The energy crisis and global warming are two major challenges confronting humanity. Since the dawn of the industrial revolution, fossil fuels have been the backbone of the world's energy system, driving the rapid development of modern human civilization and economic growth. However, fossil resources are non-renewable, and their consumption rate far exceeds their regeneration rate, leading to a potential energy crisis. Moreover, fossil fuel combustion is the dominant source of CO2 and other greenhouse gases (GHGs), and the immediate problem for humanity may not be the limitation of fossil fuel reserves but the consequence of its combustion. The versatile nature of microorganisms allows the production of a wide range of fuels and chemicals from two main groups of feedstocks, i.e., sugar or lignocellulose-based feedstocks, usually referred to the first or second biorefineries. The first or second biorefineries can substitute the production of petrochemical products and are green and low-carbon. However, feedstocks used in first and second biorefineries are continuously under criticisms, due to their requirement for arable land and fresh water, the reduction in biodiversity, and the release of N2O from fertilized soils. Thus, the first or second biorefineries cannot allow carbon-negative biomanufacturing. Some human activities inevitably produce carbon emissions, and in order to achieve the goal of carbon neutrality, there is an urgent need to develop negative emission technologies to offset these emissions. Currently, reducing or eliminating carbon dioxide emissions alone is no longer sufficient to curb the negative effects of greenhouse gases. Therefore, in order to achieve the goal of carbon neutrality, the third generation biorefinery using carbon dioxide as raw material has attracted more and more attention. Proof of concept studies of the third-generation biorefinery have been made to produce several commodity chemicals, and have suggested that this biomanufacturing can be net carbon negative. Although some microorganisms have shown their ability to convert CO2 to target products, currently these processes still suffer from limited product spectrum and difficulty in genetic modification, as well as low capture and conversion efficiencies for both CO2 and energy, making it challenging to enable the commercial production of target compounds in these hosts. Thus, researchers can focus on exploring new carbon-fixing autotrophs, enhancing carbon-fixing efficiency in current autotrophs by developing new synthetic biology tools, or re-establishment of carbon-fixing pathways in mature model industrial microorganisms. Many problems must be solved in the design and construction of the third-generation biorefining cell factory. To achieve this, current identified natural CO2 fixation pathways were first introduced, and then several artificial CO2 fixation ways that were designed based on the concept of synthetic biology were summarized. The unique features of each pathway, as well as the advantages and limitation factors of each CO2 fixation pathway, have been discussed. It has also been found that assimilation or conservation of the very stable and low energy configuration of CO2 into cellular carbon requires four reducing equivalents and a lot of energy. This demand can be obtained through energy sources such as light, chemicals, and electricity, and will require cleverly designed catalysts or high-efficient carbon fixation enzymes. Some examples of the third generation biorefinery were reviewed that were conducted with photocatalytic or electrocatalytic reduction of carbon dioxide. Meanwhile, we review how heterotrophs can be engineered for use in the third generation biorefinery and some problems in current industrial applications. Finally, the main advantages and challenges of the third generation biorefinery were prospected, such as the low carbon sequestration rate due to the low catalytic rate of carboxylase, the large amount of ATP required to convert carbon dioxide into the center carbon metabolism of cell, and the energy/carbon loss during carbon fixation. To solve these problems, synthetic biology strategies can be used to optimize the adaptation of the carbon sequestration pathway to host endogenous carbon and energy metabolism, including regulating the expression of enzymes related to the carbon sequestration pathway and balancing the distribution of carbon flux by modifying carbon sequestration pathway and related metabolic networks. In order to break through the low efficiency of biological carbon sequestration, the combination of inorganic catalytic carbon sequestration and biological-catalyzed carbon extending reaction can also be used for two-stage carbon sequestration and biological manufacturing. As a summary, the third generation biorefinery is an important milestone in carbon-negative biomanufacturing, and it is of great significance to achieve carbon neutrality. At present, by integrating biology, chemistry, materials science, the technological level of the third generation biorefinery can be significantly improved, which can shape the low-carbon circular economy of human society.
