Metabolic pathway assembly using docking domains from type I cis-AT polyketide synthases

Assembly artificial pathway in design connecting media can increase biosynthetic efficiency, but the choice of connecting media is limited. Here, the authors develop a new protein assembly strategy using a pool of docking peptides from polyketide synthase and show its application in astaxanthin biosynthesis in E. coli. Engineered metabolic pathways in microbial cell factories often have no natural organization and have challenging flux imbalances, leading to low biocatalytic efficiency. Modular polyketide synthases (PKSs) are multienzyme complexes that synthesize polyketide products via an assembly line thiotemplate mechanism. Here, we develop a strategy named mimic PKS enzyme assembly line (mPKSeal) that assembles key cascade enzymes to enhance biocatalytic efficiency and increase target production by recruiting cascade enzymes tagged with docking domains from type I cis-AT PKS. We apply this strategy to the astaxanthin biosynthetic pathway in engineered Escherichia coli for multienzyme assembly to increase astaxanthin production by 2.4-fold. The docking pairs, from the same PKSs or those from different cis-AT PKSs evidently belonging to distinct classes, are effective enzyme assembly tools for increasing astaxanthin production. This study addresses the challenge of cascade catalytic efficiency and highlights the potential for engineering enzyme assembly.

Automated summary

The authors develop a new protein assembly strategy using a pool of docking peptides from polyketide synthase and apply it to the biosynthesis of astaxanthin in E. coli, resulting in a 2.4-fold increase in production. This study addresses the challenge of improving cascade catalytic efficiency and highlights the potential for engineering enzyme assembly.