Journal of Energy Bioscience 2025, Vol.16, No.5, 238-247 http://bioscipublisher.com/index.php/jeb 243 5.2 Synthetic biology approaches to reconstruct or optimize C4 pathways. Synthetic biology has brought new ideas for the modification and optimization of the C4 photosynthetic pathway. By modularly assembling multiple enzymes and regulatory elements, the unique carbon concentration mechanism of C4 can be introduced into C3 plants, or the carbon flow distribution can be further improved in C4 crops. Synthetic biology can also design multi-enzyme complexes, introduce artificial regulatory elements, and construct new metabolic pathways to support the improvement of photosynthetic efficiency and biomass accumulation (Hagemann and Hess, 2018; Kumlehn et al., 2018; Abdelrahman et al., 2021). 5.3 Systems biology and computational modeling to predict metabolic flux changes. Systems biology utilizes genomic, transcriptomic, proteomic and metabolomic data, combined with metabolic network modeling and flux analysis, to predict the impact of genetic engineering intervention on C4 photosynthesis and metabolic processes. Under genome-wide scale modeling, metabolic bottlenecks can be identified, carbon flow allocation can be optimized, and a theoretical basis can be provided for the design of multi-gene editing and synthetic biology (Hagemann and Hess, 2018; Khan et al., 2019). 5.4 Integration of multi-omics (transcriptomics, proteomics, metabolomics) for pathway optimization. At the current stage, multi-omics analysis (transcriptome, proteome, metabolome) is the main approach to optimizing the C4 photosynthetic pathway. High-throughput data can systematically identify key genes, proteins and metabolites that affect photosynthetic efficiency and biomass accumulation. Combining CRISPR/Cas and synthetic biology tools, these omics data provide solid support for precise regulation and pathway optimization, and can accelerate the molecular design and directed improvement of C4 crops (Hagemann and Hess, 2018; Khan et al., 2019; Alamillo et al., 2023). 6 Challenges and Future Perspectives 6.1 Trade-offs between photosynthesis enhancement and plant growth/fitness. Enhancing the expression of genes related to C4 photosynthesis can increase carbon assimilation and yield. However, if the photosynthetic pathway is overly strengthened, it may lead to an imbalance in energy and resource distribution, affecting the overall growth of plants. For instance, if key enzymes such as Rubisco are not coordinated with the overall metabolic network, it may lead to a decline in growth tolerance or adaptability (Pradhan et al., 2022; Nazari et al., 2024; Prasanna et al., 2025). Furthermore, both Schuler et al. (2016) and Cui (2021) pointed out that the unique Kranz structure and cell differentiation mechanism of C4 plants are very complex, and physiological bottlenecks or adaptive losses are prone to occur during engineering modification. 6.2 Biosafety, ecological, and regulatory concerns in genetically engineered biofuel crops. Genetically modified C4 biofuel crops will encounter biosecurity and ecological risks when promoted, such as gene drift, impact on non-target organisms, and disruption of ecosystem balance, etc. The regulatory policies for genetically modified crops vary from country to country, the approval process is complex, and the public acceptance is limited. All these factors have affected the commercialization of genetically engineered C4 crops. Meanwhile, long-term ecological impacts and environmental adaptability still require large-scale field trials and continuous monitoring (Shokravi et al., 2021; Pradhan et al., 2022). 6.3 Scalability and cost-effectiveness of genetically engineered C4 crops for industrial biofuel production. Although genetically engineered C4 crops have already demonstrated higher biomass and resource utilization efficiency in both laboratory and greenhouse conditions, there are still many challenges to achieving large-scale industrialization of C4 crops, including the stability of multi-gene co-expression, consistency of traits in field environments, compatibility with planting systems, as well as the production and distribution costs of genetically modified seeds. The market price fluctuations of biofuels and the intensity of policy support will affect the economic feasibility of large-scale industrialization of C4 crops (Ambaye et al., 2021; Pradhan et al., 2022).
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