Production of highly optically active (R)-3-chloro-1,2-propanediol using a bacterium assimilating the (S)-isomer

A bacterium that assimilates (S)-3-chloro-1,2-propanediol [monochlorohydrin (MCH)] was isolated from soil by enrichment culture. The bacterium was identified as Pseudomonas sp. by taxonomic studies. The strain grew in a medium containing racemic MCH as a source of carbon and degraded (S)-MCH stereoselectively, liberating chloride ions. The residual isomer was the (R)-form [99.5% enantiomeric excess (ee)], which was obtained from the racemate in a final yield of 36% by using this strain. Subsequently, highly optically active (R)-glycidol (GLD) (99.3% ee) was prepared from the (R)-MCH obtained by reaction in alkaline solution. The cell-free extracts of the cells had both dehalogenating and epoxide-opening activities, which converted various halohydrins to the corresponding epoxides and epoxides to the corresponding diols, respectively. Correspondence to: T. Suzuki     

A novel enzymatic dehalogenation of (R)-3-chloro-1,2-propanediol inAlcaligenes sp. DS-S-7G

The mechanism of the novel enzymatic dehalogenating action of (R)-3-chloro-1,2-propanediol [monochlorohydrin (MCH)] was investigated. The (R)-MCH-dechlorinating enzyme system ofAlcaligenes sp. DS-S-7G, which stereoselectively assimilated (R)-MCH from the racemate, was composed of two components (Enzyme 1 and Enzyme 2). Enzyme 1 was a flavoprotein with a relative molecular mass (M _r) of 70,000 and was composed of two kinds of polypeptides (58,000 and 16,000). The enzyme exhibited activity for converting (R)-MCH to hydroxyacetone with the liberation of chloride ions under aerobic conditions. On the other hand, Enzyme 2 with anM _r of 86,000, which was also composed of two kinds of polypeptides (33,000 and 53,000), showed no dechlorinating activity for (R)-MCH. However, in the presence of NAD⁺, when Enzyme 1 was conjugated with Enzyme 2 in the (R)-MCH-dechlorinating reaction, the co-operative dechlorinating activity was four to five times higher than that by Enzyme 1 alone. (R)-MCH was finally degraded to acetic acid and formic acid by the joint action of the two enzymes. These facts indicate that (R)-MCH is oxidatively dechlorinated by the two enzymes in the presence of NAD⁺ inAlcaligenes sp. DS-S-7G.     

Enhanced production of (R)-1,2-propanediol by metabolically engineered Escherichia coli

1,2-Propanediol (1,2-PD) is a major commodity chemical currently derived from propylene. Previously, we have demonstrated the production of enantiomerically pure (R)-1,2-propanediol from glucose by an engineered E. coli expressing genes for NADH-linked glycerol dehydrogenase and methylglyoxal synthase. In this work, we investigate three methods to improve 1,2-PD in E. coli. First, we investigated improving the host by eliminating production of a byproduct, lactate. To do this, we constructed strains with mutations in two enzymes involved in lactate production, lactate dehydrogenase and glyoxalase I. (Surprisingly, when mutations were made in its ability to produce lactate, one strain of E. coli [MM294], produced a small amount of 1,2-PD without any added genes.) Second, we constructed a complete pathway to 1,2-PD from the glycolytic intermediate, dihydroxyacetone phosphate. Our previous 1, 2-PD producing strains relied on at least one endogenous E. coli activity and only produced 0.7 g/L of 1,2-PD. The complete pathway involved the coexpression of methylglyoxal synthase (mgs), glycerol dehydrogenase (gldA), and either yeast alcohol dehydrogenase (adhI) or E. coli 1,2-propanediol oxidoreductase (fucO). Third, we investigated bioprocessing improvements by carrying out a fed-batch fermentation with the best engineered strain (expressing mgs, gldA, and fucO). A final titer of 4.5 g/L of (R)-1,2-PD was produced, with a final yield of 0.19 g of 1,2-PD per gram of glucose consumed. This work provides a basis for further strain and process improvement.     

Synthesis of (1R,2S)-1-(3'-chloro-4' -methoxyphenyl)-1,2-propanediol (trametol) and (1R,2S)-1-(3',5'-dichloro-4'-methoxyphenyl)-1,2-propanediol,chlorinated fungal metabolites in the natural environment

(1R,2S)-1-(3'-Chloro-4'-methoxyphenyl)-1,2propanediol (Trametol, 3), a metabolite of the fungus Trametes sp. IVP-F640 and Bjerkandera sp. BOS55, was synthesized by employing Sharpless asymmetric dihydroxylation as the key step. Similarly, the (1R,2S)-isomer of 1-(3',5'-dichloro-4'-methoxyphenyl)-1,2-propanediol (4), another metabolite of Bjerkandera sp. BOS55, was synthesized by asymmetric dihydroxylation.     

Isolation of (S)-2,3-dichloro-1-propanol assimilating bacterium,its characterization,and its use in preparation of (R)-2,3-dichloro-1-propanol and (S)-epichlorohydrin

Summary Linear alkylbenzene sulfonate (LAS) is a widely used anionic surfactant. Although approximately 1 million metric tons of LAS are produced annually, relatively little is known about the bacteria or the genetic factors that control LAS degradation in the environment. The objectives of this research were to: i) compare bacterial populations in wastewater and pristine pond systems; ii) determine the frequency of plasmids in bacteria from these sites; and iii) compare the frequency of DNA sequences coding for aromatic catabolism in isolates from these two sites. Plate counts indicated that exposure to wastewater resulted in higher levels of both heterotrophic bacteria and bacteria capable of growing on LAS containing medium (LAS/YEPG). In addition to higher numbers, a higher proportion of heterotrophs from the wastewater system were capable of growth on LAS/YEPG medium. Thus, the high levels of LAS in the wastewater system apparently selected fro organisms that were able to tolerate and/or degrade, it. Mineralization of¹⁴C-ring labelled LAS in any habitat related to the presence of organisms that grew on LAS/YEPG. Although may of these isolates could carry out primary degradation, no isolate, could mineralize¹⁴C-ring LAS in pure culture. A higher incidence of plasmids was found in bacteria from the wastewater pond and among bacteria that grew on LAS containing medium. However, the presence of plasmid, DNA did not necessarily confer the ability to degrade LAS nor was the ability to degrade LAS dependent on the presence of a plasmid. The incidence of selected genotypes for aromatic catabolism was similar among isolates on LAS/YEPG at both sites, suggesting that LAS ring degradation may be present in other populations or encoded by alternative sequences. In conclusion, LAS mineralization is mediated by a consortium and the evidence that initial attack of LAS is plasmid mediated is inconclusive.     

A two-step enzymatic resolution process for large-scale production of (S)- and (R)-ethyl-3-hydroxybutyrate

An efficient two-step enzymatic process for production of (R)- and (S)-ethyl-3-hydroxybutyrate (HEB), two important chiral intermediates for the pharmaceutical market, was developed and scaled-up to a multikilogram scale. Both enantiomers were obtained at 99% chemical purity and over 96% enantiomeric excess, with a total process yield of 73%. The first reaction involved a solvent-free acetylation of racemic HEB with vinylacetate for the production of (S)-HEB. In the second reaction, (R)-enriched ethyl-3-acetoxybutyrate (AEB) was subjected to alcoholysis with ethanol to derive optically pure (R)-HEB. Immobilized Candida antarctica lipase B (CALB) was employed in both stages, with high productivity and selectivity. The type of butyric acid ester influenced the enantioselectivity of the enzyme. Thus, extending the ester alkyl chain from ethyl to octyl resulted in a decrease in enantiomeric excess, whereas using bulky groups such as benzyl or t-butyl, improved the enantioselectivity of the enzyme. A stirred reactor was found unsuitable for large-scale production due to attrition of the enzyme particles and, therefore, a batchwise loop reactor system was used for bench-scale production. The immobilized enzyme was confined to a column and the reactants were circulated through the enzyme bed until the targeted conversion was reached. The desired products were separated from the reaction mixture in each of the two stages by fractional distillation. The main features of the process are the exclusion of solvent (thus ensuring high process throughput), and the use of the same enzyme for both the acetylation and the alcoholysis steps. Kilogram quantities of (S)-HEB and (R)-HEB were effectively prepared using this unit, which can be easily scaled-up to produce industrial quantities.     

A novel enantioselective epoxide hydrolase for (R)-phenyl glycidyl ether to generate (R)-3-phenoxy-1,2-propanediol

Bacillus sp. Z018, a novel strain producing epoxide hydrolase, was isolated from soil. The epoxide hydrolase catalyzed the stereospecific hydrolysis of (R)-phenyl glycidyl ether to generate (R)-3-phenoxy-1,2-propanediol. Epoxide hydrolase from Bacillus sp. Z018 was inducible, and (R)-phenyl glycidyl ether was able to act as an inducer. The fermentation conditions for epoxide hydrolase were 35°C, pH 7.5 with glucose and NH₄Cl as the best carbon and nitrogen source, respectively. Under optimized conditions, the biotransformation yield of 45.8% and the enantiomeric excess of 96.3% were obtained for the product (R)-3-phenoxy-1,2-propanediol.     

酵母不对称催化制备R-1,2-丙二醇

研究了采用面包酵母还原丙酮醇制备R-1,2-丙二醇的工艺。采用摇瓶对转化条件进行单因素实验,确定最优转化条件：丙酮醇浓度0.3mmol/mL,pH7.0,酵母质量浓度150g/L,乙醇浓度为0.3mmol/mL,转化时间25h。在此条件下,采用分批流加策略进行1.5L规模发酵罐转化试验,转化25h后,发酵液的R-1,2-丙二醇浓度为0.27mmol/mL。     

Enantioselective preparation of (R) and (S)-3-hydroxycyclopentanone by kinetic resolution

A straightforward approach to enantiomerically enriched (R) and (S)-3-hydroxycyclopentanone is described. The key step involves a kinetic resolution of racemic 3-hydroxycyclopentanone using commercial Pseudomonas cepacia lipase immobilized on diatomite (Amano lipase PS-DI). The absolute stereochemistry of the product was determined by derivatization into (R)-3-(benzyloxy)cyclopentanone.     

Comparative toxicity of (+)-(R)- and (−)-(S)-1,2-dibromo-3-chloropropane

The haloalkane 1,2-dibromo-3-chloropropane (DBCP), an environmental pollutant that was widely used as a soil fumigant, is a carcinogen and a mutagen and displays target-organ toxicity to the testes and the kidneys. Because little is known about effects of stereochemistry on the metabolism and toxicity of halogenated alkyl compounds and because DBCP, which has a chiral center at C-2, may show enantioselectivity in its metabolism and/or toxicities, the optically pure enantiomers of DBCP were tested in vivo in rats for organ toxicity as well as for bacterial mutagenicity. Organ toxicity studies showed that (S)-DBCP was slightly more renal toxic than (R)-DBCP but was not significantly more toxic than the racemate, and that no significant differences were observed in the extents of testicular necrosis and atrophy caused by either enantiomer or the racemate. In contrast, (R)-DBCP was more mutagenic than either (S)-DBCP or the racemate to Salmonella typhimurium (S. typhimurium) strains TA 100 and TA104. However, there was little or no enantioselectivity in glutathione S-transferase (GST)-catalyzed conjugation reactions of glutathione with DBCP based on the lack of selectivity in the rates of disappearance of the enantiomers of DBCP in the presence of glutathione (GSH) and GSTs as monitored by chiral gas chromatography (GC). © 1995 Wiley-Liss, Inc.     

Biocatalytic synthesis of (S)-4-chloro-3-hydroxybutanoate ethyl ester using a recombinant whole-cell catalyst

A cofactor regeneration system for enzymatic biosynthesis was constructed by coexpressing a carbonyl reductase from Pichia stipitis and a glucose dehydrogenase from Bacillus megaterium in Escherichia coli Rosetta (DE3) PlySs. Transformants containing the polycistronic plasmid pET-PII-SD2-AS1-B exhibited an activity of 13.5 U/mg protein with 4-chloro-3-oxobutanoate ethyl ester (COBE) as the substrate and an activity of 14.4 U/mg protein with glucose as the substrate; NAD(H) was the coenzyme in both cases. Asymmetric reduction of COBE to (S)-4-chloro-3-hydroxybutanoate ethyl ester [(S)-CHBE] with more than 99% enantiomeric excess was demonstrated by transformants. Furthermore, the paper made a comparison of crude enzyme catalysis and whole-cell catalysis in an aqueous monophasic system and a water/organic solvent biphasic system. In the water/n-butyl acetate system, the coexpression system produced 1,398 mM CHBE in the organic phase, which is the highest yield ever reported for CHBE production by NADH-dependent reductases from yeasts. In this case, the molar yield of CHBE was 90.7%, and the total turnover number, defined as moles (S)-CHBE formed per mole NAD+, was 13,980.     

Serinol (2-Amino-1,3-propanediol) and 3-Amino-1,2-propanediol in Soybean Nodules

The compound X, which had previously been found to be accumulatedin the soybean nodules formed by infection with wild-type H₂-uptakenegative Bradyrhizobium japonicum strains, was identified asserinol (2-amino-1,3-propanediol) by means of elementary analysis,infrared spectrometry, ¹H-nuclear magnetic resonance, ¹³C-nuclearmagnetic resonance, high-performance liquid chromatography andgas chromatography/mass spectrometry. During the process ofpurification of compound X, it was also elucidated that 3-amino-1,2-propanediolwas present in the soybean nodules as a minor component. (Received January 6, 1986; Accepted June 16, 1986)     

Microbial production of the aromatic building-blocks (S)-styrene oxide and (R)-1,2-phenylethanediol from renewable resources

(S)-Styrene oxide and (R)-1,2-phenylethanediol are chiral aromatic molecular building blocks used commonly as precursors to pharmaceuticals and other specialty chemicals. Two pathways have been engineered in Escherichia coli for their individual biosynthesis directly from glucose. The novel pathways each constitute extensions of the previously engineered styrene pathway, developed by co-expressing either styrene monooxygenase (SMO) or styrene dioxygenase (SDO) to convert styrene to (S)-styrene oxide and (R)-1,2-phenylethanediol, respectively. StyAB from Pseudomonas putida S12 was determined to be the most effective SMO. SDO activity was achieved using NahAaAbAcAd of Pseudomonas sp. NCIB 9816-4, a naphthalene dioxygenase with known broad substrate specificity. Production of phenylalanine, the precursor to both pathways, was systematically enhanced through a number of mutations, most notably via deletion of tyrA and over-expression of tktA. As a result, (R)-1,2-phenylethanediol reached titers as high as 1.23 g/L, and at 1.32 g/L (S)-styrene oxide titers already approach their toxicity limit. As with other aromatics, product toxicity was strongly correlated with a model of membrane accumulation and disruption. This study additionally demonstrates that greater flux through the styrene pathway can be achieved if its toxicity is addressed, as achieved in this case by reacting styrene to less toxic products. See accompanying commentary by Brian F. Pfleger DOI: 10.1002/biot.201300251     

Resolution and some properties of enzymes involved in enantioselective transformation of 1,3-dichloro-2-propanol to (R)-3-chloro-1,2-propanediol by Corynebacterium sp. strain N-1074.

During the course of the transformation of 1,3-dichloro-2-propanol (DCP) into (R)-3-chloro-1,2-propanediol [(R)-MCP] with the cell extract of Corynebacterium sp. strain N-1074, epichlorohydrin (ECH) was transiently formed. The cell extract was fractionated into two DCP-dechlorinating activities (fractions Ia and Ib) and two ECH-hydrolyzing activities (fractions IIa and IIb) by TSKgel DEAE-5PW column chromatography. Fractions Ia and Ib catalyzed the interconversion of DCP to ECH, and fractions IIa and IIb catalyzed the transformation of ECH into MCP. Fractions Ia and IIa showed only low enantioselectivity for each reaction, whereas fractions Ib and IIb exhibited considerable enantioselectivity, yielding R-rich ECH and MCP, respectively. Enzymes Ia and Ib were isolated from fractions Ia and Ib, respectively. Enzyme Ia had a molecular mass of about 108 kDa and consisted of four subunits identical in molecular mass (about 28 kDa). Enzyme Ib was a protein of 115 kDa, composed of two different polypeptides (about 35 and 32 kDa). The specific activity of enzyme Ib for DCP was about 30-fold higher than that of enzyme Ia. Both enzymes catalyzed the transformation of several halohydrins into the corresponding epoxides with liberation of halides and its reverse reaction. Their substrate specificities and immunological properties differed from each other. Enzyme Ia seemed to be halohydrin hydrogen-halide-lyase which was already purified from Escherichia coli carrying a gene from Corynebacterium sp. strain N-1074.     

Enhancement of activity of lipase-displaying yeast cells and their application to optical resolution of (R,S)-1-benzyloxy-3-chloro-2-propyl monosuccinate

Rhizopus oryzae lipase (ROL) was displayed on the cell surface of Saccharomyces cerevisiae via the Flo1 N-terminal region (1100 amino acids), which corresponds to a flocculation functional domain. The activity of lipase-displaying yeast whole-cell biocatalysts was enhanced 7.3-fold by incubation of the yeast cells at 20 degrees C in distilled water for 8 days after 8 day cultivation. The amount of lipase molecules present in cell wall and intracellular fractions was found to be increased 4.5- and 1.8-fold, respectively, by incubation, which proves that ROL molecules are expressed during incubation. The ROL-displaying yeast whole-cell biocatalyst with enhanced activity was successfully catalyzed by optical resolution of the pharmaceutical precursor (R,S)-1-benzyloxy-3-chloro-2-propyl monosuccinate. Moreover, it showed stable activity through at least eight reaction cycles. These results demonstrate that ROL-displaying yeast cells with enhanced activity by incubation in distilled water are very effective in industrial bioconversion processes.     

Asymmetric synthesis of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate using a self-sufficient biocatalyst based on carbonyl reductase and cofactor co-immobilization

Bioprocess and Biosystems Engineering - tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate [(3R,5S)-CDHH] is the key chiral intermediate to synthesize the side chain of the lipid-lowering drug...     

Efficient production of recombinant aldehyde reductase and its application for asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to ethyl (R)-4-chloro-3-hydroxybutanoate

An NADPH-dependent aldehyde reductase (ALR, EC1.1.1.2) gene is cloned from Sporobolomyces salmonicolor ZJUB 105, and inserted into plasmid pQE30 to construct the expression plasmid (pQE30-ALR). A variety of E. coli strains were employed as hosts to obtain transformants with pQE30-ALR, respectively. Among these different types of transformants, the highest enzyme activity of ALR can be produced with E. coli M15 (pQE30-ALR). The bioactivity of ALR could be further improved significantly by the optimization of induction conditions. The results showed that the enzyme activity of ALR reached 6.48 U/mg protein, which is fifteen times higher than that of S. salmonicolor ZJUB 105. This recombinant strain was applied to the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3- hydroxybutanoate (CHBE). The results showed that the yield and optical purity of (R)-CHBE reached 98.5% and 99% e.e. (enantiomeric excess), respectively.     

Efficient synthesis of the ketone body ester (R)-3-hydroxybutyryl-(R)-3-hydroxybutyrate and its (S,S) enantiomer

The ketone body ester (R)-3-hydroxybutyryl-(R)-3-hydroxybutyrate and its (S,S) enantiomer were prepared in a short, operationally simple synthetic sequence from racemic β-butyrolactone. Enantioselective hydrolysis of β-butyrolactone with immobilized Candida antarctica lipase-B (CAL-B) results in (R)-β-butyrolactone and (S)-β-hydroxybutyric acid, which are easily converted to (R) or (S)-ethyl-3-hydroxybutyrate and reduced to (R) or (S)-1,3 butanediol. Either enantiomer of ethyl-3-hydroxybutyrate and 1,3 butanediol are then coupled, again using CAL-B, to produce the ketone body ester product. This is an efficient, scalable, atom-economic, chromatography-free, and low cost synthetic method to produce the ketone body esters.     

Synthesis of (3R,10R)- and (3S,10S)-Diastereomers of 3,10-Dimethylspermine

Russian Journal of Bioorganic Chemistry - A simple and practical 10-step synthesis is reported for previously unknown diastereomers of C?methylated spermine (Spm) analogue,...