Engineering of phosphoketolase from Bifidobacterium adolescentis for increased activity on non-phosphorylated substrates and application in a cell-free reaction system for ATP regeneration.

Phosphoketolases (Pkts) naturally catalyze thiamine diphosphate (TPP)-dependent C-C bond cleavage in phosphorylated ketoses through formation of a covalent TPP-bound intermediate, yielding an aldose phosphate and acetyl phosphate (AcP). Since the reaction mechanism does not directly involve the substrate-bound phosphate group, Pkts can be engineered towards efficient synthesis of the high-energy phosphoryl donor AcP from non-phosphorylated substrates in presence of inorganic phosphate. Industrial interest arises from the applicability of engineered Pkt variants in cell-free reaction systems for ATP regeneration from inexpensive substrates such as d-fructose. This chapter describes engineering of the fructose-6-phosphate phosphoketolase from Bifidobacterium adolescentis (Bad.F6Pkt) towards improved activity on d-fructose, with a focus on the establishment of a high-throughput screening (HTS) system for accelerated identification of variants with optimized catalytic performance. Detailed protocols are provided for the expression of pkt libraries in 96-well microtiter plate format and screening for enhanced activity on d-fructose. Furthermore, production of selected variants in shake-flask cultures, enzyme purification and activity assays for validation of hits are covered. The chapter additionally outlines the demonstration of cell-free ATP-regenerating enzyme cascades using engineered Bad.F6Pkt variants. In previous work, application of this workflow enabled identification of the Bad.F6Pkt H548N variant, exhibiting a 5.6-fold increased activity on d-fructose compared to the wild-type. When applied in an ATP-regenerating cascade combined with glycerol kinase, acetate kinase and erythrulose-isomerase activities, this variant enabled ATP yields of up to 2.53 molATP molFructose-1. The strategy is broadly applicable and can be adapted for screening Pkt libraries towards increased activity on other non-phosphorylated substrates.