title: Enhancement of protocatechuate decarboxylase activity for the effective production of muconate from lignin-related aromatic compounds.
abstract: The decarboxylation reaction of protocatechuate has been described as a bottleneck and a rate-limiting step in cis,cis-muconate (ccMA) bioproduction from renewable feedstocks such as sugar. Because sugars are already in high demand in the development of many bio-based products, our work focuses on improving protocatechuate decarboxylase (Pdc) activity and ccMA production in particular, from lignin-related aromatic compounds. We previously had transformed an Escherichia coli strain using aroY, which had been used as a protocatechuate decarboxylase encoding gene from Klebsiella pneumoniae subsp. pneumoniae A170-40, and inserted other required genes from Pseudomonas putida KT2440, to allow the production of ccMA from vanillin. This recombinant strain produced ccMA from vanillin, however the Pdc reaction step remained a bottleneck during incubation. In the current study, we identify a way to increase protocatechuate decarboxylase activity in E. coli through enzyme production involving both aroY and kpdB; the latter which encodes for the B subunit of 4-hydroxybenzoate decarboxylase. This permits expression of Pdc activity at a level approximately 14-fold greater than the strain with aroY only. The expression level of AroY increased, apparently as a function of the co-expression of AroY and KpdB. Our results also imply that ccMA may inhibit vanillate demethylation, a reaction step that is rate limiting for efficient ccMA production from lignin-related aromatic compounds, so even though ccMA production may be enhanced, other challenges to overcome vanilate demethylation inhibition still remain.
authors.forename: Tomonori
authors.initials: T
authors.lastname: Sonoki
authors.forename: Miyuki
authors.initials: M
authors.lastname: Morooka
authors.forename: Kimitoshi
authors.initials: K
authors.lastname: Sakamoto
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Otsuka
authors.forename: Masaya
authors.initials: M
authors.lastname: Nakamura
authors.forename: Jody
authors.initials: J
authors.lastname: Jellison
authors.forename: Barry
authors.initials: B
authors.lastname: Goodell
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001547:Benzaldehydes / Q000378:metabolism*; D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D062385:Hydroxybenzoates / Q000378:metabolism*; D007711:Klebsiella pneumoniae / Q000201:enzymology / Q000235:genetics; D008031:Lignin; D010957:Plasmids; D016958:Pseudomonas putida / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D013011:Sorbic Acid / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D001547:Benzaldehydes; D062385:Hydroxybenzoates; D011994:Recombinant Proteins; C009091:protocatechuic acid; C005939:muconic acid; D008031:Lignin; C100058:vanillin; D002262:Carboxy-Lyases; D013011:Sorbic Acid