Improvement in organic solvent tolerance by double disruptions of proV and marR genes in Escherichia coli.

AIMS: To investigate the involvement of osmoprotectant transporters in organic solvent tolerance in Escherichia coli and to construct an E. coli strain with high organic solvent tolerance. METHODS AND RESULTS: The organic solvent tolerance of ΔbetT, ΔproV, ΔproP or ΔputP single-gene knockout mutants of E. coli K-12 strain was examined. Among these mutants, the organic solvent tolerance of the ΔproV mutant remarkably increased compared with that of the parent strain. It has been known that a marR mutation confers tolerance on E. coli to organic solvents. A ΔproV and ΔmarR double-gene mutant was more tolerant to organic solvents than the ΔproV or ΔmarR single-gene mutant. The n-hexane amount accumulated in E. coli cells was examined after incubation in an n-hexane-aqueous medium two-phase system. The intracellular n-hexane level in the ΔproV and ΔmarR double-gene mutant was kept lower than those of the parent strain, ΔproV mutant and ΔmarR mutant. CONCLUSIONS: The organic solvent tolerance level in E. coli highly increased by dual disruption of proV and marR. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests a new strategy for increasing the organic solvent tolerance level in E. coli to improve the usability of the whole-cell biocatalysts in two-phase systems employing organic solvents.

Indigo production by Escherichia coli carrying the phenol hydroxylase gene from Acinetobacter sp strain ST-550 in a water-organic solvent two-phase system.

Acinetobacter sp. strain ST-550 produces indigo from indole in the presence of a large volume of diphenylmethane and a high level of indole. Particular proteins increased remarkably in strain ST-550 grown in the two-phase culture system for indigo production. One of the proteins showed a N-terminal amino acid sequence that was identical to that of the largest subunit of phenol hydroxylase (MopN) from A. calcoaceticus NCBI8250. The indigo-producing activity was strongly induced when ST-550 was grown with phenol as a sole carbon source. Genes coding for the multicomponent phenol hydroxylase were cloned, based on the homology with mopKLMOP from A. calcoaceticus NCBI8250. Escherichia coli carrying the genes produced indigo from indole. E. coli JA300 and its cyclohexane-resistant mutant, OST3410, carrying the hydroxylase genes and the NADH regeneration system were grown in the two-phase culture system for indigo production. The OST3410 recombinant produced 52 microg indigo ml(-1) of medium in the presence of diphenylmethane. This productivity was 4.3-fold higher than that of the JA300 recombinant.

Isolation of an Acinetobacter sp. ST-550 which produces a high level of indigo in a water-organic solvent two-phase system containing high levels of indole.

Acinetobacter sp. strain ST-550 was isolated from fumus soil as an efficient indigo producer in the presence of organic solvent. The minimum inhibitory concentration of indole was 0.4 mg/ml for ST-550. ST-550 produced only a small amount of indigo (less than 0.01 microg/ml) when grown in the presence of indole at concentrations of 0.05 to 0.3 mg/ml without any organic solvent. However. ST-550 produced indigo effectively when grown in the presence of a large volume of diphenylmethane and a high level of indole: optimized conditions were 3 ml of a medium containing 0.3 ml diphenylmethane and 2.7 mg indole. Under these conditions, ST-550 produced 0.88 mg indigo (292 microg/mI medium).

Cloning, sequence analysis and expression of a gene encoding an organic solvent- and detergent-tolerant cholesterol oxidase of Burkholderia cepacia strain ST-200.

Burkholderia cepacia strain ST-200 produces an extracellular cholesterol oxidase which is stable and highly active in the presence of organic solvents. This cholesterol oxidase produces 6beta-hydroperoxycholest-4-en-3-one from cholesterol, with the consumption of two moles of O2 and the formation of one mole of H2O2. The structural gene encoding the cholesterol oxidase was cloned and sequenced. The primary translation product was predicted to be 582 amino acid residues. The mature product is composed of 539 amino acid residues and is preceded by a signal sequence of 43 residues. The cloned gene was expressed as an active product in Escherichia coli and the product was localized in the periplasmic space. The cholesterol oxidase produced from E. coli was purified to homogeneity from the periplasmic fraction. The purified enzyme was highly stable in the presence of various organic solvents or detergents, as compared with the commercially available cholesterol oxidases tested.

Two moles of O2 consumption and one mole of H2O2 formation during cholesterol peroxidation with cholesterol oxidase from Pseudomonas sp. strain ST-200.

Cholesterol oxidase from Pseudomonas sp. strain ST-200 oxidized cholesterol and cholestanol to 6beta-hydroperoxycholest-4-en-3-one and 5alpha-cholestan-3-one respectively. The former was converted spontaneously to several oxysteroids such as 6-hydroxycholest-4-en-3-one and cholest-4-ene-3,6-dione, with the consumption of 2 mol of O(2) and the formation of 1 mol of H(2)O(2) for each mole of cholesterol oxidized. An oxidized form of the cholesterol oxidase dehydrogenates cholesterol, probably to the 5-en-3-one derivative. A reduced form of the enzyme, yielded from the cholesterol dehydrogenation reaction, dioxygenated cholest-5-en-3-one to 6beta-hydroperoxycholest-4-en-3-one.

Purification of Extracellular Cholesterol Oxidase with High Activity in the Presence of Organic Solvents from Pseudomonas sp. Strain ST-200

Extracellular cholesterol oxidase of Pseudomonas sp. strain ST-200 was purified from the culture supernatant. This oxidase contained bound flavin and was categorized as a 3beta-hydroxysteroid oxidase, converting 3beta-hydroxyl groups to keto groups. The molecular mass was 60 kDa. The enzyme was stable at pH 4 to 11 and active at pH 5.0 to 8.5, showing optimal activity at pH 7 at 60 degreesC. The Michaelis constant of the ST-200 cholesterol oxidase was lower than those of commercially available oxidases. The cholesterol oxidation rate was enhanced 3- to 3.5-fold in the presence of organic solvents, with log Pow values (partition coefficients of the organic solvent between n-octanol and water), in the range of 2.1 to 4.2, compared with that in the absence of organic solvents.

Lithocholic acid side-chain cleavage to produce 17-keto or 22-aldehyde steroids by Pseudomonas putida strain ST-491 grown in the presence of an organic solvent, diphenyl ether.

We devised a method to screen for microorganisms capable of growing on bile acids in the presence of organic solvents and producing organic solvent-soluble derivatives. Pseudomonas putida biovar A strain ST-491 isolated in this study produced decarboxylated derivatives from the bile acids. Strain ST-491 grown on 0.5% lithocholic acid catabolized approximately 30% of the substrate as a carbon source, and transiently accumulated in the medium androsta-1,4-diene-3,17-dione in an amount of corresponding to 5% of the substrate added. When 20% (v/v) diphenyl ether was added to the medium, 60% of the substrate was converted to 17-keto steroids (androst-4-ene-3,17-dione-like steroid, androsta-1,4-diene-3,17-dione) or a 22-aldehyde steroid (pregna-1,4-dien-3-on-20-al). Amounts of the products were responsible for 45, 10, and 5% of the substrate, respectively. In the presence of the surfactant Triton X-100 instead of diphenyl ether, 40% of the substrate was converted exclusively to androsta-1,4-diene-3,17-dione.

Effects of Organic Solvents on Indigo Formation by Pseudomonas sp. strain ST-200 Grown with High Levels of Indole.

The indole tolerance level of Pseudomonas sp. strain ST-200 was 0.25 mg/ml. The level was raised to 4 mg/ml when diphenylmethane was added to the medium to 20% by volume. ST-200 grown in this two-phase culture system containing indole (1 mg/ml) and diphenylmethane (0.2 ml/ml) produced a water-soluble yellow pigment, isatic acid, and two water-insoluble and diphenylmethane-soluble pigments, blue indigo and purple indirubin. The amounts of the water-insoluble pigments corresponded to 0.5% (indigo) and 0.2% (indirubin) of the indole added to the medium. Of the conditions tried, indigo and indirubin were formed only when ST-200 was grown in the two-phase system overlaid with organic solvents with appropriate polarity.

Biodegradation of indole at high concentration by persolvent fermentation with Pseudomonas sp. ST-200.

Pseudomonas sp. strain ST-200 grew on indole as a sole carbon source. The minimal inhibitory concentration of indole was 0.3 mg/ml for ST-200. However, ST-200 grew in a persolvent fermentation system containing a large amount of indole (a medium containing 20% by vol. diphenylmethane and 4 mg/ml indole), because most of the indole was partitioned in the organic solvent layer. When the organism was grown in the medium containing indole at 1 mg/ml in the presence of diphenylmethane, more than 98% of the indole was consumed after 48h. Isatic acid (0.4 mg/ml) and isatin (0.03 mg/ml) were produced as the metabolites in the aqueous medium layer.

Oxidative Bioconversion of Cholesterol by Pseudomonas sp. Strain ST-200 in a Water-Organic Solvent Two-Phase System.

Pseudomonas sp. strain ST-200, which is capable of conversion of cholesterol, was isolated from humus soil. This organism effectively modified cholesterol dissolved in an organic solvent by dehydrogenation and oxygenation. When the organism was grown in a medium overlaid with a 10% volume of a mixed organic solvent (p-xylene and diphenylmethane; 3:7, vol/vol) containing cholesterol (20 mg/ml), the cholesterol concentration in the organic solvent was reduced to only 0.4 mg/ml after 8 days. Although the organism did not assimilate cholesterol, 98% of the cholesterol initially present disappeared. The organic solvent layer contained two major and three minor compounds converted from cholesterol. The major compounds were 6beta-hydroxycholest-4-en-3-one (8.9 mg/ml) and cholest-4-ene-3,6-dione (7.6 mg/ml). The concentrations of these compounds were equivalent to 43 and 37% of the cholesterol initially present. This organism would provide an effective and convenient system to oxidize the C-3 and -6 positions of cholesterol by introduction of a hydroxyl or ketone group.