Presentation Information
[P02-910]From controllable enzyme assemblies to multienzyme aggregates for enhanced biocatalytic efficiency
○Tian Ma1 (1. Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences (China))
Keywords:
Spatial metabolic engineering,Enzyme assembly,Scaffold protein,Metabolic flux,Cascade catalysis
[Purpose] In nature, metabolic enzymes are spatially organized to enable efficient substrate channeling and flux regulation. However, heterologous expression in microbial hosts often disrupts this organization, causing imbalanced flux, intermediate accumulation, and suboptimal yields. This study aims to develop synthetic spatial engineering strategies that enhance metabolic efficiency by orchestrating enzyme positioning, addressing the long-standing challenge of inefficient cascade catalysis in heterologous biosynthesis.
[Method] We established three progressively complex spatial engineering platforms. First, we used a pair of short peptide tags, RIAD and RIDD, which form a high-affinity non-covalent interaction. Fusing these tags to two sequential enzymes in the carotenoid pathway created binary enzyme assemblies that promote substrate channeling. Second, we developed mPKSeal (mimic polyketide synthase enzyme assembly line), inspired by docking domains from type I cis-AT PKS. Three cascade enzymes tagged with engineered docking domains formed ordered linear multienzyme complexes. Third, we constructed a scaffold protein-mediated enzyme multimolecular aggregation (SPMEMA) system, where a synthetic scaffold protein with multiple interaction domains recruits several cascade enzymes simultaneously, forming higher-order aggregates that coordinate multi-step reactions and balance metabolic flow.
[Results] The RIAD-RIDD system increased carotenoid production by 5.7-fold, showing that simple colocalization of two sequential enzymes significantly enhances flux. The mPKSeal strategy improved astaxanthin production by 2.4-fold through ordered multienzyme assembly. Notably, SPMEMA achieved a 12.3-fold increase in valerenadiene production by recruiting and synergizing multiple cascade enzymes. These results demonstrate a clear progression in efficacy with increasing spatial organization complexity, highlighting the critical role of multienzyme coordination in optimizing metabolic flux.
[Consideration] Although these strategies dramatically improved product titers, several considerations remain. First, optimal assembly configuration—including enzyme stoichiometry, order, and linker flexibility—requires empirical determination for each pathway. Second, spatial organization must balance pathway flux to avoid toxic intermediate accumulation, necessitating careful tuning of expression levels alongside assembly. Finally, scalability to industrial fermentation and compatibility with other metabolic engineering approaches warrant further investigation.
[Conclusion] This study demonstrates that spatial metabolic engineering—from simple binary assemblies to scaffold-mediated multienzyme aggregates—provides a powerful framework for enhancing cascade catalytic efficiency in heterologous hosts. By mimicking nature’s spatial regulation, these strategies enable precise metabolic flux control and significantly improve production of high-value compounds such as terpenoids. Our findings underscore the potential of spatial engineering as a versatile tool for synthetic biology and industrial biotechnology, paving the way for more efficient and sustainable biosynthesis.
[Method] We established three progressively complex spatial engineering platforms. First, we used a pair of short peptide tags, RIAD and RIDD, which form a high-affinity non-covalent interaction. Fusing these tags to two sequential enzymes in the carotenoid pathway created binary enzyme assemblies that promote substrate channeling. Second, we developed mPKSeal (mimic polyketide synthase enzyme assembly line), inspired by docking domains from type I cis-AT PKS. Three cascade enzymes tagged with engineered docking domains formed ordered linear multienzyme complexes. Third, we constructed a scaffold protein-mediated enzyme multimolecular aggregation (SPMEMA) system, where a synthetic scaffold protein with multiple interaction domains recruits several cascade enzymes simultaneously, forming higher-order aggregates that coordinate multi-step reactions and balance metabolic flow.
[Results] The RIAD-RIDD system increased carotenoid production by 5.7-fold, showing that simple colocalization of two sequential enzymes significantly enhances flux. The mPKSeal strategy improved astaxanthin production by 2.4-fold through ordered multienzyme assembly. Notably, SPMEMA achieved a 12.3-fold increase in valerenadiene production by recruiting and synergizing multiple cascade enzymes. These results demonstrate a clear progression in efficacy with increasing spatial organization complexity, highlighting the critical role of multienzyme coordination in optimizing metabolic flux.
[Consideration] Although these strategies dramatically improved product titers, several considerations remain. First, optimal assembly configuration—including enzyme stoichiometry, order, and linker flexibility—requires empirical determination for each pathway. Second, spatial organization must balance pathway flux to avoid toxic intermediate accumulation, necessitating careful tuning of expression levels alongside assembly. Finally, scalability to industrial fermentation and compatibility with other metabolic engineering approaches warrant further investigation.
[Conclusion] This study demonstrates that spatial metabolic engineering—from simple binary assemblies to scaffold-mediated multienzyme aggregates—provides a powerful framework for enhancing cascade catalytic efficiency in heterologous hosts. By mimicking nature’s spatial regulation, these strategies enable precise metabolic flux control and significantly improve production of high-value compounds such as terpenoids. Our findings underscore the potential of spatial engineering as a versatile tool for synthetic biology and industrial biotechnology, paving the way for more efficient and sustainable biosynthesis.
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