Propane and n -Butane Oxidation by Pseudomonas putida GPo1

Abstract

Propane and n-butane inhibit methyl tertiary butyl ether oxidation by n-alkane-grown Pseudomonas putida GPo1. Here we demonstrate that these gases are oxidized by this strain and support cell growth. Both gases induced alkane hydroxylase activity and appear to be oxidized by the same enzyme system used for the oxidation of n-octane.The n-alkane-oxidizing activity of Pseudomonas putida GPo1 is a model for the bacterial oxidation of n-alkanes other than methane (11). In this strain, n-alkane oxidation is initiated by alkane hydroxylase and the n-alkane growth substrate range of the bacterium (C 5 to C 13 ) matches the n-alkane substrate range of the native hydroxylase (10-12). We recently demonstrated that alkane hydroxylase in strain GPo1 oxidizes the gasoline oxygenate MTBE to tertiary butyl alcohol (TBA) (9). Propane and n-butane inhibited this activity, and these effects appeared to be due to a competitive interaction between hydroxylase substrates. However, no evidence for oxidation of these gases was presented, and neither gas is currently recognized as an alkane hydroxylase substrate.In this study we first examined whether strain GPo1 oxidized gaseous n-alkanes (C 1 to C 4 ) during growth on a model nalkane, n-octane. No consumption of either methane or ethane occurred during growth on n-octane, while both propane (ϳ20%) and n-butane (ϳ60%) were consumed under the same conditions (Fig. 1). In abiotic incubations, Յ20% of noctane ( Fig. 1) and Յ3% of gaseous n-alkanes were lost over the same time period (not shown). In a separate experiment, we determined the final culture densities (optical densities at 600 nm [OD 600 ]) and protein contents of cultures grown on C-limiting n-octane with gaseous n-alkanes. After exhaustion of n-octane (5 days), the average culture density and protein concentration for three separate cultures were 0.22 and 46 g/ml, respectively, for cultures grown on n-octane alone. No increase in culture density was observed with methane or ethane, while the average (n ϭ 3) final culture densities and protein concentrations for cultures containing propane were 0.43 and 62 g/ml or for cultures containing n-butane were 1.1 and 148 g/ml, respectively.Propane and n-butane were also examined as potential independent n-alkane growth substrates. Cultures were established with propane, n-butane, n-pentane, or n-octane added individually as sole substrates. The time courses of n-pentane and n-octane consumption were almost identical, and changes in culture density closely reflected n-alkane consumption (Fig. 2). In contrast, no detectable n-alkane consumption or increases in culture density were initially observed in n-butanecontaining cultures. However, after ϳ4 days, n-butane consumption was initiated and accompanied by a corresponding increase in culture density. No discernible effect on this lag phase or final culture density was observed when cultures were supplemented with fourfold-higher (ϳ0.7% [vol/vol] gas phase) concentrations of the main hydrocarbon contaminant (isobutane) present in our ...

Kinetic characterization of the soluble butane monooxygenase from Thauera butanivorans, formerly ‘Pseudomonas butanovora’

“…The alkB hydroxylase from Pseudomonas putida GPo1 is capable of oxidizing large (.C 12 ) linear alkanes and substituted cyclic alkanes at similar rates (van Beilen et al, 1994), while also oxidizing the smaller growth substrates propane and butane with high affinity (K s 66 and 13 mM, respectively) (Johnson & Hyman, 2006). The alkane hydroxylase from propaneutilizing Mycobacterium vaccae JOB5 has also recently been characterized as an alkB hydroxylase (Lopes Ferreira et al, 2007), and has a low reported K s for propane of 3.3-4.4 mM but high K s values for branched substrates tertbutyl alcohol and methyl tert-butyl ether (1.36 and 1.18 mM, respectively) .…”

Monooxygenases involved in the n-alkanes metabolism by Rhodococcus sp. BCP1: molecular characterization and expression of alkB gene

“…Mycobacterium austroafricanum can grow on C 2 -C 16 and the alkB gene expression, involved in tert-butyl alcohol (TBA) co-metabolism, was induced not only after growth on n-hexane and n-hexadecane but also after growth on propane ( [67]. The involvement of AlkB-type hydroxylase in gaseous alkanes metabolism has been also reported in Pseudomonas putida GPo1 [165]. These new discoveries about AlkB activity open new frontiers of applications for this family members and will allow the continuation of AlkB hydroxylases as the central focus of alkane oxidation research.…”

“…Pseudomonas putida GPo1 grows optimally on C 5 -C 10 alkanes, but can use C 3 -C 4 and C 11 -C 13 alkanes as well, although growth is much slower and shows a long lag time [154,165] When cells are grown in the LB complete medium, the catabolite repression of the alkane-degradation genes depends on the additive effects of two global regulation systems.…”

“…The AlkG of P. putida GPo1 is unusual because it contains two rubredoxin domains, AlkG1 and AlkG2, connected by a linker, while rubredoxins from other microorganisms have only one of these domains ( Fig. 1.11 P. putida GPo1 is able to oxidize linear alkanes ranging from n-pentane to n-dodecane by virtue of the AlkB hydroxylase system [170] and can oxidize also butane and propane, even though the degradation rate is much slower [165]. In addition to the hydroxylation of aliphatic and alicyclic, the AlkB system has been shown to catalyze: oxidation of terminal alcohols to the corresponding aldehydes; demethylation of branched methyl ethers; sulfoxidation of thioethers, and epoxidation of terminal olefins [171][172][173].…”

“…In the absence of alkanes, AlkS is expressed from promoter PalkS1 and since AlkS acts as a repressor of its own promoter, it allows for low expression levels. In the presence of C 5 -C 10 alkanes, AlkS activates expression of its own gene from PalkS2, in order to achieve AlkS levels that are high enough to activate the expression of the alkBFGHJKL operon from the PalkB promoter [166,216,217,223,224].Pseudomonas putida GPo1 grows optimally on C 5 -C 10 alkanes, but can use C 3 -C 4 and C 11 -C 13 alkanes as well, although growth is much slower and shows a long lag time [154,165] When cells are grown in the LB complete medium, the catabolite repression of the alkane-degradation genes depends on the additive effects of two global regulation systems.One of them relies on the global regulatory protein Crc [218], while the other one apparently receives information from the cytochrome o ubiquinol oxidase (Cyo), a component of the electron transport chain [227,228]. However, in cells grown in a minimal salts medium containing succinate as the carbon source, the role of Crc is minor and the inhibitory effect derives mainly from the Cyo terminal oxidase [218,227].…”