Engineering formation of multiple recombinant Eut protein nanocompartments in E. coli

Compartmentalization of designed metabolic pathways within protein based nanocompartments has the potential to increase reaction efficiency in multi-step biosynthetic reactions. We previously demonstrated proof-of-concept of this aim by targeting a functional enzyme to single cellular protein nanocompartments, which were formed upon recombinant expression of the Salmonella enterica LT2 ethanolamine utilization bacterial microcompartment shell proteins EutS or EutSMNLK in Escherichia coli. To optimize this system, increasing overall encapsulated enzyme reaction efficiency, factor(s) required for the production of more than one nanocompartment per cell must be identified. In this work we report that the cupin domain protein EutQ is required for assembly of more than one nanocompartment per cell. Overexpression of EutQ results in multiple nanocompartment assembly in our recombinant system. EutQ specifically interacts with the shell protein EutM in vitro via electrostatic interactions with the putative cytosolic face of EutM. These findings lead to the theory that EutQ could facilitate multiple nanocompartment biogenesis by serving as an assembly hub for shell proteins. This work offers insights into the biogenesis of Eut bacterial microcompartments, and also provides an improved platform for the production of protein based nanocompartments for targeted encapsulation of enzyme pathways.