TY - JOUR
T1 - Discovery of novel enzyme genes involved in the conversion of an arylglycerol-β-aryl ether metabolite and their use in generating a metabolic pathway for lignin valorization
AU - Higuchi, Yudai
AU - Kato, Ryo
AU - Tsubota, Koichiro
AU - Kamimura, Naofumi
AU - Westwood, Nicholas J.
AU - Masai, Eiji
PY - 2019/9
Y1 - 2019/9
N2 - Microbial conversions known as “biological funneling” have attracted
attention for their ability to upgrade heterogeneous mixtures of
low-molecular-weight aromatic compounds obtained by chemical lignin
depolymerization. β-hydroxypropiovanillone (HPV) and its analogs can be
obtained by chemoselective catalytic oxidation of lignin using
2,3-dichloro-5,6-dicyano-1,4-benzoquinone/tert-butyl nitrite/O2, followed by cleavage of arylglycerol-β-aryl ether with zinc. Sphingobium
sp. strain SYK-6 can degrade HPV generated by the catabolism of
arylglycerol-β-aryl ether through 2-pyrone-4,6-dicarboxylate (PDC), a
promising platform chemical. Therefore, production of PDC from HPV can
be achieved using the HPV catabolic pathway. However, the pathway and
genes involved in the catabolism of vanilloyl acetic acid (VAA)
generated during HPV catabolism have not been investigated. In the
present study, we isolated SLG_24960 (vceA), which encodes an
enzyme that converts VAA into a coenzyme A (CoA) derivative of vanillate
(vanilloyl-CoA) from SYK-6, by shotgun cloning. The analysis of a vceA mutant indicated that this gene is not required for VAA conversion in vivo, but it encodes a major enzyme catalyzing CoA-dependent VAA conversion in vitro. We also identified SLG_12450 (vceB), whose product can convert vanilloyl-CoA to vanillate. Enzyme genes besides vceA and vceB, which are necessary for the conversions of HPV to VAA and of vanillate to PDC, were introduced and expressed in Pseudomonas putida.
The resulting engineered strain completely converted 1 mM HPV into PDC
after 24 h. Our results suggest that the enzyme genes that are not
required for the catabolic pathway in microorganisms but can be used for
the conversion of target substrates are buried in microbial genomes.
These genes are, thus, useful for designing metabolic pathways to
produce value-added metabolites.
AB - Microbial conversions known as “biological funneling” have attracted
attention for their ability to upgrade heterogeneous mixtures of
low-molecular-weight aromatic compounds obtained by chemical lignin
depolymerization. β-hydroxypropiovanillone (HPV) and its analogs can be
obtained by chemoselective catalytic oxidation of lignin using
2,3-dichloro-5,6-dicyano-1,4-benzoquinone/tert-butyl nitrite/O2, followed by cleavage of arylglycerol-β-aryl ether with zinc. Sphingobium
sp. strain SYK-6 can degrade HPV generated by the catabolism of
arylglycerol-β-aryl ether through 2-pyrone-4,6-dicarboxylate (PDC), a
promising platform chemical. Therefore, production of PDC from HPV can
be achieved using the HPV catabolic pathway. However, the pathway and
genes involved in the catabolism of vanilloyl acetic acid (VAA)
generated during HPV catabolism have not been investigated. In the
present study, we isolated SLG_24960 (vceA), which encodes an
enzyme that converts VAA into a coenzyme A (CoA) derivative of vanillate
(vanilloyl-CoA) from SYK-6, by shotgun cloning. The analysis of a vceA mutant indicated that this gene is not required for VAA conversion in vivo, but it encodes a major enzyme catalyzing CoA-dependent VAA conversion in vitro. We also identified SLG_12450 (vceB), whose product can convert vanilloyl-CoA to vanillate. Enzyme genes besides vceA and vceB, which are necessary for the conversions of HPV to VAA and of vanillate to PDC, were introduced and expressed in Pseudomonas putida.
The resulting engineered strain completely converted 1 mM HPV into PDC
after 24 h. Our results suggest that the enzyme genes that are not
required for the catabolic pathway in microorganisms but can be used for
the conversion of target substrates are buried in microbial genomes.
These genes are, thus, useful for designing metabolic pathways to
produce value-added metabolites.
KW - Sphingobium sp. SYK-6
KW - Lignin
KW - β-aryl ether
KW - 2-Pyrone-4,6-dicarboxylate
KW - β-keto acid cleavage enzyme
U2 - 10.1016/j.ymben.2019.08.002
DO - 10.1016/j.ymben.2019.08.002
M3 - Article
SN - 1096-7176
VL - 55
SP - 258
EP - 267
JO - Metabolic Engineering
JF - Metabolic Engineering
ER -