Presentation Information
[P01-066]Semi-Automated Modular Engineering and Whole-Broth Utilization Strategy for Scalable Cannabigerolic Acid Production in Corynebacterium glutamicum
○Eugene Kim1,2, Yubeen Heo1,2, Hanmin Woo1,2 (1. Lab of food molecular biotechnology, Sungkyunkwan University (Korea), 2. BioFoundry Research Center, Institute of Biotechnology and Bioengineering, Sungkyunkwan University (Korea))
Keywords:
Corynebacterium Glutamicum,Cannabinoids,Cannabigerolic acid,Metabolic engineering,Modular pathway optimization,Biofoundry
[Purpose]
Cannabigerolic acid (CBGA) is the central precursor of major cannabinoids and a promising target for sustainable microbial production. This study aimed to reconstruct and optimize a modular CBGA biosynthetic pathway in Corynebacterium glutamicum through hierarchical and combinatorial pathway engineering and to evaluate its scalability and downstream applicability.
[Method]
The CBGA pathway was divided into three modules: (1) an olivetolic acid (OLA) module, (2) a CBGA prenylation module using geranyl pyrophosphate (GPP), and (3) an isopentenol utilization pathway (IUP) module for isoprenoid flux enhancement. Multiple enzyme variants and promoters of varying strengths were systematically combined to construct transcriptional units (TUs), which were hierarchically assembled into sub-pathway units (SPUs) for combinatorial strain generation. The best-performing strain was selected for 2 L-scale bioreactor validation, and whole-broth stabilization and powder-based formulation strategies are currently under investigation.
[Results]
Pathway performance was strongly dependent on enzyme source and promoter strength. In the OLA module, lvaE from Pseudomonas significantly improved precursor formation, while B-dependent intermediate promoters outperformed stronger A-dependent promoters. For CBGA biosynthesis, optimized combinations of idi, erg20 mutant, and nphB enabled efficient prenylation, and the best-performing strain produced 57.4 ± 10.2 mg/L CBGA under OLA and isoprenol feeding conditions. Co-expression of the OLA, CBGA, and IUP modules further highlighted the importance of flux allocation between aromatic and isoprenoid branches.
[Consideration]
Balanced pathway expression, rather than maximal transcriptional strength, was critical for efficient CBGA biosynthesis. Flux competition between aromatic and isoprenoid branches and precursor limitation were identified as key bottlenecks, emphasizing the importance of modular pathway balancing in multi-branch biosynthetic systems.
[Conclusion]
This study demonstrates that biofoundry-assisted modular engineering is an effective and scalable strategy for CBGA biosynthesis in Corynebacterium glutamicum. Ongoing scale-up fermentation and whole-broth formulation studies may further support practical applications of microbially produced CBGA.
Cannabigerolic acid (CBGA) is the central precursor of major cannabinoids and a promising target for sustainable microbial production. This study aimed to reconstruct and optimize a modular CBGA biosynthetic pathway in Corynebacterium glutamicum through hierarchical and combinatorial pathway engineering and to evaluate its scalability and downstream applicability.
[Method]
The CBGA pathway was divided into three modules: (1) an olivetolic acid (OLA) module, (2) a CBGA prenylation module using geranyl pyrophosphate (GPP), and (3) an isopentenol utilization pathway (IUP) module for isoprenoid flux enhancement. Multiple enzyme variants and promoters of varying strengths were systematically combined to construct transcriptional units (TUs), which were hierarchically assembled into sub-pathway units (SPUs) for combinatorial strain generation. The best-performing strain was selected for 2 L-scale bioreactor validation, and whole-broth stabilization and powder-based formulation strategies are currently under investigation.
[Results]
Pathway performance was strongly dependent on enzyme source and promoter strength. In the OLA module, lvaE from Pseudomonas significantly improved precursor formation, while B-dependent intermediate promoters outperformed stronger A-dependent promoters. For CBGA biosynthesis, optimized combinations of idi, erg20 mutant, and nphB enabled efficient prenylation, and the best-performing strain produced 57.4 ± 10.2 mg/L CBGA under OLA and isoprenol feeding conditions. Co-expression of the OLA, CBGA, and IUP modules further highlighted the importance of flux allocation between aromatic and isoprenoid branches.
[Consideration]
Balanced pathway expression, rather than maximal transcriptional strength, was critical for efficient CBGA biosynthesis. Flux competition between aromatic and isoprenoid branches and precursor limitation were identified as key bottlenecks, emphasizing the importance of modular pathway balancing in multi-branch biosynthetic systems.
[Conclusion]
This study demonstrates that biofoundry-assisted modular engineering is an effective and scalable strategy for CBGA biosynthesis in Corynebacterium glutamicum. Ongoing scale-up fermentation and whole-broth formulation studies may further support practical applications of microbially produced CBGA.
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