Exploration of Acetylation as a Base-Labile Protecting Group in for an Indigo Precursor.

Biochemical protecting groups are observed in natural metabolic pathways to control reactivity and properties of chemical intermediates; similarly, they hold promise as a tool for metabolic engineers to achieve the same goals. Protecting groups come with costs: lower yields from carbon, metabolic load to the production host, deprotection catalyst costs and kinetics limitations, and wastewater treatment of the group. Compared to glycosyl biochemical protection, such as glucosyl groups, acetylation can mitigate each of these costs. As an example application where these benefits could be valuable, we explored acetylation protection of indoxyl, the reactive precursor to the clothing dye, indigo. First, we demonstrated denim dyeing with chemically sourced indoxyl acetate by deprotection with base, showing results comparable to industry-standard denim dyeing. Second, we modified an production host for improved indoxyl acetate stability by the knockout of 14 endogenous hydrolases. Cumulatively, these knockouts yielded a 67% reduction in the indoxyl acetate hydrolysis rate from 0.22 mmol/g DCW/h to 0.07 mmol/g DCW/h. To biosynthesize indoxyl acetate, we identified three promiscuous acetyltransferases which acetylate indoxyl . Indoxyl acetate titer, while low, was improved 50%, from 43 μM to 67 μM, in the hydrolase knockout strain compared to wild-type . Unfortunately, low millimolar concentrations of indoxyl acetate proved to be toxic to the production host; however, the principle of acetylation as a readily cleavable and low impact biochemical protecting group and the engineered hydrolase knockout production host should prove useful for other metabolic products.