Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jbiosc.2023.05.013

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Language：English   Publishing type：Research paper (scientific journal)  

Clostridioides difficile causes nosocomial antibiotic-associated diarrhea on a global scale. Susceptibility to C. difficile infection (CDI) is influenced by the composition and metabolism of gut microbiota, which in turn are affected by diet. However, the mechanism underlying the interplay between diet and gut microbiota that modulates susceptibility to CDI remains unclear. Here, we show that a soy protein diet increases the mortality of antibiotic-treated, C. difficile-infected mice while also enhancing the intestinal levels of amino acids (aas) and relative abundance of Lactobacillus genus. Indeed, Ligilactobacillus murinus-mediated fermentation of soy protein results in the generation of aas, thereby promoting C. difficile growth, and the process involves the anchored cell wall proteinase PrtP. Thus, mutual interaction between dietary protein and the gut microbiota is a critical factor affecting host susceptibility to CDI, suggesting that dietary protein sources can be an important determinant in controlling the disease.

DOI： 10.1016/j.celrep.2022.111332

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/cbic.202200210

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/cbic.202200210

Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

Paraburkholderia terrae strain KU-15 has been investigated for its ability to degrade 2-nitrobenzoate. Here, we report the complete 10,422,345-bp genome of this microorganism, which consists of six circular replicons containing 9,483 protein-coding sequences. The genome carries genes that are potentially responsible for 2-nitrobenzoate and 4-nitirobenzoate degradation.

DOI： 10.1128/mra.00373-22

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: There has been an increasing demand for optically pure d-lactic and l-lactic acid for the production of stereocomplex-type polylactic acid. The d-lactic acid production from lignocellulosic biomass is important owing to its great abundance in nature. Corn steep liquor (CSL) is a cheap nitrogen source used for industrial fermentation, though it contains a significant amount of l-lactic acid, which decreases the optical purity of d-lactic acid produced. METHOD AND RESULTS: To remove l-lactic acid derived from the CSL-based medium, l-lactate oxidase (LoxL) from Enterococcus sp. NBRC 3427 was expressed in an engineered Lactiplantibacillus plantarum (formally called Lactobacillus plantarum) strain KOLP7, which exclusively produces d-lactic acid from both hexose and pentose sugars. When the resulting strain was applied for d-lactic acid fermentation from the mixed sugars consisting of the major constituent sugars of lignocellulose (35 g L-1 glucose, 10 g L-1 xylose, and 5 g L-1 arabinose) using the medium containing 10 g L-1 CSL, it completely removed l-lactic acid derived from CSL (0.52 g L-1 ) and produced 41.7 g L-1 of d-lactic acid. The l-lactic acid concentration was below the detection limit, and improvement in the optical purity of d-lactic acid was observed (from 98.2% to > 99.99%) by the overexpression of LoxL. CONCLUSION AND IMPLICATIONS: The LoxL-mediated consumption of l-lactic acid would enable the production of optically pure d-lactic acid in any medium contaminated by l-lactic acid.

DOI： 10.1002/biot.202100331

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The demand for the amino acid l-cysteine is increasing in the food, cosmetic, and pharmaceutical industries. Conventionally, the commercial production of l-cysteine is achieved by its extraction from the acid hydrolysate of hair and feathers. However, this production method is associated with the release of environmentally hazardous wastewater. Additionally, l-cysteine produced from animal sources cannot be halal-certified, which limits the market size. Although recent studies have developed an alternative commercial l-cysteine production method based on microbial fermentation, the production yield was insufficient owing to the cytotoxicity of l-cysteine against the host cells. In a previous study, we had developed an in vitrol-cysteine production method with a combination of 11 thermophilic enzymes, which yielded 10.5 mM l-cysteine from 20 mM glucose. In this study, we performed re-screening for enzymes catalyzing the rate-limiting steps of the in vitro pathway. Subsequently, the genes encoding enzymes necessary for the in vitro synthesis of l-cysteine were assembled in an expression vector and co-expressed in a single strain. To prevent the synthesis of hydrogen peroxide (H2O2), which is a byproduct and inhibits the enzyme activity, the redox balance in this biosynthetic pathway was maintained by replacing the H2O2-forming NADH oxidase with another enzymatic reaction in which pyruvate was used as a sacrificial substrate. The re-designed in vitro synthetic pathway resulted in the production of 28.2 mM l-cysteine from 20 mM glucose with a molar yield of 70.5%.

DOI： 10.1016/j.jbiosc.2021.09.003

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system is a valuable genome editing tool for microorganisms. However, the commonly used Cas9 nuclease derived from Streptococcus pyogenes (SpCas9) is not applicable to many industrially relevant bacteria, due to its cytotoxicity and large size (1368 amino acids [aa]). We developed an alternative genome editing system using a miniature Cas12f1 nuclease (529 aa) derived from an uncultured archaeon, Un1Cas12f1. When editing four dispensable genes in Escherichia coli MG1655 and BW25113, the CRISPR/Un1Cas12f1 system showed higher efficiency (63%-100%) than the CRISPR/SpCas9 system (50%-79%). The CRISPR/Un1Cas12f1 genome editing system is expected to be applied to the genome editing of a wide variety of bacteria.

DOI： 10.1016/j.jbiosc.2021.04.009

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Coenzyme A (CoA) is an essential cofactor present in all domains of life and is involved in numerous metabolic pathways, including fatty acid metabolism, pyruvate oxidation through the TCA cycle, and production of secondary metabolites. This characteristic makes CoA a commercially valuable compound in the pharmaceutical, cosmetic, and clinical industries. However, CoA is difficult to accumulate in living cells at a high level as it is consumed in multiple metabolic pathways, hampering its manufacturing by typical cell cultivation and extraction approaches. The feedback inhibition by CoA to a biosynthetic enzyme, pantothenate kinase (PanK), is also a serious obstacle for high-titer production of CoA. To overcome this challenge, in vitro production of CoA, in which the CoA biosynthetic pathway was reconstructed outside of cells using recombinant thermophilic enzymes, was performed. The in vitro pathway was designed to be insensitive to the feedback inhibition of CoA using a CoA-insensitive type-III PanK from the thermophilic bacterium Thermus thermophilus Furthermore, a statistical approach using Design of Experiments was employed to rationally determine the enzyme loading ratio to maximize CoA production rate. Consequently, 0.94 mM CoA could be produced from 2 mM d-pantetheine through the designed pathway. We hypothesized that the insufficient conversion yield is attributed to the high Km value of T. thermophilus PanK towards ATP. Based on these observations, possible CoA regulation mechanisms in T. thermophilus and approaches to improve the feasibility of CoA production through the in vitro pathway have been investigated.IMPORTANCEThe biosynthesis of coenzyme A (CoA) in bacteria and eukaryotes is regulated by feedback inhibition targeting type-I and type-II pantothenate kinase (PanK). Type-III PanK is only found in bacteria and is generally insensitive to CoA. Previously, type-III PanK from the hyperthermophilic bacterium Thermotoga maritima was shown to defy this typical characteristic, and instead shows inhibition towards CoA. In the present study, phylogenetic analysis combined with functional analysis of type-III PanK from thermophiles revealed that the CoA-sensitive behavior of type-III PanK from T. maritima is uncommon. We cloned type-III PanKs from Thermus thermophilus and Geobacillus sp. 30 and showed that neither enzyme's activities were inhibited by CoA. Furthermore, we utilized type-III PanK for a one-pot cascade reaction to produce CoA.

DOI： 10.1128/AEM.00541-21

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Cupriavidus necator strain A-04 has shown 16S rRNA gene identity to the well-known industrial strain C. necator H16. Nevertheless, the cell characteristics and polyhydroxyalkanoate (PHA) production ability of C. necator strain A-04 were different from those of C. necator H16. This study aimed to express PHA biosynthesis genes of C. necator strain A-04 in Escherichia coli via an arabinose-inducible expression system. In this study, the PHA biosynthesis operon of C. necator strain A-04, consisting of three genes encoding acetyl-CoA acetyltransferase (phaAA-04, 1182 bp, 40.6 kDa), acetoacetyl-CoA reductase (phaBA-04, 741 bp, 26.4 kDa) and PHB synthase Class I (phaCA-04, 1770 bp), was identified. Sequence analysis of the phaAA-04, phaBA-04, and phaCA-04 genes revealed that phaCA-04 was 99% similar to phaCH16 from C. necator H16. The difference in amino acid residue situated at position 122 of phaCA-04 was proline, whereas that of C. necator H16 was leucine. The intact phaCABA-04 operon was cloned into the arabinose-inducible araBAD promoter and transformed into E. coli strains Top 10, JM109 and XL-1 blue. The results showed that optimal conditions obtained from shaken flask experiments yielded 6.1 ± 1.1 g/L cell dry mass (CDM), a PHB content of 93.3 ± 0.9% (w/w) and a productivity of 0.24 g/(L⋅h), whereas the wild-type C. necator strain A-04 accumulated 78% (w/w) PHB with a productivity of 0.09 g/(L⋅h). Finally, for the scaled-up studies, fed-batch cultivations by pH-stat control in a 5-L fermenter of E. coli strains XL1-Blue harboring pBAD/Thio-TOPO-phaCABA-04 and pColdTF-phaCABA-04 in MR or LB medium, leading to a PHB production of 31.4 ± 0.9 g/L at 54 h with a PHB content of 83.0 ± 3.8% (w/w), a CDM of 37.8 ± 1.2 g/L, a Y
P/S
value of 0.39 g PHB/g glucose and a productivity of 0.6 g PHB/(L⋅h) using pColdTF-phaCABA-04 in MR medium. In addition, PHB production was 29.0 ± 1.1 g/L with 60.2 ± 2.3% PHB content in the CDM of 53.1 ± 1.0 g/L, a Y
P/S
value of 0.21 g PHB/g glucose and a productivity of 0.4 g PHB/(L⋅h) using pBAD/Thio-TOPO-phaCABA-04 in LB medium. Thus, a relatively high PHB concentration and productivity were achieved, which demonstrated the possibility of industrial production of PHB.

DOI： 10.3389/fbioe.2021.661096

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1007/s00792-021-01238-9

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Other Link： https://link.springer.com/article/10.1007/s00792-021-01238-9/fulltext.html

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

S-Adenosylmethionine (SAM)-dependent methyltransferases are important tools for the biocatalytic methylation of diverse biomolecules. Methylation by a whole-cell biocatalyst allows the utilization of intrinsic SAM and its regeneration system, which consists of a cyclic and multi-step enzymatic cascade. However, low intracellular availability of 5-methyl-tetrahydrofolate (5-methyl-THF), which functions as a methyl group donor, limits SAM regeneration. Here, we integrated methanol metabolism with 5-methyl-THF formation into SAM-dependent methylation system in Escherichia coli, driven by heterologously expressed methanol dehydrogenase (MDH). The coupling of MDH-catalyzed methanol oxidation with the E. coli endogenous reactions enhances the formation of 5-methyl-THF using methanol as a source of methyl group, thereby promoting both the SAM regeneration and methylation reactions. Co-expression of the mutant MDH2 from Cupriavidus necator N-1 with the O-methyltransferase 5 from Streptomyces avermitilis MA-4680 enhanced O-methylation of esculetin 1.4-fold. Additional overexpression of the E. coli endogenous 5,10-methylene-THF reductase, which catalyzes the last step of 5-methyl-THF formation, further enhanced the methylation reaction by 1.9-fold. Together with deregulation of SAM biosynthesis, the titer of methylated compounds was increased about 20-fold (from 0.023 mM to 0.44 mM). The engineered E. coli strain with enhanced 5-methyl-THF formation is now available as a chassis strain for the production of a variety of methylated compounds.

DOI： 10.3390/catal10091001

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Authorship：Lead author,　Corresponding author   Language：English  

In vitro metabolic engineering is an emerging framework for bioproduction systems, in which synthetic metabolic pathways are constructed using a limited number of enzymes. Employment of thermophilic enzymes as catalytic elements in pathways enables the use of simple heat purification of recombinantly expressed enzymes. However, thermophilic enzymes are generally incompatible with thermo-labile substrates and intermediates. In previous work, we showed that lactate production through a non-ATP forming chimeric Embden-Meyerhof (EM) pathway required careful adjustment of the metabolic fluxes by continuous substrate feeding and optimization of enzyme ratios to prevent the accumulation and degradation of thermo-labile intermediates (Ye et al., Microb. Cell Fact., 11, 120, 2012). In the study reported here, we constructed an in vitro non-phosphorylative Entner-Doudoroff (np-ED) pathway. Because of the high thermal stability of the metabolic intermediates in the np-ED pathway, it could prevent degradation of accumulated metabolic intermediates caused by inconstant metabolic fluxes, and batch-mode production of lactate in which the concentrations of the substrate and metabolic intermediates change dynamically could be achieved. By combining the enzymes involved in the np-ED pathway and lactate dehydrogenase, 20.9 mM lactate was produced from 10 mM glucose and 1 mM gluconate in 6 h.

DOI： 10.1016/j.jbiosc.2019.09.010

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Growth temperature is one of the most representative biological parameters for characterizing living organisms. Prokaryotes have been isolated from various temperature environments and show wide diversity in their growth temperatures. We herein constructed a database of growth TEMPeratures of Usual and RAre prokaryotes (TEMPURA, http://togodb.org/db/tempura), which contains the minimum, optimum, and maximum growth temperatures of 8,639 prokaryotic strains. Growth temperature information is linked with taxonomy IDs, phylogenies, and genomic information. TEMPURA provides useful information to researchers working on biotechnological applications of extremophiles and their biomolecules as well as those performing fundamental studies on the physiological diversity of prokaryotes.

DOI： 10.1264/jsme2.ME20074

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Cysteine is a commercially valuable amino acid with an increasing demand in the food, cosmetic, and pharmaceutical industries. Although cysteine is conventionally manufactured by extraction from animal proteins, this method has several problems, such as troublesome waste-water treatment and incompatibility with some dietary restrictions. Fermentative production of cysteine from plant-derived substrates is a promising alternative for the industrial production of cysteine. However, it often suffers from low product yield as living organisms are equipped with various regulatory systems to control the intracellular cysteine concentration at a moderate level. In this study, we constructed an in vitro cysteine biosynthetic pathway by assembling 11 thermophilic enzymes. The in vitro pathway was designed to be insensitive to the feedback regulation by cysteine and to balance the intra-pathway consumption and regeneration of cofactors. A kinetic model for the in vitro pathway was built using rate equations of individual enzymes and used to optimize the loading ratio of each enzyme. Consequently, 10.5 mM cysteine could be produced from 20 mM glucose through the optimized pathway. However, the observed yield and production rate of the assay were considerably lower than those predicted by the model. Determination of cofactor concentrations in the reaction mixture indicated that the inconsistency between the model and experimental assay could be attributed to the depletion of ATP and ADP, likely due to host-derived, thermo-stable enzyme(s). Based on these observations, possible approaches to improve the feasibility of cysteine production through an in vitro pathway have been discussed.

DOI： 10.1007/s00253-019-10061-4

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DOI： 10.1186/s12934-019-1125-x

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DOI： 10.1016/j.jbiosc.2018.07.011

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DOI： 10.1093/femsle/fny215

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Wiley-VCH Verlag  

Fermentative production of optically pure lactic acid (LA) has attracted great interest because of the increased demand for plant-based plastics. For cost-effective LA production, an engineered Lactobacillus plantarum NCIMB 8826 strain, which enables the production of optically pure l-LA from raw starch, is constructed. The wild-type strain produces a racemic mixture of d- and l-LA from pyruvate by the action of the respective lactate dehydrogenases (LDHs). Therefore, the gene encoding D-LDH (ldhD) is deleted. Although no decrease in d-LA formation is observed in the ΔldhD mutant, additional disruption of the operon encoding lactate racemase (larA-E), which catalyzes the interconversion between d- and l-LA, completely abolished d-LA production. From 100 g L−1 glucose, the ΔldhD ΔlarA-E mutant produces 87.0 g L−1 of l-LA with an optical purity of 99.4%. Subsequently, a plasmid is introduced into the ΔldhD ΔlarA-E mutant for the secretion of α-amylase from Streptococcus bovis 148. The resulting strain could produce 50.3 g L−1 of l-LA from raw corn starch with a yield of 0.91 (g per g of consumed sugar) and an optical purity of 98.6%. The engineered L. plantarum strain would be useful in the production of l-LA from starchy materials.

DOI： 10.1002/biot.201700517

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

A simple technology for phosphate (P (i) ) recovery has been developed using a bifunctional adsorption-aggregation agent. The bifunctional agent was prepared by soaking calcium silicates in hydrochloric acid solution. Importantly, recyclable calcium silicates were available almost free of charge from the cement industry and also from the steel industry. The acid treatment was essential not only for enhancing the ability of calcium silicates to remove P (i) from aqueous solution but also for enabling the high settleability of removed P (i) . On-site experiments using a mobile plant showed that approximately 80% P (i) could be recovered from anaerobic sludge digestion liquor at a wastewater treatment plant. This technology has the potential to offer a simple, compact service for recycling P (i) from wastewater to farmland in rural areas.

DOI： 10.1007/s13280-017-0976-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

NAD (NAD(+)) is a cofactor related to many cellular processes. This cofactor is known to be unstable, especially at high temperatures, where it chemically decomposes to nicotinamide and ADP-ribose. Bacteria, yeast, and higher organisms possess the salvage pathway for reconstructing NAD(+) from these decomposition products; however, the importance of the salvage pathway for survival is not well elucidated, except for in pathogens lacking the NAD(+) de novo synthesis pathway. Herein, we report the importance of the NAD(+) salvage pathway in the thermophilic bacterium Thermus thermophilus HB8 at high temperatures. We identified the gene encoding nicotinamidase (TTHA0328), which catalyzes the first reaction of the NAD(+) salvage pathway. This recombinant enzyme has a high catalytic activity against nicotinamide (K-m of 17 mu M, k(cat) of 50 s(-1), k(cat)/K-m of 3.0 x 10(3) s(-1).mM(-1)). Deletion of this gene abolished nicotinamide deamination activity in crude extracts of T. thermophilus and disrupted the NAD(+) salvage pathway in T. thermophilus. Disruption of the salvage pathway led to the severe growth retardation at a higher temperature (80 degrees C), owing to the drastic decrease in the intracellular concentrations of NAD(+) and NADH.
IMPORTANCE NAD(+) and other nicotinamide cofactors are essential for cell metabolism. These molecules are unstable and decompose, even under the physiological conditions in most organisms. Thermophiles can survive at high temperatures where NAD(+) decomposition is, in general, more rapid. This study emphasizes that NAD(+) instability and its homeostasis can be one of the important factors for thermophile survival in extreme temperatures.

DOI： 10.1128/JB.00359-17

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

Chitin is the second most abundant organic compound on the planet and thus has been regarded as an alternative resource to petroleum feedstocks. One of the key challenges in the biological conversion of biomass-derived polysaccharides, such as cellulose and chitin, is to close the gap between optimum temperatures for enzymatic saccharification and microbial fermentation and to implement them in a single bioreactor. To address this issue, in the present study, we aimed to perform an in vitro, one-pot bioconversion of chitin to pyruvate, which is a precursor of a wide range of useful metabolites. Twelve thermophilic enzymes, including that for NAD(+) regeneration, were heterologously produced in Escherichia coli and semi-purified by heat treatment of the crude extract of recombinant cells. When the experimentally decided concentrations of enzymes were incubated with 0.5 mg mL(-1) colloidal chitin (equivalent to 2.5 mM N-acetylglucosamine unit) and an adequate set of cofactors at 70 degrees C, 0.62 mM pyruvate was produced in 5 h. Despite the use of a cofactor-balanced pathway, determination of the pool sizes of cofactors showed a rapid decrease in ATP concentration, most probably due to the thermally stable ATP-degrading enzyme(s) derived from the host cell. Integration of an additional enzyme set of thermophilic adenylate kinase and polyphosphate kinase led to the deceleration of ATP degradation, and the final product titer was improved to 2.1 mM. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2017.04.013

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Language：English   Publisher：KeAi Communications Co.  

Bio-based chemical production has drawn attention regarding the realization of a sustainable society. In vitro metabolic engineering is one of the methods used for the bio-based production of value-added chemicals. This method involves the reconstitution of natural or artificial metabolic pathways by assembling purified/semi-purified enzymes in vitro. Enzymes from distinct sources can be combined to construct desired reaction cascades with fewer biological constraints in one vessel, enabling easier pathway design with high modularity. Multiple modules have been designed, built, tested, and improved by different groups for different purpose. In this review, we focus on these in vitro metabolic engineering modules, especially focusing on the carbon metabolism, and present an overview of input modules, output modules, and other modules related to cofactor management.

DOI： 10.1016/j.synbio.2017.06.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

Directed evolution of enantio-selective carbonyl reductase from Ogataea minuta was conducted to improve the operational stability of the enzyme. A mutant library was constructed by an error-prone PCR and screened using a newly developed colorimetric assay. The stability of a mutant with two amino acid substitutions was significantly higher than that of the wild type at 50 degrees C in the presence of dimethyl sulfoxide. Site-directed mutagenesis analysis showed that the improved stability of the enzyme can be attributed to the amino acid substitution of V166A. The half-lives of the V166A mutant were 11- and 6.1-times longer than those of the wild type at 50 degrees C in the presence and absence, respectively, of 20% (v/v) dimethyl sulfoxide. No significant differences in the substrate specificity and enantio-selectivity of the enzyme were observed. The mutant enzyme converted 60 mM 2,2,2-trifluoroacetophenone to (R)-(-)-alpha-(trifluoromethyl)benzyl alcohol in a molar yield of 71% whereas the conversion yield with an equivalent concentration of the wild-type enzyme was 27%. (C) 2017, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2017.01.016

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Simultaneous saccharification and fermentation (SSF) of d-lactic acid was performed using brown rice as both a substrate and a nutrient source. An engineered Lactobacillus plantarum NCIMB 8826 strain, in which the EY-lactate dehydrogenase gene was disrupted, produced 97.7 g/L d-lactic acid from 20% (w/v) brown rice without any nutrient supplementation. However, a significant amount of glucose remained unconsumed and the yield of lactic acid was as low as 0.75 (g/g-glucose contained in brown rice). Interestingly, the glucose consumption was significantly improved by adapting L. plantarum cells to the low-pH condition during the early stage of SSF (8-17 h). As a result, 117.1 g/L d-lactic acid was produced with a high yield of 0.93 and an optical purity of 99.6% after 144 h of fermentation. SSF experiments were repeatedly performed for ten times and d-lactic acid was stably produced using recycled cells (118.4-129.8 g/L). On average, d-lactic acid was produced with a volumetric productivity of 2.18 g/L/h over 48 h.

DOI： 10.1007/s00253-016-7976-8

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

A mobile pilot-scale plant was developed for the in situ examination and demonstration of phosphate (P-i) recovery from wastewater. The mobile pilot plant consisted of a 1000-L reinforced-plastic reactor, a self-made filter, and ancillary equipment and was set up on a 1.5-tonne motor truck for transport. Five separate in situ experiments were carried out using the mobile pilot plant to evaluate the ability of amorphous calcium silicate hydrates (A-CSHs) to recover P-i from anaerobic sludge digestion liquor at a wastewater treatment plant. On average, approximately 80% P-i could be recovered from the anaerobic sludge digestion liquor by a process consisting of 20-min mixing, 30-min settling, and 90-min filtration. Approximately 20% of the dry weight of the recovered product was citrate-soluble P2O5. The levels of heavy metals such as Cd, As, Pb, Ni, and Cr were much lower than their regulatory standards for fertilizer. The efficacy of the recovered product as P-i fertilizer was confirmed by plant cultivation tests using the leaf vegetable Komatsuna (Brassica rapa L. var. perviridis). The present study showed that the mobile pilot plant is useful as a simple, potentially low-cost tool for the in situ examination and demonstration of Pi recovery from wastewater. (C) 2016 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.seppur.2016.06.040

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A simple technology for phosphorus (P) recovery from aqueous solution has been developed using acid treated concrete sludge (A-CS) as a bifunctional adsorption-aggregation agent. Dried particles of concrete sludge (CS), an alkaline waste containing hydrated cement and fine aggregates, were soaked in 1.3 M HCl at a concentration of 0.1 g/mL for 60 min. The HCl-soaking treatment solubilized alkaline substances such as Ca(OH)(2) and CaCO3, thereby releasing approximately 87% Ca2+ from the CS particles to the acid solution. When A-CS was added to 500-mL synthetic anaerobic sludge digestion liquor containing 273 mg/L of phosphate (Pi) at the Ca/P molar ratio of 2.5, A-CS showed 20 times higher 131 removal efficiency than that of untreated CS particles. Although A-CS could precipitate 72% P-i in 5-min free sedimentation, the Ca2+-rich liquid fraction of A-CS alone led to the precipitation of only 48% P-i. This suggests that the solid fraction of A-CS can serve as an auxiliary aggregation agent. When P-i recovery was examined using anaerobic sludge digestion liquor from a full-scale wastewater treatment, A-CS could recover 96% P-i at the Ca/P molar ratio of 2.5. Citrate-soluble P2O5 accounted for 19% of the dry weight of the recovered P-i product. The levels of heavy metals such as As, Cd, Pb, Ni, and Cr in the recovered P-i product were much below their regulatory standards for fertilizers. (C) 2016 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.seppur.2016.03.054

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Excellent thermal and operational stabilities of thermophilic enzymes can greatly increase the applicability of biocatalysis in various industrial fields. However, thermophilic enzymes are generally incompatible with thermo-labile substrates, products, and cofactors, since they show the maximal activities at high temperatures. Despite their pivotal roles in a wide range of enzymatic redox reactions, NAD(P)(+) and NAD(P)H exhibit relatively low stabilities at high temperatures, tending to be a major obstacle in the long-term operation of biocatalytic chemical manufacturing with thermophilic enzymes. In this study, we constructed an in vitro artificial metabolic pathway for the salvage synthesis of NAD(+) from its degradation products by the combination of eight thermophilic enzymes. The enzymes were heterologously produced in recombinant Escherichia coli and the heat -treated crude extracts of the recombinant cells were directly used as enzyme solutions. When incubated with experimentally optimized concentrations of the enzymes at 60 degrees C, the NAD(+) concentration could be kept almost constant for 15 h. (C) 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ymben.2016.02.005

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Acetolactate synthase and pyruvate decarboxylase are thiamine pyrophosphate-dependent enzymes that convert pyruvate into acetolactate and acetaldehyde, respectively. Although the former are encoded in the genomes of many thermophiles and hyperthermophiles, the latter has been found only in mesophilic organisms. In this study, the reaction specificity of acetolactate synthase from Thermus thermophilus was redirected to catalyze acetaldehyde formation to develop a thermophilic pyruvate decarboxylase. Error-prone PCR and mutant library screening led to the identification of a quadruple mutant with 3.1-fold higher acetaldehyde-forming activity than the wild-type. Site-directed mutagenesis experiments revealed that the increased activity of the mutant was due to H474R amino acid substitution, which likely generated two new hydrogen bonds near the thiamine pyrophosphate-binding site. These hydrogen bonds might result in the better accessibility of H+ to the substrate-cofactor-enzyme intermediate and a shift in the reaction specificity of the enzyme.

DOI： 10.1371/journal.pone.0146146

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Amorphous calcium silicate hydrates (A-CSHs) were synthesized using soluble silicates extracted from a natural siliceous shale (M-rite) and Ca(OH)(2). Simultaneous thermogravimetry and differential thermal analysis confirmed that the synthesized A-CSHs contained no detectable amount of free Ca(OH)(2). Their performance on phosphate (Pi) recovery from aqueous solutions was examined using a 3.0-L bacth reactor. A-CSHs possessed a greater ability to recover P-i from a synthetic anaerobic sludge digestion liquor than did CaCl2 and Ca(OH)(2). Si-29 magic-angle-spinning NMR analysis suggested that wet A-CSHs consisted of silicate polymers (average chain length of 3.5) that are linked to each other through ion binding with Ca2+. Based on Ca2+ release and settleability experiments, it was speculated that Ca-P-i-silicates aggregates were formed by the ionic association of P-i, Ca2+, and negatively charged silicates. This hypothesis could reasonably explain the high settleability of P-i, removed by A-CSHs. Powder X-ray diffraction analysis showed that recovered products had an amorphous structure similar to that of A-CSHs. In this study, we suggest that A-CSHs have a unique mechanism for recovering P-i, thereby enabling their high reactivity and settleability. (C) 2015 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.seppur.2015.01.043

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

In vitro reconstitution of an artificial metabolic pathway is an emerging approach for the biocatalytic production of industrial chemicals. However, several enzymes have to be separately prepared (and purified) for the construction of an in vitro metabolic pathway, thereby limiting the practical applicability of this approach. In this study, genes encoding the nine thermophilic enzymes involved in a non-ATP-forming chimeric glycolytic pathway were assembled in an artificial operon and co-expressed in a single recombinant Escherichia coli strain. Gene expression levels of the thermophilic enzymes were controlled by their sequential order in the artificial operon. The specific activities of the recombinant enzymes in the cell-free extract of the multiple-gene-expression E. coli were 5.0-1,370 times higher than those in an enzyme cocktail prepared from a mixture of single-gene-expression strains, in each of which a single one of the nine thermophilic enzymes was overproduced. Heat treatment of a crude extract of the multiple-gene-expression cells led to the denaturation of indigenous proteins and one-step preparation of an in vitro synthetic pathway comprising only a limited number of thermotolerant enzymes. Coupling this in vitro pathway with other thermophilic enzymes including the H2O-forming NADH oxidase or the malate/lactate dehydrogenase facilitated one-pot conversion of glucose to pyruvate or lactate, respectively. (C) 2014 Wiley Periodicals, Inc.

DOI： 10.1002/bit.25338

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Rhodococcus opacus B-4 cells are adhesive to and even dispersible in water-immiscible hydrocarbons owing to their highly lipophilic nature. In this study, we focused on the high operational stability of thermophilic enzymes and applied them to a biocatalytic conversion in an organic reaction medium using R. opacus B-4 as a lipophilic capsule of enzymes to deliver them into the organic medium. A novel thermo- and organic-solvent-tolerant ene reductase, which can catalyze the enantioselective reduction of ketoisophorone to (6R)-levodione, was isolated from Geobacillus sp. 30, and the gene encoding the enzyme was heterologously expressed in R. opacus B-4. Another thermophilic enzyme which catalyzes NAD(+)-dependent dehydrogenation of cyclohexanol was identified from the gene-expression library of Thermus thermophilus and the gene was coexpressed in R. opacus B-4 for cofactor regeneration. While the recombinant cells were not viable in the mixture due to high reaction temperature, 634 mM of (6R)-levodione could be produced with an enantiopurity of 89.2 % ee by directly mixing the wet cells of the recombinant R. opacus with a mixture of ketoisophorone and cyclohexanol at 50 A degrees C. The conversion rate observed with the heat-killed recombinant cells was considerably higher than that obtained with a cell-free enzyme solution, demonstrating that the accessibility between the substrates and enzymes could be improved by employing R. opacus cells as a lipophilic enzyme capsule. These results imply that a combination of thermophilic enzymes and lipophilic cells can be a promising approach for the biocatalytic production of water-insoluble chemicals.

DOI： 10.1007/s00253-014-5668-9

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Language：English   Publishing type：Part of collection (book)   Publisher：Wiley Blackwell  

Lactic acid (LA) is an important and versatile chemical that can be produced from renewable resources such as biomass. LA is used in the food, pharmaceutical, and polymers industries and is produced by microorganism fermentation
however, most microorganisms cannot directly utilize biomass such as starchy materials and cellulose. Here, we summarize LA and LA-based polymers production using several kinds of genetically modified microorganisms, such as lactic acid bacteria, Escherichia coli, Corynebacterium glutamicum, and yeasts. Using gene manipulation and metabolic engineering, the yield and optical purity of LA produced from biomass have been significantly improved, as well as LA-based polymers. The drawbacks as well as improvements of LA production by fermentation are discussed.

DOI： 10.1002/9781118845394.ch13

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

The directed evolution of the thermotolerant NADP(H)-dependent malic enzyme from Thermococcus kodakarensis was conducted to alter the cofactor preference of the enzyme from NADP(H) to NAD(H). The construction and screening of two generations of mutant libraries led to the isolation of a triple mutant that exhibited 6-fold higher K-cat/K-m with NAD(+) than the wild type. We serendipitously found that, in addition to the change in the cofactor preference, the reaction specificity of the mutant enzyme was altered. The reductive carboxylation of pyruvate to malate catalyzed by the wild type enzyme is accompanied by HCO3 (-)-independent reduction of pyruvate and gives lactate as a byproduct. The reaction specificity of the triple mutant was significantly shifted to malate production and the mutant gave a less amount of the byproduct than the wild type. When the triple mutant enzyme was used as a catalyst for pyruvate carboxylation with NADH, the enzyme gave 1.2 times higher concentration of malate than the wild type with NADPH. Single-point mutation analysis revealed that the substitution of Arg221 with Gly is responsible for the shift in reaction specificity. This finding may shed light on the catalytic mechanisms of malic enzymes and other related CO2- and/or HCO3 (-)-fixing enzymes. (C) 2013, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2013.07.005

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: Metabolic engineering has emerged as a practical alternative to conventional chemical conversion particularly in biocommodity production processes. However, this approach is often hampered by as yet unidentified inherent mechanisms of natural metabolism. One of the possible solutions for the elimination of the negative effects of natural regulatory mechanisms on artificially engineered metabolic pathway is to construct an in vitro pathway using a limited number of enzymes. Employment of thermostable enzymes as biocatalytic modules for pathway construction enables the one-step preparation of catalytic units with excellent selectivity and operational stability. Acetyl-CoA is a central precursor involved in the biosynthesis of various metabolites. In this study, an in vitro pathway to convert pyruvate to acetyl-CoA was constructed and applied to N-acetylglutamate production.
Results: A bypassed pyruvate decarboxylation pathway, through which pyruvate can be converted to acetyl-CoA, was constructed by using a coupled enzyme system consisting of pyruvate decarboxylase from Acetobacter pasteurianus and the CoA-acylating aldehyde dehydrogenase from Thermus thermophilus. To demonstrate the applicability of the bypassed pathway for chemical production, a cofactor-balanced and CoA-recycling synthetic pathway for N-acetylglutamate production was designed by coupling the bypassed pathway with the glutamate dehydrogenase from T. thermophilus and N-acetylglutamate synthase from Thermotoga maritima. N-Acetylglutamate could be produced from an equimolar mixture of pyruvate and alpha-ketoglutarate with a molar yield of 55% through the synthetic pathway consisting of a mixture of four recombinant E. coli strains having either one of the thermostable enzymes. The overall recycling number of CoA was calculated to be 27.
Conclusions: Assembly of thermostable enzymes enables the flexible design and construction of an in vitro metabolic pathway specialized for chemical manufacture. We herein report the in vitro construction of a bypassed pathway capable of an almost stoichiometric conversion of pyruvate to acetyl-CoA. This pathway is potentially applicable not only to N-acetylglutamate production but also to the production of a wide range of acetyl-CoAderived metabolites.

DOI： 10.1186/1475-2859-12-91

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

A novel technique for phosphorus (P) recovery from aqueous solutions was developed using amorphous calcium silicate hydrates (A-CSHs). A-CSHs, which have a high Ca/Si molar ratio of 2.0 or greater, could be synthesized using unlimitedly available, inexpensive materials such as siliceous shale and calcium hydroxide. A-CSHs showed high performance for P recovery from an anaerobic sludge digestion liquor (ASDL) and the synthetic model liquor (s-ASDL) containing 89 mg PO4-P/L. After 20 min mixing, 1.5 g/L A-CSHs could remove approximately 69 and 73% PO4-P from ASDL and s-ASDL, respectively. By contrast, autoclaved lightweight concrete particles, which contained crystalline calcium silicate hydrates as a principal component, removed only 10 and 6% PO4-P, from ASDL and s-ASDL, respectively, under the same experimental conditions. When A-CSHs were washed with deionized water to remove free Ca(OH)(2), P removability was significantly improved (up to 82%) despite the reduction in the amount of Ca2+ released. Unlike in the case of Ca(OH)(2), no significant carbonate inhibition was observed with P removal by A-CSHs. Moreover, P removed by A-CSHs showed better settleability, filterability, and dewaterability than P precipitated with conventional CaCl2 and Ca(OH)(2). The present study demonstrated that A-CSHs have great potential as a novel, beneficial material for P recovery and recycling. (C) 2013 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.watres.2013.01.052

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Synthetic metabolic engineering enables us to construct an in vitro artificial synthetic pathways specialized for chemical manufacturing through the simple heat-treatment of the recombinant mesophiles having thermophilic enzymes, followed by rational combination of those biocatalytic modules. In this work, we constructed a synthetic pathway capable of direct conversion of glucose to malate. The reversible carboxylation of pyruvate catalyzed by a malic enzyme derived from Thermococcus kodakarensis (TkME) (ΔG°'=+7.3kJmol-1) was coupled with a thermodynamically favorable non-ATP-forming Embden-Meyerhof pathway to balance the consumption and regeneration of redox cofactors and to shift the overall equilibrium toward malate production (glucose+2HCO3-+2H→2 malate+2H2O
ΔG°'=-121.4kJmol-1). TkME exhibited both pyruvate carboxylation (malate-forming) and pyruvate reduction (lactate-forming) activities. By increasing HCO3- concentration, the reaction specificity could be redirected to malate production. As a result, the direct conversion of glucose to malate was achieved with a molar yield of 60%. © 2012 Elsevier B.V.

DOI： 10.1016/j.jbiotec.2012.11.011

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The heat treatment of recombinant mesophilic cells having heterologous thermophilic enzymes results in the denaturation of indigenous mesophilic enzymes and the elimination of undesired side reactions
therefore, highly selective whole-cell catalysts comparable to purified enzymes can be readily prepared. However, the thermolysis of host cells leads to the heat-induced leakage of thermophilic enzymes, which are produced as soluble proteins, limiting the exploitation of their excellent stability in repeated and continuous reactions. In this study, Escherichia coli cells having the thermophilic fumarase from Thermus thermophilus (TtFTA) were treated with glutaraldehyde to prevent the heat-induced leakage of the enzyme, and the resulting cells were used as a whole-cell catalyst in repeated and continuous reactions. Interestingly, although electron microscopic observations revealed that the cellular structure of glutaraldehyde-treated E. coli was not apparentlychanged by the heat treatment, the membrane permeability of the heated cells to relatively small molecules (up to at least3 kDa) was significantly improved. By applying the glutaraldehyde-treated E. coli having TtFTA to a continuous reactor equipped with a cell-separation membrane filter, the enzymatic hydration of fumarate to malate could be operated for more than 600 min with a molar conversion yield of 60% or higher. © 2013, American Society for Microbiology.

DOI： 10.1128/AEM.03752-12

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Academic Press Inc.  

The heat treatment of recombinant mesophiles having heterologous thermotolerant enzymes results in the one-step preparation of highly selective biocatalytic modules. The assembly of these modules enables us to readily construct an artificial metabolic pathway in vitro. In this work, we constructed a non-natural, cofactor-balanced, and oxygen-insensitive pathway for n-butanol production using 16 thermotolerant enzymes. The whole pathway was divided into 7 parts, in each of which NAD(H)-dependent enzymes were assigned to be the last step, and the fluxes through each part were spectrophotometrically determined. This real-time monitoring technique enabled the experimental optimization of enzyme level to achieve a desired production rate. Through the optimized pathway, n-butanol could be produced from glucose with a molar yield of 82% at a rate of 8.2μmoll-1min-1. Our approach would be widely applicable to the rational optimization of artificial metabolic pathways as well as to the in vitro production of value-added biomolecules. © 2013 Elsevier Inc.

DOI： 10.1016/j.ymben.2013.09.006

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: The integration of biotechnology into chemical manufacturing has been recognized as a key technology to build a sustainable society. However, the practical applications of biocatalytic chemical conversions are often restricted due to their complexities involving the unpredictability of product yield and the troublesome controls in fermentation processes. One of the possible strategies to overcome these limitations is to eliminate the use of living microorganisms and to use only enzymes involved in the metabolic pathway. Use of recombinant mesophiles producing thermophilic enzymes at high temperature results in denaturation of indigenous proteins and elimination of undesired side reactions; consequently, highly selective and stable biocatalytic modules can be readily prepared. By rationally combining those modules together, artificial synthetic pathways specialized for chemical manufacturing could be designed and constructed.
Results: A chimeric Embden-Meyerhof (EM) pathway with balanced consumption and regeneration of ATP and ADP was constructed by using nine recombinant E. coli strains overproducing either one of the seven glycolytic enzymes of Thermus thermophilus, the cofactor-independent phosphoglycerate mutase of Pyrococcus horikoshii, or the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase of Thermococcus kodakarensis. By coupling this pathway with the Thermus malate/lactate dehydrogenase, a stoichiometric amount of lactate was produced from glucose with an overall ATP turnover number of 31.
Conclusions: In this study, a novel and simple technology for flexible design of a bespoke metabolic pathway was developed. The concept has been testified via a non-ATP-forming chimeric EM pathway. We designated this technology as "synthetic metabolic engineering". Our technology is, in principle, applicable to all thermophilic enzymes as long as they can be functionally expressed in the host, and thus would be potentially applicable to the biocatalytic manufacture of any chemicals or materials on demand.

DOI： 10.1186/1475-2859-11-120

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

The replication region of the 111-kb circular plasmid pKNR from Rhodococcus opacus B-4 was identified. A PCR-based deletion analysis using the lambda Red recombination technique followed by restriction digestion and PCR-amplification analyses revealed that a 2.5-kb fragment covering one putative open reading frame (ORF) was involved in the replication of pKNR. The product of this ORF showed significant similarity to a functionally unknown protein encoded in the replication region of the 70-kb circular plasmid of Clavibacter michiganensis and to ones in other bacterial large circular plasmids. These observations suggest that the product of the identified ORF and its orthologs can serve as novel replication proteins for large circular bacterial plasmids.

DOI： 10.1271/bbb.120362

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

We previously demonstrated the stoichiometric conversion of glycerol to glycerol-3-phosphate (G3P) using Escherichia coli recombinants producing the ATP-dependent glycerol kinase of the hyperthermophile Thermococcus kodakaraensis (TkGK) and the polyphosphate kinase of Therm us thermophilus HB27 (TtPPK). TtPPK was associated with the membrane fraction of E. coli recombinants, whereas TkGK was released from the cells during the reaction at 70 degrees C. In this study, TUCK was fused with either TtPPK or an E. coli membrane-intrinsic protein, YedZ, to minimize the heat-induced leakage of TkGK. When the E. coli recombinants having these fusion proteins were incubated at 70 degrees C for 2 h, more than 80% of TkGK activity was retained in the heated E. coli cells. However, the yields of G3P production by E. coli having the fusion proteins of TtPPK and TkGK were only less than 35%. Polyphosphate is a strong chelator for metal ions and has an inhibitory effect on TkGK which requires magnesium. Insufficient space between TtPPK and TkGK might enhance the inhibitory effect of polyphosphate on TkGK activity of the fusion protein. The mixture of E. con cells having TtPPK and those having TkGK fused with YedZ converted 80% of glycerol into G3P. These recombinant cells could be easily recovered from the reaction mixture by centrifugation and repeatedly used without a significant loss of enzyme activities. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2011.11.016

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Other Link： http://orcid.org/0000-0001-9069-7574

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE SA  

The potential of thermal analysis for differentiating between oleaginous and non-oleaginous microorganisms was investigated using thermogravimetry (TG) and differential thermal analysis (DTA). The model oleaginous microorganisms used in the present study were the fungi, Mortierella alpina IFO32281 and Mortierella alliacea YN-15, the unicellular alga. Aurantiochytrium sp. CB 15-5, and the yeast, Rhodosporidium toruloides DMKU3-TK 16. Escherichia coli JM109, Rhodococcus opacus B-4, and Saccharomyces cerevisiae were used as the control non-oleaginous microorganisms. In simultaneous TG and DTA, the furnace temperature was linearly increased from 30 to 280 degrees C, decreased to 30 degrees C, linearly increased from 30 to 360 degrees C, and then isothermally held at 360 degrees C for 30 min. This two-step linear temperature program was effective in resolving overlapping exothermic peaks in the DTA curves in the temperature range from 280 to 360 degrees C. Heat evolved from a microbial sample was estimated from the area under the exothermic peak between 280 and 360 degrees C using indium as a standard material. There was a linear relationship between the exothermic heat and total lipid content of the tested microorganisms. Exothermic heat per dry sample mass (kJ/g) in the temperature range from 280 to 360 degrees C is a promising measure for differentiating between oleaginous and non-oleaginous microorganisms. (C) 2011 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bej.2011.08.002

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In order to achieve efficient d-lactic acid fermentation from a mixture of xylose and glucose, the xylose-assimilating xylAB operon from Lactobacillus pentosus (PXylAB) was introduced into an l-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum (Delta ldhL1-xpk1::tkt-Delta xpk2) strain in which the phosphoketolase 1 gene (xpk1) was replaced with the transketolase gene (tkt) from Lactococcus lactis, and the phosphoketolase 2 (xpk2) gene was deleted. Two copies of xylAB introduced into the genome significantly improved the xylose fermentation ability, raising it to the same level as that of Delta ldhL1-xpk1::tkt-Delta xpk2 harboring a xylAB operon-expressing plasmid. Using the two-copy xylAB integrated strain, successful homo-d-lactic acid production was achieved from a mixture of 25 g/l xylose and 75 g/l glucose without carbon catabolite repression. After 36-h cultivation, 74.2 g/l of lactic acid was produced with a high yield (0.78 g per gram of consumed sugar) and an optical purity of d-lactic acid of 99.5%. Finally, we successfully demonstrated homo-d-lactic acid fermentation from a mixture of three kinds of sugar: glucose, xylose, and arabinose. This is the first report that describes homo-d-lactic acid fermentation from mixed sugars without carbon catabolite repression using the xylose-assimilating pathway integrated into lactic acid bacteria.

DOI： 10.1007/s00253-011-3356-6

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In order to achieve efficient homo L-lactic acid fermentation from xylose, we first carried out addition of xylose assimilation ability to Lactococcus lactis IL 1403 by introducing a plasmid carrying the xylRAB genes from L. lactis IO-1 (pXylRAB). Then modification of xylose assimilation pathway was carried out. L. lactis has two pathways for xylose assimilation called the phosphoketolase pathway (PK pathway) that produces both lactic acid and acetic acid and the pentose phosphate pathway (PP pathway) that produces only lactic acid as a final product. Thus a mutant strain that disrupted its phosphokeolase gene (ptk) was constructed. The Delta ptk mutant harboring pXylRAB lacked the PK pathway and produced predominantly lactic acid from xylose via the PP pathway, although its fermentation rate slightly decreased. Further introduction of the transketolase gene (tkt) to disrupted ptk locus led restoration of fermentation rate and this was attributed to enhancement of the PP pathway. As a result, ptk::tkt strain harboring pXylRAB produced 50.1 g/l of L-lactic acid from xylose with a high optical purity of 99.6% and a high yield of 1.58 (moles per mole xylose consumed) that is close to theoretical value of 1.67 from xylose.

DOI： 10.1007/s00253-011-3342-z

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In order to achieve direct fermentation of an optically pure d-lactic acid from cellulosic materials, an endoglucanase from a Clostridium thermocellum (CelA)-secreting plasmid was introduced into an l-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum (a dagger ldhL1) bacterial strain. CelA expression and its degradation of beta-glucan was confirmed by western blot analysis and enzyme assay, respectively. Although the CelA-secreting a dagger ldhL1 assimilated cellooligosaccharides up to cellohexaose (although not cellotetraose), the main end product was acetic acid, not lactic acid, due to the conversion of lactic acid to acetic acid. Cultivation under anaerobic conditions partially suppressed this conversion resulting in the production of 1.27 g/l of D-lactic acid with a high optical purity of 99.5% from a medium containing 2 g/l of cellohexaose. Subsequently, D-lactic acid fermentation from barley beta-glucan was carried out with the addition of Aspergillus aculeatus beta-glucosidase produced by recombinant Aspergillus oryzae and 1.47 g/l of D-lactic was produced with a high optical purity of 99.7%. This is the first report of direct lactic acid fermentation from beta-glucan and a cellooligosaccharide that is a more highly polymerized sugar than cellotriose.

DOI： 10.1007/s00253-009-2111-8

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Lactic acid (LA) is an important and versatile chemical that can be produced from renewable resources such as biomass. LA is used in the food, pharmaceutical, and polymers industries and is produced by microorganism fermentation; however, most microorganisms cannot directly utilize biomass such as starchy materials and cellulose. Here, we summarize LA production using several kinds of genetically modified microorganisms, such as LA bacteria, Escherichia coli, Corynebacterium glutamicum, and yeast. Using gene manipulation and metabolic engineering, the yield and optical purity of LA produced from biomass has been significantly improved. In this review, the drawbacks as well as improvements of LA production by fermentation is discussed.

DOI： 10.1007/s00253-009-2280-5

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

The production of optically pure D-lactic acid via xylose fermentation was achieved by using a Lactobacillus plantarum NCIMB 8826 strain whose L-lactate dehydrogenase gene was deficient and whose phosphoketolase genes were replaced with a heterologous transketolase gene. After 60 h of fermentation, 41.2 g/liter of D-lactic acid was produced from 50 g/liter of xylose.

DOI： 10.1128/AEM.01692-09

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Optically pure D-lactic acid fermentation from arabinose was achieved by using the Lactobacillus plantarum NCIMB 8826 strain whose L-lactate dehydrogenase gene was deficient and whose phosphoketolase gene was substituted with a heterologous transketolase gene. After 27 h of fermentation, 38.6 g/liter of D-lactic acid was produced from 50 g/liter of arabinose.

DOI： 10.1128/AEM.00573-09

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In order to achieve direct and efficient fermentation of optically pure D-lactic acid from raw corn starch, we constructed L-lactate dehydrogenase gene (ldhL1)-deficient Lactobacillus plantarum and introduced a plasmid encoding Streptococcus bovis 148 alpha-amylase (AmyA). The resulting strain produced only D-lactic acid from glucose and successfully expressed amyA. With the aid of secreting AmyA, direct D-lactic acid fermentation from raw corn starch was accomplished. After 48 h of fermentation, 73.2 g/liter of lactic acid was produced with a high yield (0.85 g per g of consumed sugar) and an optical purity of 99.6%. Moreover, a strain replacing the ldhL1 gene with an amyA-secreting expression cassette was constructed. Using this strain, direct D-lactic acid fermentation from raw corn starch was accomplished in the absence of selective pressure by antibiotics. This is the first report of direct D-lactic acid fermentation from raw starch.

DOI： 10.1128/AEM.01514-08

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Here, we established a system for displaying heterologous protein to the C terminus of the peptidoglycan-binding domain (cA domain) of AcmA (a major autolysin from Lactococcus lactis). Western blot and flow cytometric analyses revealed that the fusion proteins (cA-AmyA) of the cA domain and alpha-amylase from Streptococcus bovis 148 (AmyA) are efficiently expressed and successfully displayed on the surfaces of L. lactis cells. AmyA was also displayed on the cell surface while retaining its activity. Moreover, with an increase in the number of cA domains, the quantity of cA-AmyA fusion proteins displayed on the cell surface increased. When three repeats of the cA domain were used as an anchor protein, 82% of alpha-amylase activity was detected on the cells. The raw starch-degrading activity of AmyA was significantly higher when AmyA was fused to the C terminus of the cA domain than when it was fused to the N terminus. In addition, cA-AmyA fusion proteins were successfully displayed on the cell surfaces of Lactobacillus plantarum and Lactobacillus casei.

DOI： 10.1128/AEM.02012-07

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

To achieve direct and efficient lactic acid production from starch, a genetically modified Lactococcus lactis IL 1403 secreting alpha-amylase, which was obtained from Streptococcus bovis 148, was constructed. Using this strain, the fermentation of soluble starch was achieved, although its rate was far from efficient (0.09 g l(-1) h(-1) lactate). High-performance liquid chromatography revealed that maltose accumulated during fermentation, and this was thought to lead to inefficient fermentation. To accelerate maltose consumption, starch fermentation was examined using L. lactis cells adapted to maltose instead of glucose. This led to a decrease in the amount of maltose accumulation in the culture, and, as a result, a more rapid fermentation was accomplished (1.31 g l(-1) h(-1) lactate). Maximum volumetric lactate productivity was further increased (1.57 g l(-1) h(-1) lactate) using cells adapted to starch, and a high yield of lactate (0.89 g of lactate per gram of consumed sugar) of high optical purity (99.2% of L-lactate) was achieved. In this study, we propose a new approach to lactate production by alpha-amylase-secreting L. lactis that allows efficient fermentation from starch using cells adapted to maltose or starch before fermentation.

DOI： 10.1007/s00253-007-0905-0

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The 5'-untranslated leader sequence (UTLS) of the slpA gene from Lactobacillus acidophilus contributes to mRNA stabilization by producing a 5' stem and loop structure, and a high-level expression system for the lactic acid bacteria was developed using the UTLS in this study. A plasmid, which expresses alpha-amylase under the control of the ldh promoter, was constructed by integrating the core promoter sequence with the UTLS. The role of the UTLS in increasing the copies of the alpha-amylase mRNA was proved by measuring alpha-amylase activity in the culture supernatant and the relative expression of alpha-amylase mRNA was determined by the quantitative real-time PCR analysis. Moreover, several expression systems were constructed by combining the core promoter sequence with the UTLS or with the partially deleted UTLS and the expression level was evaluated. The use of the UTLS led to the success in improving alpha-amylase expression in the two strains of Lactobacillus casei and Lactococcus lactis. The current study showed that the improvement in protein production using the UTLS could be applied to the expression system in the lactic acid bacteria.

DOI： 10.1007/s00253-006-0477-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

We have developed a novel Escherichia coli cell surface display system by employing PgsA as an anchoring motif. In our display system, C-terminal fusion to PgsA anchor protein from Bacillus subtilis was used. The enzymes selected for display were alpha-amylase (AmyA) from Streptococcus bovis 148 and lipase B (CALB) from Candida antarctica. The molecular mass values of AmyA and CALB are approximately 77 and 34 kDa, respectively. The enzymes were displayed on the surface as a fusion protein with a FLAG peptide tag at the C terminus. Both the PgsA-AmyA-FLAG and PgsA-CALB-FLAG fusion proteins were shown to be displayed by immunofluorescence labeling using anti-FLAG antibody. The displayed enzymes were active forms, and AmyA and CALB activities reached 990 U/g (dry cell weight) and 4.6 U/g (dry cell weight), respectively. AmyA-displaying E. coli cells grew utilizing cornstarch as the sole carbon source, while CALB-displaying E. coli cells catalyzed enantioselective transesterification, indicating that they are effective whole-cell biocatalysts. Since a target enzyme with a size of 77 kDa and an industrially useful lipase have been successfully displayed on the cell surface of E. coli for the first time, PgsA protein is probably a useful anchoring motif to display various enzymes.

DOI： 10.1007/s00253-005-0111-x

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PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

We developed a new cell surface engineering system based on the PgsA anchor protein from Bacillus subtilis. In this system, the N terminus of the target protein was fused to the PgsA protein and the resulting fusion protein was expressed on the cell surface. Using this new system, we constructed a novel starch-degrading strain of Lactobacillus casei by genetically displaying a-amylase from the Streptococcus bovis strain 148 with a FLAG peptide tag (AmyAF). Localization of the PgsA-AmyA-FLAG fusion protein on the cell surface was confirmed by immunofluorescence microscopy and flow cytometric analysis. The lactic acid bacteria which displayed AmyAF showed significantly elevated hydrolytic activity toward soluble starch. By fermentation using AmyAF-displaying L. casei cells, 50 g/liter of soluble starch was reduced to 13.7 g/liter, and 21.8 g/liter of lactic acid was produced within about 24 h. The yield in terms of grams of lactic acid produced per gram of carbohydrate utilized was 0.60 g per g of carbohydrate consumed at 24 h. Since AmyA was immobilized on the cells, cells were recovered after fermentation and used repeatedly. During repeated utilization of cells, the lactic acid yield was improved to 0.81 g per g of carbohydrate consumed at 72 h. These results indicate that efficient simultaneous saccharification and fermentation from soluble starch to lactic acid were carried out by recombinant L. casei cells with cell surface display of AmyA.

DOI： 10.1128/AEM.72.1.269-275.2006

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PubMed

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Total pages：227   Responsible for pages：141-149  

ASIN

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Total pages：776   Responsible for pages：519-524  

ASIN

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Language：Japanese  

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/2016045869

Language：Japanese  

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Language：Japanese   Publisher：シーエムシー出版  

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Other Link： http://search.jamas.or.jp/link/ui/2014247613

Language：Japanese   Publisher：日本生物工学会  

CiNii Books

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Language：Japanese   Publisher：公益社団法人 日本農芸化学会  

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Language：Japanese  

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Language：Japanese  

CiNii Books

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Language：English  

CiNii Books

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CiNii Books

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Language：Japanese  

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Language：Japanese  

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Language：English   Publisher：The Society for Biotechnology, Japan  

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Language：English   Publisher：The Society for Biotechnology, Japan  

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Language：Japanese   Publisher：化学工業社  

CiNii Books

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Language：Japanese   Publisher：日本バイオプラスチック協会  

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Language：Japanese  

CiNii Books

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Language：Japanese  

CiNii Books

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Language：Japanese   Publisher：日本生物工学会  

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Language：Japanese   Publisher：シーエムシー出版  

CiNii Books

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Language：Japanese  

CiNii Books

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Language：English   Presentation type：Oral presentation (invited, special)  

Venue：Chuncheon, Korea  

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Language：English   Presentation type：Oral presentation (invited, special)  

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Language：English   Presentation type：Poster presentation  

Venue：Chiayi, Taiwan  

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Language：English   Presentation type：Oral presentation (general)  

Venue：Keio University, Kanagawa  

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Language：English   Presentation type：Poster presentation  

Venue：Tokushima University, Tokushima  

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Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Venue：徳島大学  

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Language：English   Presentation type：Poster presentation  

Venue：Philadelphia, USA  

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Language：English   Presentation type：Oral presentation (general)  

Venue：Osaka  

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Language：English   Presentation type：Poster presentation  

Venue：Kaohsiung, Taiwan  

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