Stepwise optimization of genetic RuBisCO-equipped Escherichia coli for low carbon-footprint protein and chemical production

Bio-mitigation of carbon dioxide has recently attracted more research focus because it is a sustainable and ecofriendly way of carbon conversion to a variety of indispensable chemicals. However, studies regarding the recombinant strain development toward simultaneous chemical production and CO2 assimilation in E. coli are limited and the reported studies are challenged with critically low efficiency. In this context, we first expressed ribulose-1,5-bisphosphate carboxylase/oxygenases (RuBisCOs) and phosphoribulokinases (PRKs) from the Calvin cycle to optimize the CO2 assimilation capability in E. coli with a value of -4.9 g-CO2 per g-DCW. To enhance the CO2 assimilation capability and broaden the applicability of RuBisCO-equipped E. coli, we conducted stepwise integration of the recombinant genes (RuBisCO and PRK) as well as introduced the CRISPRi system for redirecting the carbon flux to the RuBisCO pathway. The final strain, SSCI with CRISPRi targeted on zwf and pfkAB, enhanced the CO2 assimilation capability to -1.58 g-CO2 per g-DCW. Finally, RuBisCO-equipped E. coli was applied for simultaneous CO2 recycling and production of high-value molecules from various proteins, including 5-aminoleuvinic acid (ALA) by ALA synthetase, carbon dioxide sequestration via carbonic anhydrase (CA), and 1,5-diaminopentane (DAP) through lysine decarboxylase. This genomic RuBisCO-equipped E. coli neutralizer is denoted as a GREEN workhorse, with the great opportunity to be a low-carbon footprint microbial cell factory.

Automated summary

The study focuses on enhancing CO2 assimilation capability in E. coli by introducing RuBisCO and PRK genes along with the CRISPRi system. This led to improved efficiency in simultaneous CO2 recycling and production of high-value molecules. The genetically engineered RuBisCO-equipped E. coli has the potential to be a low-carbon microbial cell factory with significant environmental benefits.