Presentation Information
[4Ferm-05-KL]From Digital Design to Ecological Resilience: A Systems-Level Framework for Engineering Uncommon Metabolic Pathways in Prokaryotic Factories
○Han-Jia Lin1, Hung-Yun Lin1, Chun-Ting Lee1, Sin-Wei Lin1, Man-Chun Hsu1 (1. National Taiwan Ocean University (Taiwan))
Keywords:
Metabolic Engineering,Prokaryotic Cell Factory,Predictive Design,Higher-order Polyamines,Ecological Resilience
[Purpose] Digital biology is shifting metabolic engineering from trial-and-error to a predictive discipline. Higher-order polyamines (PAs), like the pentaamine homocaldopentamine (HCPA), are vital for stress adaptation but difficult to produce. This study establishes a systems-level framework for deploying uncommon pathways in prokaryotic factories. Using marine diatoms as a genetic resource and E. coli as a programmable platform, we demonstrate how predictive modeling guides complex network optimization to address ecological challenges.
[Method] Our framework integrates three pillars: First, AI-driven structural analysis (AlphaFold3) to decipher non-canonical functions of atypical thermospermine synthases (PtTSMS) from P. tricornutum. Second, protein engineering via C-terminal truncation to enable pentaamine biosynthetic activity in typical synthases. Third, a predictive "Ori-tuning" strategy to manage metabolic burden. By modulating gene dosage of PtTSMS, PtSDS1, and EcSAMDC using distinct plasmid origins (BRL322/RSF), we balanced the dcSAM pool. Finally, engineered cells were used as nutrient carriers to validate osmotic stress tolerance in copepods.
[Results] Structural analysis identified a C-terminal loop in typical TSMS acting as a gatekeeping lid; its removal accommodates longer PA chains. In E. coli, the PtTSMS-dominant strain (PT_04-1) achieved an HCPA yield of 6.3 mg/L (77.3% purity) without compromising host growth. Crucially, this optimal equilibrium achieved via Ori-tuning perfectly aligned with metabolic load simulations. In application, dietary exposure to HCPA-enriched strains significantly enhanced copepod survival under osmotic stress, increasing rates to 70% and 55% in high- and low-salinity conditions, respectively.
[Consideration] Pentaamine synthesis imposes a heavy metabolic burden due to high dcSAM and ATP consumption. While Ori-tuning provides a robust method for flux balancing, future efforts must transition to dynamic regulation. Integrating tools like AlphaGenome will allow prediction of whole-genome responses to metabolic loads, moving optimization from protein to genome levels. Additionally, using engineered bacteria as trophic carriers necessitates developing sophisticated genetic kill-switches for ecological safety.
[Conclusion] This study provides a roadmap for translating molecular discovery into ecological resilience. We proved that even fundamental genetic tools like plasmid origin manipulation can implement complex pathways when guided by precise simulations. This framework facilitates scalable production of high-value rare metabolites, contributing to a resilient and sustainable bio-economy.
[Method] Our framework integrates three pillars: First, AI-driven structural analysis (AlphaFold3) to decipher non-canonical functions of atypical thermospermine synthases (PtTSMS) from P. tricornutum. Second, protein engineering via C-terminal truncation to enable pentaamine biosynthetic activity in typical synthases. Third, a predictive "Ori-tuning" strategy to manage metabolic burden. By modulating gene dosage of PtTSMS, PtSDS1, and EcSAMDC using distinct plasmid origins (BRL322/RSF), we balanced the dcSAM pool. Finally, engineered cells were used as nutrient carriers to validate osmotic stress tolerance in copepods.
[Results] Structural analysis identified a C-terminal loop in typical TSMS acting as a gatekeeping lid; its removal accommodates longer PA chains. In E. coli, the PtTSMS-dominant strain (PT_04-1) achieved an HCPA yield of 6.3 mg/L (77.3% purity) without compromising host growth. Crucially, this optimal equilibrium achieved via Ori-tuning perfectly aligned with metabolic load simulations. In application, dietary exposure to HCPA-enriched strains significantly enhanced copepod survival under osmotic stress, increasing rates to 70% and 55% in high- and low-salinity conditions, respectively.
[Consideration] Pentaamine synthesis imposes a heavy metabolic burden due to high dcSAM and ATP consumption. While Ori-tuning provides a robust method for flux balancing, future efforts must transition to dynamic regulation. Integrating tools like AlphaGenome will allow prediction of whole-genome responses to metabolic loads, moving optimization from protein to genome levels. Additionally, using engineered bacteria as trophic carriers necessitates developing sophisticated genetic kill-switches for ecological safety.
[Conclusion] This study provides a roadmap for translating molecular discovery into ecological resilience. We proved that even fundamental genetic tools like plasmid origin manipulation can implement complex pathways when guided by precise simulations. This framework facilitates scalable production of high-value rare metabolites, contributing to a resilient and sustainable bio-economy.
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