Materials and methods for protein production

Patent metadata

Field	Value
Publication number	US-11965167-B2
Application number	US-202016858443-A
Country	US
Kind code	B2
Filing date	Apr 24, 2020
Priority date	Apr 25, 2019
Publication date	Apr 23, 2024
Grant date	Apr 23, 2024

Abstract

This document relates to materials and methods for the production of protein. In one aspect, this document provides a first exogenous nucleic acid construct including a nucleotide sequence encoding an aminolevulinate synthase (ALAS) protein operably linked to a first promoter element, wherein the ALAS includes at least a first heme responsive motif (HRM), and wherein the ALAS includes a mutation in the first HRM, and a second exogenous nucleic acid construct comprising a nucleotide sequence encoding a heme-binding protein, wherein the second exogenous nucleic acid construct including a nucleotide sequence encoding the heme-binding protein is operably linked to the first promoter element or is operably linked to a second promoter element.

First claim

What is claimed is:

1. A cell comprising:
a first exogenous nucleic acid construct comprising a nucleotide sequence encoding an aminolevulinate synthase (ALAS) protein operably linked to a first promoter element, wherein the ALAS protein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 29, wherein the ALAS protein comprises at least a first heme responsive motif (HRM), wherein the first HRM comprises the amino acid sequence of SEQ ID NO:32 except for a mutation relative to the amino acid sequence of SEQ ID NO:32, wherein the mutation relative to the amino acid sequence of SEQ ID NO:32 in the first HRM corresponds to a mutation in residue 12 of SEQ ID NO: 29, and wherein the ALAS protein catalyzes the transformation of glycine and succinyl-CoA to aminolevulinic acid; and
a second exogenous nucleic acid construct comprising a nucleotide sequence encoding a heme-binding protein,
wherein the second exogenous nucleic acid construct comprising a nucleotide sequence encoding the heme-binding protein is operably linked to the first promoter element or is operably linked to a second promoter element.

2. The cell of claim 1 , wherein the cell is a bacterial cell, a fungal cell, an algal cell, a plant cell, an insect cell, or a mammalian cell.

3. The cell of claim 1 , wherein the mutation relative to the amino acid sequence of SEQ ID NO:32 in the first HRM is a mutation from a cysteine to a different amino acid.

4. The cell of claim 3 , wherein the ALAS protein comprises a second HRM, wherein the second HRM comprises the amino acid sequence of SEQ ID NO:32 except for a mutation relative to the amino acid sequence of SEQ ID NO:32, and wherein the mutation relative to the amino acid sequence of SEQ ID NO:32 in the second HRM corresponds to a mutation in residue 39 of SEQ ID NO: 29.

5. The cell of claim 4 , wherein the mutation relative to the amino acid sequence of SEQ ID NO:32 in the second HRM is a mutation from a cysteine to a different amino acid.

6. The cell of claim 5 , wherein the different amino acid is the same for the mutation relative to the amino acid sequence of SEQ ID NO:32 in the first HRM and the mutation relative to the amino acid sequence of SEQ ID NO:32 in the second HRM.

7. The cell of claim 3 , wherein the different amino acid is serine, alanine, phenylalanine, or histidine.

8. The cell of claim 1 , wherein the ALAS protein comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 29.

9. The cell of claim 1 , wherein the heme-binding protein is selected from the group consisting of a globin, a cytochrome, a cytochrome c oxidase, a ligninase, a catalase, and a peroxidase.

10. The cell of claim 1 , wherein the first HRM has the amino acid sequence of any one of SEQ ID NOs: 33-36 except for a mutation relative to the amino acid sequence of any one of SEQ ID NOs:33-36.

11. The cell of claim 4 , wherein the second HRM has the amino acid sequence of any one of SEQ ID NOs: 33-36 except for a mutation relative to the amino acid sequence of any one of SEQ ID NOs:33-36.

12. The cell of claim 1 , wherein the ALAS protein comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 29.

13. The cell of claim 1 , wherein the cell is a bacterial cell.

14. The cell of claim 1 , wherein the cell is a fungal cell.

15. The cell of claim 1 , wherein the cell is an algal cell.

16. The cell of claim 1 , wherein the cell is a mammalian cell.

17. The cell of claim 5 , wherein the different amino acid in the second HRM is serine, alanine, phenylalanine, or histidine.

18. The cell of claim 5 , wherein the different amino acid is different for the mutation relative to the amino acid sequence of SEQ ID NO:32 in the first HRM and the mutation relative to the amino acid sequence of SEQ ID NO:32 in the second HRM.

19. The cell of claim 6 , wherein the different amino acid is serine, alanine, phenylalanine, or histidine.

Assignees

• Impossible Foods Inc

Inventors

• Roy Chaudhuri Biswajoy
• Shankar Smita

Classifications

• C12N15/52 Primary

  Genes encoding for enzymes or proenzymes · CPC title

• C12Y203/01037 Primary

  5-Aminolevulinate synthase (2.3.1.37) · CPC title

• C12N15/81 Primary

  for yeasts · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title