Design of a New Multienzyme Complex Synthesis System Based on Yarrowia lipolytica Simultaneously Secreted and Surface Displayed Fusion Proteins for Sustainable Production of Fatty Acid-Derived Hydrocarbons

In order to address the challenges of relative ratio and relative position among individual enzymes and substrate channeling in multienzyme biocatalysis, we propose a new one-pot genetically encoded self-assembly system as an alternative to the heavy procedure of separately preparing individual enzymes and subsequently co-immobilizing them by physicochemical means, allowing us to construct cell-associated two- or three-enzyme complexes, through the highly specific interaction of cohesin and dockerin domains. Our method for multienzyme complexes (MECs) assembled on Yarrowia lipolytica yeast cells is based on simultaneous surface display of a synthetic multiple cohesins backbone (scaffoldin) and extracellular secretion of dockerin-fusions of individual enzymes. This methodology was applied to fatty acid-derived hydrocarbon (alkenes, alkanes) production. The genetically tailored cell-associated MECs exhibited different proportional and positional effects through genetically encoded customization of the scaff...