Isolation of a Gene Responsible for the Oxidation of trans -Anethole to para -Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

Abstract

A plasmid, pTA163, in Escherichia coli contained an approximately 34-kb gene fragment from Pseudomonas putida JYR-1 that included the genes responsible for the metabolism of trans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyze trans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter in E. coli catalyzed oxidative cleavage of a propenyl group of trans-anethole to an aldehyde group, resulting in the production of para-anisaldehyde, and this gene was designated tao (trans-anethole oxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein of Agrobacterium vitis S4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water into p-anisaldehyde using 18 Olabeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase from Pseudomonas putida IE27 and Pseudomonas nitroreducens Jin1, TAO from P. putida JYR-1 catalyzed isoeugenol, O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts of E. coli (pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future. Bacterial metabolism as a means of producing value-added compounds from natural resources has been given much attention as an alternative method to replace conventional chemical syntheses (21,27). If natural resources can be continuously supplied as the starting materials, biotransformation employing bacterial systems may become practical from the points of view of sustainability and applicability. One of the major groups of natural compounds targeted for the production of value-added compounds includes the group of chemicals that occur in plant phenylpropanoid pathways, which are involved in the production of lignin, flavonoids, anthocyanins, etc. (5, 16-19). For example, isoeugenol, eugenol, and ferulic acid produced by the phenylpropanoid pathway have often been attempted as the starting materials to produce vanillin, one of the most extensively used aromatic flavor compounds (25-27, 32).trans-Anethole (p-methoxy propenylbenzene), the major component present in the essential oils of anise, fennel, and star anise, is also a type of phenylpropanoid compound formed by terpene synthesis in plants (10). trans-Anethole is used commercially as a flavor substance in baked goods, candy, ice cream, chewing gum, and alcoholic beverages (21). However, there have been few research reports regarding its metabolism (8,12,21,22,31). Indeed, to date, the only two bacterial strains isolated that are known to use trans-anethole as a sole carbon source are Arthrobacter sp. strain TA13 and Pseudomonas putida JYR-1 ...

Oxidation of a non-phenolic lignin model compound by two Irpex lacteus manganese peroxidases: evidence for implication of carboxylate and radicals

“…These ions could also be observed in the MS/MS analysis of Il MnP2-VA reaction products, confirming that Il MnP2 could indeed convert VA into veratraldehyde (Fig. 4) [27].

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Characterization of a Self-sufficient Trans-Anethole Oxygenase from Pseudomonas putida JYR-1

“…The catalytic kinetics of GST-TAO were determined by measuring the amount of p -anisaldehyde produced from the substrate trans -anethole [18]. The conversion of trans -anethole to p -anisaldehyde followed Mechaelis-Menton kinetics, with an affinity K m of 64.70±2.40 µM and a turnover number k cat of 0.49 s −1 (Table 3).…”

“…Compared to the extremely narrow substrate range of isoeugenol monooxygenases, Iem, from Pseudomonas nitroreducens Jin1 [20], and Iso from Pseudomonas putida IE27 [21], that only use isoeugenol as a substrate, TAO exhibited a relatively broad substrate range. TAO is likely to be NAD(P)H-dependent, even though there was no conserved NAD(P)H binding domain found from the deduced amino acid sequence [18]. Since the TAO from Pseudomonas putida JYR-1 displayed very low similarity to the deduced amino acid sequences of other enzymes in currently available databases, it was thought to be a novel enzyme, worthy of further characterization.…”

“…We previously reported [17], [18] the isolation of the tao gene from Pseudomonas putida JYR-1 encoding trans -anethole oxygenase (TAO) activity. The enzyme catalyzed the oxidation of trans -anethole, a type of phenylpropanoid compound formed via terpene biosynthesis in plants [19], to p -anisaldehyde.…”

“…The enzyme catalyzed the oxidation of trans -anethole, a type of phenylpropanoid compound formed via terpene biosynthesis in plants [19], to p -anisaldehyde. Interestingly, whole cell assays done with TAO heterologously expressed in E. coli showed that the enzyme also acted on isoeugenol, O -methyl isoeugenol, and isosafrole as substrates, all of which contain the propenyl functional group on the aromatic ring structure [18]. Compared to the extremely narrow substrate range of isoeugenol monooxygenases, Iem, from Pseudomonas nitroreducens Jin1 [20], and Iso from Pseudomonas putida IE27 [21], that only use isoeugenol as a substrate, TAO exhibited a relatively broad substrate range.…”