Biomedical subjects

B Witholt

Publications and source records attributed to B Witholt.

At least 19 recordsLinked to original sources

PubMed2003

Biotransformation of limonene by bacteria, fungi, yeasts, and plants.

The past 5 years have seen significant progress in the field of limonene biotransformation, especially with regard to the regiospecificity of microbial biocatalysts. Whereas earlier only regiospecific biocatalysts for the 1,2 position (limonene-1,2-diol) and the 8-position (alpha-terpineol) were available, recent reports describe microbial biocatalysts specifically hydroxylating the 3-position (isopiperitenol), 6-position (carveol and carvone), and 7-position (perillyl alcohol, perillylaaldehyde, and perillic acid). The present review also includes the considerable progress made in the characterization of plant P-450 limonene hydroxylases and the cloning of the encoding genes.

Bacteria↗

PubMed2002

Gene cloning and characterization of multiple alkane hydroxylase systems in Rhodococcus strains Q15 and NRRL B-16531.

The alkane hydroxylase systems of two Rhodococcus strains (NRRL B-16531 and Q15, isolated from different geographical locations) were characterized. Both organisms contained at least four alkane monooxygenase gene homologs (alkB1, alkB2, alkB3, and alkB4). In both strains, the alkB1 and alkB2 homologs were part of alk gene clusters, each encoding two rubredoxins (rubA1 and rubA2; rubA3 and rubA4), a putative TetR transcriptional regulatory protein (alkU1; alkU2), and, in the alkB1 cluster, a rubredoxin reductase (rubB). The alkB3 and alkB4 homologs were found as separate genes which were not part of alk gene clusters. Functional heterologous expression of some of the rhodococcal alk genes (alkB2, rubA2, and rubA4 [NRRL B-16531]; alkB2 and rubB [Q15]) was achieved in Escherichia coli and Pseudomonas expression systems. Pseudomonas recombinants containing rhodococcal alkB2 were able to mineralize and grow on C(12) to C(16) n-alkanes. All rhodococcal alkane monooxygenases possessed the highly conserved eight-histidine motif, including two apparent alkane monooxygenase signature motifs (LQRH[S/A]DHH and NYXEHYG[L/M]), and the six hydrophobic membrane-spanning regions found in all alkane monooxygenases related to the Pseudomonas putida GPo1 alkane monooxygenase. The presence of multiple alkane hydroxylases in the two rhodococcal strains is reminiscent of other multiple-degradative-enzyme systems reported in Rhodococcus.

Amino Acid Sequence↗

PubMed2001

Preparation of (R)- and (S)-N-protected 3-hydroxypyrrolidines by hydroxylation with Sphingomonas sp. HXN-200, a highly active, regio- and stereoselective, and easy to handle biocatalyst.

Hydroxylation of N-benzylpyrrolidine 8 with resting cells of Sphingomonas sp. HXN-200 gave N-benzyl-3-hydroxypyrrolidine 15 in 53% ee (S) with an activity of 5.8 U/g CDW. By changing the "docking/protecting group" in pyrrolidines, hydroxylation activity and enantioselectivity were further improved and the enantiocomplementary formation of 3-hydroxypyrrolidines was achieved: hydroxylation of N-benzoyl-, N-benzyloxycarbonyl-, N-phenoxycarbonyl-, and N-tert-butoxycarbonyl-pyrrolidines 9-12 gave the corresponding 3-hydroxypyrrolidines 16-19 in ee of 52% (R), 75% (R), 39% (S), and 23% (R), respectively, with an activity of 2.2, 16, 14, and 24 U/g CDW, respectively. Simple crystallizations increased the ee of 16-18 to 95% (R), 98% (R), and 96% (S), respectively. Hydroxylation of pyrrolidines 8-12 with soluble cell-free extracts of Sphingomonas sp. HXN-200 and equimolar NADH gave 3-hydroxypyrrolidines 15-19 in nearly the same ee as the products generated by whole cell transformation, suggesting that this strain possesses a novel soluble alkane monooxygenase. Cells of Sphingomonas sp. HXN-200 were produced in large amounts and could be stored at -80 degrees C for 2 years without significant loss of activity. The frozen cells can be thawed and resuspended for biohydroxylation, providing a highly active and easy to handle biocatalyst for the regio- and stereoselective hydroxylation of nonactivated carbon atoms. These cells were used to prepare 1.0-3.2 g (66.4-93.5% yield) of 3-hydroxypyrrolidines 16-19 by hydroxylation of pyrrolidines 9-12 on 0.9-2 L scale. Preparative hydroxylation was also achieved with growing cells as biocatalysts; hydroxylation of pyrrolidine 11 on 1 L scale gave 1.970 g (79.7% yield) of 3-hydroxypyrrolidine 18.

Catalysis↗

PubMed2001

Occurrence, synthesis and medical application of bacterial polyhydroxyalkanoate.

Polyhydroxyalkanoates (PHAs) comprise a large class of polyesters that are synthesized by many bacteria as an intracellular carbon and energy compound. Analysis of isolated PHAs reveal interesting properties such as biodegradability and biocompatibility. Research was focused only recently on the application of PHA in implants, scaffolds in tissue engineering, or as drug carriers. Such applications require that PHA be produced at a constant and reproducible quality. To date this can be achieved best through bacterial production in continuous culture where growth conditions are kept constant (chemostat). Recently, it was found that PHA producing bacteria are able to grow simultaneously limited by carbon and nitrogen substrates. Thus, it became possible to produce PHA at high yields on toxic substrate and also control its composition accurately (tailor-made synthesis). Finally, applications of PHA in medicine are discussed.

Acyltransferases↗

PubMed2001

Factors involved in the regulatory network of polyhydroxyalkanoate metabolism.

Polyhydroxyalkanoates (PHAs) are bacterial storage materials which are accumulated by various bacteria under unbalanced growth conditions. Although PHAs are produced in larger amounts and are studied because of their plastic material properties, not much is known about the regulation of PHA accumulation and the regulatory interactions with the general cell metabolism. In this report, we point out the diversity of regulatory mechanisms involved in PHA metabolism, and present examples for factors acting at the transcription or enzymatic level.

Bacteria↗

PubMed2001

Accumulation of poly[(R)-3-hydroxyalkanoates] in Pseudomonas oleovorans during growth in batch and chemostat culture with different carbon sources.

Pseudomonas oleovorans (ATCC 29347) was grown in batch and chemostat cultures with citrate, hexanoate, heptanoate, octanoate, and nonanoate as single carbon substrates. The growth medium for batch cultures was adjusted such that nitrogen (NH(4)(+)) limitation terminated the exponential-growth phase. During batch cultivation with octanoate or nonanoate the biomass continued to increase after depletion of ammonium due to the accumulation of medium-chain-length poly[(R)-3-hydroxyalkanoates] (mcl-PHAs). Additionally, a significant rate of mcl-PHA accumulation was also observed in the exponential-growth phase of batch cultures. It is well known that the accumulation of reserve materials is strongly dependent on the ratio of nutrients (here of carbon, C, and of nitrogen, N) and that in a batch culture the ratio of C:N is continuously changing. Therefore, we have also investigated the effect of defined ratios of C:N under constant cultivation conditions, namely at a fixed dilution rate (D) in a chemostat fed with different medium C:N ratios. These experiments were performed at a constant D of 0.2 h(-1). The concentration of the nitrogen source in the inflowing medium (N()) was kept constant, while its carbon concentration (C()) was increased stepwise, resulting in an increase of the medium carbon to nitrogen ratio (C()/N() ratio). The culture parameters and the cell composition of steady-state cultures were determined as a function of the C()/N() ratio in the feed medium. Mcl-PHA accumulation was detected during growth with the fatty acids, and three distinct regimes of growth limitation were discovered: In addition to carbon limitation at low, and nitrogen limitation at high C()/N() ratios, an intermediate growth regime of simultaneous limitation by carbon and nitrogen was detected where both substrates were used to completion. The width of this dual-nutrient-limited growth regime was dependent on the change in the yield factors for carbon and nitrogen (Y(X/C), Y(X/N)) measured during single-nutrient-limited growth.

Alkanes↗

PubMed2001

Industrial biocatalysis today and tomorrow.

The use of biocatalysis for industrial synthetic chemistry is on the verge of significant growth. Biocatalytic processes can now be carried out in organic solvents as well as aqueous environments, so that apolar organic compounds as well as water-soluble compounds can be modified selectively and efficiently with enzymes and biocatalytically active cells. As the use of biocatalysis for industrial chemical synthesis becomes easier, several chemical companies have begun to increase significantly the number and sophistication of the biocatalytic processes used in their synthesis operations.

Catalysis↗

PubMed2001

Two-stage continuous process development for the production of medium-chain-length poly(3-hydroxyalkanoates).

Pseudomonas oleovorans forms medium-chain-length poly(3-hydroxyalkanoate) (PHA) most effectively at growth rates below the maximum specific growth rate. Under adequate conditions, PHA accumulates in inclusion bodies in cells up to levels higher than half of the cell mass, which is a time-consuming process. For PHA production, a two-stage continuous cultivation system with two fermentors connected in series is a potentially useful system. It offers production of cells at a specific growth rate in a first compartment at conditions that lead cells to generate PHA at higher rates in a second compartment, with a relatively long residence time. In such a system, dilution rates of 0.21 h(-1) in the first fermentor (D(1)) and 0.16 h(-1) in the second fermentor (D(2)) were found to yield the highest volumetric PHA productivity. Transient-state experiments allowed investigation of D(1) and D(2) over a wide dilution rate range at high resolution in time-saving experiments. Furthermore, the influence of temperature, pH, nutrient limitation, and carbon source on PHA productivity was investigated and results similar to optimum conditions in single-stage chemostat cultivations of P. oleovorans were found. With all culture parameters optimized, a volumetric PHA productivity of 1.06 g L(-1) h(-1) was determined. Under these conditions, P. oleovorans cells contained 63% (dry weight) PHA in the effluent of the second fermentor. This is the highest PHA productivity and PHA content reported thus far for P. oleovorans cultures grown on alkanes.

Alkanes↗

PubMed2001

New alkane-responsive expression vectors for Escherichia coli and pseudomonas.

We have developed Escherichia coli and Pseudomonas expression vectors based on the alkane-responsive Pseudomonas putida (oleovorans) GPo1 promoter PalkB. The expression vectors were tested in several E. coli strains, P. putida GPo12 and P. fluorescens KOB2Delta1 with catechol-2,3-dioxygenase (XylE). Induction factors ranged between 100 and 2700 for pKKPalk in E. coli and pCom8 in Pseudomonas strains, but were clearly lower for pCom8, pCom9, and pCom10 in E. coli. XylE expression levels of more than 10% of total cell protein were obtained for E. coli as well as for Pseudomonas strains.

Alkanes↗

PubMed2001

Production of microbial polyesters: fermentation and downstream processes.

Poly(3-hydroxyalkanoates) (PHAs) constitute a large and versatile family of polyesters produced by various bacteria. PHAs are receiving considerable attention because of their potential as renewable and biodegradable plastics, and as a source of chiral synthons since the monomers are chiral. Industrial PHA production processes have been developed for poly(3-hydroxybutyrate) (poly(3HB)) and poly(3-hydroxybutyrate-co-3-valerate) (poly(3HB-co-3HV). More than 100 other poly(3HAMCL)s, characterized by monomers of medium chain length, have been identified in the past two decades. These monomers typically contain 6-14 carbon atoms, are usually linked via-3-hydroxy ester linkages, but can occasionally also exhibit 2-, 4-, 5-, or 6-hydroxy ester linkages. Such polyesters are collectively referred to as medium chain length PHAs poly(3HAMCL)s. The vast majority of these interesting biopolyesters have been studied and produced only on the laboratory scale. However, there have been several attempts to develop pilot scale processes, and these provide some insight into the production economics of poly(3HAMCL)s other than poly(3HB) and poly(3HB-co-3HV). These processes utilize diverse fermentation strategies to control the monomer composition of the polymer, enabling the tailoring of polymer material properties to some extent. The best studied of these is poly(3-hydroxyoctanoate) (poly(3HO)), which contains about 90% 3-hydroxyoctanoate. This biopolyester has been produced on the pilot scale and is now being used in several experimental applications.

Chemical Engineering↗

PubMed2001

Using proteins in their natural environment: potential and limitations of microbial whole-cell hydroxylations in applied biocatalysis.

The unique catalytic properties of oxygenases (the regio-specific and/or enantio-specific hydroxylation of non-activated carbons) are of undisputed biosynthetic value. Factors that govern the economics of their industrial use include a low k(cat), a frequently decreased k(cat) in recombinant strains, limiting oxygen transfer rates in bioreactors, product inhibition, and the demanding discovery (screening) process.

Bacteria↗

PubMed2001

Effectiveness of orbital shaking for the aeration of suspended bacterial cultures in square-deepwell microtiter plates.

Growth of heterogeneous culture collections in microtiter plates is advantageous for logistic reasons and also in enabling significant savings in medium costs, labor input and use of equipment during large screening projects. The main hurdles to overcome for aerobic microbial strains are the prevention of cross-contamination and excessive evaporation while assuring sufficient aeration rates. For this purpose we developed a sandwich spongy silicone/cotton wool cover to close the wells of square-deepwell microtiter plates. Oxygen transfer rates were derived from growth curves of Pseudomonas putida and were shown to be threefold higher during orbital shaking at a shaking diameter of 5cm at 300rpm (24mmolO(2)l(-1)h(-1) at a culture volume of 0.75ml) in comparison to a shaking diameter of 2.5cm. Photographic analysis showed a clear influence of the shaking diameter on the hydrodynamic behavior in the wells; during shaking at a 2.5cm amplitude, out-of-phase conditions occurred resulting in poor vertical mixing, while a 5cm shaking amplitude led to an optimal surface to volume ratio and a turbulent flow.

Journal Article↗

PubMed2001

Biotransformation of D-limonene to (+) trans-carveol by toluene-grown Rhodococcus opacus PWD4 cells.

The toluene-degrading strain Rhodococcus opacus PWD4 was found to hydroxylate D-limonene exclusively in the 6-position, yielding enantiomerically pure (+) trans-carveol and traces of (+) carvone. This biotransformation was studied using cells cultivated in chemostat culture with toluene as a carbon and energy source. The maximal specific activity of (+) trans-carveol formation was 14.7 U (g of cells [dry weight])(-1), and the final yield was 94 to 97%. Toluene was found to be a strong competitive inhibitor of the D-limonene conversion. Glucose-grown cells did not form any trans-carveol from D-limonene. These results suggest that one of the enzymes involved in toluene degradation is responsible for this allylic monohydroxylation. Another toluene degrader (Rhodococcus globerulus PWD8) had a lower specific activity but was found to oxidize most of the formed trans-carveol to (+) carvone, allowing for the biocatalytic production of this flavor compound.

Biotransformation↗

PubMed2001

p-Hydroxyphenylacetic Acid Metabolism in Pseudomonas putida F6.

Pseudomonas putida F6 was found to metabolize p-hydroxyphenylacetic acid through 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxymandelic acid, and 3,4-dihydroxybenzaldehyde. Cell extracts of P. putida F6 catalyze the NAD(P)H-independent hydroxylation of p-hydroxyphenylacetic acid to 3,4-dihydroxyphenylacetic acid which is further oxidized to 3,4-dihydroxymandelic acid. Oxidation and decarboxylation of the latter yields 3,4-dihydroxybenzaldehyde. A red-brown color accompanies all of the above enzyme activities and is probably due to the polymerization of quinone-like compounds. 3,4-Dihydroxybenzaldehyde is further metabolized through extradiol ring cleavage.

3,4-Dihydroxyphenylacetic Acid↗

PubMed2000

Preparation of (S)-N-substituted 4-hydroxy-pyrrolidin-2-ones by regio- and stereoselective hydroxylation with Sphingomonas sp. HXN-200.

[reaction: see text] Enantiopure (S)-N-substituted 4-hydroxy-pyrrolidin-2-ones have been prepared for the first time by regio- and stereoselective hydroxylation of the corresponding pyrrolidin-2-ones by use of a biocatalyst. Hydroxylation of 6 and 8 with Sphingomonas sp. HXN-200 afforded 68% of (S)-7 in >99.9% ee and 46% of (S)-9 in 92% ee, respectively. Simple crystallization increased the ee of (S)-9 to 99. 9% in 82% yield.

Chromatography, High Pressure Liquid↗

PubMed2000

Recovery of active medium-chain-length-poly-3-hydroxyalkanoate polymerase from inactive inclusion bodies using ion-exchange resin.

A novel process for the purification of active medium-chain-length-polyhydroxyalkanoate (mcl-PHA) polymerase was developed. This process is based on solubilization and activation of inactive polymerase inclusion bodies by incubation with ion-exchange resin. The mcl-PHA polymerase 1 from Pseudomonas oleovorans was overproduced from the Palk promoter. Most of the polymerase produced was sequestered in the cytoplasm as an inactive form in insoluble aggregates. By incubating the protein aggregates with S-Sepharose ion-exchange resin in the presence of dithiothreitol and glycerol, the mcl-PHA polymerase could be extracted in an active and soluble form with a final yield of about 5.2 mg/g of cell dry weight. The solubilized polymerase was able to catalyse the in vitro synthesis of mcl-PHA without any additional cell components, suggesting its potential application for production of biopolymer. The procedure used here may be of general value in solubilizing and activating purified inactive labile enzymes.

Acyltransferases↗

PubMed2000

Production of enantiopure styrene oxide by recombinant Escherichia coli synthesizing a two-component styrene monooxygenase.

A whole cell biocatalytic process was developed to enable the efficient oxidation of styrene to chiral (S)-styrene oxide with an enantiomeric excess better than 99%. Recombinant Escherichia coli cells were employed to express the genes styAB encoding the styrene monooxygenase of Pseudomonas sp. strain VLB120 from an expression plasmid utilizing the alk regulatory system of P. oleovorans GPo1. The strains reached specific activities of up to 70 U* (g cell dry weight)(-1) in shake-flask experiments with glucose as the carbon source. An efficient two-liquid phase fed-batch process was established for the production of (S)-styrene oxide with hexadecane as an apolar carrier solvent and a nutrient feed consisting of glucose, magnesium sulfate, and yeast extract. Engineering of the phase fraction and the composition of organic phase and feed led to a 2-L scale process with maximal volumetric productivities of 2.2 g (S)-styrene oxide per liter liquid volume per hour. This optimized process was based completely on defined medium and used bis(2-ethylhexyl)phthalate as the apolar carrier solvent, which together with substrate and inducer consisted of 50% of the total liquid volume. Using this system, we were able to produce per liter liquid volume 11 g of enantiopure (S)-styrene oxide in 10 h.

Base Sequence↗

PubMed2000

Xylene monooxygenase catalyzes the multistep oxygenation of toluene and pseudocumene to corresponding alcohols, aldehydes, and acids in Escherichia coli JM101.

Xylene monooxygenase of Pseudomonas putida mt-2 catalyzes the methylgroup hydroxylation of toluene and xylenes. To investigate the potential of xylene monooxygenase to catalyze multistep oxidations of one methyl group, we tested recombinant Escherichia coli expressing the monooxygenase genes xylM and xylA under the control of the alk regulatory system of Pseudomonas oleovorans Gpo1. Expression of xylene monooxygenase genes could efficiently be controlled by n-octane and dicyclopropylketone. Xylene monooxygenase was found to catalyze the oxygenation of toluene, pseudocumene, the corresponding alcohols, and the corresponding aldehydes. For all three transformations (18)O incorporation provided stong evidence for a monooxygenation type of reaction, with gem-diols as the most likely reaction intermediates during the oxygenation of benzyl alcohols to benzaldehydes. To investigate the role of benzyl alcohol dehydrogenase (XylB) in the formation of benzaldehydes, xylB was cloned behind and expressed in concert with xylMA. In comparison to E. coli expressing only xylMA, the presence of xylB lowered product formation rates and resulted in back formation of benzyl alcohol from benzaldehyde. In P. putida mt-2 XylB may prevent the formation of high concentrations of the particularly reactive benzaldehydes. In the case of high fluxes through the degradation pathways and low aldehyde concentrations, XylB may contribute to benzaldehyde formation via the energetically favorable dehydrogenation of benzyl alcohols. The results presented here characterize XylMA as an enzyme able to catalyze the multistep oxygenation of toluenes.

Alcohols↗