Expression constructs and methods of genetically engineering methylotrophic yeast
Inventors
Shankar, Smita • Hoyt, Martin Andrew
Assignees
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Publication Number
US-11319544-B2
Publication Date
2022-05-03
Expiration Date
Abstract
Methods and materials for genetically engineering methylotrophic yeast are provided.
Core Innovation
The invention provides nucleic acid constructs that include a first nucleic acid encoding a transcriptional activator operably linked to a promoter element, where the promoter element comprises a second nucleic acid that binds to the transcriptional activator. The transcriptional activator is selected from methanol expression regulator 1 (Mxr1), alcohol dehydrogenase regulator 1 (Adr1), transcriptional regulation of methanol induction 1 (Trm1), and transcriptional regulation of methanol induction 2 (Trm2). The constructs are configured so the promoter element can be regulated through binding of the second nucleic acid to the selected transcriptional activator.
The invention further provides methylotrophic yeast cells containing such nucleic acid constructs. In the yeast cells, the nucleic acid construct is stably integrated into the genome of the methylotrophic yeast cell, enabling expression under promoter control involving activator-bound promoter elements.
In a Pichia yeast embodiment, the invention specifies a methanol expression regulator 1 (Mxr1) transcriptional activator construct operably linked to an alcohol oxidase 1 (AOX1) promoter element. The AOX1 promoter element includes a plurality of nucleic acid sequences that bind to the Mxr1 transcriptional activator, and a second nucleic acid construct encoding an exogenous protein is operably linked to a second AOX1 promoter element that likewise includes a plurality of nucleic acid sequences that bind to the Mxr1 transcriptional activator.
Claims Coverage
The provided independent claims include three inventive claim scopes. Across the independent claims, the coverage centers on nucleic acid constructs using methanol-responsive transcriptional activators (Mxr1, Adr1, Trm1, Trm2) operably linked to promoter elements containing activator-binding nucleic acids, including stably genome-integrated methylotrophic yeast cells and a dual AOX1/Mxr1 promoter arrangement in Pichia.
Transcriptional activator operably linked to activator-binding promoter element
A nucleic acid construct comprising a first nucleic acid encoding a transcriptional activator operably linked to a promoter element, wherein the promoter element comprises a second nucleic acid that binds to the transcriptional activator; and wherein the transcriptional activator is selected from methanol expression regulator 1 (Mxr1), alcohol dehydrogenase regulator 1 (Adr1), transcriptional regulation of methanol induction 1 (Trm1), and transcriptional regulation of methanol induction 2 (Trm2).
Stably genome-integrated activator-binding construct in a methylotrophic yeast cell
A methylotrophic yeast cell comprising a nucleic acid construct comprising a first nucleic acid encoding a transcriptional activator operably linked to a promoter element, wherein the promoter element comprises a second nucleic acid that binds to the transcriptional activator, wherein the transcriptional activator is selected from methanol expression regulator 1 (Mxr1), alcohol dehydrogenase regulator 1 (Adr1), transcriptional regulation of methanol induction 1 (Trm1), and transcriptional regulation of methanol induction 2 (Trm2), wherein the nucleic acid construct is stably integrated into the genome of the methylotrophic yeast cell.
Dual AOX1 promoter elements with Mxr1-binding sequences driving activator-linked and exogenous-protein expression
A methylotrophic Pichia yeast cell comprising a nucleic acid construct encoding a methanol expression regulator 1 (Mxr1) transcriptional activator operably linked to an alcohol oxidase 1 (AOX1) promoter element, wherein the AOX1 promoter element comprises a second nucleic acid, wherein the second nucleic acid comprises a plurality of nucleic acid sequences that bind to the Mxr1 transcriptional activator; and a nucleic acid construct encoding an exogenous protein operably linked to a second AOX1 promoter element, wherein the second AOX1 promoter element comprises a third nucleic acid, wherein the third nucleic acid comprises a plurality of nucleic acid sequences that bind to the Mxr1 transcriptional activator.
Across the independent claims, the inventive concept is centered on promoter elements that contain nucleic acid sequences binding to methanol-regulated transcriptional activators, including genome-integrated embodiments in methylotrophic yeast cells and a Pichia dual AOX1 promoter arrangement in which both an Mxr1-related construct and an exogenous-protein construct are controlled by AOX1 promoter elements containing Mxr1-binding sequence pluralities.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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