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

Abstract

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 Escherichia coli 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 in vivo. Indoxyl acetate titer, while low, was improved 50%, from 43 μM to 67 μM, in the hydrolase knockout strain compared to wild-type E. coli. Unfortunately, low millimolar concentrations of indoxyl acetate proved to be toxic to the E. coli 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.

Eco‐Friendly Bio‐Based Solvents for the Acetylation of the Amino Group of Amino Acids

“…Moreover, it is generally considered a low-impact protective agent. [35] In the literature, several methods have been reported for the acetylation of amino groups. For amino acids, the most frequently applied procedure involves the use of acetic anhydride in methanol.…”

“…Among the available options, the acetyl group can be particularly convenient because it is small, offers a good protection against a variety of side reactions, and is readily cleavable under mild conditions. Moreover, it is generally considered a low‐impact protective agent [35] . In the literature, several methods have been reported for the acetylation of amino groups.…”

Structure-guided engineering of a flavin-containing monooxygenase for the efficient production of indirubin

“…Unfortunately, indirubin is only a minor byproduct of the reaction, and indigo, an isomer of indirubin, is usually the major product. Most of the previous studies mainly focused on indigo production because of the low yield and inefficiency of indirubin production (Latimer et al 2020 ). Attempts have been carried out to improve the production of indirubin by selecting and engineering new enzymes (Choi et al 2003 ) and metabolic pathways (Du et al 2018 ; Hu et al 2010 ).…”