Alkane hydroxylases involved in microbial alkane degradation

This review focuses on the role and distribution in the environment of alkane hydroxylases and their (potential) applications in bioremediation and biocatalysis. Alkane hydroxylases play an important role in the microbial degradation of oil, chlorinated hydrocarbons, fuel additives, and many other compounds. Environmental studies demonstrate the abundance of alkane degraders and have lead to the identification of many new species, including some that are (near)-obligate alkanotrophs. The availability of a growing collection of alkane hydroxylase gene sequences now allows estimations of the relative abundance of the different enzyme systems and the distribution of the host organism

In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6

Enzymes of the AlkB and CYP153 families catalyze the first step in the catabolism of medium-chain-length alkanes, selective oxidation of the alkane to the 1-alkanol, and enable their host organisms to utilize alkanes as carbon sources. Small, gaseous alkanes, however, are converted to alkanols by evolutionarily unrelated methane monooxygenases. Propane and butane can be oxidized by CYP enzymes engineered in the laboratory, but these produce predominantly the 2-alkanols. Here we report the in vivo-directed evolution of two medium-chain-length terminal alkane hydroxylases, the integral membrane di-iron enzyme AlkB from Pseudomonas putida GPo1 and the class II-type soluble CYP153A6 from Mycobacterium sp. strain HXN-1500, to enhance their activity on small alkanes. We established a P. putida evolution system that enables selection for terminal alkane hydroxylase activity and used it to select propane- and butane-oxidizing enzymes based on enhanced growth complementation of an adapted P. putida GPo12(pGEc47{Delta}B) strain. The resulting enzymes exhibited higher rates of 1-butanol production from butane and maintained their preference for terminal hydroxylation. This in vivo evolution system could be useful for directed evolution of enzymes that function efficiently to hydroxylate small alkanes in engineered hosts

Functional characterization of genes involved in alkane oxidation by Pseudomonas aeruginosa

Most clinical isolates identified as Pseudomonas aeruginosa grow on long-chain n-alkanes, while environmental P. aeruginosa isolates often grow on medium- as well as long-chain n-alkanes. Heterologous expression showed that the two alkane hydroxylase homologs of P. aeruginosa PAO1 (AlkB1 and AlkB2) oxidize C12-C16 n-alkanes, while two rubredoxin (RubA1 and RubA2) and a rubredoxin reductase (RubB) homologs can replace their P. putida GPo1 counterparts in n-octane oxidation. The two long-chain alkane hydroxylase genes are present in all environmental and clinical isolates of P. aeruginosa strains tested in this stud

Biocatalysis. Biological systems for the production of chemicals

Biocatalysis harnesses the catalytic potential of enzymes to produce building blocks and end-products for the pharmaceutical and chemical industry. Located at the interface between fermentation processes and petrol-based chemistry, biotransformation processes broaden the toolbox for bioconversion of organic compounds to functionalized product

Expression of PHA polymerase genes of Pseudomonas putida in Escherichia coli and its effect on PHA formation

Poly-3-hydroxyalkanoates (PHAs) are synthesized by many bacteria as intracellular storage material. The final step in PHA biosynthesis is catalyzed by two PHA polymerases (phaC) in Pseudomonas putida. The expression of these two phaC genes (phaC1 and phaC2)was studied in Escherichia coli, either under control of the native promoter or under control of an external promoter. It was found that the two phaC genes are not expressed in E. coli without an external promoter. During heterologous expression of phaC from Plac on a high copy number plasmid, a rapid reduction of the number of colony forming units was observed, especially for phaC2. It appears that the plasmid instability was partially caused by high-level production of PHA polymerase. Subsequently, tightly regulated phaC2 expression systems on a low copy number vector were applied in E. coli. This resulted in PHA yields of over 20 of total cell dry weight, which was 2 fold higher than that obtained from the system where phaC2 is present on a high copy number vector. In addition, the PHA monomer composition differed when different gene expression systems or different phaC genes were applie

Abnormal Parietal Function in Conversion Paresis

The etiology of medically unexplained symptoms such as conversion disorder is poorly understood. This is partly because the interpretation of neuroimaging results in conversion paresis has been complicated by the use of different control groups, tasks and statistical comparisons. The present study includes these different aspects in a single data set. In our study we included both normal controls and feigners to control for conversion paresis. We studied both movement execution and imagery, and we contrasted both within-group and between-group activation. Moreover, to reveal hemisphere-specific effects that have not been reported before, we performed these analyses using both flipped and unflipped data. This approach resulted in the identification of abnormal parietal activation which was specific for conversion paresis patients. Patients also showed reduced activity in the prefrontal cortex, supramarginal gyrus and precuneus, including hemisphere-specific activation that is lateralized in the same hemisphere, regardless of right- or left-sided paresis. We propose that these regions are candidates for an interface between psychological mechanisms and disturbed higher-order motor control. Our study presents an integrative neurophysiological view of the mechanisms that contribute to the etiology of this puzzling psychological disorder, which can be further investigated with other types of conversion symptoms

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 40U/g polymerase, whereas in E. coli, the maximal specific polymerase activity was found in the early stationary growth phase (2U/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. col

Prevalence of alkane monooxygenase genes in Arctic and Antarctic hydrocarbon-contaminated and pristine soils

The prevalence of four alkane monooxygenase genotypes (Pseudomonas putida GPo1, Pp alkB; Rhodococcus sp. strain Q15, Rh alkB1 and Rh alkB2; and Acinetobacter sp. strain ADP-1, Ac alkM) in hydrocarbon-contaminated and pristine soils from the Arctic and Antarctica were determined by both culture-independent (PCR hybridization analyses) and culture-dependent (colony hybridization analyses) molecular methods, using oligonucleotide primers and DNA probes specific for each of the alk genotypes. PCR hybridization of total soil community DNA detected the rhodococcal alkB genotypes in most of the contaminated (Rh alkB1, 18/20 soils; Rh alkB2, 13/20) and many pristine soils (Rh alkB1, 9/10 soils; Rh alkB2, 7/10), while Pp alkB was generally detected in the contaminated soils (15/20) but less often in pristine soils (5/10). Ac alkM was rarely detected in the soils (1/30). The colony hybridization technique was used to determine the prevalence of each of the alk genes and determine their relative abundance in culturable cold-adapted (5°C) and mesophilic populations (37°C) from eight of the polar soils. The cold-adapted populations, in general, possessed relatively higher percentages of the Rh alkB genotypes (Rh alkB1, 1.9% (0.55); Rh alkB2, 2.47% (0.89)), followed by the Pp alkB (1.13% (0.50)), and then the Ac alkM (0.53% (0.36)). The Rh alkB1 genotype was clearly more prevalent in culturable cold-adapted bacteria (1.9% (0.55)) than in culturable mesophiles (0.41 (0.55)), suggesting that cold-adapted bacteria are the predominant organisms possessing this genotype. Overall, these results indicated that (i) Acinetobacter spp. are not predominant members of polar alkane degradative microbial communities, (ii) Pseudomonas spp. may become enriched in polar soils following contamination events, and (iii) Rhodococcus spp. may be the predominant alkane-degradative bacteria in both pristine and contaminated polar soil