Jun Ogawa

Name:

Organization: Kyoto University

Department: 1 Industrial Microbiology, Graduate School of Agriculture

Title:

Chemicals
References

Enzymatic synthesis of 2′-O-methylribonucleosides with a nucleoside hydrolase family enzyme from Lactobacillus buchneri LBK78

Co-reporter:Yuuki Mitsukawa, Makoto Hibi, Narihiro Matsutani, Nobuyuki Horinouchi, ... Jun Ogawa

Journal of Bioscience and Bioengineering 2017 Volume 123, Issue 6(Volume 123, Issue 6) pp:

Publication Date(Web):1 June 2017

DOI:10.1016/j.jbiosc.2017.01.005

2′-O-Methylribonucleosides (2′-OMe-NRs) are promising raw materials for the production of nucleic acid drugs. We previously reported that LbNH, a nucleoside hydrolase from Lactobacillus buchneri LBK78 (NITE P-01581), was the first enzyme found to act on 2′-OMe-NRs. In the present study, we determined that LbNH also has the transribosylation activity between 2′-OMe-NRs and nucleobases, in addition to the hydrolyzing activity towards 2′-OMe-NRs. When 2′-O-methyluridine (2′-OMe-UR) and adenine were reacted with LbNH, 2′-O-methyladenosine (2′-OMe-AR) was produced. LbNH preferred purine nucleobases as its acceptor substrates for the transribosylation with 2′-OMe-UR as a donor substrate. Kinetic analysis of LbNH revealed that adenine behaved as a mixed inhibitor of the hydrolysis of 2′-OMe-UR. Under the optimal reaction conditions, the maximum molar yield of enzymatic 2′-OMe-AR produced reached 0.97% towards 2′-OMe-UR, corresponding to 0.16 g/L.

Cover Picture: Multi-Enzymatic Synthesis of Optically Pure -Hydroxy -Amino Acids (Adv. Synth. Catal. 4/2015)

Co-reporter:Makoto Hibi;Takuya Kasahara;Takashi Kawashima;Hiroko Yajima;Shoko Kozono;Sergey V. Smirnov;Tomohiro Kodera;Masakazu Sugiyama;Sakayu Shimizu;Kenzo Yokozeki

Advanced Synthesis & Catalysis 2015 Volume 357( Issue 4) pp:

Publication Date(Web):

DOI:10.1002/adsc.201500107

Multi-Enzymatic Synthesis of Optically Pure -Hydroxy -Amino Acids

Co-reporter:Makoto Hibi;Takuya Kasahara;Takashi Kawashima;Hiroko Yajima;Shoko Kozono;Sergey V. Smirnov;Tomohiro Kodera;Masakazu Sugiyama;Sakayu Shimizu;Kenzo Yokozeki

Advanced Synthesis & Catalysis 2015 Volume 357( Issue 4) pp:767-774

Publication Date(Web):

DOI:10.1002/adsc.201400672

Eicosapentaenoic acid (EPA) production by an oleaginous fungus Mortierella alpina expressing heterologous the 17-desaturase gene under ordinary temperature

Co-reporter:Tomoyo Okuda;Akinori Ando;Hiroaki Negoro;Tatsuya Muratsubaki;Hiroshi Kikukawa;Takaiku Sakamoto;Eiji Sakuradani;Sakayu Shimizu

European Journal of Lipid Science and Technology 2015 Volume 117( Issue 12) pp:1919-1927

Publication Date(Web):

DOI:10.1002/ejlt.201400657

Abstract

The oleaginous fungus Mortierella alpina is known to accumulate eicosapentaenoic acid (EPA) only when cultivated at a low temperature (below 15°C). Here, we investigated EPA production at an ordinary temperature (28°C) by expressing the Saprolegnia diclina Δ17 desaturase gene (sdd17m) in M. alpina ST1358, an ω3-desaturation activity-defective mutant derived from M. alpina 1S-4. Expression of the exogenous gene was confirmed by EPA accumulation in transformants at both 28 and 12°C. The EPA content in total lipids produced by transformants was over 20% at 28°C. Bench-scale fermentation with a 5-L jar fermentor showed that EPA content reached 26.4% of total fatty acids, and EPA production reached 1.8 g/L. This is the first study to report the accumulation of EPA in M. alpina at an ordinary temperature, and provide a platform technology for the industrial production of EPA using M. alpina as a promising source for EPA.

Practical applications: This achievement shows the potential of M. alpina for industrial production of EPA at lower cost compared to conventional transgenic organisms such as plants and yeasts. The composition of fatty acids produced by M. alpina transformants was simple and did not contain unusual fatty acids compared with that of fish oils, so it may be acceptable for general consumers. Low-cost, large-scale, and high-purity EPA supply by M. alpina will also lead to development of academic research on physiological functions of EPA and its derivatives.

The oleaginous fungus Mortierella alpina accumulates eicosapentaenoic acid (EPA) only when cultivated at a low temperature (below 15°C). Here, we investigated EPA production at an ordinary temperature (28°C) by expressing the Saprolegnia diclina Δ17 desaturase gene (sdd17m) in M. alpina. The transformants with the sdd17m gene produced EPA (>20% in total lipids) at 28°C.

Imidase catalyzing desymmetric imide hydrolysis forming optically active 3-substituted glutaric acid monoamides for the synthesis of gamma-aminobutyric acid (GABA) analogs

Co-reporter:Masutoshi Nojiri;Makoto Hibi;Hiroaki Shizawa

Applied Microbiology and Biotechnology 2015 Volume 99( Issue 23) pp:9961-9969

Publication Date(Web):2015 December

DOI:10.1007/s00253-015-6812-x

The recent use of optically active 3-substituted gamma-aminobutyric acid (GABA) analogs in human therapeutics has identified a need for an efficient, stereoselective method of their synthesis. Here, bacterial strains were screened for enzymes capable of stereospecific hydrolysis of 3-substituted glutarimides to generate (R)-3-substituted glutaric acid monoamides. The bacteria Alcaligenes faecalis NBRC13111 and Burkholderia phytofirmans DSM17436 were discovered to hydrolyze 3-(4-chlorophenyl) glutarimide (CGI) to (R)-3-(4-chlorophenyl) glutaric acid monoamide (CGM) with 98.1 % enantiomeric excess (e.e.) and 97.5 % e.e., respectively. B. phytofirmans DSM17436 could also hydrolyze 3-isobutyl glutarimide (IBI) to produce (R)-3-isobutyl glutaric acid monoamide (IBM) with 94.9 % e.e. BpIH, an imidase, was purified from B. phytofirmans DSM17436 and found to generate (R)-CGM from CGI with specific activity of 0.95 U/mg. The amino acid sequence of BpIH had a 75 % sequence identity to that of allantoinase from A. faecalis NBRC13111 (AfIH). The purified recombinant BpIH and AfIH catalyzed (R)-selective hydrolysis of CGI and IBI. In addition, a preliminary investigation of the enzymatic properties of BpIH and AfIH revealed that both enzymes were stable in the range of pH 6–10, with an optimal pH of 9.0, stable at temperatures below 40 °C, and were not metalloproteins. These results indicate that the use of this class of hydrolase to generate optically active 3-substituted glutaric acid monoamide could simplify the production of specific chiral GABA analogs for drug therapeutics.

Gene targeting in the oil-producing fungus Mortierella alpina 1S-4 and construction of a strain producing a valuable polyunsaturated fatty acid

Co-reporter:Hiroshi Kikukawa;Eiji Sakuradani;Masato Nakatani;Akinori Ando

Current Genetics 2015 Volume 61( Issue 4) pp:579-589

Publication Date(Web):2015 November

DOI:10.1007/s00294-015-0481-2

To develop an efficient gene-targeting system in Mortierella alpina 1S-4, we identified the ku80 gene encoding the Ku80 protein, which is involved in the nonhomologous end-joining pathway in genomic double-strand break (DSB) repair, and constructed ku80 gene-disrupted strains via single-crossover homologous recombination. The Δku80 strain from M. alpina 1S-4 showed no negative effects on vegetative growth, formation of spores, and fatty acid productivity, and exhibited high sensitivity to methyl methanesulfonate, which causes DSBs. Dihomo-γ-linolenic acid (DGLA)-producing strains were constructed by disruption of the Δ5-desaturase gene, encoding a key enzyme of bioconversion of DGLA to ARA, using the Δku80 strain as a host strain. The significant improvement of gene-targeting efficiency was not observed by disruption of the ku80 gene, but the construction of DGLA-producing strain by disruption of the Δ5-desaturase gene was succeeded using the Δku80 strain as a host strain. This report describes the first study on the identification and disruption of the ku80 gene in zygomycetes and construction of a DGLA-producing transformant using a gene-targeting system in M. alpina 1S-4.

Selection and characterization of promoters based on genomic approach for the molecular breeding of oleaginous fungus Mortierella alpina 1S-4

Co-reporter:Tomoyo Okuda;Akinori Ando;Eiji Sakuradani;Hiroshi Kikukawa

Current Genetics 2014 Volume 60( Issue 3) pp:183-191

Publication Date(Web):2014 August

DOI:10.1007/s00294-014-0423-4

To express a foreign gene effectively, a good expression system is required. In this study, we investigated various promoters as useful tools for gene manipulation in oleaginous fungus Mortierella alpina 1S-4. We selected and cloned the promoter regions of 28 genes in M. alpina 1S-4 on the basis of expression sequence tag abundance data. The activity of each promoter was evaluated using the β-glucuronidase (GUS) reporter gene. Eight of these promoters were shown to enhance GUS expression more efficiently than a histone promoter, which is conventionally used for the gene manipulation in M. alpina. Especially, the predicted protein 3 and the predicted protein 6 promoters demonstrated approximately fivefold higher activity than the histone promoter. The activity of some promoters changed along with the cultivation phase of M. alpina 1S-4. Seven promoters with constitutive or time-dependent, high-level expression activity were selected, and deletion analysis was carried out to determine the promoter regions required to retain activity. This is the first report of comprehensive promoter analysis based on a genomic approach for M. alpina. The promoters described here will be useful tools for gene manipulation in this strain.

Characterization of galactose-dependent promoters from an oleaginous fungus Mortierella alpina 1S-4

Co-reporter:Tomoyo Okuda;Akinori Ando;Eiji Sakuradani;Hiroshi Kikukawa

Current Genetics 2014 Volume 60( Issue 3) pp:175-182

Publication Date(Web):2014 August

DOI:10.1007/s00294-014-0422-5

An inducible promoter is a useful tool for the controlled expression of a given gene. In this report, we describe galactose-dependent promoters for potential use in an oleaginous fungus Mortierella alpina. We cloned the putative promoter regions of two genes encoding galactose metabolic enzymes, GAL1 and GAL10, from the genome of M. alpina 1S-4. The β-glucuronidase (GUS) reporter gene assay in M. alpina 1S-4 revealed that regulation of these promoters was dependent on the presence of galactose in the medium both with and without other sugars. With the GAL10 promoter, an approximately 50-fold increase of GUS activity was demonstrated by addition of galactose into the culture media at any cultivation phase. The 5′ deletion analysis of the GAL10 promoter revealed that a promoter region of over 2,000 bp length was required for its high-level activity and sufficient inducible response. Significantly, this is the first report of inducible promoters of zygomycetes. The GAL10 promoter will be a valuable tool for gene manipulation in M. alpina 1S-4.

β-Glucuronidase from Lactobacillus brevis useful for baicalin hydrolysis belongs to glycoside hydrolase family 30

Co-reporter:Haruko Sakurama;Shigenobu Kishino

Applied Microbiology and Biotechnology 2014 Volume 98( Issue 9) pp:4021-4032

Publication Date(Web):2014 May

DOI:10.1007/s00253-013-5325-8

Baicalin (baicalein 7-O-β-d-glucuronide) is one of the major flavonoid glucuronides found in traditional herbal medicines. Because its aglycone, baicalein, is absorbed more quickly and shows more effective properties than baicalin, the conversion of baicalin into baicalein by β-glucuronidase (GUS) has drawn the attention of researchers. Recently, we have found that Lactobacillus brevis subsp. coagulans can convert baicalin to baicalein. Therefore, we aimed to identify and characterize the converting enzyme from L. brevis subsp. coagulans. First, we purified this enzyme from the cell-free extracts of L. brevis subsp. coagulans and cloned its gene. Surprisingly, this enzyme was found to be a GUS belonging to glycoside hydrolase (GH) family 30 (designated as LcGUS30), and its amino acid sequence has little similarity with any GUS belonging to GH families 1, 2, and 79 that have been reported so far. We then established a high-level expression and simple purification system of the recombinant LcGUS30 in Escherichia coli. The detailed analysis of the substrate specificity revealed that LcGUS30 has strict specificity toward glycon but not toward aglycones. Interestingly, LcGUS30 prefers baicalin rather than estrone 3-(β-d-glucuronide), one of the human endogenous steroid hormones. These results indicated that L. brevis subsp. coagulans and LcGUS30 should serve as powerful tools for the construction of a safe bioconversion system for baicalin. In addition, we propose that this novel type of GUS forms a new group in subfamily 3 of GH family 30.

Characteristics and biotechnology applications of aliphatic amino acid hydroxylases belonging to the Fe(II)/α-ketoglutarate-dependent dioxygenase superfamily

Co-reporter:Makoto Hibi

Applied Microbiology and Biotechnology 2014 Volume 98( Issue 9) pp:3869-3876

Publication Date(Web):2014 May

DOI:10.1007/s00253-014-5620-z

The asymmetric hydroxylation of inactive carbon atoms is still an important reaction in the industrial synthesis of valuable chiral compounds such as pharmaceuticals and fine chemicals. Applications of monooxygenation enzymes, like cytochrome P450 monooxygenases, flavin-containing monooxygenases, and Fe(II)/α-ketoglutarate-dependent dioxygenases (Fe/αKG-DOs), are strongly desired as hydroxylation biocatalysts because they have great advantages in regio- and stereoselectivity of the reactions. Recently, several novel Fe/αKG-DOs have been found to catalyze the asymmetric hydroxylation of aliphatic amino acids. Depending on their amino acid sequences, these Fe/αKG-DOs catalyze different types of regioselective hydroxylations, or C3–, C4–, and C5-hydroxylation. Additionally, most also have stereoselective sulfoxidation activities. Here, we have reviewed the characterization and process development of this novel functioning group of Fe/αKG-DOs.

Hydroxy fatty acid production by Pediococcus sp.

Co-reporter:Michiki Takeuchi;Shigenobu Kishino;Kaori Tanabe;Akiko Hirata;Si-Bum Park;Sakayu Shimizu

European Journal of Lipid Science and Technology 2013 Volume 115( Issue 4) pp:386-393

Publication Date(Web):

DOI:10.1002/ejlt.201200414

Abstract

Through the screening of about 300 strains of lactic acid bacteria, Pediococcus sp. AKU 1080 was selected as a strain with the ability to hydrate linoleic acid (cis-9,cis-12-octadecadienoic acid) to three hydroxy fatty acids, i.e., 10-hydroxy-cis-12-octadecenoic acid, 13-hydroxy-cis-9-octadecenoic acid, and 10,13-dihydroxyoctadecanoic acid. The strain hydrated one of two cis double bonds at Δ9 and Δ12 positions to produce 10-hydroxy-cis-12-octadecenoic acid and 13-hydroxy-cis-9-octadecenoic acid, respectively, then further hydrated these two mono-hydroxy fatty acids to 10,13-dihydroxyoctadecanoic acid. The growing cells of this strain were applied to the production of 13-hydroxy-cis-9-octadecenoic acid, that is potential as polymer substrates and functional foods but its specific and efficient production was not established. Under the optimum conditions, 2.3 mg/mL of 13-hydroxy-cis-9-octadecenoic acid was produced from 12.3 mg/mL of linoleic acid with 0.04 mg/mL 10-hydroxy-cis-12-octadecenoic acid (HYA) and 0.05 mg/mL 10,13-dihydroxyoctadecanoic acid in the cultivation medium. Specific production of 13-hydroxy-cis-9-octadecenoic acid was attained using cell-free extracts of the strain as the catalyst. Under the optimum conditions, 0.4 mg/mL of 13-hydroxy-cis-9-octadecenoic acid was produced from 2.0 mg/mL of linoleic acid without HYA and 10,13-dihydroxyoctadecanoic acid.

Practical applications: Hydroxy fatty acids are useful as starting materials for industrial chemicals, functional foods, and pharmaceuticals. Regioselective introduction of hydroxyl group to unsaturated fatty acids by microorganisms was applied to hydroxy fatty acid production. Especially, specific production of 13-hydroxy-cis-9-octadecenoic acid, which is useful for the production of 13-oxo-fatty acids with anti-obesity activity, was established in this work.

l-Leucine 5-hydroxylase of Nostoc punctiforme is a novel type of Fe(II)/α-ketoglutarate-dependent dioxygenase that is useful as a biocatalyst

Co-reporter:Makoto Hibi;Takashi Kawashima;Pavel M. Sokolov

Applied Microbiology and Biotechnology 2013 Volume 97( Issue 6) pp:2467-2472

Publication Date(Web):2013 March

DOI:10.1007/s00253-012-4136-7

l-Leucine 5-hydroxylase (LdoA) previously found in Nostoc punctiforme PCC 73102 is a novel type of Fe(II)/α-ketoglutarate-dependent dioxygenase. LdoA catalyzed regio- and stereoselective hydroxylation of l-leucine and l-norleucine into (2S,4S)-5-hydroxyleucine and (2S)-5-hydroxynorleucine, respectively. Moreover, LdoA catalyzed sulfoxidation of l-methionine and l-ethionine in the same manner as previously described l-isoleucine 4-hydroxylase. Therefore LdoA should be a promising biocatalyst for effective production of industrially useful amino acids.

Polyunsaturated fatty acid saturation by gut lactic acid bacteria affecting host lipid composition

Co-reporter:Shigenobu Kishino;Jun Kunisawa;Michiki Takeuchi;Akiko Hirata;Hiroyuki Arai;Hiroshi Kiyono;Yosuke Isobe;Kazumitsu Ueda;Satomi Takahashi;Ryo Iwamoto;Jun Shima;Kenzo Yokozeki;Makoto Arita;Si-Bum Park;Nahoko Kitamura;Sakayu Shimizu

PNAS 2013 Volume 110 (Issue 44 ) pp:17808-17813

Publication Date(Web):2013-10-29

DOI:10.1073/pnas.1312937110

In the representative gut bacterium Lactobacillus plantarum, we identified genes encoding the enzymes involved in a saturation metabolism of polyunsaturated fatty acids and revealed in detail the metabolic pathway that generates hydroxy fatty acids, oxo fatty acids, conjugated fatty acids, and partially saturated trans-fatty acids as intermediates. Furthermore, we observed these intermediates, especially hydroxy fatty acids, in host organs. Levels of hydroxy fatty acids were much higher in specific pathogen-free mice than in germ-free mice, indicating that these fatty acids are generated through polyunsaturated fatty acids metabolism of gastrointestinal microorganisms. These findings suggested that lipid metabolism by gastrointestinal microbes affects the health of the host by modifying fatty acid composition.

Enzymatic synthesis of chiral amino acid sulfoxides by Fe(II)/α-ketoglutarate-dependent dioxygenase

Co-reporter:Makoto Hibi, Takashi Kawashima, Hiroko Yajima, Sergey V. Smirnov, Tomohiro Kodera, Masakazu Sugiyama, Sakayu Shimizu, Kenzo Yokozeki, Jun Ogawa

Tetrahedron: Asymmetry 2013 Volume 24(Issue 17) pp:990-994

Publication Date(Web):15 September 2013

DOI:10.1016/j.tetasy.2013.07.017

Asymmetric sulfoxidation of sulfur-containing l-amino acids was successfully achieved through bioconversion using IDO, which is an Fe(II)/α-ketoglutarate-dependent dioxygenase previously found in Bacillus thuringiensis strain 2e2. The IDO catalyzed sulfoxidation of l-methionine, l-ethionine, S-methyl-l-cysteine, S-ethyl-l-cysteine, and S-allyl-l-cysteine into the corresponding (S)-configured sulfoxides such as (+)-methiin and (+)-alliin, which are responsible for valuable physiological activities in mammals, and have high stereoselectivity. Herein we have established an effective preparative laboratory scale production method to obtain enantiomerically pure chiral sulfoxides using an IDO biocatalyst.S-Allyl-l-cysteine (S)-sulfoxideC6H11NO3S[α]D25=+31.3 (c 0.1, H2O)Source of chirality: S-Allyl-l-cysteine, enzymatic sulfoxidationAbsolute configuration: (2S,Ss)S-Ethyl-l-cysteine (S)-sulfoxideC5H11NO3S[α]D25=+40.0 (c 0.1, H2O)Source of chirality: S-Ethyl-l-cysteine, enzymatic sulfoxidationAbsolute configuration: (2S,Ss)l-Ethionine (S)-sulfoxideC6H13NO3S[α]D25=+30.9 (c 0.1, H2O)Source of chirality: l-Ethionine, enzymatic sulfoxidationAbsolute configuration: (2S,Ss)S-Methyl-l-cysteine (S)-sulfoxideC4H9NO3S[α]D25=+96.1 (c 0.1, H2O)Source of chirality: S-methyl-l-cysteine, enzymatic sulfoxidationAbsolute configuration: (2S,Ss)l-Methionine (S)-sulfoxideC5H11NO3S[α]D25=+67.3 (c 0.1, H2O)Source of chirality: l-Methionine, enzymatic sulfoxidationAbsolute configuration: (2S,Ss)

Isolation and Characterization of a Docosahexaenoic Acid-Phospholipids Producing Microorganism Crypthecodinium sp. D31

Co-reporter:Tomoyo Okuda;Akinori Ando;Eiji Sakuradani

Journal of the American Oil Chemists' Society 2013 Volume 90( Issue 12) pp:1837-1844

Publication Date(Web):2013 December

DOI:10.1007/s11746-013-2337-6

Thirty-four strains of docosahexaenoic acid (DHA)-producing microorganisms were newly isolated from brackish areas in Japan. These strains showing various compositions of fatty acids. Especially, the fatty acids produced by one of the strains, named D31, had a high DHA content (over 60 % of the total fatty acids) and the simple fatty acid composition (16:0, 18:0, 18:1 and DHA without any other polyunsaturated acids). Although most oleaginous microorganisms accumulate DHA as triacylglycerol, the strain D31 accumulated DHA mainly as a polar lipid (79.4 % of total DHA), especially as phosphatidylcholine (71.4 % of polar DHA). This strain D31 was identified as a related species of Crypthecodinium cohnii on the basis of phylogenetic analysis. Crypthecodinium sp. D31 showed high DHA productivity when cultivated in a medium containing glycerol as the carbon source and a mixture of yeast extract and polypeptone as the nitrogen sources, with a salinity that was equivalent to 50 % of that of seawater and a pH in the acidic range (

Polyunsaturated fatty acids production and transformation by Mortierella alpina and anaerobic bacteria

Co-reporter:Jun Ogawa;Eiji Sakuradani;Shigenobu Kishino;Akinori Ando;Kenzo Yokozeki;Sakayu Shimizu

European Journal of Lipid Science and Technology 2012 Volume 114( Issue 10) pp:1107-1113

Publication Date(Web):

DOI:10.1002/ejlt.201200069

Abstract

Microorganisms are promising as producers of various polyunsaturated fatty acids (PUFAs) and as catalysts transforming them into unique molecular species beyond common PUFAs. This article describes PUFA production through chemical mutant- and molecular-breeding of an oleaginous filamentous fungus Mortierella alpina 1S-4 and PUFA transformation by anaerobic bacteria. M. alpina 1S-4 and its mutants and transformants produce oils containing not only common n − 6 and n − 3 PUFAs but also rare PUFAs. Unique PUFA-transforming activities were found in anaerobic bacteria. They isomerized PUFA to conjugated fatty acids and further transformed to partially saturated fatty acids with hydroxyl fatty acids as intermediates. The functions of these unique PUFAs have been attracting much attention for improving our health and for developing new chemical materials.

Metabolic diversity in biohydrogenation of polyunsaturated fatty acids by lactic acid bacteria involving conjugated fatty acid production

Co-reporter:Shigenobu Kishino;Kenzo Yokozeki

Applied Microbiology and Biotechnology 2009 Volume 84( Issue 1) pp:87-97

Publication Date(Web):2009 August

DOI:10.1007/s00253-009-1949-0

Lactobacillus plantarum AKU 1009a effectively transforms linoleic acid to conjugated linoleic acids of cis-9,trans-11-octadecadienoic acid (18:2) and trans-9,trans-11–18:2. The transformation of various polyunsaturated fatty acids by washed cells of L. plantarum AKU 1009a was investigated. Besides linoleic acid, α-linolenic acid [cis-9,cis-12,cis-15-octadecatrienoic acid (18:3)], γ-linolenic acid (cis-6,cis-9,cis-12–18:3), columbinic acid (trans-5,cis-9,cis-12–18:3), and stearidonic acid [cis-6,cis-9,cis-12,cis-15-octadecatetraenoic acid (18:4)] were found to be transformed. The fatty acids transformed by the strain had the common structure of a C18 fatty acid with the cis-9,cis-12 diene system. Three major fatty acids were produced from α-linolenic acid, which were identified as cis-9,trans-11,cis-15–18:3, trans-9,trans-11,cis-15–18:3, and trans-10,cis-15–18:2. Four major fatty acids were produced from γ-linolenic acid, which were identified as cis-6,cis-9,trans-11–18:3, cis-6,trans-9,trans-11–18:3, cis-6,trans-10–18:2, and trans-10-octadecenoic acid. The strain transformed the cis-9,cis-12 diene system of C18 fatty acids into conjugated diene systems of cis-9,trans-11 and trans-9,trans-11. These conjugated dienes were further saturated into the trans-10 monoene system by the strain. The results provide valuable information for understanding the pathway of biohydrogenation by anaerobic bacteria and for establishing microbial processes for the practical production of conjugated fatty acids, especially those produced from α-linolenic acid and γ-linolenic acid.

Omega-3 eicosatetraenoic acid production by molecular breeding of the mutant strain S14 derived from Mortierella alpina 1S-4

Co-reporter:Tomoyo Okuda, Akinori Ando, Hiroaki Negoro, Hiroshi Kikukawa, ... Jun Ogawa

Journal of Bioscience and Bioengineering (September 2015) Volume 120(Issue 3) pp:299-304

Publication Date(Web):1 September 2015

DOI:10.1016/j.jbiosc.2015.01.014

We investigated the omega-3 eicosatetraenoic acid (ETA) production by molecular breeding of the oleaginous fungus Mortierella alpina, which can slightly accumulate ETA only when cultivated at a low temperature. The endogenous ω3-desaturase gene or the heterologous Saprolegnia diclina Δ17 (sdd17m) desaturase gene were overexpressed in M. alpina S14, a Δ5-desaturation activity-defective mutant derived from M. alpina 1S-4. M. alpina S14 transformants introduced with the endogenous ω3-desaturase gene showed ETA at 42.1% content in the total lipids that was 84.2-fold and 3.2-fold higher than that of the wild-type strain 1S-4 and host strain S14, respectively, when cultivated at 12°C. No accumulation of ETA was observed at 28°C. In contrast, transformants with the heterologous sdd17m gene showed 24.9% of the content of total lipids at 28°C. These results indicated that these M. alpina S14 transformants are promising strains for the production of ETA, which is hard to obtain from natural sources.

Lactic acid bacteria-containing chocolate as a practical probiotic product with increased acid tolerance

Co-reporter:Yasunori Yonejima, Keiko Hisa, Marina Kawaguchi, Hiroaki Ashitani, Toshiyuki Koyama, Yoko Usamikrank, Nayumi Kishida, Shigenobu Kishino, Jun Ogawa

Biocatalysis and Agricultural Biotechnology (October 2015) Volume 4(Issue 4) pp:773-777

Publication Date(Web):October 2015

DOI:10.1016/j.bcab.2015.09.001

Enantioselective ester hydrolase from Sphingobacterium sp. 238C5 useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis

Co-reporter:Jun Ogawa, Junichi Mano, Tairo Hagishita, Sakayu Shimizu

Journal of Molecular Catalysis B: Enzymatic (October 2009) Volume 60(Issues 3–4) pp:138-144

Publication Date(Web):1 October 2009

DOI:10.1016/j.molcatb.2009.04.011

A novel enzyme, β-phenylalanine ester hydrolase, useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis was characterized. The enzyme purified from the cell free-extract of Sphingobacterium sp. 238C5 well hydrolyzed β-phenylalanine esters (S)-stereospecifically. Besides β-phenylalanine esters, the enzyme catalyzed the hydrolysis of several α-amino acid esters with l-stereospecificity, while the deduced 369 amino acid sequence of the enzyme exhibited homology to alkaline d-stereospecific peptide hydrolases from Bacillus strains. Escherichia coli transformant expressing the β-phenylalanine ester hydrolase gene exhibited an about 8-fold increase in specific (S)-β-phenylalanine ethyl ester hydrolysis as compared with that of Sphingobacterium sp. 238C5. The E. coli transformant showed (S)-enantiomer specific esterase activity in the reaction with a low concentration (30 mM) of β-phenylalanine ethyl ester, while it showed both esterase and transpeptidase activity in the reaction with a high concentration (170 mM) of β-phenylalanine ethyl ester and produced β-phenylalanyl-β-phenylalanine ethyl ester. This transpeptidase activity was useful for β-phenylalanine β-peptide synthesis.

Characterization of the linoleic acid Δ9 hydratase catalyzing the first step of polyunsaturated fatty acid saturation metabolism in Lactobacillus plantarum AKU 1009a

Co-reporter:Michiki Takeuchi, Shigenobu Kishino, Akiko Hirata, Si-Bum Park, ... Jun Ogawa

Journal of Bioscience and Bioengineering (June 2015) Volume 119(Issue 6) pp:636-641

Publication Date(Web):1 June 2015

DOI:10.1016/j.jbiosc.2014.10.022

Linoleic acid Δ9 hydratase, which is involved in linoleic acid saturation metabolism of Lactobacillus plantarum AKU 1009a, was cloned, expressed as a his-tagged recombinant enzyme, purified with an affinity column, and characterized. The enzyme required FAD as a cofactor and its activity was enhanced by NADH. The maximal activities for the hydration of linoleic acid and for the dehydration of 10-hydroxy-cis-12-octadecenoic acid (HYA) were observed at 37 °C in buffer at pH 5.5 containing 0.5 M NaCl. Free C16 and C18 fatty acids with cis-9 double bonds and 10-hydroxy fatty acids served as substrates for the hydration and dehydration reactions, respectively. The apparent Km value for linoleic acid was estimated to be 92 μM, with a kcat of 2.6∙10−2 s−1 and a Hill factor of 3.3. The apparent Km value for HYA was estimated to be 98 μM, with a kcat of 1.2∙10−3 s−1.

Microbial production of dihomo-γ-linolenic acid by Δ5-desaturase gene-disruptants of Mortierella alpina 1S-4

Co-reporter:Hiroshi Kikukawa, Eiji Sakuradani, Akinori Ando, Tomoyo Okuda, ... Jun Ogawa

Journal of Bioscience and Bioengineering (July 2016) Volume 122(Issue 1) pp:22-26

Publication Date(Web):1 July 2016

DOI:10.1016/j.jbiosc.2015.12.007

We constructed dihomo-γ-linolenic acid (DGLA)-producing strains with disruption of the Δ5-desaturase (Δ5ds) gene, which encodes a key enzyme catalyzing the bioconversion of DGLA to arachidonic acid (ARA), by efficient gene-targeting, using Δlig4 strain of Mortierella alpina 1S-4 as the host. In previous study, we had already identified and disrupted the lig4 gene encoding DNA ligase 4, which involves in non-homologous end joining, in M. alpina 1S-4, and the Δlig4 strain had showed efficient gene-targeting. In this study, the uracil auxotroph of Δlig4 strain was constructed, and then transformed for disruption of Δ5ds. The isolation of nine Δ5ds-disruptants out of 18 isolates indicated that the disruption efficiency was 50%. The ratio of DGLA among the total fatty acids of the Δ5ds-disruptants reached 40.1%; however, no ARA was detected. To our knowledge, this is the first study to report the construction of DGLA-producing transformants by using the efficient gene-targeting system in M. alpina 1S-4.

Characterization of hydroxy fatty acid dehydrogenase involved in polyunsaturated fatty acid saturation metabolism in Lactobacillus plantarum AKU 1009a

Co-reporter:Michiki Takeuchi, Shigenobu Kishino, Si-Bum Park, Nahoko Kitamura, Jun Ogawa

Journal of Molecular Catalysis B: Enzymatic (July 2015) Volume 117() pp:7-12

Publication Date(Web):1 July 2015

DOI:10.1016/j.molcatb.2015.03.020

•Hydroxy fatty acid dehydrogenase (CLA-DH) properties from L. plantarum were studied.•CLA-DH showed wide substrate specificity toward hydroxy fatty acids preferring those with an internal hydroxy group.•CLA-DH was involved in polyunsaturated fatty acid saturation metabolism.Hydroxy fatty acid dehydrogenase, which is involved in polyunsaturated fatty acid saturation metabolism in Lactobacillus plantarum AKU 1009a, was cloned, expressed, purified, and characterized. The enzyme preferentially catalyzed NADH-dependent hydrogenation of oxo fatty acids over NAD+-dependent dehydrogenation of hydroxy fatty acids. In the dehydrogenation reaction, fatty acids with an internal hydroxy group such as 10-hydroxy-cis-12-octadecenoic acid, 12-hydroxy-cis-9-octadecenoic acid, and 13-hydroxy-cis-9-octadecenoic acid served as better substrates than those with α- or β-hydroxy groups such as 3-hydroxyoctadecanoic acid or 2-hydroxyeicosanoic acid. The apparent Km value for 10-hydroxy-cis-12-octadecenoic acid (HYA) was estimated to be 38 μM with a kcat of 7.6 × 10−3 s−1. The apparent Km value for 10-oxo-cis-12-octadecenoic acid (KetoA) was estimated to be 1.8 μM with a kcat of 5.7 × 10−1 s−1. In the hydrogenation reaction of KetoA, both (R)- and (S)-HYA were generated, indicating that the enzyme has low stereoselectivity. This is the first report of a dehydrogenase with a preference for fatty acids with an internal hydroxy group.Download full-size image

Oxidative pyrimidine metabolism in Rhodococcus erythropolis useful for valuable nucleoside synthesis: Discovery of a novel amidohydorolase, ureidomalonase

Co-reporter:Nobuyuki Horinouchi, Chee-Leong Soon, Sakayu Shimizu, Jun Ogawa

Biocatalysis and Agricultural Biotechnology (July 2012) Volume 1(Issue 3) pp:264-266

Publication Date(Web):July 2012

DOI:10.1016/j.bcab.2012.03.011

β-Aryl-β-amino acid aminotransferase from Variovorax sp. JH2 is useful for enantioselective β-phenylalanine production

Co-reporter:Makoto Hibi, Junichi Mano, Tairo Hagishita, Jun Shima, Sakayu Shimizu, Jun Ogawa

Biocatalysis and Agricultural Biotechnology (July 2012) Volume 1(Issue 3) pp:253-258

Publication Date(Web):July 2012

DOI:10.1016/j.bcab.2012.04.001

Extracellular oxidases of Cerrena sp. complementarily functioning in artificial dye decolorization including laccase, manganese peroxidase, and novel versatile peroxidases

Co-reporter:Makoto Hibi, Satoko Hatahira, Masato Nakatani, Kenzo Yokozeki, Sakayu Shimizu, Jun Ogawa

Biocatalysis and Agricultural Biotechnology (July 2012) Volume 1(Issue 3) pp:220-225

Publication Date(Web):July 2012

DOI:10.1016/j.bcab.2012.03.003

Characterization of starch-accumulating duckweeds, Wolffia globosa, as renewable carbon source for bioethanol production

Co-reporter:Tomohiro Fujita, Eiji Nakao, Michiki Takeuchi, Ayumi Tanimura, Akinori Ando, Shigenobu Kishino, Hiroshi Kikukawa, Jun Shima, Jun Ogawa, Sakayu Shimizu

Biocatalysis and Agricultural Biotechnology (April 2016) Volume 6() pp:123-127

Publication Date(Web):April 2016

DOI:10.1016/j.bcab.2016.03.006

Two laccase isoenzymes and a peroxidase of a commercial laccase-producing basidiomycete, Trametes sp. Ha1

Co-reporter:Masato Nakatani, Makoto Hibi, Masashi Minoda, Jun Ogawa, Kenzo Yokozeki, Sakayu Shimizu

New Biotechnology (30 September 2010) Volume 27(Issue 4) pp:317-323

Publication Date(Web):30 September 2010

DOI:10.1016/j.nbt.2010.02.008

Three extracellular ligninolytic oxidoreductases that are produced by a commercial laccase-producing Trametes sp. Ha1 were purified and characterized. This fungus showed strong ligninolytic oxidoreductase activity with and without hydrogen peroxide present in the reaction mixture. The oxidoreductase activity was found to be derived from two laccases and a peroxidase. One of the two laccases represents a main component of the commercial laccase preparation from Trametes sp. Ha1. This enzyme had a high thermostability, which makes it attractive for practical applications. The second laccase was induced by the addition of p-xylidine into the culture medium and showed unique characteristics with respect to pI value and substrate specificity. The peroxidase showed wide oxidation activity against aromatic compounds.

Disruption of lig4 improves gene targeting efficiency in the oleaginous fungus Mortierella alpina 1S-4

Co-reporter:Hiroshi Kikukawa, Eiji Sakuradani, Akinori Ando, Tomoyo Okuda, Misa Ochiai, Sakayu Shimizu, Jun Ogawa

Journal of Biotechnology (20 August 2015) Volume 208() pp:63-69

Publication Date(Web):20 August 2015

DOI:10.1016/j.jbiotec.2015.05.020

•We identified a lig4 gene encoding DNA ligase 4 in Mortierella alpina 1S-4.•We disrupted the lig4 gene in M. alpina 1S-4 by homologous recombination.•We improved gene targeting efficiency dramatically in M. alpina 1S-4 by disruption of the lig4 gene.The oil-producing zygomycete Mortierella alpina 1S-4 is known to accumulate beneficial polyunsaturated fatty acids. We identified the lig4 gene that encodes for a DNA ligase 4 homolog, which functions to repair double strand breaks by non-homologous end joining. We disrupted the lig4 gene to improve the gene targeting efficiency in M. alpina. The M. alpina 1S-4 Δlig4 strains showed no defect in vegetative growth, formation of spores, and fatty acid production, but exhibited high sensitivity to methyl methansulfonate, an agent that causes DNA double-strand breaks. Importantly, gene replacement of ura5 marker by CBXB marker occurred in 67% of Δlig4 strains and the gene targeting efficiency was 21-fold greater than that observed in disruption of the lig4 gene in the M. alpina 1S-4 host strain. Further metabolic engineering of the Δlig4 strains is expected to result in strains that produce higher levels of rare and beneficial polyunsaturated fatty acids and contribute to basic research on the zygomycete.

Screening and characterization of a phosphopentomutase useful for enzymatic production of 2′-deoxyribonucleoside

Co-reporter:Nobuyuki Horinouchi, Takako Kawano, Takafumi Sakai, Seiichiro Matsumoto, Mie Sasaki, Yoichi Mikami, Jun Ogawa, Sakayu Shimizu

New Biotechnology (1 October 2009) Volume 26(Issues 1–2) pp:75-82

Publication Date(Web):1 October 2009

DOI:10.1016/j.nbt.2009.03.015

Bacillus sphaericus AKU 229 was found to produce an acetaldehyde-tolerant and phosphorylated compound-tolerant phosphopentomutase useful for enzymatic 2′-deoxyribonucleoside production. The gene encoding the phosphopentomutase was cloned and expressed in Escherichia coli. The E. coli expressing B. sphaericus phosphopentomutase was an excellent catalyst as to production of 2′-deoxyribonucleoside in the presence of acetaldehyde and phosphorylated compounds such as fructose 1,6-diphosphate, and d-glyceraldehyde 3-phosphate, which are derived from glucose through glycolysis with yeast cells, and exist abundantly in the practical reaction mixture for enzymatic 2′-deoxyribonucleoside production.