Contributions of mutations in acrR and marR genes to organic solvent tolerance in Escherichia coli

The AcrAB-TolC efflux pump is involved in maintaining intrinsic organic solvent tolerance in Escherichia coli. Mutations in regulatory genes such as marR, soxR, and acrR are known to increase the expression level of the AcrAB-TolC pump. To identify these mutations in organic solvent tolerant E. coli, eight cyclohexane-tolerant E. coli JA300 mutants were isolated and examined by DNA sequencing for mutations in marR, soxR, and acrR. Every mutant carried a mutation in either marR or acrR. Among all mutants, strain CH7 carrying a nonsense mutation in marR (named marR109) and an insertion of IS5 in acrR, exhibited the highest organic solvent-tolerance levels. To clarify the involvement of these mutations in improving organic solvent tolerance, they were introduced into the E. coli JA300 chromosome by site-directed mutagenesis using λ red-mediated homologous recombination. Consequently, JA300 mutants carrying acrR::IS5, marR109, or both were constructed and named JA300 acrRIS, JA300 marR, or JA300 acrRIS marR, respectively. The organic solvent tolerance levels of these mutants were increased in the following order: JA300 < JA300 acrRIS < JA300 marR < JA300 acrRIS marR. JA300 acrRIS marR formed colonies on an agar plate overlaid with cyclohexane and p-xylene (6:4 vol/vol mixture). The organic solvent-tolerance level and AcrAB-TolC efflux pump-expression level in JA300 acrRIS marR were similar to those in CH7. Thus, it was shown that the synergistic effects of mutations in only two regulatory genes, acrR and marR, can significantly increase organic solvent tolerance in E. coli

Semi-pilot scale-up of a continuous packed-bed bioreactor system developed for the lipase-catalyzed production of pseudo-ceramides

Ceramides are sphingolipid compounds that are very attractive as active components in both the pharmaceutical and the cosmetic industries. In this study, the synthesis of 1-O,3-N-diacyl 3-amino-1,2-propanediol-type pseudo-ceramides was developed at the semi-pilot scale, starting from a two-step continuous enzymatic process with immobilized Candida antarctica lipase B (Novozym® 435) in a packed-bed bioreactor, previously optimized at the laboratory scale. This process involved the selective N-acylation of 3-amino-1,2-propanediol (step 1), followed by the selective O-acylation of the N-acyl 3-amino-1,2-propanediol synthesized in the first step, with various fatty acids as acyl donors, to produce N,O-diacyl 3-amino-1,2-propanediol-type pseudo-ceramides (step 2). Under partially optimized operating conditions, high synthesis yields and production rates were obtained, within the ranges 76–92% and 3.7–4.6 g h−1 (step 1), or 23–36% and 1–1.4 g h−1 (step 2), respectively, depending on the fatty acids used as acyl donors. The overall synthesis yields varied from 20 to 33%: the best yield was obtained using palmitic acid and lauric acid as first and second acyl donors, respectively. Together with the high production rates also obtained with these acyl donors, this confirms that this two-step process has great potential for the production of differently functionalized 1-O,3-N-diacyl 3-amino-1,2-propanediol-type pseudo-ceramides on an industrial scale

Enzymatic degradation of granular potato starch by Microbacterium aurum strain B8.A

Microbacterium aurum strain B8.A was isolated from the sludge of a potato starch-processing factory on the basis of its ability to use granular starch as carbon- and energy source. Extracellular enzymes hydrolyzing granular starch were detected in the growth medium of M. aurum B8.A, while the type strain M. aurum DSMZ 8600 produced very little amylase activity, and hence was unable to degrade granular starch. The strain B8.A extracellular enzyme fraction degraded wheat, tapioca and potato starch at 37 °C, well below the gelatinization temperature of these starches. Starch granules of potato were hydrolyzed more slowly than of wheat and tapioca, probably due to structural differences and/or surface area effects. Partial hydrolysis of starch granules by extracellular enzymes of strain B8.A resulted in large holes of irregular sizes in case of wheat and tapioca and many smaller pores of relatively homogeneous size in case of potato. The strain B8.A extracellular amylolytic system produced mainly maltotriose and maltose from both granular and soluble starch substrates; also, larger maltooligosaccharides were formed after growth of strain B8.A in rich medium. Zymogram analysis confirmed that a different set of amylolytic enzymes was present depending on the growth conditions of M. aurum B8.A. Some of these enzymes could be partly purified by binding to starch granules

Hot or not? Discovery and characterization of a thermostable alditol oxidase from Acidothermus cellulolyticus 11B

We describe the discovery, isolation and characterization of a highly thermostable alditol oxidase from Acidothermus cellulolyticus 11B. This protein was identified by searching the genomes of known thermophiles for enzymes homologous to Streptomyces coelicolor A3(2) alditol oxidase (AldO). A gene (sharing 48% protein sequence identity to AldO) was identified, cloned and expressed in Escherichia coli. Following 6xHis tag purification, characterization revealed the protein to be a covalent flavoprotein of 47 kDa with a remarkably similar reactivity and substrate specificity to that of AldO. A steady-state kinetic analysis with a number of different polyol substrates revealed lower catalytic rates but slightly altered substrate specificity when compared to AldO. Thermostability measurements revealed that the novel AldO is a highly thermostable enzyme with an unfolding temperature of 84 °C and an activity half-life at 75 °C of 112 min, prompting the name HotAldO. Inspired by earlier studies, we attempted a straightforward, exploratory approach to improve the thermostability of AldO by replacing residues with high B-factors with corresponding residues from HotAldO. None of these mutations resulted in a more thermostable oxidase; a fact that was corroborated by in silico analysis

Engineering of cyclodextrin glucanotransferases and the impact for biotechnological applications

Cyclodextrin glucanotransferases (CGTases) are industrially important enzymes that produce cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. Cyclodextrin glucanotransferases are also applied as catalysts in the synthesis of glycosylated molecules and can act as antistaling agents in the baking industry. To improve the performance of CGTases in these various applications, protein engineers are screening for CGTase variants with higher product yields, improved CD size specificity, etc. In this review, we focus on the strategies employed in obtaining CGTases with new or enhanced enzymatic capabilities by searching for new enzymes and improving existing enzymatic activities via protein engineering

Indole and 7‐hydroxyindole diminish Pseudomonas aeruginosa virulence

Indole is an extracellular biofilm signal for Escherichia coli, and many bacterial oxygenases readily convert indole to various oxidized compounds including 7‐hydroxyindole (7HI). Here we investigate the impact of indole and 7HI on Pseudomonas aeruginosa PAO1 virulence and quorum sensing (QS)‐regulated phenotypes; this strain does not synthesize these compounds but degrades them rapidly. Indole and 7HI both altered extensively gene expression in a manner opposite that of acylhomoserine lactones; the most repressed genes encode the mexGHI‐opmD multidrug efflux pump and genes involved in the synthesis of QS‐regulated virulence factors including pyocyanin (phz operon), 2‐heptyl‐3‐hydroxy‐4(1H)‐quinolone (PQS) signal (pqs operon), pyochelin (pch operon) and pyoverdine (pvd operon). Corroborating these microarray results, indole and 7HI decreased production of pyocyanin, rhamnolipid, PQS and pyoverdine and enhanced antibiotic resistance. In addition, indole affected the utilization of carbon, nitrogen and phosphorus, and 7HI abolished swarming motility. Furthermore, 7HI reduced pulmonary colonization of P. aeruginosa in guinea pigs and increased clearance in lungs. Hence, indole‐related compounds have potential as a novel antivirulence approach for the recalcitrant pathogen P. aeruginosa