Mutant firefly luciferases with improved specific activity and dATP discrimination constructed by yeast cell surface engineering

Abstract

Pyrosequencing system utilizing luciferase is one of the next-generation DNA sequencing systems. However, there is a crucial problem with the current pyrosequencing system: luciferase cannot discriminate between ATP and dATP completely, and dATPαS must be used as the dATP analogue. dATPαS is expensive and has low activity for the enzyme. If luciferase can clearly recognize the difference between ATP and dATP, dATP could be used instead of the expensive dATPαS in the pyrosequencing system. We attempted to prepare a novel luciferase with improved specific activity and dATP discrimination with the molecular display method. First, we selected two amino acid residues, Ser440 and Ser456, as target residues for mutation from the whole sequence of Photinus pyralis luciferase; we comprehensively mutated these two amino acids. A mutant luciferase library was constructed using yeast cell surface engineering. Through three step-wide screenings with individual conditions, we easily and speedily isolated three candidate mutants from 1,152 candidates and analyzed the properties of these mutants. Consequently, we succeeded in obtaining interesting mutant luciferases with improved specific activity and dATP discrimination more conveniently than with other methods.

Rapid and highly sensitive detection of Escherichia coli O157:H7 in food with loop‐mediated isothermal amplification coupled to a new bioluminescent assay

“…In the novel bioluminescent pyrophosphate assay, dATP was replaced with dATPαS in LAMP to avoid a high background signal because the light intensity produced by dATPαS as the substrate of luciferin is about 99 arbitrary units in Figure 3 that only 0.048% of that produced by dATP [24,25]. Therefore, although dATPαS is also incorporated into the LAMP product, the amount of dATPαS in the product has little effect on the intensity of light detected in the bioluminescence assay.…”

Applications of Yeast Surface Display for Protein Engineering

“…In another example, a general strategy for evolving bond-forming enzymes was developed (Figure 4) and applied to identify a bacterial transpeptidase sortase A enzyme with a 140-fold enhancement in catalytic activity 88 . Yeast display has been applied to enhance the activity and/or substrate selectivity of a variety of other enzymes, including firefly luciferase 89 , Rhizomucor miehei lipase 90 , the adenylation domain of a nonribosomal peptide synthetase 91 , E. coli lipoic acid ligase 92 , and a Tobacco Etch virus protease 93 . In general, the success of these strategies was contingent on the enzyme substrate being labeled with an affinity handle or fluorescent probe.…”

Establishment of cell surface engineering and its development

“…A mutant luciferase library was constructed by cell surface engineering, and interesting mutant luciferases with improved specific activity and dATP discrimination were obtained through only three stepwise screenings. 106,107) Further technological innovation of the system for single-cell analysis including development of single-cell chips 108) and isolation would lead to more efficient protein engineering and directed evolution by cell surface engineering.…”