Biocatalytic synthesis of non-standard amino acids by a decarboxylative aldol reaction

Enantioselective C-C bond-forming reactions are underdeveloped in the biocatalysis toolbox. Now, engineering an efficient and promiscuous decarboxylative aldolase enzyme provides a solution to facilitate the convenient synthesis of non-standard gamma-hydroxy amino acids from simple building blocks. Enzymes are renowned for their catalytic efficiency and selectivity. Despite the wealth of carbon-carbon bond-forming transformations in traditional organic chemistry and nature, relatively few C-C bond-forming enzymes have found their way into the biocatalysis toolbox. Here we show that the enzyme UstD performs a highly selective decarboxylative aldol addition with diverse aldehyde substrates to make non-standard gamma-hydroxy amino acids. We increased the activity of UstD through three rounds of classic directed evolution and an additional round of computationally guided engineering. The enzyme that emerged, UstD(v2.0), is efficient in a whole-cell biocatalysis format. The products are highly desirable, functionally rich bioactive gamma-hydroxy amino acids that we demonstrate can be prepared stereoselectively on the gram scale. The X-ray crystal structure of UstD(v2.0) at 2.25 angstrom reveals the active site and provides a foundation for probing the UstD mechanism.

Automated summary

Enantioselective C-C bond-forming reactions are underdeveloped in biocatalysis. This study presents an engineered decarboxylative aldolase enzyme that facilitates the synthesis of non-standard gamma-hydroxy amino acids. The enzyme shows improved activity through directed evolution and engineering, and demonstrates high efficiency in whole-cell biocatalysis reactions.