Materials and methods for protein production
Inventors
Hoyt, Martin Andrew • Guo, Xiao • Shankar, Smita
Assignees
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Publication Number
US-11427932-B2
Publication Date
2022-08-30
Expiration Date
Abstract
This document relates to materials and methods for the production of protein. In one aspect, this document provides a nucleic acid construct including a first alcohol oxidase promoter element, wherein the first alcohol oxidase promoter element includes a mutation at one or more nucleotide positions corresponding to any of nucleotide positions 668-734 relative to SEQ ID NO: 28.
Core Innovation
The invention relates to nucleic acid constructs that include a first alcohol oxidase promoter element comprising one or more mutations at nucleotide positions selected from specified positions relative to SEQ ID NO: 28. In particular, the document describes mutated alcohol oxidase 1 promoter elements for methylotrophic yeasts such as Pichia pastoris and Komagataella species, with enumerated mutation positions including T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862.
The promoter element is used as a promoter to drive expression of a nucleotide sequence operably linked to it. Yeast cells containing the constructs are used to produce proteins, including heme-binding proteins and leghemoglobin, and the disclosure also identifies exogenous and heterologous products, transcription factors, and heme biosynthesis enzymes among the potential products associated with the operably linked nucleotide sequence.
Embodiments are described that enable expression in the absence of added methanol. The disclosure further relates to construct, cell, and method embodiments for producing a product in a cell by expressing a nucleic acid construct containing the mutated alcohol oxidase promoter element.
Claims Coverage
The independent claims are clm-00001 and clm-00012. Across these independent claims, the core coverage centers on a mutated alcohol oxidase promoter element defined by one or more mutations at enumerated nucleotide positions relative to SEQ ID NO: 28, together with a method of producing a product in a cell by expressing a construct with that promoter-linked nucleotide sequence.
Mutated alcohol oxidase promoter element with specified nucleotide positions relative to SEQ ID NO: 28
A nucleic acid construct comprising a first alcohol oxidase promoter element, wherein the first alcohol oxidase promoter element comprises one or more mutations at a nucleotide position selected from the group consisting of nucleotide positions corresponding to T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862 relative to SEQ ID NO: 28.
Expressing a promoter-linked nucleic acid construct in a cell for producing a product
A method of producing a product in a cell comprising expressing a nucleic acid construct comprising a nucleotide sequence operably linked to a first alcohol oxidase promoter element, wherein the first alcohol oxidase promoter element comprises one or more mutations at a nucleotide position selected from the group consisting of nucleotide positions corresponding to T146, C154, T303, T426, A433, A435, T530, C572, T596, T617, T688, A696, T702, A709, A712, T714, A790, A841, and T862 relative to SEQ ID NO: 28.
Overall, the claim set protects nucleic acid constructs that use an alcohol oxidase promoter element carrying one or more mutations at enumerated positions relative to SEQ ID NO: 28, and it protects methods of producing a product in a cell by expressing a promoter-linked nucleotide sequence using the same mutated promoter element definition.
Stated Advantages
Improved expression phenotype reported for selected AOX1 promoter variants.
Improved GFP expression on media lacking methanol.
Higher titer protein production is described in the partial content.
Expression in methylotrophic yeast is described in the partial content, including protein and heme-binding protein production without added methanol.
Enables expression in the absence of added methanol.
Documented Applications
Use of engineered AOX1 promoter variants to drive GFP expression in methylotrophic yeast (Pichia pastoris) via AOX1-GFP reporter/episomal vectors.
Production of a product in a cell by expressing a nucleic acid construct having a mutated first alcohol oxidase promoter element operably linked to a nucleotide sequence.
Expression that is described as methanol-independent (without adding methanol) for the reporter context described.
Producing a product in a cell by expressing a nucleic acid construct that includes an engineered alcohol oxidase promoter element.
Expression in methylotrophic yeast, including Pichia pastoris and Komagataella species, to produce proteins, heme-binding proteins, and leghemoglobin.
Producing proteins and heme-binding proteins potentially without added methanol.
Producing proteins, including leghemoglobin, by expressing a nucleotide sequence operably linked to the mutated alcohol oxidase promoter element in yeast cells.
Protein production in methylotrophic yeasts using mutated AOX1 promoter elements.
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