Identification of a di-glucose conjugate of 4-hydroxybenzoic acid in bamboo cells expressing bacterial 4-hydroxycinnamoyl-CoA hydratase/lyase.

Rational metabolic-flow switching is an effective strategy that we proposed for producing exogenous high-value natural products using transformed plant cells. In an earlier proof-of-concept study, we generated bamboo (; Pn) cells expressing the 4-hydroxycinnamoyl-CoA hydratase/lyase gene of KT2440 (). The encoded enzyme catalyzes the formation of 4-hydroxybenzaldehyde and vanillin from -coumaroyl-CoA and feruloyl-CoA, respectively. The PpHCHL-transformed Pn cells accumulated mono-glucose conjugates (glucoside and glucose ester) of 4-hydroxybenzoic acid and vanillic acid, indicating that the products (aldehydes) of the PpHCHL-catalyzed reaction were oxidized by endogenous enzyme(s) in Pn cells. In this study, we re-examined the extracts of PpHCHL-transformed Pn cells to screen for additional 4-hydroxybenzoic acid derivatives. An unidentified compound was detected exclusively in the PpHCHL-transformed Pn cells. This compound was purified via column chromatography and then identified as a di-glucose conjugate of 4-hydroxybenzoic acid (i.e., β-D-glucopyranosyl 4--β-D-glucopyranosylbenzoate), implying that some of the mono-glucose conjugates of 4-hydroxybenzoic acid were converted to the di-glucose conjugate by endogenous enzyme(s) in Pn cells. The maximum production titer of this di-glucose conjugate in the suspension-cultured cells was 0.38 g l, which was the second highest titer among the four glucose conjugates produced by the PpHCHL-transformed Pn cells. The study findings further support the utility of PpHCHL-transformed Pn cells for the bioproduction of 4-hydroxybenzoic acid and its derivatives.