Characterization of the catabolic pathway for a phenylcoumaran-type lignin-derived biaryl in Sphingobium sp. strain SYK-6

Abstract

Sphingobium sp. strain SYK-6 is capable of degrading various lignin-derived biaryls. We determined the catabolic pathway of a phenylcoumaran-type compound, dehydrodiconiferyl alcohol (DCA) in SYK-6, and identified some of the DCA catabolism genes. In SYK-6 cells, the alcohol group of DCA was oxidized to the carboxyl group, first at the B-ring side chain and then at the A-ring side chain. The resultant metabolite was degraded to 5-formylferulate and vanillin through the decarboxylation and the Cα-Cβ cleavage of the A-ring side chain. Based on the DCA catabolic pathway, alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) genes are thought to be involved in the conversion of DCA into an aldehyde intermediate (DCA-L) and the conversion of DCA-L into a carboxylic acid intermediate (DCA-C), respectively. SLG_05620 and SLG_24930, which belong to quinohemoprotein ADH and aryl ADH, respectively, were isolated as the genes responsible for the oxidation of DCA. In addition to these genes, multiple genes similar to SLG_05620 and SLG_24930 were found to confer DCA oxidation activities on Escherichia coli cells. In order to identify the DCA-L dehydrogenase genes, the DCA-L oxidation activities of the SYK-6 gene products of putative twenty-one ALDH genes were examined. Significant activities were observed in the four ALDH gene products, including the SLG_27910 product, which showed the highest activity. The disruption of SLG_27910 caused a decreased conversion of DCA-L, suggesting that SLG_27910 plays an important role in the DCA-L oxidation. In conclusion, no specific gene seems to be solely responsible for the conversion of DCA and DCA-L, however, the multiple genes encoding quinohemoprotein ADH and aryl ADH genes, and four ALDH genes are probably involved in the conversion processes.

Membrane-Associated Glucose-Methanol-Choline Oxidoreductase Family Enzymes PhcC and PhcD Are Essential for Enantioselective Catabolism of Dehydrodiconiferyl Alcohol

“…We later found a similar catabolic route for DCA in SYK-6, and provided a detailed characterization ( Fig. 1) (33). In SYK-6 cells, the alcohol group of the B-ring side chain of DCA was first oxidized to the carboxyl group to generate 3-(2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-7-methoxy-2,3-dihydrobenzofuran-5-yl) acrylic acid (DCA-C) via an aldehyde derivative, 3-(2-(4-hydroxy-3-methoxyphenyl)-3-(hydroxymethyl)-7-methoxy-2,3-dihydrobenzofuran-5-yl) acrylaldehyde (DCA-L).…”

“…In addition, since the activities of extracts of the SYK-6 cells grown with or without DCA were almost the same, the genes encoding DCA-converting enzymes seem to be constitutively expressed. In our previous study, we suggested the involvement of multiple quinohemoprotein ADHs and aryl ADHs in the DCA oxidation (33). However, since quinohemoprotein ADHs are usually localized in the membrane or periplasm (42), aryl ADHs appear to play an important role in the oxidation of DCA.…”

“…Preparation of substrates. DCA, DCA-L, DCA-C, DCA-CC, and DCA-S were prepared as described previously (33). Guaiacylglycerol-␤-guaiacyl ether (GGE), coniferyl alcohol, and vanillyl alcohol were purchased from Tokyo Chemical Industry Co., Ltd., or Wako Pure Chemical Industries, Ltd. 2-(4-Hydroxy-3-methoxyphenyl)-5-(3-hydroxyprop-1-enyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylic acid (DCA-AC) was prepared by selective reduction of DCA-CC.…”

“…Then the resulting DCA-C was converted to 5-(2-carboxyvinyl)-2-(4-hydroxy-3-methoxyphenyl)-7-methoxy-2,3-dihydrobenzofuran-3-carboxylic acid (DCA-CC) by the twostep oxidations of the alcohol group of the A-ring side chain of DCA-C. DCA-CC was decarboxylated to produce 3-(4-hydroxy-3-(4-hydroxy-3-methoxystyryl)-5-methoxyphenyl) acrylic acid (DCA-S), and this lignostilbene was further cleaved between C␣ and C␤ of the A-ring side chain to form vanillin and 5-formylferulate. Our previous study also suggested that the multiple genes encoding quinohemoprotein alcohol dehydrogenases (quinohemoprotein ADH) and aryl ADH, as well as four aldehyde dehydrogenase (ALDH) genes, are involved in the oxidation of DCA and DCA-L, respectively (33). In addition, Kamoda et al reported that four isozymes of different lignostilbene ␣,␤-dioxygenases of TMY1009 had the ability to cleave the interphenyl ␣, ␤ double bond of DCA-S (34,35).…”

“…Nevertheless, no reports on the genes and enzymes involved in the conversions of DCA-C to DCA-S have been documented to date. Interestingly, quinohemoprotein ADH and aryl ADH, which were able to oxidize the alcohol group of the B-ring side chain of DCA, showed little or no activity in the oxidation of the alcohol group of the A-ring side chain of DCA-C (33). These facts suggested that other types of enzymes participate in the oxidation of DCA-C in SYK-6.…”