Discovery of enzymes from the alpha-keto acid decarboxylase family
Inventors
Siegel, Justin • Bertolani, Steve • Mak, Wai Shun • Liao, James • Tran, Stephen • Marcheschi, Ryan • Thompson, James • Baker, David
Assignees
University of Washington • University of California, San Diego
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Publication Number
US-10829756-B2
Publication Date
2020-11-10
Expiration Date
Abstract
2-ketoacid decarboxylase enzymes, compositions encoding for 2 ketoacid decarboxylase enzymes, and host cells comprising such enzymes or compositions are provided.
Core Innovation
The invention relates to an enzyme comprising 2-ketoacid decarboxylase activity, where the enzyme comprises a 3-layer alpha-beta-alpha sandwich and a thiamine pyrophosphate (TPP) binding pocket. The enzyme is characterized by at least a 100-fold greater catalytic efficiency for C8 2-ketoacid substrates as measured by kcat/Km compared to C3 or isoC5 2-ketoacid substrates.
The enzyme further requires a primary amino acid sequence that differs from the primary amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 at least at an amino acid residue within 8 A of a 2-ketoacid decarboxylase active site of the enzyme. The enzyme comprises mutations corresponding to G402V, M538L, and F542V of SEQ ID NO:1.
In the disclosed summary, these variants are engineered and evaluated for catalytic efficiency improvements with long-chain alcohol targets, including long-chain (≥C5, especially C8) alcohols. The described discovery and engineering includes integrative genomic enzyme mining that identifies enzymes such as GEO 175 with high kcat/Km on C8 2-ketoacid substrates and >100-fold C8/C5 specificity improvement.
Claims Coverage
The independent claim is clm-00001. It requires an enzyme with 2-ketoacid decarboxylase activity having a 3-layer alpha-beta-alpha sandwich and a TPP binding pocket, plus a specified sequence difference within 8 A of a defined active site and inclusion of mutations corresponding to G402V, M538L, and F542V, with at least a 100-fold increase in catalytic efficiency for C8 2-ketoacid substrates over C3 or isoC5 substrates measured by kcat/Km.
2-ketoacid decarboxylase enzyme with 3-layer alpha-beta-alpha sandwich and TPP binding pocket
An enzyme comprising 2-ketoacid decarboxylase activity wherein the enzyme comprises a 3-layer alpha-beta-alpha sandwich and a thiamine pyrophosphate (TPP) binding pocket.
C8 catalytic efficiency at least 100-fold greater by kcat/Km
At least a 100-fold greater catalytic efficiency for C8 2-ketoacid substrates as compared to C3 or isoC5 2-ketoacid substrates, wherein the catalytic efficiency is measured by kcat/Km.
Active-site-proximal sequence variation within 8 A of the 2-ketoacid decarboxylase active site
The primary amino acid sequence differs from the primary amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:2 at least at an amino acid residue within 8 A of a 2-ketoacid decarboxylase active site.
Mutations corresponding to G402V, M538L, and F542V of SEQ ID NO:1
The enzyme comprises mutations corresponding to G402V, M538L, and F542V of SEQ ID NO:1.
Across the independent claim, the coverage centers on a 2-ketoacid decarboxylase enzyme with a 3-layer alpha-beta-alpha sandwich and a TPP binding pocket, providing at least 100-fold improved kcat/Km for C8 2-ketoacid substrates over C3 or isoC5 substrates, with required active-site-proximal sequence differences and specific mutations corresponding to G402V, M538L, and F542V.
Stated Advantages
At least a 100-fold greater catalytic efficiency for C8 2-ketoacid substrates as compared to C3 or isoC5 2-ketoacid substrates, measured by kcat/Km.
C8 2-ketoacid specificity improvement relative to C5 (reported for GEO 175 in the summary).
C8/C5 specificity improvement reported as >600-fold for KIVD_VLV (as described in the summary).
In vivo alcohol profile switching toward long-chain alcohols, with heptanol and hexanol dominance (as described in the summary).
Documented Applications
Use in a synthetic recursive+1 pathway for producing long-chain (≥C5, especially C8) alcohols (as described in the summary).
In vivo alcohol profile switching toward long-chain alcohols, with heptanol and hexanol dominance (as described in the summary).
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