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US-20260125432-A1 - MALTOSE DEPENDENT DEGRONS, MALTOSE-RESPONSIVE PROMOTERS, STABILIZATION CONSTRUCTS, AND THEIR USE IN PRODUCTION OF NON-CATABOLIC COMPOUNDS

US20260125432A1US 20260125432 A1US20260125432 A1US 20260125432A1US-20260125432-A1

Abstract

The present disclosure relates to the use of a maltose dependent degron to control stability of a protein of interest fused thereto at the post-translational level. The present disclosure also relates to the use of a maltose dependent degron in combination with a maltose-responsive promoter to control gene expression at the transcriptional level and to control protein stability at the post-translational level. The present disclosure also relates to the use of a stabilization construct that couples expression of a cell-growth-affecting protein with the production of non-catabolic compounds. The present disclosure further relates to the use of a synthetic maltose-responsive promoter. The present disclosure further provides compositions and methods for using a maltose dependent degron, a maltose-responsive promoter, and a stabilization construct, either alone or in various combinations, for the production of non-catabolic compounds in genetically modified host cells.

Inventors

  • Penelope R. CHUA
  • Hanxiao Jiang
  • Adam Leon Meadows

Assignees

  • AMYRIS, INC

Dates

Publication Date
20260507
Application Date
20250812

Claims (20)

  1. 1 . A method of modulating protein stability, the method comprising contacting, with a maltose based inducer, a fusion protein comprising a protein of interest fused in frame to a maltose dependent degron, wherein the fusion protein is more stable when the maltose dependent degron is in contact with the maltose based inducer compared to when the maltose dependent degron is not in contact with the maltose based inducer.
  2. 2 . The method of claim 1 , further comprising removing the maltose based inducer from contact with the fusion protein.
  3. 3 . The method of claim 1 or 2 , wherein a half-life of the fusion protein after removing the maltose based inducer from contact with the fusion protein is at least about 50% less compared to a half-life of a fusion protein comprising a wild-type MBP fused in frame to the protein of interest.
  4. 4 . The method of any one of claims 1 to 3 , the method further comprising providing a host cell comprising a heterologous nucleic acid encoding the fusion protein, wherein the contacting the fusion protein with the maltose based inducer comprises culturing the host cell expressing the fusion protein in a culture medium comprising the maltose based inducer.
  5. 5 . The method of any one of claims 1 to 4 , wherein the fusion protein directly or indirectly modulates the levels of one or more target molecules.
  6. 6 . The method of claim 4 or 5 , wherein the host cell further comprises a biomolecule that interacts with the fusion protein in the host cell to modulate the levels of the one or more target molecules.
  7. 7 . The method of any one of claims 1 to 6 , wherein the protein of interest is a transcriptional regulator.
  8. 8 . The method of claim 7 , wherein the protein of interest comprises Gal80p.
  9. 9 . The method of any one of claims 6 to 8 , wherein the biomolecule is a transcriptional activator.
  10. 10 . The method of any one of claims 5 to 9 , wherein the one or more target molecules are enzymes in a biosynthetic pathway that produces a non-catabolic compound.
  11. 11 . The method of any one of claims 5 to 9 , wherein the one or more target molecules comprise a non-catabolic compound.
  12. 12 . The method of any one of claims 4 to 11 , wherein the heterologous nucleic acid sequence encoding the fusion protein is integrated into a genome of the host cell.
  13. 13 . The method of any one of claims 4 to 12 , wherein the heterologous nucleic acid encoding the fusion protein is operably linked to an endogenous promoter of a gene encoding the protein of interest.
  14. 14 . The method of any one of claims 4 to 12 , wherein the heterologous nucleic acid encoding the fusion protein is operably linked to a maltose-responsive promoter.
  15. 15 . The method of any one of claims 1 to 14 , wherein the maltose dependent degron in the fusion protein comprises an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 28 and comprises one or more variant amino acid residues compared to SEQ ID NO: 2 or SEQ ID NO: 28.
  16. 16 . The method of any one of claims 1 to 14 , wherein the maltose dependent degron in the fusion protein comprises an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 28 and comprises one or more variant amino acid residues that are located at one or more positions selected from 7, 10, 11, 21, 24, 28, 42, 43, 64, 68, 83, 88, 92, 95, 98, 101, 110, 117, 134, 135, 136, 149, 168, 177, 186, 187, 193, 198, 210, 216, 217, 229, 236, 237, 242, 263, 291, 304, 321, 322, 339, 351, 357, 367, 370, and 374, wherein the positions of these variant amino acid residues correspond to amino acid positions of SEQ ID NO: 2 or SEQ ID NO: 28.
  17. 17 . The method of any one of claims 1 to 14 , wherein the maltose dependent degron in the fusion protein comprises an amino acid sequence that has at least about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 2 or SEQ ID NO: 28, and comprises one or more variant amino acid residues selected from the group consisting of K7R, I10T, W11G, L21S, V24A, F28Y, D42V, K43E, A64T, F68S, D83G, D88N, P92T, W95R, V98I, N101I, A110 T, I117V, P134S, A135T, L136M, M149I, Y168C, Y168N, Y177H, N186S, A187P, L193S, D198V, D210E, A216V, A217D, G229C, I236N, D237N, N242D, L263M, L291V, A304S, T321N, M322L, A339T, A351T, T357S, T367S, S370P, and N374S, wherein positions of the one or more variant amino acid residues correspond to amino acid positions of SEQ ID NO: 2 or SEQ ID NO: 28.
  18. 18 . The method of any one of claims 15 to 17 , wherein the maltose dependent degron is truncated after position 365 compared to SEQ ID NO: 2.
  19. 19 . The method of claim 17 or 18 , wherein the one or more variant amino acid residues comprise I10T, V24A, D42V, M149I, and A216V, wherein positions of the one or more variant amino acid residues correspond to amino acid positions of SEQ ID NO: 2.
  20. 20 . The method of any one of claims 17 to 19 , wherein the maltose dependent degron comprises at least one set of variant amino acid residues compared to SEQ ID NO: 2 or SEQ ID NO: 28, wherein the at least one set of variant amino acid residues is selected from the group of sets of variant amino acid residues consisting of: (a) I10T, V24A, D42V, K43E, D83G, P92T, M149I, Y168N, N186S, A216V, and T357S; (b) I10T, V24A, D42V, K43E, D83G, M149I, Y168N, N186S, A216V, and D237N; (c) I10T, V24A, D42V, K43E, D83G, M149I, Y168N, N186S, A216V, and A339T; (d) I10T, V24A, D42V, K43E, D83G, M149I, Y168N, N186S, A216V, and N242D; (e) I10T, V24A, D42V, A110T, M149I, and A216V; (f) I10T, V24A, D42V, K43E, D83G, M149I, Y168N, N186S, and A216V; (g) L21S, A64T, L136M, Y177H, A187P, A304S, T321N, and A351T; (h) K7R, D83G, V98I, L193S, I236N, and N374S; (i) W11G, D88N, P134S, A135T, D210E, and M322L; (j) I117V, Y168N, G229C, L263M, T367S, and S370P; (k) F68S, W95R, N186S, and D198V; and (l) F28Y, K43E, N101I, Y168C, A217D, and L291V.

Description

1. CROSS REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. patent application Ser. No. 17/013,246, fled on Sep. 4, 2020, which is a divisional of U.S. patent application Ser. No. 15/738,555, filed on Dec. 20, 2017, which is the U.S. 371 National Stage Application of PCT International Application No. PCT/US2016/039386, filed on Jun. 24, 2016, which claims benefit of priority to U.S. Provisional Application No. 62/184,793, filed Jun. 25, 2015, and U.S. Provisional Patent Application No. 62/266,436, filed Dec. 11, 2015, all of which are incorporated herein by reference. REFERENCE TO ELECTRONIC SEQUENCE LISTING The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on Dec. 29, 2025, is named “107345.00969.xml” and is 235,676 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety. 2. FIELD OF THE INVENTION The present disclosure generally relates to maltose dependent degrons, maltose-responsive promoters, and stabilization constructs. The present disclosure also relates to their use in controlling gene expression, protein stability, and production of non-catabolic compounds by genetically mod fed host cells. 3. BACKGROUND The advent of synthetic biology has brought about the promise of fermentative microbial production of biofuels, chemicals and biomaterials from renewable sources at industrial scale and quality. For example, functional non-native biological pathways have been successfully constructed in microbial hosts for the production of precursors to the antimalarial drug artemisinin (see, e.g., Martin et al., Nat Biotechnol 21:796-802 (2003)); fatty ad d derived fuels and chemicals (e.g., fatty esters, fatty alcohols and waxes; see, e.g., Steen et al., Nature 463: 559-562 (2010)); polyketide synthases that make cholesterol lowering drugs (see, e.g., Ma et al., Science 326:589-592 (2009)); and polyketides (see, e.g., Kodumal, Proc Natl Acad Sci USA 101:15573-15578 (2004)). However, the commercial success of synthetic biology will depend largely on whether the production cost of renewable products can be made to compete with, or out-compete, the production costs of their respective non-renewable counterparts. Strain stability can be a major driver of the cost of industrial fermentations, as it affects the length of time that a continuous fermentation can be run productively. Strain stability generally refers to the ability of a microbe to maintain favorable production characteristics (e.g., high yield (grams of compound per gram of substrate) and productivity (grams per liter of fermentation broth per hour) of a non-catabolic fermentation product over extended cultivation times. In particular, genetic stability, which is the propensity of the producing microbial population to have little to no alteration of the intended allelic. frequency of genes relevant to the production of product over time, plays a major role in the sustained output of product. For non-catabolic fermentation of products other than biomass (which products, by definition, consume metabolic energy and carbon that could otherwise be used in the production of more cells), the basis of instability is two-fold: evolutionary mutation and selection. First, loss-of-production mutations arise spontaneously and randomly. Second, a growth rate or “fitness” advantage of cells with reduced product yields leads to an eventual population sweep by low producers, and thereby decreases the overall culture performance. This phenomenon can be referred to as “strain degeneration.” Brazilian fuel ethanol fermentations achieve extremely high yields of ethanol from sugar for long periods of time, i.e., about 90% of maximum theoretical yield. This is in part because the production of ethanol is catabolic: it generates 2 ATP per molecule of sugar produced and is redox balanced without the involvement of oxygen. A cell that mutates to not produce ethanol is less fit under the low oxygen conditions of the fermentor and will not sweep the population. This allows industrial ethanol fermentations to recycle the majority of yeast biomass throughout the season, thereby minimizing conversion of sugar into yeast cell biomass and directing nearly all of the sugar to ethanol production. This extended propagation and re-use of biomass increases the efficiencies of ethanol production: operational expenditures are reduced because less sugar goes to biomass during each cycle (i.e., the yield increases); and capital expenditures are reduced because fewer and smaller fermentors are needed to build biomass for inoculations. By contrast, the production of acetyl-CoA derived hydrocarbons (e.g., isoprenoids, fatty acids, and polyketides) are generally non-catabolic in nature; they usually require a net input of ATP, NADPH, and carbon, o