Presentation Information
[P01-118]Plasmid-free pathway construction and metabolic rewiring enable high-titer serinol production in Escherichia coli
○shuai Xu1, Zhixian Xu1, Yan Feng1 (1. Shanghai Jiao Tong University (China))
Keywords:
Serinol,Artificial biosynthetic pathway,Metabolic engineering,Escherichia coli,Fed-batch fermentation
Purpose: Serinol (2-amino-1,3-propanediol) is a highly versatile platform chemical serving as an indispensable precursor for critical pharmaceuticals and a valuable bio-based monomer for high-performance polyimide materials. As current industrial manufacturing fundamentally relies on chemical synthesis involving hazardous raw materials, harsh conditions, and complex downstream processing, developing a green and scalable biomanufacturing alternative is highly imperative. This study aimed to construct and systematically optimize an artificial biosynthetic pathway in Escherichia coli for the high-level production of serinol from glucose.
Methods: In silico-driven phosphatase identification; CRISPR/Cas9-mediated gene editing; Multicopy genomic integration; Fed-batch fermentation; HPLC-based quantification of serinol.
Results: An efficient artificial serinol biosynthesis pathway was successfully established in E. coli, utilizing the glycolytic intermediate dihydroxyacetone phosphate as the starting node. A previously engineered aminotransferase variant, RtxA-D9, was employed to direct the carbon flux toward serinol phosphate. To eliminate downstream bottlenecks, screening endogenous phosphatases identified HisB as an exceptionally efficient enzyme for the specific dephosphorylation of serinol phosphate. For genetic stability and maximize metabolic flux, a robust, plasmid-free strategy was implemented via multicopy genomic integration of the rtxA-D9 and hisB genes. Furthermore, the host metabolic network was systematically rewired by disrupting competitive branch pathways and intermediate degradation routes, while concurrently optimizing amino donor supply and cofactor regeneration cycles. This precise carbon flux regulation culminated in a genetically stable and highly efficient strain that achieved a serinol titer of 51.3 g/L with a productivity of 0.71 g/L/h, representing the highest production level reported to date.
Consideration: While systemic metabolic rewiring significantly improved carbon flux, future efforts could employ dynamic metabolic control strategies to better balance cell growth and product synthesis, thereby minimizing trace by-products and achieving even higher productivity. Furthermore, exploring the utilization of low-cost, non-food feedstocks will be essential to enhance the economic sustainability of this bioprocess.
Conclusion: By systematic metabolic network rewiring, this study achieved a record-breaking serinol titer of 51.3 g/L. This robust and scalable platform provides a superior, sustainable alternative to traditional chemical synthesis, effectively overcoming metabolic bottlenecks to facilitate the rapid industrial commercialization of bio-based serinol.
Methods: In silico-driven phosphatase identification; CRISPR/Cas9-mediated gene editing; Multicopy genomic integration; Fed-batch fermentation; HPLC-based quantification of serinol.
Results: An efficient artificial serinol biosynthesis pathway was successfully established in E. coli, utilizing the glycolytic intermediate dihydroxyacetone phosphate as the starting node. A previously engineered aminotransferase variant, RtxA-D9, was employed to direct the carbon flux toward serinol phosphate. To eliminate downstream bottlenecks, screening endogenous phosphatases identified HisB as an exceptionally efficient enzyme for the specific dephosphorylation of serinol phosphate. For genetic stability and maximize metabolic flux, a robust, plasmid-free strategy was implemented via multicopy genomic integration of the rtxA-D9 and hisB genes. Furthermore, the host metabolic network was systematically rewired by disrupting competitive branch pathways and intermediate degradation routes, while concurrently optimizing amino donor supply and cofactor regeneration cycles. This precise carbon flux regulation culminated in a genetically stable and highly efficient strain that achieved a serinol titer of 51.3 g/L with a productivity of 0.71 g/L/h, representing the highest production level reported to date.
Consideration: While systemic metabolic rewiring significantly improved carbon flux, future efforts could employ dynamic metabolic control strategies to better balance cell growth and product synthesis, thereby minimizing trace by-products and achieving even higher productivity. Furthermore, exploring the utilization of low-cost, non-food feedstocks will be essential to enhance the economic sustainability of this bioprocess.
Conclusion: By systematic metabolic network rewiring, this study achieved a record-breaking serinol titer of 51.3 g/L. This robust and scalable platform provides a superior, sustainable alternative to traditional chemical synthesis, effectively overcoming metabolic bottlenecks to facilitate the rapid industrial commercialization of bio-based serinol.
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