US 11,866,744 B2
Gene construct encoding mutant Thioesterase, mutant Thioesterase encoded thereby, transformed host cell containing the gene construct, and method of using them to produce medium-chain fatty acids
Brian F. Pfleger, Madison, WI (US); Nestor Jose Hernandez-Lozada, Jena (DE); Costas Maranas, State College, PA (US); and Matthew Grisewood, Mountaintop, PA (US)
Assigned to Wisconsin Alumni Research Foundation, Madison, WI (US); and The Penn State Research Foundation, University Park, PA (US)
Filed by WISCONSIN ALUMNI RESEARCH FOUNDATION, Madison, WI (US); and The Penn State Research Foundation, University Park, PA (US)
Filed on May 27, 2021, as Appl. No. 17/332,186.
Application 17/332,186 is a continuation of application No. 15/930,147, filed on May 12, 2020, granted, now 11,053,480.
Application 15/930,147 is a continuation of application No. 16/535,305, filed on Aug. 8, 2019, granted, now 10,689,631, issued on Jun. 23, 2020.
Application 16/535,305 is a continuation of application No. 15/630,442, filed on Jun. 22, 2017, granted, now 10,421,951, issued on Sep. 24, 2019.
Claims priority of provisional application 62/353,069, filed on Jun. 22, 2016.
Prior Publication US 2021/0292720 A1, Sep. 23, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 9/16 (2006.01)
CPC C12N 9/16 (2013.01) [C12Y 301/02002 (2013.01)] 15 Claims
 
1. An unnatural, mutated protein comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:1 and comprises one or more substitutions with respect to SEQ ID NO:1, wherein the protein has at least one of enhanced thioesterase activity and enhanced thioesterase specificity in catalyzing the hydrolysis of a medium-chain acyl-acyl carrier protein substrate or a medium-chain acyl-CoA substrate to yield a free fatty acid or a free fatty acid derivative compared to an unaltered protein of SEQ ID NO:1, wherein the one or more substitutions comprise one or more of:
a lysine at a position aligning to R108 of SEQ ID NO:1;
a tyrosine at a position aligning to F139 of SEQ ID NO:1;
a valine at a position aligning to 142 of SEQ ID NO:1;
a glycine at a position aligning to A111 of SEQ ID NO:1;
a serine at a position aligning to P138 of SEQ ID NO:1;
an isoleucine at a position aligning to M170 of SEQ ID NO:1;
a serine at a position aligning to T35 of SEQ ID NO:1; and
an aspartate as a position aligning to E142 of SEQ ID NO:1.