Three-dimensional structure of isoprene synthase and its use thereof for generating variants
AssigneesDanisco US Inc., The Goodyear Tire & Rubber Company
InventorsZachary Q. Beck, Richard R. Bott, Jeffrey V. Miller, Christopher L. Rife, Derek H. Wells
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Abstract
The present invention provides a three-dimensional structures of P. tremuloides isoprene synthase and P. alba isoprene synthase. The invention also provides methods of using the three-dimensional structure to design isoprene synthases with improved activity for increased isoprene production in microbial host cells. Biosynthetically produced isoprene of the present invention finds use in the manufacture of rubber and elastomers.
Claims18
An isolated host cell comprising an isoprene synthase variant, wherein the isoprene synthase variant is produced by:
- a) identifying amino acid residues in the flexible loops of isoprene synthase based on the three dimensional structure of P. alba isoprene synthase;
- b) introducing one or more amino acid substitutions in the flexible loop amino acids of said isoprene synthase to produce an isoprene synthase variant;
- and
- c) identifying said isoprene synthase variant with substitutions that improve activity, expression or stability of the isoprene synthase,
- wherein said isoprene synthase variant comprises one or more amino acid substitution(s) in the flexible loops corresponding to an amino acid position of SEQ ID NO:45 selected from the group consisting of A443, A453, N454, H515 and A519;
- and
- wherein said isoprene synthase variant has improved activity, expression or stability as compared to an isoprene synthase without said one or more amino acid substitutions.
An isolated host cell comprising an isoprene synthase variant, wherein the variant comprises one or more substitution(s) in the flexible loops corresponding to an amino acid position of SEQ ID NO:45 selected from the group consisting of A443, A453, N454, H515 and A519, wherein said isoprene synthase variant has improved activity, expression or stability as compared to an isoprene synthase without said one or more amino acid substitutions.
Dependencies are inferred from the claim text.