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Improving the organic solvent tolerance of bacteria is beneficial for the bioproduction of various valuable chemicals. In this study, we found that 1,4-dihydroxy-2-naphthoic acid (DHNA), known as a prebiotic, increased organic solvent tolerance in Escherichia coli. The AcrAB-TolC multidrug efflux pump contributes to the intrinsic organic solvent tolerance of E. coli. The addition of DHNA increased this pump's expression level. Transcriptional activators MarA, SoxS, and Rob proteins are known to control the expression of marA/soxS/rob regulon genes, including acrAB and tolC. Evaluation of the organic solvent tolerances of ΔmarA mutant, ΔsoxS mutant, and Δrob mutant showed that ΔmarA mutant and ΔsoxS mutant did not improve organic solvent tolerance by the addition of DHNA. In addition, DHNA increased the promoter activities of both marA and soxS. These results indicated that DHNA induces the AcrAB-TolC pump through both the marRAB system and the soxRS system., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Vanillin is used as a flavor ingredient in a diverse range of food categories and is known to inhibit microbial fermentation. In this study, we found that vanillin improved the hydrophobic organic solvent tolerance of Escherichia coli and showed that the AcrAB-TolC efflux pump is implicated in that tolerance. The expression level of the pump was enhanced by the addition of vanillin. AcrAB-TolC efflux pump expression is known to be regulated by transcription activators such as MarA, SoxS, and Rob. Among these three transcription factors, marA transcription was significantly elevated by the addition of vanillin. We found that the AcrAB-TolC efflux pump is involved also in vanillin tolerance. The ΔacrB mutant was more sensitive to vanillin than the parent strain. A complementation test revealed that the introduction of the acrB gene recovered the vanillin tolerance of the ΔacrB mutant., (Copyright © 2021 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The AcrAB-TolC efflux pump is involved in the organic solvent tolerance of Escherichia coli . Most E. coli strains are highly sensitive to organic solvents such as n -hexane and cyclohexane. Here, a recombinant E. coli transformed with an expression plasmid containing acrAB and tolC became tolerant to n -hexane and cyclohexane. The levels of AcrA, AcrB, and TolC in the recombinant increased by 3- to 5-fold compared to those in the control strain without the plasmid for acrAB or tolC . To investigate the usability of the recombinant as a biocatalyst in an aqueous-organic solvent two-phase system, we further introduced xylMA xylene monooxygenase genes from Pseudomonas putida mt-2 into the recombinant and examined the production of styrene oxide from styrene. The resulting recombinant produced 1.8 mg and 1.0 mg styrene oxide mL -1 of medium in a medium overlaid with a 25% volume of n -hexane and cyclohexane containing 10% (wt vol -1 ) styrene, respectively.

Organic solvent-tolerant Pseudomonas aeruginosa LST-03 secretes an organic solvent-stable lipase, LST-03 lipase. The gene of the LST-03 lipase (Lip9) and the gene of the lipase-specific foldase (Lif9) were cloned and expressed in Escherichia coli. In the cloned 2.6 kbps DNA fragment, two open reading frames, Lip9 consisting of 933 nucleotides which encoded 311 amino acids and Lif9 consisting of 1,020 nucleotides which encoded 340 amino acids, were found. The overexpression of the lipase gene (lip9) was achieved when T7 promoter was used and the signal peptide of the lipase was deleted. The expressed amount of the lipase was greatly increased and overexpressed lipase formed inclusion body in E. coli cell. The collected inclusion body of the lipase from the cell was easily solubilized by urea and activated by using lipase-specific foldase of which 52 or 58 amino acids of N-terminal were deleted. Especially, the N-terminal methionine of the lipase of which the signal peptide was deleted was released in E. coli and the amino acid sequence was in agreement with that of the originally-produced lipase by P. aeruginosa LST-03. Furthermore, the overexpressed and solubilized lipase of which the signal peptide was deleted was more effectively activated by lipase-specific foldase.

A time-course gene-expression profile was generated for Escherichia coli TK31 when it was exposed to an organic solvent mixture, and classified by fuzzy adaptive resonance theory (Fuzzy ART). It was found that the purR regulon plays an important role in the organic solvent tolerance (OST) of E. coli.

Cholesterol oxidases (COXases) have a diverse array of applications including analysis of blood cholesterol levels, synthesis of steroids, and utilization as an insecticidal protein. The COXase gene from Janthinobacterium agaricidamnosum was cloned and expressed in Escherichia coli. The purified COXase showed an optimal temperature of 60 °C and maintained about 96 and 72% of its initial activity after 30 min at 60 and 70 °C, respectively. In addition, the purified COXase exhibited a pH optimum at 7.0 and high pH stability over the broad pH range of 3.0–12.0. The pH stability of the COXase at pH 12.0 was higher than that of highly stable COXase from Chromobacterium sp. DS-1. The COXase oxidized cholesterol and β-cholestanol at higher rates than other 3β-hydroxysteroids. The Km, Vmax, and kcat values for cholesterol were 156 μM, 13.7 μmol/min/mg protein, and 14.4 s−1, respectively. These results showed that this enzyme could be very useful in the clinical determination of cholesterol in serum and the production of steroidal compounds. This is the first report to characterize a COXase from the genus Janthinobacterium. [ABSTRACT FROM AUTHOR]

Chromobacterium sp. strain DS-1 produces an extracellular cholesterol oxidase that is very stable at high temperatures and in the presence of organic solvents and detergents. In this study, we cloned and sequenced the structural gene encoding the cholesterol oxidase. The primary translation product was predicted to be 584 amino acid residues. The mature product is composed of 540 amino acid residues. The amino acid sequence of the product showed significant similarity (53–62%) to the cholesterol oxidases from Burkholderia spp. and Pseudomonas aeruginosa. The DNA fragment corresponding to the mature enzyme was subcloned in the pET-21d(+) expression vector and expressed as an active product in Escherichia coli. The cholesterol oxidase produced from the recombinant E. coli was purified to homogeneity. The physicochemical properties were similar to those of native enzyme purified from strain DS-1. Km and Vmax values of the cholesterol oxidase were estimated from Lineweaver–Burk plots. The Vmax/ Km ratio of the enzyme was higher than those of commercially available cholesterol oxidases. The circular dichroism spectral analysis of the recombinant DS-1 enzyme and Burkholderia cepacia ST-200 cholesterol oxidase showed that the conformational stability of the DS-1 enzyme was higher than that of B. cepacia ST-200 enzyme at higher temperatures. [ABSTRACT FROM AUTHOR]

The article explores brachybacterium strain LB25 produces a malto-oligosaccharide-forming amylase. This content improves product selectivity in water-miscible organic solvents. The enzyme hydrolyzed starch to produce maltotriose primarily. The structural gene encoding the amylase from strain LB25 was cloned and sequenced.

Abstract: Overexpression of the organic solvent-stable LST-03 lipase from organic solvent-tolerant Pseudomonas aeruginosa LST-03 was attempted using a recombinant Escherichia coli system. It was achieved by deleting the signal peptide of the lipase and using T7 promoter. Although, the expressed lipase formed an inclusion body in E. coli, it was completely solubilized with 4M urea and 0.05mM dithiothreitol. The denaturants used for solubilization of the lipase inclusion body were easily removed by dilution or dialysis. The solubilized lipase was effectively activated by incubation with a lipase-specific foldase. The purity of the lipase recovered from the inclusion body was high. Use of a recombinant E. coli system improved the productivity of the highly pure lipase by about 807-fold, compared to the original system using P. aeruginosa LST-03. [Copyright &y& Elsevier]

Gene expression profiles were collected from Escherichia coli strains (OST3410, TK33, and TK31) before and after exposure to organic solvents, and the six genes that showed higher gene expression were selected. Among these genes, glpC encoding the anaerobic glycerol-3-phosphate dehydrogenase subunit C remarkably increased the organic solvent tolerance. [ABSTRACT FROM AUTHOR]