Isolation and Functional Analysis of Cytochrome P450 CYP153A Genes from Various Environments

Kubota, Mitsutoshi; Nodate, Miho; Yasumoto-Hirose, Mina; Uchiyama, Taku; Kagami, Osamu; Shizuri, Yoshikazu; Misawa, Norihiko

doi:10.1271/bbb.69.2421

Cited by 99 publications

(79 citation statements)

References 28 publications

(23 reference statements)

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“…Metagenomic analyses have showed that only a few enzymes which degrade aromatic hydrocarbons are produced in most microorganisms in natural environments 13,27) . Therefore, a microbial consortium containing a number of microorganisms which synthesize the degradative enzymes for different parts of the decomposition pathway is considered to be well suited to the degradation of aromatic hydrocarbons.…”

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confidence: 99%

Change in the Predominant Bacteria in a Microbial Consortium Cultured on Media Containing Aromatic and Saturated Hydrocarbons as the Sole Carbon Source

2007

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A No.22 consortium was isolated from contaminated soils at oilfields by repeated transfer on a mineral salts medium with the aromatic hydrocarbon fraction from crude petroleum. The consortium was able to degrade aromatic hydrocarbons at high rates. Fourteen major DNA bands were detected in the consortium by PCR-DGGE. Three strains were capable of forming colonies on agarose plates containing mineral salts and aromatic hydrocarbons, and identified as Pandoraea sp. Y1, Hyphomicrobium facile Y3, and Burkholderia multivorans Y4 by 16S rRNA gene sequencing. The consortia of three cultures; an aromatic subculture, a saturated culture transferred from the subculture, and an aromatic obtained culture from the transferred culture, were compared using PCR-DGGE, a clone library, and quantitative real-time PCR. Burkholderia species containing strain Y4 became the dominant species in the saturated culture whereas its abundance decreased in the aromatic culture. Pandoraea sp. Y1, which was of low abundance in the saturated culture, and an unisolated bacterium, Brachymonas sp. F, in contrast, increased in the aromatic culture. The aromatic hydrocarbons were degraded by Pandoraea sp. Y1 and B. multivorans Y4, whereas the saturates were only degraded by B. multivorans Y4. The results indicate that the No.22 consortium adapts well to different carbon sources through a change in its predominant species. This study provides some basic information regarding bioremediation using the consortium.

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confidence: 99%

Change in the Predominant Bacteria in a Microbial Consortium Cultured on Media Containing Aromatic and Saturated Hydrocarbons as the Sole Carbon Source

2007

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“…Complementation of P. putida GPo12(pGEc47⌬B) with various CYP153 wild-type enzymes resulted in growth on alkanes ranging from pentane to decane (31), but never on butane (unpublished results). And, until now, biotransformations using CYP153 enzymes have only demonstrated activity for hexane and longer alkanes (9,10,14). Here we demonstrated that CYP153A6 wild-type and CYP153A6-BMO1 act on butane and even propane, but only the BMO1 variant complemented growth of Pcyp2 on butane.…”

Section: Discussionmentioning

confidence: 66%

“…CYP153 enzymes are soluble, and accurate determination of functional expression and concentration is easier due to observable CO binding. Furthermore, CYP153 proteins have been shown to function in wholecell bioconversions as single-component, self-sufficient fusion proteins with the P450RhF reductase domain (14,21) as well as with the CYP102A reductase domain (unpublished results). Further directed evolution of terminal alkane hydroxylases like …”

Section: Discussionmentioning

confidence: 94%

“…These soluble class II-type three-component P450 enzymes and the AlkB enzymes are the main actors in medium-chain-length alkane hydroxylation by the cultivated bacteria analyzed to date (31). CYP153 monooxygenases have been the subject of biochemical studies (9,16,19), and their substrate range has been explored (10,14). Known substrates include C 5 to C 11 alkanes.…”

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confidence: 99%

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

Koch

Chen

Beilen

et al. 2009

Appl Environ Microbiol

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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 mediumchain-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⌬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.Microbial utilization and degradation of alkanes was discovered almost a century ago (27). Since then, several enzyme families capable of hydroxylating alkanes to alkanols, the first step in alkane degradation, have been identified and categorized based on their preferred substrates (30). The soluble and particulate methane monooxygenases (sMMO and pMMO) and the related propane monooxygenase and butane monooxygenase (BMO) are specialized on gaseous small-chain alkanes (C 1 to C 4 ), while medium-chain (C 5 to C 16 ) alkane hydroxylation seems to be the domain of the CYP153 and AlkB enzyme families.Conversion of C 1 to C 4 alkanes to alkanols is of particular interest for producing liquid fuels or chemical precursors from natural gas. The MMO-like enzymes that catalyze this reaction in nature, however, exhibit limited stability or poor heterologous expression (30) and have not been suitable for use in a recombinant host that can be engineered to optimize substrate or cofactor delivery. Alkane monooxygenases often cometabolize a wider range of alkanes than those which support growth (12). We wished to determine whether it is possible to engineer a medium-chain alkane monooxygenase to hydroxylate small alkanes, thereby circumventing difficulties associated with en-

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“…6). Die Alkanhydroxylasen der CYP153-Familie stehen im Zentrum mehrerer Studien, deren Ziel die Charakterisierung von existierenden oder neuen Mitglieder dieser Familie ist [31,[71][72][73][74] (Tabelle 1, Nr. 7), um ihre Substratbandbreite zu ändern [75] oder ihre Effizienz für In-vitro-Anwendungen zu verbessern.…”

Section: P450-enzymeunclassified

Katalytische, milde und selektive Oxyfunktionalisierung von linearen Alkanen: aktuelle Herausforderungen

2012

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Es besteht eine große Nachfrage nach selektiven Katalysatoren für die nachhaltige Oxidation von Alkanen, denn die in der Umwelt sehr häufig vorkommenden Alkane kçnnen dadurch in hçherwertige Verbindungen, z.B. Chemikalien oder synthetische Brennstoffe, umgewandelt werden. Für diese – kinetisch gesehen – schwierige Reaktion wurde in den letzten Jahrzehnten eine Vielzahl von chemischen und biologischen Katalysatoren entwickelt, was einen Uberblick über das Gebiet erschwert. Dieser Kurzaufsatz gibt zunächst eine Definitiondes idealen Katalysators für die Alkan-Oxyfunktionalisierung und behandelt dann die heute verfügbaren Katalysatoren, die einem solchen idealen Katalysator am nächsten kommen

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Isolation and Functional Analysis of Cytochrome P450 CYP153A Genes from Various Environments

Cited by 99 publications

References 28 publications

Change in the Predominant Bacteria in a Microbial Consortium Cultured on Media Containing Aromatic and Saturated Hydrocarbons as the Sole Carbon Source

Change in the Predominant Bacteria in a Microbial Consortium Cultured on Media Containing Aromatic and Saturated Hydrocarbons as the Sole Carbon Source

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

Katalytische, milde und selektive Oxyfunktionalisierung von linearen Alkanen: aktuelle Herausforderungen

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