The Gene Cluster styABCDE of the Upper Styrene Degradation Pathway

The number and arrangement of genes within the styABCD(E) clusters of pseudomonads reflect the necessity and sequence of encoded enzymes, respectively,

Steam Piping Design

Fig. 3.3 Comparison of the organization of a the styABCDE-operon from Pseudomonas sp. Y2, b other (incomplete) styrene-catabolic gene clusters from pseudomonads, and c the genetic location of flavin monooxygenases with (# hypothetical) function as styrene monooxygenases. The upper pathway of styrene degradation by side-chain oxygenation is shown at the top and involved gene products are given. Identical types of filling of ORFs indicate similar function of encoded proteins

Fig. 3.3 Comparison of the organization of a the styABCDE-operon from Pseudomonas sp. Y2, b other (incomplete) styrene-catabolic gene clusters from pseudomonads, and c the genetic location of flavin monooxygenases with (# hypothetical) function as styrene monooxygenases. The upper pathway of styrene degradation by side-chain oxygenation is shown at the top and involved gene products are given. Identical types of filling of ORFs indicate similar function of encoded proteins to catalyze the conversion of styrene into phenylacetic acid (Fig. 3.3a, b). The genes styA and styB encode the two-component flavin-dependent styrene mono-oxygenase (SMO, or StyA/StyB) which initially converts styrene to styrene oxide at the expense of NADH. A single gene styC encodes a styrene oxide isomerase (SOI, or StyC) which catalyzes the intramolecular rearrangement of styrene oxide to phenylacetaldehyde. Finally, styD which is located directly downstream to styC encodes an NAD- or phenanzine methosulfate-dependent phenylacetaldehyde dehydrogenase (PAD or StyD) oxidizing phenylacetaldehyde into phenylacetic acid, the final product of the upper styrene pathway. An additional gene styE

(or porA) was found in few pseudomonads to be located directly downstream to the catabolic cluster styABCD and high similarities to several membrane-associated ATPase-dependent kinase proteins (Velasco et al. 1998; Mooney et al. 2006a) suggested a function as styrene transporter. This hypothesis was strengthened by the detection of (i) a co-expression of styE (P. putida CA-3) with styABCD, (ii) a styrene-dependent transcription, (iii) a membrane association, and (iv) an increased styrene degradation rate in the presence of additionally overexpressed styE copies (Mooney et al. 2006a). Furthermore, basic necessity of the transporter was shown by a styE-negative mutant of strain CA-3 which lost its ability to grow on styrene. However, Nikodinovic-Runic and coworkers detected only minor levels of StyE from a styrene-grown pseudomonad under both non-limiting and nitrogen-limiting conditions (Nikodinovic-Runic et al. 2009). These findings implicate that both, membrane diffusion as well as active styrene transport, are important for the uptake of styrene. In addition, it cannot be excluded that other transporters support active styrene uptake.

It is supposed that the upper pathway of styrene degradation may have evolved in a different way as the lower route necessary for phenylacetic acid conversion to TCA cycle intermediates (Ferrandez et al. 1998; Olivera et al. 1998; Alonso et al. 2003a; Di Gennaro et al. 2007).

Considering the low number of identified styrene-catabolic gene clusters and their apparently restricted occurrence in pseudomonads, styrene degradation by side-chain oxygenation seems to be not widely distributed in nature. This assumption is currently strengthened by available genome data which did not allow the identification of styABCD-like gene clusters from other bacterial phyla. However, taking into account on one hand the widespread ability of styrene utilization (Table 3.2), and on the other hand the preferred isolation of pseudomonads during classical enrichment procedures, current knowledge probably does not reflect the true distribution. In fact, single enzymatic activities or genes with hypothetical function in upper styrene degradation could be found in several other organisms than pseudomonads including gram-positive bacteria.

Starting with the initial flavin-dependent styrene monooxygenase StyA/StyB, the distribution frequency among bacteria is supposed to be generally low (based on representatives per genome, van Berkel et al. 2006). The only gene products showing significant homology to StyA/StyB from pseudomonads and activity on styrene or analogous compounds are found in two metagenoms (Guan et al. 2007; van Hellemond et al. 2007), as well as in the actinobacterium Rhodococcus opacus 1CP (Tischler et al. 2009, 2010) (Fig. 3.3c). In silico-screening for further representatives yielded several homologous proteins with similarities either to SMOs from pseudomonads or to that one from Rhodococcus opacus strain 1CP which was exemplarily shown by van Hellemond et al. (2007) and Tischler et al. (2009) (Fig. 3.3c). Interestingly, only in few Actinobacteria, a novel type of styrene monooxygenase was found so far which will be discussed later.

Biochemical evidence for styrene oxide isomerases was provided for several bacteria like Xanthobacter sp. 124X (Hartmans et al. 1989), Corynebacterium spp. (Itoh et al. 1996, 1997a), Rhodococcus opacus 1CP (Tischler et al. 2009),

Rhodococcus sp. S5, and others (Hartmans et al. 1990). However, none of the corresponding genes has been described so far and homology search of StyC towards available databases did not indicate significant similarities to gene products others than StyC-homologs from pseudomonads.

It should be mentioned that in Rhodococcus opacus 1CP, no styC-homologous gene could be found in direct neighborhood to the styrene monooxygenase genes styA1/styA2B (Fig. 3.3c), indicating that the genetic organization of styrene catabolism is different to that in pseudomonads (Tischler et al. 2009).

Phenylacetaldehyde dehydrogenases should be much more common in bacteria, since their substrate phenylacetaldehyde originates from different catabolic pathways apart from styrene degradation. Precursors are phenylpyruvic acid, 2-phenylethylamine, 2-phenylethanol, and phenylmalonic semialdehyde (Ferran-dez et al. 1997; Long et al. 1997). This assumption is strengthened by homology search of styD genes and gene products to the non-redundant genome database which reveals many high-score hits. However, only a few reports about functionally characterized phenylacetaldehyde dehydrogenases are available so far.

Continue reading here: The Regulatory System of the Sty Operon

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