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.