Carboxysomes Research Articles

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.

Molecular simulations unravel the molecular principles that mediate selective permeability of carboxysome shell protein

Bacterial microcompartments (BMCs) are nanoscale proteinaceous organelles that encapsulate enzymes from the cytoplasm using an icosahedral protein shell that resembles viral capsids. Of particular interest are the carboxysomes (CBs), which sequester the CO2-fixing enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) to enhance carbon assimilation. The carboxysome shell serves as a semi-permeable barrier for passage of metabolites in and out of the carboxysome to enhance CO2 fixation. How the protein shell directs influx and efflux of molecules in an effective manner has remained elusive. Here we use molecular dynamics and umbrella sampling calculations to determine the free-energy profiles of the metabolic substrates, bicarbonate, CO2 and ribulose bisphosphate and the product 3-phosphoglycerate associated with their transition through the major carboxysome shell protein CcmK2. We elucidate the electrostatic charge-based permeability and key amino acid residues of CcmK2 functioning in mediating molecular transit through the central pore. Conformational changes of the loops forming the central pore may also be required for transit of specific metabolites. The importance of these in-silico findings is validated experimentally by site-directed mutagenesis of the key CcmK2 residue Serine 39. This study provides insight into the mechanism that mediates molecular transport through the shells of carboxysomes, applicable to other BMCs. It also offers a predictive approach to investigate and manipulate the shell permeability, with the intent of engineering BMC-based metabolic modules for new functions in synthetic biology.

Carboxysomes in a Row

Carboxysomes in a Row