Pseudomonas Putida GPo1 Research Articles

Understanding the Limitations in the Biosynthesis of Polyhydroxyalkanoate (PHA) from Lignin Derivatives

To investigate the conversion of lignin derivatives to biopolyesters, 11 polyhydroxyalkanoate (PHA)-accumulating strains were cultured on mineral salt media containing each of the 18 lignin derivatives and hydroxybenzoic acids, including intermediates derived from the metabolism of lignin derivatives in bacteria. Most of the strains grew poorly in media containing lignin derivatives such as p-coumaric acid, caffeic acid, ferulic acid, and sinapinic acid. One of the strains, Pseudomonas putida Gpo1, grew in the presence of p-coumaric acid, ferulic acid, vanillic acid, 4-hydroxybenzoic acid (4-HBA), and 3,4-dihydroxybenzoic acid (3,4-DHBA). Pseudomonas putida JCM 13063 also grew in the presence of 4-HBA, 3,4-DHBA, and vanillic acid. Another strain, Ralstonia eutropha H16, synthesized PHA from 4-HBA, 2,5-DHBA, and 3,4-DHBA. On the basis of the data obtained from these experiments, we suggest that the conversion of lignin derivatives into intermediates such as 4-HBA, 2,5-DHBA, 3,4-DHBA, and vanillic acid repr...

Controlled oxidation of aliphatic C[sbnd]H bonds in metallo-monooxygenases: Mechanistic insights derived from studies on deuterated and fluorinated hydrocarbons

The control over the regio- and/or stereo-selective aliphatic CH oxidation by metalloenzymes is of great interest to scientists. Typically, these enzymes invoke host–guest chemistry to sequester the substrates within the protein pockets, exploiting sizes, shapes and specific interactions such as hydrogen-bonding, electrostatic forces and/or van der Waals interactions to control the substrate specificity, regio-specificity and stereo-selectivity. Over the years, we have developed a series of deuterated and fluorinated variants of these hydrocarbon substrates as probes to gain insights into the controlled CH oxidations of hydrocarbons facilitated by these enzymes. In this review, we illustrate the application of these designed probes in the study of three monooxygenases: (i) the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath), which oxidizes straight-chain C1–C5 alkanes and alkenes to form their corresponding 2-alcohols and epoxides, respectively; (ii) the recombinant alkane hydroxylase (AlkB) from Pseudomonas putida GPo1, which oxidizes the primary CH bonds of C5–C12 linear alkanes; and (iii) the recombinant cytochrome P450 from Bacillus megaterium, which oxidizes C12–C20 fatty acids at the ω-1, ω-2 or ω-3 CH positions.

Critical residues of class II PHA synthase for expanding the substrate specificity and enhancing the biosynthesis of polyhydroxyalkanoate

This study describes protein model of type II Pseudomonas putida GPo1 synthase (PhaC1Pp) and using single or multiple points mutagenesis to identify the beneficial amino acid residues that change the PHA accumulation and the substrate chain-length specificity of type II PHA synthase. The P. putida GPp104 PHA− was used as a host for evaluating the substrate specificity and PHA yield of the mutated PhaC1Pp. The evolved PhaC1Pp were coexpressed with β-ketothiolase (phbARe) and the acetoacetyl-CoA reductase (phbBRe) to supply sufficient short-chain length (R)-3-hydroxyacyl-CoA as a substrate. A single point mutation at L484V remarkably enhanced the monomer ratio of (R)-3-hydroxybutyrate in a PHA accumulation experiment. Saturation mutagenesis experiment at 484 concluded that Val is the most favorable amino acid in PhaC1Pp for incorporating (R)-3-hydroxybutyrate unit synthesis. In addition, a single mutation at Q481M, S482G and A547V obviously increased PHA yields. Q481M and S482G enhanced the (R)-3-hydroxyhexanoate monomer composition in the PHA accumulation by P. putida GPp104 PHA−. This is the first data that spotlighted the important effect of Leu484 on substrate specificity of PHA synthase and Ala547 on the PHA accumulation.

Characterization and Two-Dimensional Crystallization of Membrane Component AlkB of the Medium-Chain Alkane Hydroxylase System from Pseudomonas putida GPo1

The alkane hydroxylase system of Pseudomonas putida GPo1 allows it to use alkanes as the sole source of carbon and energy. Bacterial alkane hydroxylases have tremendous potential as biocatalysts for the stereo- and regioselective transformation of a wide range of chemically inert unreactive alkanes into valuable reactive chemical precursors. We have produced and characterized the first 2-dimensional crystals of the integral membrane component of the P. putida alkane hydroxylase system, the nonheme di-iron alkane monooxygenase AlkB. Our analysis reveals for the first time that AlkB reconstituted into a lipid bilayer forms trimers. Addition of detergents that do not disrupt the AlkB oligomeric state (decyl maltose neopentyl glycol [DMNG], lauryl maltose neopentyl glycol [LMNG], and octaethylene glycol monododecyl ether [C(12)E(8)]) preserved its activity at a level close to that of the detergent-free control sample. In contrast, the monomeric form of AlkB produced by purification in n-decyl-β-D-maltopyranoside (DM), n-dodecyl-β-D-maltopyranoside (DDM), octyl glucose neopentyl glycol (OGNG), and n-dodecyl-N,N-dimethylamine-N-oxide (LDAO) was largely inactive. This is the first indication that the physiologically active form of membrane-embedded AlkB may be a multimer. We present for the first time experimental evidence that 1-octyne acts as a mechanism-based inhibitor of AlkB. Therefore, despite the lack of any significant full-length sequence similarity with members of other monooxygenase classes that catalyze the terminal oxidation of alkanes, AlkB is likely to share a similar catalytic mechanism.

Kinetic Analysis of Terminal and Unactivated CH Bond Oxyfunctionalization in Fatty Acid Methyl Esters by Monooxygenase‐Based Whole‐Cell Biocatalysis

AbstractThe alkane monooxygenase AlkBGT from Pseudomonas putida GPo1 constitutes a versatile enzyme system for the ω‐oxyfunctionalization of medium chain‐length alkanes. In this study, recombinant Escherichia coli W3110 expressing alkBGT was investigated as whole‐cell catalyst for the regioselective biooxidation of fatty acid methyl esters to terminal alcohols. The ω‐functionalized products are of general economic interest, serving as building blocks for polymer synthesis. The whole‐cell catalysts proved to functionalize fatty acid methyl esters with a medium length alkyl chain specifically at the ω‐position. The highest specific hydroxylation activity of 104 U gCDW−1 was obtained with nonanoic acid methyl ester as substrate using resting cells of E. coli W3110 (pBT10). In an optimized set‐up, maximal 9‐hydroxynonanoic acid methyl ester yields of 95% were achieved. For this specific substrate, apparent whole‐cell kinetic parameters were determined with a Vmax of 204±9 U gCDW−1, a substrate uptake constant (KS) of 142±17 μM, and a specificity constant Vmax/KS of 1.4 U gCDW−1 μM−1 for the formation of the terminal alcohol. The same E. coli strain carrying additional alk genes showed a different substrate selectivity. A comparison of biocatalysis with whole cells and enriched enzyme preparations showed that both substrate availability and enzyme specificity control the efficiency of the whole‐cell bioconversion of the longer and more hydrophobic substrate dodecanoic acid methyl ester. The efficient coupling of redox cofactor oxidation and product formation, as determined in vitro, combined with the high in vivo activities make E. coli W3110 (pBT10) a promising biocatalyst for the preparative synthesis of terminally functionalized fatty acid methyl esters.

Growth and accumulation dynamics of poly(3‐hydroxyalkanoate) (PHA) in Pseudomonas putida GPo1 cultivated in continuous culture under transient feed conditions

It has been shown that Pseudomonas putida GPo1 is able to grow in continuous culture simultaneously limited by ammonium (N source) and octanoate (C source), and concomitantly accumulate poly([R]-3-hydroxyalkanoate) (PHA). Under such growth conditions the material properties of PHA can be fine-tuned if a second PHA precursor substrate is supplied. To determine the range of dual carbon and nitrogen (C, N)-limited growth conditions, tedious chemostat experiments need to be carried out for each carbon source separately. To determine the growth regime, the C/N ratio of the feed (f) to a chemostat was changed in a stepwise manner at a constant dilution rate of 0.3/h. Dual-(C, N)-limited growth was observed between C(f) /N(f) ≤ 6.4 g/g and C(f) /N(f) >9.5 g/g. In the following, we analyzed alternative approaches, using continuous medium gradients at the same dilution rate, that do not require time consuming establishments of steady states. Different dynamic approaches were selected in which the C(f) /N(f) ratio was changed continuously through a convex increase of C(f) , a convex increase of N(f) , or a linear decrease of C(f) (gradients 1, 2, and 3, respectively). In these experiments, the dual-(C, N)-limited growth regime was between 7.2 and 11.0 g/g for gradient 1, 4.3 and 6.9 g/g for gradient 2, and 5.1 and 8.9 g/g for gradient 3. A mathematical equation was developed that compensated a time delay of the gradient that was caused by the wash-in/wash-out effects of the medium feed.

An Improved Procedure for the Purification of Catalytically Active Alkane Hydroxylase from Pseudomonas putida GPo1

Bacterial alkane hydroxylases are of high interest for bioremediation applications as they allow some bacteria to grow in oil-contaminated environments. Furthermore, they have tremendous biotechnological potential as they catalyse the stereo- and regio-specific hydroxylation of chemically inert alkanes, which can then be used in the synthesis of pharmaceuticals and other high-cost chemicals. Despite their potential, progress on the detailed characterization of these systems has so far been slow mainly due to the lack of a robust procedure to purify its membrane protein component, monooxygenase AlkB, in a stable and active form. This study reports a new method for isolating milligramme amounts of recombinant Pseudomonas putida GPo1 AlkB in a folded, catalytically active form to purity levels above 90%. AlkB solubilised and purified in the detergent lauryldimethylamine oxide was demonstrated to be active in catalysing the epoxidation reaction of 1-octene with an estimated K (m) value of 0.2 mM.

Regio-selective hydroxylation of gem-difluorinated octanes by alkane hydroxylase (AlkB)

gem-Difluoromethylene substituted molecules constitute a distinct class of fluorinated compounds. In this study, special chemistry has been developed for their preparation based on the highly selective terminal hydroxylation of these gem-difluorinated octanes by AlkB (alkane hydroxylase) from Pseudomonas putida Gpo1 to form gem-difluorinated octan-1-ols. The hydroxylation reaction is performed by whole-cell catalysis. Identification of the distal- and proximal-hydroxylation products was made by 1H, 13C, and 19F NMR; GC and GC/MSD; and/or by comparison with authentic standards in GC. To the best of our knowledge, we have obtained the first synthesis of 2,2-, 3,3- and 4,4-difluorooctan-1-ols, from simple and inexpensive starting materials.

Whole-cell bio-oxidation of n-dodecane using the alkane hydroxylase system of P. putida GPo1 expressed in E. coli

The alkane-1-monoxygenase (alkB) complex of Pseudomonas putida GPo1 has been extensively studied in the past and shown to be capable of oxidising aliphatic C 5–C 12 alkanes to primary alcohols both in the wild-type organism by growth on C 5–C 12 alkanes as sole carbon source and in vitro. Despite this, successful n-dodecane oxidation for the production of 1-dodecanol or dodecanoic acid has proven elusive in the past when using alkB-expressing recombinants. This article demonstrates, for the first time in vivo, by using the Escherichia coli GEC137 pGEc47ΔJ strain, that n-dodecane oxidation using this enzyme for the production of primary alcohols and carboxylic acids is feasible and in fact potentially more promising than n-octane oxidation due to lower product and substrate toxicity. Yields are reported of 1-dodecanol of up to 2 g/L organic and dodecanoic acid up to 19.7 g/L organic in a 2 L stirred tank reactor with 1 L aqueous phase and 200 mL of n-dodecane as a second phase. The maximum volumetric rate of combined alcohol and acid production achieved was 1.9 g/L organic/h (0.35 g/L total/h). The maximum specific activity of combined alcohol and acid production was 7-fold lower on n-dodecane (3.5 μmol/min/g dcw) than on n-octane (21 μmol/min/g dcw); similar to the 5-fold difference observed between wild-type growth rates using the two respective alkanes as sole carbon source. Despite this, both total volumetric rate and final yield exceeded n-octane oxidation by 3.5-fold under the same conditions, due to the lower toxicity of n-dodecane and its oxidation products to E. coli compared to the 8-carbon equivalents. Substrate access limitations and the overoxidation of 1-dodecanol to dodecanoic acid were identified as the most important limitations to be addressed.

Simultaneous Biosynthesis of Two Copolymers in Pseudomonas putida GPo1 Using a Two-Stage Continuous Culture System

The bacterial production of medium-chain-length polyhydroxyalkanoate (PHA) in continuous two-stage cultivation was investigated. Two chemostats were linked in sequence and Pseudomonas putida GPo1 was supplied with different concentrations of octanoic acid in the first and 10-undecenoic acid in the second reactor. PHA isolated from the second bioreactor was a blend of two copolymers typically consisting of poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate) (PHO) and poly(3-hydroxy-10-undecenoate-co-3-hydroxy-8-nonenoate-co-3-hydroxy-6-heptenoate) (PHUE) with structural (monomeric) purities of 85-95 mol %, indicating simultaneous PHA accumulation and degradation inside the cell. The maximum PHA content was found to be 53.8% of total cell dry weight, which is very high for mcl-PHA.

Simultaneous Accumulation and Degradation of Polyhydroxyalkanoates: Futile Cycle or Clever Regulation?

The regulation of medium-chain-length polyhydroxyalkanoates (mcl-PHA) metabolism in Pseudomonas putida GPo1 was studied by analysis of enzymes bound to PHA granules and enzymes involved in fatty acid oxidation. N-terminal sequencing of granule-bound enzymes revealed the presence of PHA polymerase (PhaC) and PHA depolymerase (PhaZ) and an acyl-CoA synthetase (ACS1), which recently was found to be associated with PHA granules by in vivo study. The acs1 knockout mutant accumulated 30-50% less PHA than its parental strain, confirming the involvement of ACS1 in PHA metabolism. Isolated PHA granules showed both PhaC and PhaZ activities. PhaC activity was found to be sensitive to the ratio of [R-3-hydroxyacyl-CoA]/[CoA] in which free CoA was a mild competitive inhibitor. Fatty acid oxidation was regulated by the [acetyl-CoA]/[CoA] and [NADH]/[NAD] ratios, with high ratios resulting in accumulation and low ratios leading to rapid oxidation of 3-hydroxyacyl-CoA. These results suggest that PHA metabolism is likely to be controlled by the [acetyl-CoA]/[CoA] and [NADH]/[NAD] ratios. The physiological roles of simultaneous PHA accumulation and degradation are also discussed.

Overexpression and characterization of medium-chain-length polyhydroxyalkanoate granule bound polymerases from Pseudomonas putida GPo1

BackgroundPolyhydroxyalkanoates (PHA) are synthesized by many bacteria in the cytoplasm as storage compounds for energy and carbon. The key enzymes for PHA biosynthesis are PHA polymerases, which catalyze the covalent linkage of 3-hydroxyacyl coenzymeA thioesters by transesterification with concomitant release of CoA. Pseudomonas putida GPo1 and many other Pseudomonas species contain two different class II polymerases, encoded by phaC1 and phaC2. Although numerous studies have been carried out on PHA polymerases and they are well characterized at the molecular level, the biochemical properties of the class II polymerases have not been studied in detail. Previously we and other groups purified the polymerases, however, the activities of the purified enzymes were several magnitude lower than the granule-bound enzymes. It is problematic to study the intrinsic properties of these enzymes with such low activities, although they are pure.ResultsPHA polymerase 1 (PhaC1) and PHA polymerase 2 (PhaC2) from P. putida GPo1 were overexpressed in the PHA-negative host P. putida GPp104 and purified from isolated PHA granules. Only minor activity (two to three orders of magnitude lower than that of the granule bound proteins) could be recovered when the enzymes were purified to homogeneity. Therefore, kinetic properties and substrate ranges were determined for the granule bound polymerases. The polymerases differed significantly with respect to their association with PHA granules, enzyme kinetics and substrate specificity. PhaC2 appeared to bind PHA granules more tightly than PhaC1. When R-3-hydroxyoctanoic acid was used as substrate, the granule-bound PhaC1 exhibited a Km of 125 (± 35) μM and a Vmax of 40.8 (± 6.2) U/mg PhaC1, while a Km of 37 (± 10) μM and a Vmax of 2.7 (± 0.7) U/mg PhaC2 could be derived for the granule-bound PhaC2. Granule-bound PhaC1 showed a strong preference for medium chain length (mcl-) 3-hydroxyacly-CoAs, with highest affinity towards 3-hydroxydecanoyl-CoA (40 U/mg PhaC1). Granule-bound PhaC2 demonstrated a far broader specificity ranging from short chain length up to long chain length substrates. Activity increased with increasing chain length with a maximum activity for 3-hydroxyacyl-CoAs containing 12 or more C-atoms.ConclusionThe kinetic properties and substrate ranges were determined for both granule bound polymerases. Evidence was provided for the first time that two PHA polymerases exhibited significant differences in granule release and in vitro activity profiles, suggesting that there are substantial functional differences between granule bound PhaC1 and PhaC2.

Synthesis and Characterization of a Cationic Poly(β-hydroxyalkanoate)

Poly(beta-hydroxyalkanoates) (PHAs) are biodegradable polyesters produced by a wide range of bacteria. The structures of these polymers may be tuned by controlling the carbon source composition in the feed stock, but the range of functional groups accessible in this manner is limited to those that the organism is able to metabolize. Much effort has been made to chemically modify the side chains of these polymers to achieve new materials. Here, we report the synthesis of the first cationic PHA, poly(beta-hydroxy-octanoate)- co-(beta-hydroxy-11-(bis(2-hydroxyethyl)-amino)-10-hydroxyundecanoate) (PHON). Pseudomonas putida Gpo1 was used to produce poly(beta-hydroxy-octanoate)- co-(beta-hydroxy-10-undecenoate) (PHOU), whose vinyl-terminated side chains were first converted to terminal epoxides and then modified with diethanolamine. The modification of PHOU was examined using (1)H, COSY, and HSQC NMR and GPC and resulted in a loss of molecular weight due to aminolysis and also in the introduction of side chains terminated with tertiary amine groups, which are protonated at physiological pH. The polycationic PHA is soluble in polar solvents such as DMSO, DMF, and water. The new biodegradable cationic polymers are envisioned as nucleic acid delivery systems.

Identification of Two Acyl-CoA Synthetases from Pseudomonas putida GPo1: One is Located at the Surface of Polyhydroxyalkanoates Granules

Pseudomonas putida GPo1 is able to accumulate polyhydroxyalkanoates (PHA) in the form of intracellular granules as storage materials. PHA granules were isolated and analyzed for protein activities. An acyl-CoA-synthetase (ACS1) activity was detected from the purified PHA granules. The corresponding gene acs1 was then cloned from P. putida GPo1. With the genomic walking technique, a homologue acs2 located upstream of acs1 was discovered and cloned. Fusions of both acs1 and acs2 with the gene encoding the green fluorescent protein (GFP) were constructed and expressed in GPo1. In vivo fluorescence microscopy studies showed that the fluorescence generated from the ACS1-GFP was mainly associated with the PHA granules, whereas that from ACS2-GFP was mainly with the membrane of the cells. In the control strain (containing GFP alone) fluorescence was distributed evenly in the cytoplasm. We concluded that ACS1 is located on the PHA granules and may play a central role in mobilization of PHA, for example, conversion of hydroxycarboxylic acid monomers to hydroxycarboxyl-CoA, which can be further utilized by the cells.

Efficient recovery of low endotoxin medium-chain-length poly([ R]-3-hydroxyalkanoate) from bacterial biomass

Bacterial polyhydroxyalkanoate (PHA) is an attractive biopolyester for medical applications due to its biocompatibility. However, inappropriate extraction of PHA from bacterial biomass results in contamination by pyrogenic compounds (e.g. lipopolysaccharides) and thus influences medical testing. This problem was solved by a temperature-controlled method for the recovery of poly(3-hydroxyoctanoate- co-3-hydroxyhexanaote) (PHO) from Pseudomonas putida GPo1. In contrast to other methods, precipitation of PHO was triggered by cooling the hot solution to a particular temperature. N-hexane and 2-propanol were found to be optimal solvents for such procedure. Quantitative extraction with n-hexane took place at 50 °C and optimal precipitation occurred between 0 and 5 °C. The purity was > 97% (w/w) and the endotoxicity between 10 and 15 EU/g PHO. Additional re-dissolution in 2-propanol at 45 °C and precipitation at 10 °C resulted in a purity of close to 100% (w/w) and the minimal endotoxicity of 2 EU/g PHO. The polydispersity ( M w/ M n) of PHO was decreased from 2.0 to 1.5 for this optimized procedure.

Efficient Production of (R)-3-Hydroxycarboxylic Acids by Biotechnological Conversion of Polyhydroxyalkanoates and Their Purification

An efficient method to prepare enantiomerically pure (R)-3-hydroxycarboxylic acids from bacterial polyhydroxyalkanoates (PHAs) accumulated by Pseudomonas putida GPo1 is reported in this study. (R)-3-Hydroxycarboxylic acids from whole cells were obtained when conditions were provided to promote in vivo depolymerization of intracellular PHA. The monomers were secreted into the extracellular environment. They were separated and purified by acidic precipitation, preparative reversed-phase column chromatography, and subsequent solvent extraction. Eight (R)-3-hydroxycarboxylic acids were isolated: (R)-3-hydroxyoctanoic acid, (R)-3-hydroxyhexanoic acid, (R)-3-hydroxy-10-undecenoic acid, (R)-3-hydroxy-8-nonenoic acid, (R)-3-hydroxy-6-heptenoic acid, (R)-3-hydroxyundecanoic acid, (R)-3-hydroxynonanoic acid, and (R)-3-hydroxyheptanoic acid. The overall yield based on released monomers was around 78 wt % for (R)-3-hydroxyoctanoic acid. All obtained monomers had a purity of over 95 wt %. The physical properties of the purified monomers and their antimicrobial activities were also investigated.

CYP153A6, a soluble P450 oxygenase catalyzing terminal-alkane hydroxylation.

The first and key step in alkane metabolism is the terminal hydroxylation of alkanes to 1-alkanols, a reaction catalyzed by a family of integral-membrane diiron enzymes related to Pseudomonas putida GPo1 AlkB, by a diverse group of methane, propane, and butane monooxygenases and by some membrane-bound cytochrome P450s. Recently, a family of cytoplasmic P450 enzymes was identified in prokaryotes that allow their host to grow on aliphatic alkanes. One member of this family, CYP153A6 from Mycobacterium sp. HXN-1500, hydroxylates medium-chain-length alkanes (C6 to C11) to 1-alkanols with a maximal turnover number of 70 min(-1) and has a regiospecificity of > or =95% for the terminal carbon atom position. Spectroscopic binding studies showed that C6-to-C11 aliphatic alkanes bind in the active site with Kd values varying from approximately 20 nM to 3.7 microM. Longer alkanes bind more strongly than shorter alkanes, while the introduction of sterically hindering groups reduces the affinity. This suggests that the substrate-binding pocket is shaped such that linear alkanes are preferred. Electron paramagnetic resonance spectroscopy in the presence of the substrate showed the formation of an enzyme-substrate complex, which confirmed the binding of substrates observed in optical titrations. To rationalize the experimental observations on a molecular scale, homology modeling of CYP153A6 and docking of substrates were used to provide the first insight into structural features required for terminal alkane hydroxylation.

Tailor-made olefinic medium-chain-length poly[(R)-3-hydroxyalkanoates] byPseudomonas putida GPo1: Batch versus chemostat production

Functionalized medium-chain-length polyhydroxyalkanoates (mclPHAs) have gained much interest in research on biopolymers because of their ease of chemical modification. Tailored olefinic mclPHA production from mixtures of octanoic acid and 10-undecenoic acid was investigated in batch and dual (C,N) nutrient limited chemostat cultures of Pseudomonas putida GPo1 (ATCC 29347). In a batch culture, where P. putida GPo1 was grown on a mixture of octanoic acid (58 mol%) and 10-undecenoic acid (42 mol%), it was found that the fraction of aliphatic monomers was slightly lower in mclPHA produced during exponential growth than during late stationary phase. Thus, the total monomeric composition changed over time indicating different kinetics for the two carbon substrates. Chemostat experiments showed that the dual (C,N) nutrient limited growth regime (DNLGR) for 10-undecenoic acid coincided with the one for octanoic acid. Five different chemostats on equimolar mixtures of octanoic acid and 10-undecenoic acid within the DNLGR revealed that the monomeric composition of mclPHA was not a function of the carbon to nitrogen (C(0)/N(0)) ratio in the feed medium but rather of the dilution rate. The fraction of aliphatic monomers in the accumulated mclPHA was slightly lower at high dilution rates and increased towards low dilution rates, again indicating different kinetics for the two carbon substrates in P. putida GPo1.

Poly(3-hydroxyalkanoate) polymerase synthesis and in vitro activity in recombinant Escherichia coli and Pseudomonas putida

We tested the synthesis and in vitro activity of the poly(3-hydroxyalkanoate) (PHA) polymerase 1 from Pseudomonas putida GPo1 in both P. putida GPp104 and Escherichia coli JMU193. The polymerase encoding gene phaC1 was expressed using the inducible PalkB promoter. It was found that the production of polymerase could be modulated over a wide range of protein levels by varying inducer concentrations. The optimal inducer dicyclopropylketone concentrations for PHA production were at 0.03% (v/v) for P. putida and 0.005% (v/v) for E. coli. Under these concentrations the maximal polymerase level synthesized in the E. coli host (6% of total protein) was about three- to fourfold less than that in P. putida (20%), whereas the maximal level of PHA synthesized in the E. coli host (8% of total cell dry weight) was about fourfold less than that in P. putida (30%). In P. putida, the highest specific activity of polymerase was found in the mid-exponential growth phase with a maximum of 40 U/g polymerase, whereas in E. coli, the maximal specific polymerase activity was found in the early stationary growth phase (2 U/g polymerase). Our results suggest that optimal functioning of the PHA polymerase requires factors or a molecular environment that is available in P. putida but not in E. coli.

Assessing the role of alkane hydroxylase genotypes in environmental samples by competitive PCR

A molecular tool for extensive detection of prokaryotic alkane hydroxylase genes (alkB) was developed. AlkB genotypes involved in the degradation of short-chain alkanes were quantified in environmental samples in order to assess their occurrence and ecological importance. Four primer pairs specific for distinct clusters of alkane hydroxylase genes were designed, allowing amplification of alkB-related genes from all tested alkane-degrading strains and from six of seven microcosms. For the primer pair detecting alkB genes related to the Pseudomonas putida GPo1 alkB gene and the one targeting alkB genes of Gram-positive strains, both involved in short-chain alkane degradation (<C10), a quantitative competitive PCR (cPCR) assay was developed and validated on alkB-containing strains. AlkB genes of the two groups were then quantified in hydrocarbon-contaminated and pristine freshwater and soil samples, and their respective frequency was compared to degradation rates of short-chain n-alkanes. Pseudomonas putida-related alkB genes were prevalent in freshwater samples, but Gram-positive alkB-containing strains were more consistently related to alkane degradation activities. The latter genotype was more abundant in soils, although both genotypes increased in the most contaminated soils studied. Predominance of alkB genotypes depends on the ecosystem and environmental conditions, but alkane exposure generally leads to an increase of both studied genotypes. The study illustrates the distribution of two different alkB genotypes in two types of ecosystems, and highlights their respective roles in the environment.