Chloroplast reconstitution of heterologous Rubisco and carboxysome enables photoautotrophic growth of Chlamydomonas reinhardtii.

The model green alga Chlamydomonas reinhardtii has a pyrenoid within chloroplast for photosynthetic CO2 fixation under limiting CO2 conditions and its chloroplast is an ideal chassis for engineering photosynthetic modules. In contrast, carboxysomes are bacterial microcompartments that encapsulate Rubisco for CO2 fixation. The aim of the present study was to determine if a bacterial carboxysome or its components can replace pyrenoid and function to allow Chlamydomonas growth powered by photosynthesis. We replaced the Chlamydomonas endogenous RbcL gene with cbbL and cbbS from the chemoautotrophic bacterium Halothiobacillus neapolitanus, resulting in proper assembly of the heterologous Rubisco with slightly lower enzymatic activity than the endogenous Rubisco. We next expressed 4 or 6 carboxysome-related genes and observed the formation of carboxysome-like structures in chloroplast. Photoautotrophic growth of transformants were enabled under high CO2 (∼5%) conditions, but not in ambient air (∼0.04% CO2). Our study serves as a demonstration on how carboxysomes can be partially reconstituted in eukaryotic algae, which have great uses in bioindustry. The single-cell nature of Chlamydomonas allows it to be used as a testbed for optimizing the sequences of the genes required for heterologous carboxysome formation and for future improvements in CO2-fixation efficiency and incorporation of diverse metabolic pathways in chloroplasts.