US 12,084,667 B2
Expression constructs and methods of genetically engineering methylotrophic yeast
Smita Shankar, Millbrae, CA (US); and Martin Andrew Hoyt, San Francisco, CA (US)
Assigned to Impossible Foods Inc., Redwood City, CA (US)
Filed by Impossible Foods Inc., Redwood City, CA (US)
Filed on Jan. 25, 2022, as Appl. No. 17/584,028.
Application 17/584,028 is a continuation of application No. 16/123,121, filed on Sep. 6, 2018, granted, now 11,319,544.
Application 16/123,121 is a continuation of application No. 15/678,342, filed on Aug. 16, 2017, granted, now 10,273,492, issued on Apr. 30, 2019.
Application 15/678,342 is a continuation of application No. 15/678,891, filed on Aug. 16, 2017, granted, now 9,938,327, issued on Apr. 10, 2018.
Application 15/678,891 is a continuation of application No. PCT/US2016/031797, filed on May 11, 2016.
Claims priority of provisional application 62/313,491, filed on Mar. 25, 2016.
Claims priority of provisional application 62/236,506, filed on Oct. 2, 2015.
Claims priority of provisional application 62/222,388, filed on Sep. 23, 2015.
Claims priority of provisional application 62/220,366, filed on Sep. 18, 2015.
Claims priority of provisional application 62/203,052, filed on Aug. 10, 2015.
Claims priority of provisional application 62/185,921, filed on Jun. 29, 2015.
Claims priority of provisional application 62/183,074, filed on Jun. 22, 2015.
Claims priority of provisional application 62/159,899, filed on May 11, 2015.
Prior Publication US 2022/0290166 A1, Sep. 15, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 15/81 (2006.01); C07K 14/39 (2006.01); C07K 14/415 (2006.01); C07K 14/805 (2006.01); C12N 1/16 (2006.01); C12N 15/63 (2006.01)
CPC C12N 15/815 (2013.01) [C07K 14/39 (2013.01); C07K 14/415 (2013.01); C07K 14/805 (2013.01); C12N 1/16 (2013.01); C12N 15/635 (2013.01); C12N 15/81 (2013.01)] 38 Claims
 
1. A method of producing a heme-containing protein, the method comprising:
fermenting a recombinant Pichia pastoris cell lacking a recombinant nucleic acid encoding an antibiotic resistance gene in a fermentation broth, and
inducing expression of the heme-containing protein with methanol,
wherein the recombinant Pichia pastoris cell comprises:
one or more stably integrated recombinant nucleic acids encoding one or more heme biosynthesis polypeptides selected from ALA synthase, ALA dehydratase, porphobilinogen deaminase, uroporphyrinogen III synthase, uroporphyrinogen III decarboxylase, coproporphyrinogen oxidase, protoporphyrinogen III oxidase, and ferrochelatase,
a stably integrated recombinant nucleic acid encoding the heme-containing protein operably linked to a first methanol-inducible promoter, and
a recombinant nucleic acid encoding a transcriptional activator.