Auxiliary Module Promotes the Synthesis of Carboxysomes in E. coli to Achieve High-Efficiency CO2 Assimilation.

Carboxysomes (CBs) are protein organelles in cyanobacteria, and they play a central role in assimilation of CO2. Heterologous synthesis of CBs in E. coli provides an opportunity for CO2-organic compound conversion under controlled conditions but remains challenging; specifically, the CO2 assimilation efficiency is insufficient. In this study, an auxiliary module was designed to assist self-assembly of CBs derived from a model species cyanobacteria Prochlorococcus marinus (P. marinus) MED4 for synthesizing in E. coli. The results indicated that the structural integrity of synthetic CBs is improved through the transmission electron microscope images and that the CBs have highly efficient CO2-concentrating ability as revealed by enzyme kinetic analysis. Furthermore, the bacterial growth curve and 13C-metabolic flux analysis not only consolidated the fact of CO2 assimilation by synthetic CBs in E. coli but also proved that the engineered strain could efficiently convert external CO2 to some metabolic intermediates (acetyl-CoA, malate, fumarate, tyrosine, etc.) of the central metabolic pathway. The synthesis of CBs of P. marinus MED4 in E. coli provides prospects for understanding their CO2 assimilation mechanism and realizing their modular application in synthetic biology.