Carbon fixation lies in the heart of sustainability for alleviating global climate change and supporting lives on Earth. The ever-increasing atmospheric concentration of carbon dioxide (CO2) keeps reaching new alarming levels [1], further jeopardizing the global climate, while the growing human population just surpassed 8 billion [2], requesting more food and chemicals. These challenges stimulate biological innovations to enhance carbon fixation. Synthetic biology and metabolic engineering are enabling new biological designs for CO2 fixation and sustainable bioproduction 3, 4. Given these advances, autotrophic microorganisms, such as cyanobacteria, acetogens, and methanogens, have been reprogrammed to assimilate CO2 for next-generation biorefinery 5, 6, 7. However, autotrophs sometimes suffer from slow growth rates, limited product yields, and inefficient methodologies for genetic manipulation, which are often excelled by heterotrophic organisms. Thus, a rational alternative is the introduction of CO2 fixation pathways into heterotrophs for efficient carbon fixation, elevated carbon utilization efficiency, and a broader spectrum of bioproducts with higher titer and yield.

Three necessities are required to build a synthetic carbon fixation pathway in heterotrophs: (1) installing or awakening a CO2 fixation route, (2) supplying adequate energy and reducing power, and (3) optimizing the endogenous metabolism to accommodate carbon fixation. Hitherto, synthetic carbon fixation pathways have been successfully introduced into heterotrophs, including Escherichia coli 8••, 9••, 10••, Saccharomyces cerevisiae 11, 12, and Pichia pastoris [13••]. The recycled CO2 can be used as additional carbon sources for cell growth or bioproduction and as electron sinks to balance the redox cofactors. Notably, CO2 fixation can also be combined with the utilization of C1 compounds, presenting a sustainable scheme that combines green biorefinery and renewable electric power 3, 14. Despite the inspiring advances, the reconstruction of carbon fixation, which is the basic distinction in defining autotrophs and heterotrophs, is still challenging. Herein, we review the current progress in constructing synthetic CO2 fixation pathways in heterotrophs and discuss the principles and challenges in developing highly efficient carbon fixation systems. Then, we highlight the application scenarios of these pathways and how sustainable bioproduction can be enhanced. Finally, we envision the future of designing and applying synthetic carbon fixation pathways.