Journal of Energy Bioscience 2025, Vol.16, No.5, 238-247 http://bioscipublisher.com/index.php/jeb 244 6.4 Potential synergy with C3-to-C4 engineering in non-C4 biofuel candidates. Introducing the C4 photosynthetic mechanism into C3 biofuel crops (such as rice and wheat) is an important direction for enhancing global biofuel production capacity in the future. Such projects can not only enhance the efficiency of carbon fixation and nitrogen utilization, but also improve the adaptability of crops in high-temperature and arid environments. However, the C3-C4 project still faces many technical challenges at present, such as differences in anatomical structures, the complexity of gene regulatory networks, and the difficulty of multi-gene collaborative expression. However, with the development of systems biology, synthetic biology and multi-omics techniques, these problems are expected to be broken through (Schuler et al., 2016; Cui, 2021; Pradhan et al., 2022; Prasanna et al., 2025). 7 Conclusion Over the years, with the unremitting efforts of researchers in related fields, the C4 photosynthesis project has made considerable progress. Through techniques such as molecular breeding, gene editing and synthetic biology, researchers have identified and regulated key genes within plants that affect biomass accumulation, cell cycle, hormone action and cell wall synthesis, significantly increasing the yield and resource utilization efficiency of C4 energy crops such as sugarcane, corn and sorghum. New-generation gene editing tools such as CRISPR/Cas make it possible to precisely modify key enzymes and regulatory factors of photosynthesis. Many studies have shown that introducing C4-related genes into other crops can not only enhance the carbon assimilation of the crops but also improve their stress resistance and adaptability. Now, researchers have attempted to introduce the C4 photosynthetic mechanism into C3 crops and found that this measure can also enhance the photosynthetic efficiency and biomass of the target plants. The C4 photosynthesis project has laid a molecular foundation for the sustainable development of biofuels. C4 crops have an inherent high efficiency in carbon fixation and resource utilization, which enables them to better cope with adverse situations such as climate change, reducing greenhouse gas emissions and ensuring energy security. In the future, as technologies such as multi-gene editing, synthetic biology and multi-omics integration gradually mature, the photosynthetic efficiency and biomass yield of C4 crops will be further enhanced, thereby promoting the biofuel industry to be more efficient, low-carbon and economical. The new breakthrough from C3 to C4 project can enable more crops to be used for biofuel production and enhance the global bioenergy production capacity. The continuous progress of C4 photosynthesis engineering is inseparable from the integration of genetics, biotechnology, systems biology and agriculture. Future research needs to pay more attention to aspects such as gene regulatory networks, metabolic flow modeling, as well as field phenotypes and environmental interactions. Only when a complete innovation chain is formed from the laboratory to the field, from molecules to populations, can the efficient molecular design, precise breeding and large-scale application of C4 crops be truly achieved. This interdisciplinary collaboration will also provide solid support for the global production of sustainable biofuels. Acknowledgments The author expresses the gratitude to the two anonymous peer researchers for their constructive suggestions on the manuscript. Conflict of Interest Disclosure The author affirms that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest. References Abdelrahman M., Wei Z., Rohila J., and Zhao K., 2021, Multiplex genome-editing technologies for revolutionizing plant biology and crop improvement, Frontiers in Plant Science, 12: 721203. https://doi.org/10.3389/fpls.2021.721203 Aggarwal P., Muthamilarasan M., and Choudhary P., 2024, Millet as a promising C4 model crop for sustainable biofuel production, Journal of Biotechnology, 395: 110-121. https://doi.org/10.1016/j.jbiotec.2024.09.019
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