Biotransformation of Various Alkanes Using the<i>Escherichia coli</i>Expressing an Alkane Hydroxylase System from<i>Gordonia</i>sp. TF6

Fujii, Tadashi; Narikawa, Tatsuya; Takeda, Koji; Kato, Junichi

doi:10.1271/bbb.68.2171

Cited by 42 publications

(43 citation statements)

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“…Also present are two rubredoxin (Rd) genes (MpeB0603 and MpeB0602), whose products are 76 and 78% similar to rubredoxin 3 and 4, respectively, in Gordonia sp. strain TF6 (31). The Rd encoded by MpeB0603 is an AlkG1-type Rd, whereas that encoded by MpeB0602 is an AlkG2-type Rd, based on the CXXCG motif criterion described by van Beilen et al (80).…”

Section: Resultsmentioning

confidence: 99%

Whole-Genome Analysis of the Methyl tert -Butyl Ether-Degrading Beta-Proteobacterium Methylibium petroleiphilum PM1

et al. 2007

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Methylibium petroleiphilum PM1 is a methylotroph distinguished by its ability to completely metabolize the fuel oxygenate methyl tert-butyl ether (MTBE). Strain PM1 also degrades aromatic (benzene, toluene, and xylene) and straight-chain (C 5 to C 12 ) hydrocarbons present in petroleum products. Whole-genome analysis of PM1 revealed an ϳ4-Mb circular chromosome and an ϳ600-kb megaplasmid, containing 3,831 and 646 genes, respectively. Aromatic hydrocarbon and alkane degradation, metal resistance, and methylotrophy are encoded on the chromosome. The megaplasmid contains an unusual t-RNA island, numerous insertion sequences, and large repeated elements, including a 40-kb region also present on the chromosome and a 29-kb tandem repeat encoding phosphonate transport and cobalamin biosynthesis. The megaplasmid also codes for alkane degradation and was shown to play an essential role in MTBE degradation through plasmid-curing experiments. Discrepancies between the insertion sequence element distribution patterns, the distributions of best BLASTP hits among major phylogenetic groups, and the G؉C contents of the chromosome (69.2%) and plasmid (66%), together with comparative genome hybridization experiments, suggest that the plasmid was recently acquired and apparently carries the genetic information responsible for PM1's ability to degrade MTBE. Comparative genomic hybridization analysis with two PM1-like MTBE-degrading environmental isolates (ϳ99% identical 16S rRNA gene sequences) showed that the plasmid was highly conserved (ca. 99% identical), whereas the chromosomes were too diverse to conduct resequencing analysis. PM1's genome sequence provides a foundation for investigating MTBE biodegradation and exploring the genetic regulation of multiple biodegradation pathways in M. petroleiphilum and other MTBE-degrading beta-proteobacteria.Methylibium petroleiphilum strain PM1, which belongs to a newly described genus and species (57), is a motile bacterium belonging to the Comamonadaceae family of the Betaproteobacteria and is an important member of subsurface microbial communities in many gasoline-contaminated aquifers. Furthermore, PM1 is a methylotroph that can grow aerobically on the fuel oxygenate methyl tert-butyl ether (MTBE) and oxidize it completely to carbon dioxide (9, 34). MTBE is a suspected carcinogen that has contaminated drinking water wells throughout the United States due to the preponderance of underground leaking storage tanks, the widespread usage of MTBE, and its recalcitrance and mobility in groundwater. PM1 can also oxidize aromatic hydrocarbons (toluene, benzene, oxylene, and phenol) (20) and n-alkanes (C 5 to C 12 ) (57; K. Hristova, unpublished data) and has been used in two bioaugmentation field trials in gasoline-contaminated aquifers in California (67) and Montana (18,73). In contaminated sites amended with oxygen, in situ MTBE degradation was observed and corresponded to increases in native populations of Methylibium sp. (ϳ99% similarity to PM1, based on the 16S rRNA gene) (38,67,82). PM1-l...

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Section: Resultsmentioning

confidence: 99%

Whole-Genome Analysis of the Methyl tert -Butyl Ether-Degrading Beta-Proteobacterium Methylibium petroleiphilum PM1

et al. 2007

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“…strains Q15 and NRRL B-16531 (31) was obtained. Sequence analysis revealed six consecutive open reading frames (ORFs) ( Table 3) which were designated as encoding Orf1 (a conserved hypothetical protein), AlkB (alkane 1-monooxygenase), RubA3 (rubredoxin), RubA4 (rubredoxin), RubB (rubredoxin reductase), and AlkU (putative TetR-like regulator) according to the sequence homology with other known genes (6,31).…”

Section: Resultsmentioning

confidence: 99%

“…Moreover, genes encoding an AH system (alkane 1-monooxygenase, rubredoxins, and rubredoxin reductase) from Gordonia sp. strain TF6 were cloned, sequenced, and expressed in E. coli, where they were found to be the minimum components required to confer alkane hydroxylase activity in this strain on n-alkanes up to C 13 (6). Alkanes longer than C 16 support growth of many microorganisms, but the identity of enzymes involved in their oxidation is known for only a restricted number of isolates (27,30), among which is only one Gram-positive strain, belonging to the genus Geobacillus (LadA) (5).…”

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confidence: 99%

Involvement of an Alkane Hydroxylase System of Gordonia sp. Strain SoCg in Degradation of Solid n -Alkanes

Piccolo

Pasquale

Fodale

et al. 2011

Appl Environ Microbiol

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Enzymes involved in oxidation of long-chain n-alkanes are still not well known, especially those in Grampositive bacteria. This work describes the alkane degradation system of the n-alkane degrader actinobacterium Gordonia sp. strain SoCg, which is able to grow on n-alkanes from dodecane (C 12 ) to hexatriacontane (C 36 ) as the sole C source. SoCg harbors in its chromosome a single alk locus carrying six open reading frames (ORFs), which shows 78 to 79% identity with the alkane hydroxylase (AH)-encoding systems of other alkane-degrading actinobacteria. Quantitative reverse transcription-PCR showed that the genes encoding AlkB (alkane 1-monooxygenase), RubA3 (rubredoxin), RubA4 (rubredoxin), and RubB (rubredoxin reductase) were induced by both n-hexadecane and n-triacontane, which were chosen as representative long-chain liquid and solid n-alkane molecules, respectively. Biotransformation of n-hexadecane into the corresponding 1-hexadecanol was detected by solid-phase microextraction coupled with gas chromatography-mass spectrometry (SPME/GC-MS) analysis. The Gordonia SoCg alkB was heterologously expressed in Escherichia coli BL21 and in Streptomyces coelicolor M145, and both hosts acquired the ability to transform n-hexadecane into 1-hexadecanol, but the corresponding long-chain alcohol was never detected on n-triacontane. However, the recombinant S. coelicolor M145-AH, expressing the Gordonia alkB gene, was able to grow on n-triacontane as the sole C source. A SoCg alkB disruption mutant that is completely unable to grow on n-triacontane was obtained, demonstrating the role of an AlkB-type AH system in degradation of solid n-alkanes.

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“…Inspection of the Alcanivorax genomic context of ABO_0190 revealed a putative operon of four genes encoding caprolactone hydrolase (ABO_0191), cyclohexanone monooxygenase (ABO_0190), cyclohexanol dehydrogenase (ABO_0189), and metal-dependent hydrolase (ABO_0188) that are probably involved in the metabolism of cycloalkanes and convert cyclohexanol to 6-hydroxyhexanoic acid. We did not find a gene encoding cyclohexane monooxygenase in the A. borkumensis genome and therefore suspect that another enzyme mediates the initial attack of cyclic alkanes in Alcanivorax and that this enzyme may in fact be alkane hydroxylase, encoded by either alkB1 or alkB2, since Fujii et al (15) recently showed oxidation of cycloalkanes by the alkane hydroxylase system (comprising alkane 1-monooxygenase AlkB, rubredoxin AlkG, and rubredoxin AlkT) of Gordonia sp. TF6.…”

Section: Vol 188 2006 Insights Into Metabolic Adaptations In a Bormentioning

confidence: 87%

Proteomic Insights into Metabolic Adaptations inAlcanivorax borkumensisInduced by Alkane Utilization

et al. 2006

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Alcanivorax borkumensis is a ubiquitous marine petroleum oil-degrading bacterium with an unusual physiology specialized for alkane metabolism. This "hydrocarbonoclastic" bacterium degrades an exceptionally broad range of alkane hydrocarbons but few other substrates. The proteomic analysis presented here reveals metabolic features of the hydrocarbonoclastic lifestyle. Specifically, hexadecane-grown and pyruvate-grown cells differed in the expression of 97 cytoplasmic and membrane-associated proteins whose genes appeared to be components of 46 putative operon structures. Membrane proteins up-regulated in alkane-grown cells included three enzyme systems able to convert alkanes via terminal oxidation to fatty acids, namely, enzymes encoded by the well-known alkB1 gene cluster and two new alkane hydroxylating systems, a P450 cytochrome monooxygenase and a putative flavin-binding monooxygenase, and enzymes mediating ␤-oxidation of fatty acids. Cytoplasmic proteins up-regulated in hexadecane-grown cells reflect a central metabolism based on a fatty acid diet, namely, enzymes of the glyoxylate bypass and of the gluconeogenesis pathway, able to provide key metabolic intermediates, like phosphoenolpyruvate, from fatty acids. They also include enzymes for synthesis of riboflavin and of unsaturated fatty acids and cardiolipin, which presumably reflect membrane restructuring required for membranes to adapt to perturbations induced by the massive influx of alkane oxidation enzymes. Ancillary functions up-regulated included the lipoprotein releasing system (Lol), presumably associated with biosurfactant release, and polyhydroxyalkanoate synthesis enzymes associated with carbon storage under conditions of carbon surfeit. The existence of three different alkane-oxidizing systems is consistent with the broad range of oil hydrocarbons degraded by A. borkumensis and its ecological success in oil-contaminated marine habitats.Alcanivorax borkumensis is a key marine oil-degrading bacterium that can dramatically increase in numbers after an oil spill and become the most abundant microbe in oil-polluted waters (21,26,27,43). The list of sites where it has been isolated and shown to be involved in oil degradation grows with microbiological investigations of oil spills (9, 37; M. M. Yakimov, personal communication). The physiology of A. borkumensis is characterized by oligotrophy and a highly restricted growth substrate profile, namely, petroleum hydrocarbons plus a few organic acids, though the spectrum of hydrocarbons degraded is exceptionally broad. Its unusual metabolic features, presumed global importance in the natural biological removal of oil entering marine systems, and biotechnological potential for mitigation of the ecological damage caused by oil spills stimulated recent and current functional genomic studies of this organism. A key question concerning the ubiquity and competitive success of A. borkumensis in many marine locations is the genomic and biochemical basis of its physiological specialization and broad hydrocarbon su...

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Biotransformation of Various Alkanes Using theEscherichia coliExpressing an Alkane Hydroxylase System fromGordoniasp. TF6

Cited by 42 publications

References 15 publications

Whole-Genome Analysis of the Methyl tert -Butyl Ether-Degrading Beta-Proteobacterium Methylibium petroleiphilum PM1

Whole-Genome Analysis of the Methyl tert -Butyl Ether-Degrading Beta-Proteobacterium Methylibium petroleiphilum PM1

Involvement of an Alkane Hydroxylase System of Gordonia sp. Strain SoCg in Degradation of Solid n -Alkanes

Proteomic Insights into Metabolic Adaptations inAlcanivorax borkumensisInduced by Alkane Utilization

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