion, which was 4% (w/v). As a result, the conversion of SI reached to 27.6 g/l within 48 h of cultivation. The B. subtilis Cell Factory was improved to yield a SI production rate of 27.6 g/l/48 h by simultaneous overexpression of IolT and PntAB, and by addition of 4% (w/v) Bacto soytone in the conversion medium. The concentration of SI was increased even in the stationary phase perhaps due to nutrients in the Bacto soytone that contribute to the conversion process. Thus, MI conversion to SI may be further optimized via identification and control of these unknown nutrients.
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A second-generation Bacillus Cell Factory for rare inositol production.
Bioengineered, 2014Co-Authors: Kosei Tanaka, Shinji Takanaka, Ken-ichi YoshidaAbstract:Some rare inositol stereoisomers are known to exert specific health-promoting effects, including scyllo-inositol (SI), which is a promising therapeutic agent for Alzheimer disease. We recently reported a Bacillus subtilis Cell Factory that performed the efficient production of SI from the cheapest and most abundant isomer myo-inositol (MI). In the Cell Factory all “useless” genes involved in MI and SI metabolism were deleted and overexpression of the key enzymes, IolG and IolW, was appended. It converted 10 g/L MI into the same amount of SI in 48 h of cultivation. In this addendum, we discuss further improvement in the Cell Factory and its possible applications.
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An improved Bacillus subtilis Cell Factory for producing scyllo-inositol, a promising therapeutic agent for Alzheimer's disease.
Microbial cell factories, 2013Co-Authors: Kosei Tanaka, Shinji Takenaka, Shintaro Tajima, Ken-ichi YoshidaAbstract:Bacillus subtilis 168 possesses an efficient pathway to metabolize some of the stereoisomers of inositol, including myo-inositol (MI) and scyllo-inositol (SI). Previously we reported a prototype of a B. subtilis Cell Factory with modified inositol metabolism that converts MI into SI in the culture medium. However, it wasted half of initial 1.0% (w/v) MI, and the conversion was limited to produce only 0.4% (w/v) SI. To achieve a more efficient SI production, we attempted additional modifications. All “useless” genes involved in MI and SI metabolism were deleted. Although no elevation in SI production was observed in the deletion strain, it did result in no wastage of MI anymore. Thus additionally, overexpression of the key enzymes, IolG and IolW, was appended to demonstrate that simultaneous overexpression of them enabled complete conversion of all MI into SI. The B. subtilis Cell Factory was improved to yield an SI production rate of 10 g/L/48 h at least. The improved conversion was achieved only in the presence of enriched nutrition in the form of 2% (w/v) Bacto soytone in the medium, which may be due to the increasing demand for regeneration of cofactors.