Jan A. M. de Bont

Mechanisms of resistance of whole cells to toxic organic solvents

Abstract Many processes in modern biotechnology, particularly biotransformations and environmental bioremediation, are hindered by the toxic effects of organic solvents on whole cells. These compounds dissolve in the cell membrane, disturbing its integrity and effecting specific permeabilization. The hydrophobicity of a compound, expressed as its log P value, is a good indicator of toxicity. Substances with a log P value in the range 1–5 are, in general, toxic to whole cells. However, in recent years, there have been several reports of bacteria exhibiting resistance to toxic solvents. The main adaptative reactions are alterations in the composition of the membrane, particularly changes in fatty-acid composition, phospholipid headgroups, and in the protein content. One of the key processes in the adaptation of some Pseudomonas strains, enabling them to tolerate organic solvents appears to be the isomerization of cis - into trans-unsaturated fatty acids. A greater understanding of these adaptations should eventually allow biotransformation reactions to be carried out in inhospitable two-phase systems incorporating an organic phase.

Screening for ligninolytic fungi applicable to the biodegradation of xenobiotics

Woody tissues are composed mainly of three biopoly- mers: cellulose; hemicellulose; and lignin. Lignin, a highly irregular aromatic polymer which serves to pro- vide strength and structure to the tissue, is synthesized in plants by a random peroxidase-catalysed polym- erization of substituted p-hydroxy-cinnamyl alcohols. Only a few groups of microorganisms are capable of degrading complex lignin polymers, and they are best exemplified by the white-rot fungi, which cause the greatest degree of mineralization. The white-rot fungus

Engineering of Solvent-Tolerant Pseudomonas putida S12 for Bioproduction of Phenol from Glucose

ABSTRACT Efficient bioconversion of glucose to phenol via the central metabolite tyrosine was achieved in the solvent-tolerant strain Pseudomonas putida S12. The tpl gene from Pantoea agglomerans, encoding tyrosine phenol lyase, was introduced into P. putida S12 to enable phenol production. Tyrosine availability was a bottleneck for efficient production. The production host was optimized by overexpressing the aroF-1 gene, which codes for the first enzyme in the tyrosine biosynthetic pathway, and by random mutagenesis procedures involving selection with the toxic antimetabolites m-fluoro-dl-phenylalanine and m-fluoro-l-tyrosine. High-throughput screening of analogue-resistant mutants obtained in this way yielded a P. putida S12 derivative capable of producing 1.5 mM phenol in a shake flask culture with a yield of 6.7% (mol/mol). In a fed-batch process, the productivity was limited by accumulation of 5 mM phenol in the medium. This toxicity was overcome by use of octanol as an extractant for phenol in a biphasic medium-octanol system. This approach resulted in accumulation of 58 mM phenol in the octanol phase, and there was a twofold increase in the overall production compared to a single-phase fed batch.

Structure of Rhodococcus erythropolis limonene-1,2-epoxide hydrolase reveals a novel active site

Epoxide hydrolases are essential for the processing of epoxide‐containing compounds in detoxification or metabolism. The classic epoxide hydrolases have an α/β hydrolase fold and act via a two‐step reaction mechanism including an enzyme–substrate intermediate. We report here the structure of the limonene‐1,2‐epoxide hydrolase from Rhodococcus erythropolis, solved using single‐wavelength anomalous dispersion from a selenomethionine‐substituted protein and refined at 1.2 Å resolution. This enzyme represents a completely different structure and a novel one‐step mechanism. The fold features a highly curved six‐stranded mixed β‐sheet, with four α‐helices packed onto it to create a deep pocket. Although most residues lining this pocket are hydrophobic, a cluster of polar groups, including an Asp–Arg–Asp triad, interact at its deepest point. Site‐directed mutagenesis supports the conclusion that this is the active site. Further, a 1.7 Å resolution structure shows the inhibitor valpromide bound at this position, with its polar atoms interacting directly with the residues of the triad. We suggest that several bacterial proteins of currently unknown function will share this structure and, in some cases, catalytic properties.

Isolation and characterisation of fungi growing on volatile aromatic hydrocarbons as their sole carbon and energy source

Five fungal strains that are able to grow on toluene were isolated from enrichment cultures. Three different techniques were used: solid state-like batches, air biofilters and liquid cultures. Fungal growth in the latter systems was favoured by combining low pH and low water activity. Soil and groundwater samples from gasoline-polluted environments were used as inocula. The isolates were identified as deuteromycetes belonging to the genera Cladophialophora, Exophiala and Leptodontium and the ascomycete Pseudeurotium zonatum. The previously isolated toluene-degrading fungus Cladosporium sphaerospermum was included in the present study. Results showed that fungi grew on toluene with doubling times of about 2 to 3 days. Some of the strains also grew on ethylbenzene and styrene. The apparent half-saturation constant ( K m ) for toluene oxidation ranged from 5 to 22 μM. Degradation activity was inhibited by 50% at toluene concentrations ranging from 2.4 to 4.7 mM. These kinetic parameters are comparable to analogous data reported for toluene-degrading bacteria. The ability of fungi to grow at low water activities and low pH suggest that they may be used for the purification of gas streams containing aromatic hydrocarbons in air biofilters.

Isolation and screening of basidiomycetes with high peroxidative activity

Sixty-seven Poly R-478 decolorizing basidiomycetes were isolated with a selective medium (containing hemp ( Cannabis sativa ) stem wood, guaiacol and benomyl). Several of the new isolates seemed to be promising manganese peroxidase-containing white-rot fungi. Enzyme assays indicated that either glyoxal or veratryl alcohol oxidase were present in the culture fluids of peroxidative strains. In contrast, lignin peroxidase was only detected in Phanerochaete chrysosporium , despite attempts to induce this enzyme in other strains with oxygen and oxygen/veratryl alcohol additions. A highly significant correlation was found between two ligninolytic indicators: ethene formation from α-keto-γ-methylthiolbutyric acid and the decolorization of a polymeric dye, Poly R. Three of the new isolates had significantly higher Poly R decolorizing activities compared to P. chrysosporium . The Poly R decolorization rate is a good assay when trying to optimize culture conditions for peroxidase/H 2 O 2 production.

Evidence for a new extracellular peroxidase. Manganese-inhibited peroxidase from the white-rot fungus Bjerkandera sp. BOS 55.

A novel enzyme activity was detected in the extracellular fluid of Bjerkandera sp, BOS 55. The purified enzyme could oxidize several compounds, such as Phenol red, 2,6‐dimethoxyphenol (DMP), Poly R‐478, ABTS and guaiacol, with H2O2 as an electron acceptor. In contrast, veratryl alcohol was not a substrate. This enzyme also had the capacity to oxidize DMP in the absence of H2O2. With some substrates, a strong inhibition of the peroxidative activity by Mn2+ was observed. Phenol red oxidation was inhibited by 84% with only 1 mM of this metal ion. Because DMP oxidation by this enzyme is only slightly inhibited by Mn2+, this substrate should not be used in assays to detect manganese peroxidase. The enzyme is tentatively named ‘Manganese‐Inhibited Peroxidase’.

CORN FIBER, COBS AND STOVER: ENZYME-AIDED SACCHARIFICATION AND CO-FERMENTATION AFTER DILUTE ACID PRETREATMENT

Three corn feedstocks (fibers, cobs and stover) available for sustainable second generation bioethanol production were subjected to pretreatments with the aim of preventing formation of yeast-inhibiting sugar-degradation products. After pretreatment, monosaccharides, soluble oligosaccharides and residual sugars were quantified. The size of the soluble xylans was estimated by size exclusion chromatography. The pretreatments resulted in relatively low monosaccharide release, but conditions were reached to obtain most of the xylan-structures in the soluble part. A state of the art commercial enzyme preparation, Cellic CTec2, was tested in hydrolyzing these dilute acid-pretreated feedstocks. The xylose and glucose liberated were fermented by a recombinant Saccharomyces cerevisiae strain. In the simultaneous enzymatic saccharification and fermentation system employed, a concentration of more than 5% (v/v) (0.2g per g of dry matter) of ethanol was reached.

Identification and molecular characterization of an efflux system involved in Pseudomonas putida S12 multidrug resistance

The authors previously described srpABC, an operon involved in proton-dependent solvent efflux in the solvent-tolerant Pseudomonas putida S12. Recently, it was shown that organic solvents and not antibiotics induce this operon. In the present study, the authors characterize a new efflux pump, designated ArpABC, on the basis of two isolated chloramphenicol-sensitive transposon mutants. The arpABC operon is involved in the active efflux of multiple antibiotics, such as tetracycline, chloramphenicol, carbenicillin, streptomycin, erythromycin and novobiocin. The deduced amino acid sequences encoded by the three genes involved show a striking resemblance to proteins of the resistance/nodulation/cell division family, which are involved in both organic solvent and multiple drug efflux. These findings demonstrate that ArpABC is highly homologous to the MepABC and TtgABC efflux systems for organic solvents and multiple antibiotics. However, ArpABC does not contribute to organic solvent tolerance in P. putida S12 but is solely involved in multidrug resistance.

Transcriptome Analysis of a Phenol-Producing Pseudomonas putida S12 Construct: Genetic and Physiological Basis for Improved Production

The unknown genetic basis for improved phenol production by a recombinant Pseudomonas putida S12 derivative bearing the tpl (tyrosine-phenol lyase) gene was investigated via comparative transcriptomics, nucleotide sequence analysis, and targeted gene disruption. We show upregulation of tyrosine biosynthetic genes and possibly decreased biosynthesis of tryptophan caused by a mutation in the trpE gene as the genetic basis for the enhanced phenol production. In addition, several genes in degradation routes connected to the tyrosine biosynthetic pathway were upregulated. This either may be a side effect that negatively affects phenol production or may point to intracellular accumulation of tyrosine or its intermediates. A number of genes identified by the transcriptome analysis were selected for targeted disruption in P. putida S12TPL3. Physiological and biochemical examination of P. putida S12TPL3 and these mutants led to the conclusion that the metabolic flux toward tyrosine in P. putida S12TPL3 was improved to such an extent that the heterologous tyrosine-phenol lyase enzyme had become the rate-limiting step in phenol biosynthesis.