Production of (S)-(+)-citramalic acid from itaconic acid by resting cells of Alcaligenes denitrificans strain MCI2775

(S)-(+)-Citramalic-acid-producing activity in microorganisms was studied with resting cells in a reaction mixture containing itaconic acid. Itaconic-acid-utilizing bacteria were found to produce (S)-(+)-citramalic acid from itaconic acid. The strain, which showed the best productivity among those studied, was identified taxonomically as Alcaligenes denitrificans strain MCI2775. (S)-(+)-Citramalic acid produced by this strain was present in a 99.9% enantiometric excess. The culture and reaction conditions for the production were optimized for this strain. Addition of Mn²⁺, d-pantothenic acid and l-leucine to the culture medium enhanced the (S)-(+)-citramalic acid-producing activity. Under optimal conditions, 27 g (S)-(+)-citramalic acid/l was produced in 30 h. The yield to itaconic acid added was 69.0 mol%. Correspondence to: Y. Asano     

Stereoselective Synthesis of Ectocarpene and Its Antipode via Microbiological Asymmetric Hydrolysis

Biological asymmetric hydrolysis of ethyl (±)-cycloheptadienecarboxylate with Rhodotorula minuta var. texensis IFO 1102 and chemical resolution of the corresponding carboxylic acid with (?)-quinine provided (R)-(+)-ethyl 2,5-cycloheptadienecarboxylate (78% e.e.) and (S)-(+)-2,5-cycloheptadienecarboxylic acid (95% e.e.), respectively. The (R)-(+)-carboxylate was converted to (R)-(?)-2,5-cycloheptadienylcarbaldehyde and the (S)-(+)-carboxylic acid to (S)-(+)-2,5-cycloheptadienylcarbaldehyde. Ectocarpene (78% e.e.), male-gamete attractant of marine brown alga, and its antipode (95% e.e.) were synthesized by stereoselective Wittig reaction between the (R)-(?)- and (S)-(+)-aldehydes and propyltriphenylphosphonium bromide in a liquid-solid two phase system using 18-crown ether-t-BuOK, respectively.     

Microbial asymmetric oxygenation of an organometallic sulfide

Summary Penicillium frequentans IFO 5692 oxidized enantioselectively (methylthiomethyl)ferrocene (1) to (R)-(–)-(methylsulfinylmethyl)ferrocene (2) (98 %e.e.) andCorynebacterium equi IFO 3730 to (S)-(+)-2 (69 %e.e.).     

Rapid PaperMicrobial Oxygenation of Organo-metallic Bissulfides Leading to Formation of Planar Chirality and C2 Symmetry

1,2-Bis(methylthiomethyl)ferrocene (3) was oxidized by Corynebacterium equi IFO 3730 to give monosulfoxide 4 in two diastereomeric forms with (1S,2R,S_S) and (1S,2R,R_S) configurations in a ratio of 4:1, while 1,1′-bis(methylthiomethyl)ferrocene (5) was oxidized by Penicillium frequentans IFO 5692 to (R)-monosulfoxide 6 and then preferentially to (R,R)-bissulfoxide 7. Thus, the bacterial monooxygenase generated specific planar chirality in the metallocenic monosulfoxide, and the fungal enzyme formed C₂ symmetry in the bissulfoxide.     

Highly efficient conversion of (±)-mandelic acid to its (R)-(−)-enantiomer by combination of enzyme-mediated oxidation and reduction

Summary A novel method for total conversion of racemic mandelic acid into its (R)-enantiomer was developed. The method consists of enantioselective oxidation of (S)-(+)-mandelic acid byAlcaligenes bronchisepticus KU 1201 and NADH-dependent asymmetric reduction of resulting benzoylformic acid to (R)-mandelic acid with cell-free extract ofStreptococcus faecalis IFO 12964.     

A Gene (tmpA) for an Efflux Protein of the Transporter Family III from Brevibacterium linens OC2, an Antibacterial Substance-Producing Strain

A gene (tmpA) encoding a putative transmembrane protein has been cloned from B. linens OC2, an antibacterial substance-producing strain. The deduced TmpA protein sequence shares similarities to members of the transporter family III exploiting the transmembrane proton gradient to provide export of toxic compounds such as antiseptics or antibiotics. Northern blot analysis indicated that tmpA gene is expressed. Length of RNA messenger and overlapping of ORFs upstream tmpA gene suggested that it might belong to an operon. The tmpA gene is unusual among B. linens species since it was not detected among eight B. linens collection strains and 40 B. linens industrial strains. Received: 16 February 1999 / Accepted: 17 February 1999     

The role of quinate and shikimate in the metabolism of lactobacilli

The metabolism of (–)-quinate and shikimate by one heterofermentative strain,actobacillus pastorianus, and by one homofermentative strain,Lactobacillus plantarum, has been studied using growing and washed cells. Both organisms reduced quinate and shikimate under anaerobic conditions in the presence of suitable hydrogen donors including fructose, glucose andd(–) andl(+)-lactates. The end-product ofL.pastorianus metabolism was dihydroshikimate butL.plantarum carried the reduction a stage further tocis-3,4-dihydroxycyclohexanecarboxylate and formed, simultaneously, catechol. The enzymes involved in these reductions are induced; their importance in the metabolism of lactobacilli is discussed.     

Purification and characterization of a lactonase from<Emphasis Type="Italic"> Erwinia cypripedii</Emphasis> 314B that hydrolyzes (<Emphasis Type="Italic">S</Emphasis>)-5-oxo-2-tetrahydrofurancarboxylic acid

A bacterium, strain 314B, able to assimilate (S)-5-oxo-2-tetrahydrofurancarboxylic acid was isolated from soil and identified as Erwinia cypripedii. A lactonase hydrolyzing (S)-5-oxo-2-tetrahydrofurancarboxylic acid to l--hydroxyglutaric acid was purified 63-fold with 2% recovery from crude extracts of this bacterium to homogeneity as judged by SDS-PAGE. The molecular masses estimated by SDS-PAGE and gel filtration were 41 kDa and 79 kDa, respectively. The maximum activity was observed at pH 6.5–7.5 and 35–45 °C. The enzyme showed lower activity toward dl-2-oxotetrahydrofuran-4,5-dicarboxylic acid, but did not act on (R)-5-oxo-2-tetrahydrofurancarboxylic acid and other natural and synthetic lactones tested.     

Expression of a Novel Sphingosine 1-Phosphate Receptor Motif Protein Gene in Maturing Rat Testes

(S)-1,3-Butanediol (BOO) oxidizing enzyme was purified from Candida parapsilosis IFO 1396, which could produce (R)-1,3-BDO from the racemate. The purified enzyme was an NAO⁺ -dependent secondary alcohol dehydrogenase that oxidized (S)-1,3-BDO to 4-hydroxy-2-butanone stereo-specifically.     

Synthesis and Antimicrobial Activity of Chiral cis-Cycloheximide Isomers

Such (+)- and (?)-cis-cycloheximide isomers as isocyclohcximide (1a, 1b), α-epiisocycloheximide (2a, 2b) and neocycloheximide (3a, 3b) were synthesized by aldol condensation of (?)-(2R, 4R)- and (+)-(2S, 4S)-cis-2,4-dimethyl-1-cyclohexanone (5a, 5b). obtained by microbial resolution, with 4-(2-oxoethyl)-2,6-piperidinedione (7). The absolute configuration of the (?)-cis-ketone 5a was confirmed by chemical correlation with natural (2S, 4S, 6S, αR)-cycloheximide (4). The newly synthesized isomer, (?)-α-epiisocycloheximide (2b), showed strong antimicrobial activity against S. cerevisiae andP. oryzae close to that of natural cycloheximide (4).     

Neopeptins A and B,New Antifungal Antibiotics

An enzyme that reduces benzoylformate with NADH to form (R)-mandelate was extracted from cells of Streptococcus faecalis IFO 12964 and purified to more than 95% purity as evidenced by gel electrophoresis. Physicochemical and enzymic properties were studied. From the substrate specificity, we concluded that the enzyme was a kind of (R)-2-hydroxyisocaproate dehydrogenase. Optically pure (R)-(—)-mandelic acid was prepared with the enzyme, NADH, and alcohol, formate or glucose dehydrogenase in 84~93% yield. Five (R)-2-hydroxyalkanoic acids (C₄~C₆) or their Ba salts, (R)-(+)-3-phenyllactic acid and (S)-(—)-3-chlorolactic acid were also prepared with the enzyme.     

A phylogenetic analysis of the lichen familySphaerophoraceae (Caliciales); a new generic classification and notes on character evolution

A phylogenetic analysis of the familySphaerophoraceae (Caliciales, lichenized ascomycetes) has resulted in a new generic classification. Notes on character evolution are given. The generaSphaerophorus s. str.,Bunodophoron andLeifidium, gen. nov., are accepted.Pleurocybe andPseudosphaerophorus are considered synonyms ofBunodophoron andThysanophoron is considered synonym toSphaerophorus. The following new combinations are proposed:Bunodophoron coomerense (Ohlsson)Wedin,B. diplotypum (Vain.)Wedin,B. dodgei (Ohlsson)Wedin,B. flaccidum (Kantvilas & Wedin)Wedin,B. formosanum (Zahlbr.)Wedin,B. imshaugii (Ohlsson)Wedin,B. insigne (Laurer)Wedin,B. kinabaluense (M. Satô)Wedin,B. macrocarpum (Ohlsson)Wedin,B. madagascareum (Nyl.)Wedin,B. microsporum (Ohlsson)Wedin,B. murrayi (Ohlsson)Wedin,B. notatum (Tibell)Wedin,B. ohlssonii (Wedin)Wedin,B. patagonicum (C. W. Dodge)Wedin,B. ramuliferum (I. M. Lamb)Wedin,B. scrobiculatum (C. Bab.)Wedin,B. tibellii (Wedin)Wedin,B. whakapapaense (Wedin)Wedin, andLeifidium tenerum (Laurer)Wedin.     

Reduction of Ketopantoyl Lactone to D-(—)-Pantoyl Lactone by Microorganisms

The ability to reduce ketopantoyl lactone added to the culture medium to pantoyl lactone was surveyed in a variety of microorganisms. Many of the microorganisms including molds, yeasts, bacteria, actinomycetes and basidiomycetes exhibited this ability. The ratios of D-(—)- and L-(+)-isomers of the yielded pantoyl lactone, however, showed no relation to the genera or sources of strains. Among them, Rhodotorula minuta IFO 0920, Candida parapsilosis IFO 0708 and Aspergillus niger IFO 4415 were found to convert ketopantoyl lactone (45mg/ml) completely and almost specifically to D-(—)-pantoyl lactone. The main enzyme catalyzing this asymmetric reduction was suggested to be ketopantoyl lactone reductase (EC 1.1.1.168).     

R-(−)-mandelic acid production from racemic mandelic acids by Pseudomonas polycolor with asymmetric degrading activity

The microbial asymmetric degradation of S-(+)-mandelic acid was investigated in order to develop a practical process for R-(−)-mandelic acid production from racemic mandelic acids. Among the 790 culture strains tested, microorganisms belonging to the Brevibacterium, Pseudomonas, Rhodococcus, Rhodotorula, Rhodosporidium, Sporobolomyces and Gibberella genera exhibited high S-(+)-mandelic acid degrading activity. Pseudomonas polycolor IFO 3918 was determined to be the best strain and used as a biocatalyst for eliminating the S-(+)-isomer. The maximum rate of S-(+)-isomer degradation was obtained at 30°C and pH 7.0. Under these optimal conditions, the S-(+)-isomer in a racemic mandelic acid 45 g/l mixture was completely degraded within 24 h, with 20 g of R-(−)-mandelic acid per liter remaining in the reaction mixture. Crystalline R-(−)-mandelic acid with a chemical purity greater than 99% and optical purity of 99.9% enantiomeric excess was obtained at a yield of 35% by acidification of the reaction mixture, extraction with ethyl acetate and subsequent concentration.     

3-Methylaspartate ammonia-lyase from a facultative anaerobe,strain YG-1002

3-Methylaspartase was purified 24-fold and crystallized from the crude extract of the cells of a facultative anaerobic bacterium from soil, strain YG-1002. The molecular mass of the native enzyme was about 84 kDa and that of the subunit was about 42 kDa. The pH optimum for the deamination reaction of (2S, 3S)-3-methylaspartic acid and those for the amination reaction of mesaconic acid were 9.7 and 8.5; its optimum temperature was 50°C. The enzyme was stable at pH 5.5–11.0 and up to 50°C. The enzyme required both divalent and monovalent cations such as Mg²⁺ and K⁺. The enzyme was inhibited by sulfhydryl reagents, metal-chelating reagents and some divalent cations. The enzyme catalyzed the reversible amination/deamination reactions between several 3-substituted (S)-aspartic acids and their corresponding fumaric acid derivatives. The enzyme preferentially acted on (2S, 3S)-3-methylaspartic acid and mesaconic acid in the deamination and the amination reactions respectively. The enzyme showed high similarities in several enzymological properties and N-terminal amino acid sequence with 3-methylaspartase from an obligate anaerobic bacteriumClostridium tetanomorphum.     

Biotransformation of electron-poor alkenes by yeasts: Asymmetric reduction of (4S)-(+)-carvone by yeast enoate reductases

Within the framework of a large-scale screening carried out on 146 yeasts of environmental origin, 16 strains (11% of the total) exhibited the ability to biotransform (4S)-(+)-carvone. Such positive yeasts, belonging to 14 species of 6 genera (Candida, Cryptococcus, Hanseniaspora, Kluyveromyces, Pichia and Saccharomyces), were thus used under different physiological state (growing, resting and lyophilised cells). Yields (expressed as% of biotransformation) varied from 0.14 to 30.04%, in dependence of both the strain and the physiological state of the cells. Products obtained from reduction of (4S)-(+)-carvone were 1S,4S- and 1R,4S-dihydrocarvone, (1S,2S,4S)-, (1S,2R,4S)- and (1R,2S,4S)-dihydrocarveol. Only traces of (1R,2R,4S)-dihydrocarveol were observed in a few strains. As far as the stereoselectivity of the biocatalysis, with the sole exception of a few strains, the use of yeasts determined the prevalent accumulation of 1S,4S-isomers [(1S,4S)-dihydrocarvone + (1S,2S,4S)-dihydrocarveol + (1S,2R,4S)-dihydrocarveol].The addition of glucose (acting as auxiliary substrate for cofactor-recycling system) to lyophilised yeast cells determined a considerable increase of biocatalytic activity: in particular, two strains showed a surprising increase of the% of biotransformation of (4S)-(+)-carvone (to values >98%).     

Stereoselectivity of the Enzymatic Reduction of Aldehydic and Ketonic Carbonyl Groups in Planar Chiral Organomanganese Complexes

(±)-Tricarbonyl(η⁵-1-formyl-2-methylcyclopentadienyl)manganese (1) was optically resolved with horse liver alcohol dehydrogenase (HLADH) and two species of yeasts, Saccharomyces sp. H-1 and Rhodotorula rubra IFO 889. Usually, (1R)-1 was preferentially reduced to give (?)-alcohol 2 of ≥ 97% e.e. ? 84% e.e. Ketone analogue (±)-tricarbonyl(η⁵-1-acetyl-2-methylcyclopentadienyl)-manganese (4) was reduced by the yeasts. The major product by S. sp. H-1 was the (1S,2R,1′S)-(+)-alcohol (5) (≥ 98% e.e.) and the minor product, the (1R,2S,1′S)-(?)-alcohol (6) (86% e.e.). R. rubra gave only the latter alcohol (≥ 99 % e.e.). The Stereodifferentiation mechanism for these bioreductions is discussed in terms of the Prelog rule. The mechanism for HLADH reduction was examined with computer graphics.     

Biotransformation of glucose to 5-keto-<Emphasis Type="SmallCaps">d</Emphasis>-gluconic acid by recombinant<Emphasis Type="Italic"> Gluconobacter oxydans</Emphasis> DSM 2343

For the conversion of glucose to 5-keto-d-gluconate (5-KGA), a precursor of the industrially important l-(+)-tartaric acid, Gluconobacter strains were genetically engineered. In order to increase 5-KGA formation, a plasmid-encoded copy of the gene encoding the gluconate:NADP-5 oxidoreductase (gno) was overexpressed in G. oxydans strain DSM 2434. This enzyme is involved in the nonphosphorylative ketogenic oxidation of glucose and oxidizes gluconate to 5-KGA. As the 5-KGA reductase activity depends on the cofactor NADP⁺, the sthA gene (encoding Escherichia coli transhydrogenase) was cloned and overexpressed in the GNO-overproducing G. oxydans strain. Growth of the sthA-carrying strains was indistinguishable from the G. oxydans wild-type strain and therefore they were chosen for the coupled overexpression of sthA and gno. G. oxydans strain DSM 2343/pRS201-gno-sthA overproducing both enzymes showed an enhanced accumulation of 5-KGA.     

Synthesis and Absolute Configuration of Natural Gregatin B

(S)-(+)-Gregatin B and racemic gregatin B were synthesized from tetrahydro-2-methyl-5-oxo-2-furancarboxylic acid and the absolute configuration of natural gregatin B was determined as (S).     

Interspecific hybridizations among species of theElymus semicostatus andElymus tibeticus groups (Poaceae)

Interspecific hybridizations were made between species of theE. semicostatus group, viz.,E. semicostatus (Nees exSteud.)Meld.,E. validus (Meld.)B. Salomon,E. abolinii (Drob.)Tzvel., andE. fedtschenkoi Tzvel., and species of theE. tibeticus group, viz.,E. pendulinus (Nevski)Tzvel.,E. tibeticus (Meld.)Singh,E. shandongensis B. Salomon, andE. gmelinii (Ledeb.)Tzvel., as well as among species within theE. tibeticus group. All species are tetraploid (2n = 4x = 28) and possess SY genomes. Meiotic pairing data from 24 hybrids involving 17 interspecific combinations are presented. The average number of chiasmata per cell ranged from 17.91 to 26.20 in hybrids within theE. tibeticus group, compared with 7.26 to 22.04 in hybrids between the two species groups. Despite the extensive collection of cytological data, there was no definite evidence for confirming or disproving the separate existence of the two groups.