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Designed high-redox potential laccases exhibit high functional diversity

ABSTRACT: White-rot fungi secrete an impressive repertoire of high-redox potential laccases (HRPLs) and peroxidases for efficient oxidation and utilization of lignin. Laccases are attractive enzymes for green-chemistry applications due to their broad substrate range and low environmental impact. Since expression of functional recombinant HRPLs is challenging, iterative directed evolution protocols have been applied to improve their expression, activity and stability. We implement a rational, stabilize-and-diversify strategy to two HRPLs that we could not functionally express: first, we use the PROSS stability-design algorithm to allow functional expression in yeast. Second, we use the stabilized enzymes as starting points for FuncLib active-site design to improve their activity and substrate diversity. Four of the FuncLib designed HRPLs and their PROSS progenitor exhibit substantial diversity in reactivity profiles against high-redox potential substrates, including lignin monomers. Combinations of 3-4 subtle mutations that change the polarity, solvation and sterics of the substrate-oxidation site explain the differences in reactivity profiles. These stable and versatile HRPLs are a step towards the generation of an effective lignin-degrading consortium of enzymes that can be secreted from yeast. More broadly, the stabilize-and-diversify strategy can be applied to other challenging enzyme families to expand the utility of natural enzymes.

INSTRUMENT(S): maXis

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Meital Kupervaser

LAB HEAD: Sarel J. Fleishman

PROVIDER: PXD034630 | Pride | 2023-03-16

REPOSITORIES: Pride

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			Action	DRS
	TTP1LC5_SF_16049_HighMW_3uL_07-Apr-22_21-45-54.d.zip	Other
	TTP1LC5_SF_16049_HighMW_3uL_07-Apr-22_21-45-54.d_20220410.mgf	Mgf
	TTP1LC5_SF_16049_HighMW_3uL_07-Apr-22_21-45-54.d_20220410.mzid.gz	Mzid
	TTP1LC5_SF_16049_HighMW_3uL_07-Apr-22_21-45-54.d_20220410.xlsx	Xlsx
	TTP1LC5_SF_16049_LowMW_3uL_07-Apr-22_19-27-18.d.zip	Other

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Designed high-redox potential laccases exhibit high functional diversity

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