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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Role of phaD in accumulation of medium-chain-length Poly(3-hydroxyalkanoates) in Pseudomonas oleovorans.

Pseudomonas oleovorans is capable of producing poly(3-hydroxyalkanoates) (PHAs) as intracellular storage material. To analyze the possible involvement of phaD in medium-chain-length (MCL) PHA biosynthesis, we generated a phaD knockout mutant by homologous recombination. Upon disruption of the phaD gene, MCL PHA polymer accumulation was decreased. The PHA granule size was reduced, and the number of granules inside the cell was increased. Furthermore, mutant cells appeared to be smaller than wild-type cells. Investigation of MCL PHA granules revealed that the pattern of granule-associated proteins was changed and that the predominant protein PhaI was missing in the mutant. Complementation of the mutant with a phaD-harboring plasmid partially restored the wild-type characteristics of MCL PHA production and fully restored the granule and cell sizes. Furthermore, PhaI was attached to the granules of the complemented mutant. These results indicate that the phaD gene encodes a protein which plays an important role in MCL PHA biosynthesis. However, although its main effect seems to be the stabilization of MCL PHA granules, we found that the PhaD protein is not a major granule-associated protein and therefore might act by an unknown mechanism involving the PhaI protein.

Accumulation of Poly[(R)-3-hydroxyalkanoates] in Pseudomonas oleovorans during growth with octanoate in continuous culture at different dilution rates.

Pseudomonas oleovorans ATCC 29347 was grown in chemostat culture at different dilution rates with mineral media varying in their ratios of octanoate to ammonia (C(0)/N(0) ratio). At all dilution rates tested, three distinct growth regimes were observed: (i) carbon limitation with NH(4)(+) in excess at low C(0)/N(0) ratios, (ii) purely nitrogen-limited growth conditions at high C(0)/N(0) ratios with residual octanoate in the culture supernatant, and (iii) an intermediate zone of dual-nutrient-limited growth conditions where both the concentration of octanoate and that of ammonia were very low. The dual-nutrient-limited growth zone shifted to higher C(0)/N(0) ratios with decreasing dilution rates, and the extension of the dual-nutrient-limited growth zone was inversely proportional to the growth rate. The cells accumulated the storage compound medium-chain-length poly[(R)-3-hydroxyalkanoate] (mcl-PHA) during dual (C and N)-nutrient-limited and N-limited growth conditions. Within the dual-nutrient-limited growth zone, the cellular mcl-PHA contents increased when the C(0)/N(0) ratio in the feed was increased, whereas the cellular mcl-PHA level was independent from the feed C(0)/N(0) ratio during N-limited growth. The monomeric composition of the accumulated mcl-PHA was independent of both the dilution rate and the feed C(0)/N(0) ratio and consisted of 12 mol% 3-hydroxyhexanoic acid and 88 mol% 3-hydroxyoctanoic acid. Accumulation of mcl-PHA led to an increase in the cellular C/N ratio and to changes in elemental growth yields for nitrogen and carbon.

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.

Methods for intense aeration, growth, storage, and replication of bacterial strains in microtiter plates.

Miniaturized growth systems for heterogeneous culture collections are not only attractive in reducing demands for incubation space and medium but also in making the parallel handling of large numbers of strains more practicable. We report here on the optimization of oxygen transfer rates in deep-well microtiter plates and the development of a replication system allowing the simultaneous and reproducible sampling of 96 frozen glycerol stock cultures while the remaining culture volume remains frozen. Oxygen transfer rates were derived from growth curves of Pseudomonas putida and from rates of oxygen disappearance due to the cobalt-catalyzed oxidation of sulfite. Maximum oxygen transfer rates (38 mmol liter(-1) h(-1), corresponding to a mass transfer coefficient of 188 h(-1)) were measured during orbital shaking at 300 rpm at a shaking diameter of 5 cm and a culture volume of 0.5 ml. A shaking diameter of 2.5 cm resulted in threefold-lower values. These high oxygen transfer rates allowed P. putida to reach a cell density of approximately 9 g (dry weight) liter(-1) during growth on a glucose mineral medium at culture volumes of up to 1 ml. The growth-and-replication system was evaluated for a culture collection consisting of aerobic strains, mainly from the genera Pseudomonas, Rhodococcus, and Alcaligenes, using mineral media and rich media. Cross-contamination and excessive evaporation during vigorous aeration were adequately prevented by the use of a sandwich cover of spongy silicone and cotton wool on top of the microtiter plates.

Development of an improved in vitro activity assay for medium chain length PHA polymerases based on CoenzymeA release measurements.

An improved activity assay for polyhydroxyalkanoate (PHA) polymerases from Pseudomonas oleovorans GPo1 was developed. The activity assay is based on the detection of released Coenzyme A (CoA) using 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), a compound which specifically reacts with thiol groups. The formed adduct was measured spectrophotometrically with high sensitivity and accuracy. The assay was used to study the effect of several additives on the activity of granule-associated PHA polymerase. Mild non-ionic detergents such as Tween-20, Triton X-100, CHAPS and Hecameg all appeared to be strongly inhibitory. In contrast, bovine serum albumin (BSA) had a strong stimulatory effect on the activity and stability of the PHA polymerases. Using optimized conditions, activities up to 5.8 U/mg granule-bound polymerase have been measured.