Improved xylose uptake in Saccharomyces cerevisiae due to directed evolution of galactose permease Gal2 for sugar co-consumption.

AIMS: Saccharomyces cerevisiae does not express any xylose-specific transporters. To enhance the xylose uptake of S. cerevisiae, directed evolution of the Gal2 transporter was performed. METHODS AND RESULTS: Three rounds of error-prone PCR were used to generate mutants with improved xylose-transport characteristics. After developing a fast and reliable high-throughput screening assay based on flow cytometry, eight mutants were obtained showing an improved uptake of xylose compared to wild-type Gal2 out of 41 200 single yeast cells. Gal2 variant 2·1 harbouring five amino acid substitutions showed an increased affinity towards xylose with a faster overall sugar metabolism of glucose and xylose. Another Gal2 variant 3·1 carrying an additional amino acid substitution revealed an impaired growth on glucose but not on xylose. CONCLUSIONS: Random mutagenesis of the S. cerevisiae Gal2 led to an increased xylose uptake capacity and decreased glucose affinity, allowing improved co-consumption. SIGNIFICANCE AND IMPACT OF THE STUDY: Random mutagenesis is a powerful tool to evolve sugar transporters like Gal2 towards co-consumption of new substrates. Using a high-throughput screening system based on flow-through cytometry, various mutants were identified with improved xylose-transport characteristics. The Gal2 variants in this work are a promising starting point for further engineering to improve xylose uptake from mixed sugars in biomass.

Liquid-liquid extraction of fermentation inhibiting compounds in lignocellulose hydrolysate.

Several compounds that are formed or released during hydrolysis of lignocellulosic biomass inhibit the fermentation of the hydrolysate. The use of a liquid extractive agent is suggested as a method for removal of these fermentation inhibitors. The method can be applied before or during the fermentation. For a series of alkanes and alcohols, partition coefficients were measured at low concentrations of the inhibiting compounds furfural, hydroxymethyl furfural, vanillin, syringaldehyde, coniferyl aldehyde, acetic acid, as well as for ethanol as the fermentation product. Carbon dioxide production was measured during fermentation in the presence of each organic solvent to indicate its biocompatibility. The feasibility of extractive fermentation of hydrolysate was investigated by ethanolic glucose fermentation in synthetic medium containing several concentrations of furfural and vanillin and in the presence of decanol, oleyl alcohol and oleic acid. Volumetric ethanol productivity with 6 g/L vanillin in the medium increased twofold with 30% volume oleyl alcohol. Decanol showed interesting extractive properties for most fermentation inhibiting compounds, but it is not suitable for in situ application due to its poor biocompatibility.

Model description of bacterial 3-methylcatechol production in one- and two-phase systems.

Pseudomonas putida MC2 produces 3-methylcatechol from toluene in aqueous medium. A second phase of 1-octanol may improve total product accumulation. To optimise the design of such a biphasic process, a process model was developed, both for one- and two-phase applications. The insights obtained by the model predictions showed the importance of different process parameters (like growth substrate concentration and partition coefficient) on growth of biomass, accumulation of 3-methylcatechol and processing time. For future applications, the process model can be used to ensure enough extraction capacity from aqueous to octanol phase. It is a useful tool to define the optimum process conditions, depending on the desired optimisation parameter: product concentration or processing time.

Anaerobic oxidation of 2-chloroethanol under denitrifying conditions by Pseudomonas stutzeri strain JJ.

A bacterium that uses 2-chloroethanol as sole energy and carbon source coupled to denitrification was isolated from 1,2-dichloroethane-contaminated soil. Its 16 S rDNA sequence showed 98% similarity with the type strain of Pseudomonas stutzeri (DSM 5190) and the isolate was tentatively identified as Pseudomonas stutzeri strain JJ. Strain JJ oxidized 2-chloroethanol completely to CO(2) with NO(3)(- )or O(2) as electron acceptor, with a preference for O(2) if supplied in combination. Optimum growth on 2-chloroethanol with nitrate occurred at 30 degrees C with a mu(max) of 0.14 h(-1) and a yield of 4.4 g protein per mol 2-chloroethanol metabolized. Under aerobic conditions, the mu(max) was 0.31 h(-1). NO(2)(-) also served as electron acceptor, but reduction of Fe(OH)(3), MnO(2), SO(4)(2-), fumarate or ClO(3)(-) was not observed. Another chlorinated compound used as sole energy and carbon source under aerobic and denitrifying conditions was chloroacetate. Various different bacterial strains, including some closely related Pseudomonas stutzeri strains, were tested for their ability to grow on 2-chloroethanol as sole energy and carbon source under aerobic and denitrifying conditions, respectively. Only three strains, Pseudomonas stutzeri strain LMD 76.42, Pseudomonas putida US2 and Xanthobacter autotrophicus GJ10, grew aerobically on 2-chloroethanol. This is the first report of oxidation of 2-chloroethanol under denitrifying conditions by a pure bacterial culture.

High-rate 3-methylcatechol production in Pseudomonas putida strains by means of a novel expression system.

The bioconversion of toluene into 3-methylcatechol was studied as a model system for the production of valuable 3-substituted catechols in general. For this purpose, an improved microbial system for the production of 3-methylcatechol was obtained. Pseudomonas putida strains containing the todC1C2BAD genes involved in the conversion of toluene into 3-methylcatechol were used as hosts for introducing extra copies of these genes by means of a novel integrative expression system. A construct was made containing an expression cassette with the todC1C2BAD genes cloned under the control of the inducible regulatory control region for naphthalene and phenanthrene degradation, nagR. Introducing this construct into wild-type P. putida F1, which degrades toluene via 3-methylcatechol, or into mutant P. putida F107, which accumulates 3-methylcatechol, yielded biocatalysts carrying multiple copies of the expression cassette. As a result, up to 14 mM (1.74 g l(-1)) of 3-methylcatechol was accumulated and the specific production rate reached a level of 105 micromol min(-1) g(-1) cell dry weight, which is four times higher than other catechol production systems. It was shown that these properties were kept stable in the biocatalysts without the need for antibiotics in the production process. This is an important step for obtaining designer biocatalysts.

Transposon mutations in the flagella biosynthetic pathway of the solvent-tolerant Pseudomonas putida S12 result in a decreased expression of solvent efflux genes.

Fourteen solvent-sensitive transposon mutants were generated from the solvent-tolerant Pseudomonas putida strain S12 by applying the TnMod-KmO mutagenesis system. These mutants were unable to grow in the presence of octanol and toluene. By cloning the region flanking the transposon insertion point a partial sequence of the interrupted genes was determined. Comparison of the deduced amino acid sequences with a protein database revealed the following interrupted putative gene products: organic solvent efflux proteins SrpA and SrpB, the flagellar structural proteins FlgK, FlaG, FliI, FliC, and FliH, the transcriptional activator FleQ, the alternative RNA polymerase sigma factor RpoN, and the flagellum-specific RNA polymerase sigma factor FliA (RpoF). The transposon mutants, except for the organic solvent efflux mutants, were nonmotile as determined by a swarm assay and the formation of the flagellum was totally impaired. Expression studies with a srp promoter probe showed a decreased expression of the SrpABC efflux pump in the nonmotile mutants.

Production of 3-nitrocatechol by oxygenase-containing bacteria: optimization of the nitrobenzene biotransformation by Nocardia S3.

Twenty-one microorganisms were screened for their ability to convert nitroaromatics into 3-nitrocatechol as a result of the action of an oxygenase. Cultures containing toluene dioxygenases and phenol monooxygenases accumulated 3-nitrocatechol during incubation with nitrobenzene and nitrophenol, respectively. Nocardia S3 was selected and studied in more detail. Toluene-pregrown cultures were able to degrade nitrobenzene with a concomitant formation of 3-nitrocatechol. The rates of nitrobenzene utilization decreased throughout the biotransformation period and finally the accumulation ceased. The gradual deterioration of the biotransformation rates was not a consequence of depletion of the NADH pool, but was due to the accumulation of 3-nitrocatechol. The inhibition of nitrobenzene biotransformation by 3-nitrocatechol greatly impacts 3-nitrocatechol production processes.

Integrated bioproduction and extraction of 3-methylcatechol.

Pseudomonas putida MC2 is a solvent-tolerant strain that accumulates 3-methylcatechol. In aqueous media, 10 mM of 3-methylcatechol was produced and production was limited by 3-methylcatechol toxicity to the biocatalyst. Production levels increased by introduction of a second, organic phase that provides the substrate toluene and extracts the product from the culture medium. Octanol was shown to be an appropriate second phase with respect to tolerance of the strain for this solvent and with respect to partitioning of both substrate and product. Per unit of overall reactor volume (octanol and water), best results were obtained with 50% (v/v) of octanol: an overall 3-methylcatechol concentration of 25 mM was reached with 96% of the product present in the octanol phase. These product concentrations are much higher than in aqueous media without organic solvent, indicating that biocatalysis in an organic/aqueous two-phase system is an improved set-up for high production levels of 3-methylcatechol.

Fungal metabolism of toluene: monitoring of fluorinated analogs by (19)F nuclear magnetic resonance spectroscopy.

We used isomeric fluorotoluenes as model substrates to study the catabolism of toluene by five deuteromycete fungi and one ascomycete fungus capable of growth on toluene as the sole carbon and energy source, as well as by two fungi (Cunninghamella echinulata and Aspergillus niger) that cometabolize toluene. Whole cells were incubated with 2-, 3-, and 4-fluorotoluene, and metabolites were characterized by (19)F nuclear magnetic resonance. Oxidation of fluorotoluene by C. echinulata was initiated either at the aromatic ring, resulting in fluorinated o-cresol, or at the methyl group to form fluorobenzoate. The initial conversion of the fluorotoluenes by toluene-grown fungi occurred only at the side chain and resulted in fluorinated benzoates. The latter compounds were the substrate for the ring hydroxylation and, depending on the fluorine position, were further metabolized up to catecholic intermediates. From the (19)F nuclear magnetic resonance metabolic profiles, we propose that diverse fungi that grow on toluene assimilate toluene by an initial oxidation of the methyl group.

Effects of detergents on specific activity and enantioselectivity of the epoxide hydrolase from Rhodotorula glutinis.

The yeast Rhodotorula glutinis contains an enantioselective epoxide hydrolase. Previous work showed that the enzyme is a membrane-associated enzyme that can be solubilised from the membranes by a detergent treatment. Now, the effect of detergents on reaction rate and particularly enantioselectivity was investigated. Three types of detergents were tested: non-ionic, anionic and zwitterionic. Non-ionic detergents stimulated the specific activity of the enzyme. Enantioselectivity of the enzyme was strongly affected by several detergents. Thesit and sucrosemonolaurate had the most pronounced effects and enantiomeric ratios were strongly enhanced. The effects are most likely due to the ability of detergents to stabilise membrane-proteins by forming micelles and thus mimicking the membrane structure.

An insertion sequence prepares Pseudomonas putida S12 for severe solvent stress.

The novel insertion sequence ISS12 plays a key role in the tolerance of Pseudomonas putida S12 to sudden toluene stress. Under normal culturing conditions the P. putida S12 genome contained seven copies of ISS12. However, a P. putida S12 population growing to high cell density after sudden addition of a separate phase of toluene carried eight copies. The survival frequency of cells in this variant P. putida S12 population was 1000 times higher than in "normal" P. putida S12 populations. Analysis of the nucleotide sequence flanking the extra ISS12 insertion revealed integration into the srpS gene. srpS forms a gene cluster with srpR and both are putative regulators of the solvent resistance pump SrpABC. SrpABC makes a major contribution to solvent tolerance in P. putida S12 and is induced by toluene. The basal level of srp promoter activity in the P. putida S12 variant was seven times higher than in wild-type P. putida S12. Introduction of the intact srpRS gene cluster in the variant resulted in a dramatic decrease of survival frequency after a toluene shock. These findings strongly suggest that interruption of srpS by ISS12 up-regulates expression of the solvent pump, enabling the bacterium to tolerate sudden exposure to lethal concentrations of toxic solvents. We propose that P. putida S12 employs ISS12 as a mutator element to generate diverse mutations to swiftly adapt when confronted with severe adverse conditions.

Regiospecific effect of 1-octanol on cis-trans isomerization of unsaturated fatty acids in the solvent-tolerant strain Pseudomonas putida S12.

The solvent-tolerant bacterium Pseudomonas putida S12, which adapts its membrane lipids to the presence of toxic solvents by a cis to trans isomerization of unsaturated fatty acids, was used to study possible in vivo regiospecificity of the isomerase. Cells were supplemented with linoleic acid (C18:2delta9-cis,delta12-cis), a fatty acid that cannot be synthesized by this bacterium, but which was incorporated into membrane lipids up to an amount of 15% of total fatty acids. After addition of 1-octanol, which was used as an activator of the cis-trans isomerase, the linoleic acid was converted into the delta9-trans,delta12-cis isomer, while the delta9-cis,delta12-trans and delta9-trans,epsilon12-trans isomers were not synthesized. Thus, for the first time, regiospecific in vivo formation of novel, mixed cis/trans isomers of dienoic fatty acid chains was observed. The maximal conversion (27-36% of the chains) was obtained at 0.03-0.04% (v/v) octanol, after 2 h. The observed regiospecificity of the enzyme, which is located in the periplasmic space, could be due to penetration of the enzyme to a specific depth in the membrane as well as to specific molecular recognition of the substrate molecules.

Continuous production of enantiopure 1,2-epoxyhexane by yeast epoxide hydrolase in a two-phase membrane bioreactor.

A two-phase membrane bioreactor was developed to continuously produce enantiopure epoxides using the epoxide hydrolase activity of Rhodotorula glutinis. An aqueous/organic cascade, hydrophilic, hollow-fiber membrane bioreactor was used: (1) to carry out large-scale resolution of epoxides, (2) to continuously extract residual enantiopure epoxides from the aqueous phase, and (3) to separate inhibitory formed diol from the yeast cells contained in the aqueous phase. Dodecane was employed to dissolve-feed epoxide as well as to extract residual epoxide. 1,2-Epoxyhexane was used as a model substrate. By use of this membrane bioreactor, enantiopure (S)-1,2-epoxyhexane (>98% enantiomeric excess) was obtained with a volumetric productivity of 3.8 g l(-1) h(-1). The continuous-production system was operated for 12 days and resulted in 38 g enantiopure (S)-1,2-epoxyhexane.

The solvent efflux system of Pseudomonas putida S12 is not involved in antibiotic resistance.

The active efflux system contributing to the solvent tolerance of Pseudomonas putida S12 was characterized physiologically. The mutant P. putida JK1, which lacks the active efflux system, was compared with the wild-type organism. None of 20 known substrates of common multi-drug-resistant pumps had a stronger growth-inhibiting effect on the mutant than on the wild type. The amount of [14C]toluene accumulating in P. putida S12 increased in the presence of the solvent xylene and in the presence of uncouplers. The effect of uncouplers confirms the proton dependency of the efflux system in P. putida S12. Other compounds, potential substrates for the solvent pump, did not affect the accumulation of [14C]toluene. These results show that the efflux system in P. putida S12 is specific for organic solvents and does not export antibiotics or other known substrates of multi-drug-resistant pumps.

A genetically modified solvent-tolerant bacterium for optimized production of a toxic fine chemical.

The aim of the study was to investigate whether toxic fine chemical production can be improved using the solvent-tolerant Pseudomonas putida S12 in a two-liquid-phase system consisting of aqueous media and a water-immiscible octanol phase with production of 3-methylcatechol from toluene as the model conversion. For this purpose the genes involved in this conversion, todC1C2BAD from P. putida F1, were introduced into P. putida S12 with high stable expression. Production of 3-methylcatechol was monitored in batch incubations with different media using a single medium and a two-liquid medium-octanol system. The maximum concentration of 3-methylcatechol increased two-fold using the two-liquid medium-octanol system, irrespective of the selected medium.

Cloning and characterization of an epoxide hydrolase-encoding gene from Rhodotorula glutinis.

We cloned and characterized the epoxide hydrolase gene, EPH1, from Rhodotorula glutinis. The EPH1 open reading frame of 1230 bp was interrupted by nine introns and encoded a polypeptide of 409 amino acids with a calculated molecular mass of 46.3 kDa. The amino acid sequence was similar to that of microsomal epoxide hydrolase, which suggests that the epoxide hydrolase of R. glutinis also belongs to the alpha/beta hydrolase fold family. EPH1 cDNA was expressed in Escherichia coli and resting cells showed a specific activity of 200 nmol min(-1)(mg protein)(-1) towards 1,2-epoxyhexane.

Isolation and characterization of the epoxide hydrolase-encoding gene from Xanthophyllomyces dendrorhous.

The epoxide hydrolase (EH)-encoding gene (EPH1) from the basidiomycetous yeast Xanthophyllomyces dendrorhous was isolated. The genomic sequence has a 1,236-bp open reading frame which is interrupted by eight introns that encode a 411-amino-acid polypeptide with a calculated molecular mass of 46.2 kDa. The amino acid sequence is similar to that of microsomal EH and belongs to the alpha/beta hydrolase fold family. The EPH1 gene was not essential for growth of X. dendrorhous in rich medium under laboratory conditions. The Eph1-encoding cDNA was functionally expressed in Escherichia coli. A sixfold increase in specific activity was observed when we used resting cells rather than X. dendrorhous. The epoxides 1,2-epoxyhexane and 1-methylcyclohexene oxide were substrates for both native and recombinant Eph1. Isolation and characterization of the X. dendrorhous EH-encoding gene are essential steps in developing a yeast EH-based epoxide biotransformation system.

Involvement of manganese in conversion of phenylalanine to benzaldehyde by lactic acid bacteria.

We examined the involvement of Mn(II) in the conversion of phenylalanine to benzaldehyde in cell extracts of lactic acid bacteria. Experiments performed with Lactobacillus plantarum demonstrated that Mn(II), present at high levels in this strain, is involved in benzaldehyde formation by catalyzing the conversion of phenylpyruvic acid. Experiments performed with various lactic acid bacterial strains belonging to different genera revealed that benzaldehyde formation in a strain was related to a high Mn(II) level.

Stereoselective carveol dehydrogenase from Rhodococcus erythropolis DCL14. A novel nicotinoprotein belonging to the short chain dehydrogenase/reductase superfamily.

A novel nicotinoprotein, catalyzing the dichlorophenolindophenol-dependent oxidation of carveol to carvone, was purified to homogeneity from Rhodococcus erythropolis DCL14. The enzyme is specifically induced after growth on limonene and carveol. Dichlorophenolindophenol-dependent carveol dehydrogenase (CDH) is a homotetramer of 120 kDa with each subunit containing a tightly bound NAD(H) molecule. The enzyme is optimally active at pH 5.5 and 50 degrees C and displays a broad substrate specificity with a preference for substituted cyclohexanols. When incubated with a diastereomeric mixture of (4R)- or (4S)-carveol, CDH stereoselectively catalyzes the conversion of the (6S)-carveol stereoisomers only. Kinetic studies with pure stereoisomers showed that this is due to large differences in V(max)/K(m) values and simultaneous product inhibition by (R)- or (S)-carvone. The R. erythropolis CDH gene (limC) was identified in an operon encoding the enzymes involved in limonene degradation. The CDH nucleotide sequence revealed an open reading frame of 831 base pairs encoding a 277-amino acid protein with a deduced mass of 29,531 Da. The CDH primary structure shares 10-30% sequence identity with members of the short chain dehydrogenase/reductase superfamily. Structure homology modeling with trihydroxynaphthalene reductase from Magnaporthe grisea suggests that CDH from R. erythropolis DCL14 is an alpha/beta one-domain protein with an extra loop insertion involved in NAD binding and a flexible C-terminal part involved in monoterpene binding.