IP Library › Granted Patent US 12,331,302

Granted Patent B2

US 12,331,302 · App. 17/092,031 · Granted Jun 17, 2025

Fungal autoinducible expression system

Inventors: Amin Zargar (Emeryville, CA); Jenny Landberg (Berkeley, CA); Jay D. Keasling (Berkeley, CA)

Assignee: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA

C12N15/81 C12P21/00 C12N2800/102 C12N2830/002

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Quick Facts

Patent No.: US 12,331,302
App. No.: 17/092,031
Granted: Jun 17, 2025
Kind: B2

External Links

USPTO Patent Center↗ Full Patent PDF↗

Abstract

The present invention provides for a system comprising: (a) a first nucleic acid encoding an α-factor receptor operatively linked to a first promoter, (b) a second nucleic acid encoding a recombinase operatively linked to a promoter which is activated by an α-factor receptor bound to an α-factor, and (c) a third nucleic acid encoding a gene of interest (GOI) flanked by a pair of recombinase recognition sequences, recognized by the recombinase, operatively linked to a second promoter. The present invention provides for a genetically modified fungal cell comprising the system of the present invention.

Claims (330)

1. A system comprising: (a) a first nucleic acid encoding an α-factor receptor operatively linked to a first promoter, (b) a second nucleic acid encoding a recombinase operatively linked to a promoter which is activated by the α-factor receptor bound to a yeast α-factor, and (c) a third nucleic acid comprising a gene of interest (GOI) flanked by a pair of recombinase recognition sequences, recognized by the recombinase, operatively linked to a second promoter; wherein the yeast α-factor is MFα1 gene product and the α-factor receptor is STE2, and the recombinase is one selected from the following:

Name

Host

BSu_xerC

Bacillus subtilis

BSu_xerD

Bacillus subtilis

BSu_ydcL

Bacillus subtilis

CBu_tnpA

Clostridium butyricum

Col1D

Escherichia coli

CP4-57

Escherichia coli

Cre

Escherichia coli

D29

Mycobacterium smegmatis

DLP12

Escherichia coli

DNo_int

Dichelobacter nodosus

ECo_fimB

Escherichia coli

ECo_fimE

Escherichia coli

ECo_orf

Escherichia coli

ECo_xerC

Escherichia coli

ECo_xerD

Escherichia coli

HIn_orf

Haemophilus influenzae

HIn_rci

Haemophilus influenzae

HIn_xerC

Haemophilus influenzae

HIn_xerD

Haemophilus influenzae

HK22

Escherichia coli

HP1

Haemophilus influenzae

Acholeplasma sp.

Mycobacterium tuberculosis

L54

Staphylococcus aureus

Lambda

Escherichia coli

LLe_orf

Lactobacillus leichmannii

LLe_xerC

Lactobacillus leichmannii

phi10MC

Oenococcus oeni

MJa_orf

Methanococcus jannaschi

MLe_xerD

Mycobacterium leprae

MPa_int

Mycobacterium paratuberculosis

MTu_int

Mycobacterium tuberculosis

MTu_xerC

Mycobacterium tuberculosis

MV4

Lactobacillus delbrueckii

MX8

Myxococcus xanthus

pAE1

Alcaligenes eutrophus

pCL1

Chlorobium limicola

pDU1

Nostoc sp.

pMEA

Amycolatopsis methanolica

RSp_EF

Rhizobium sp.

RSp_GC

Rhizobium sp.

RSp_QK

Rhizobium sp.

RSp_RA

Rhizobium sp.

RSp_RB

Rhizobium sp.

RSp_RC

Rhizobium sp.

RSp_RD

Rhizobium sp.

RSp_RE

Rhizobium sp.

RSp_RF

Rhizobium sp.

pSAM2

Streptomyces ambofaciens

pSDL2

Salmonella dublin

pSE101

Saccharopolyspora erythraea

pSE211

Saccharopolyspora erythraea

pWS58

Lactobacillus delbrueckii

phi-11

Staphylococcus aureus

phi-13

Staphylococcus aureus

phi-80

Escherichia coli phage

phi-adh

Lactobacillus gasseri

phi-CTX

Pseudomonas aeruginosa

phi-g1e

Lactobacillus sp.

phi-LC3

Lactococcus lactis

phi-R73

Escherichia coli

P186

Escherichia coli

P21

Escherichia coli

P22

Salmonella typhimurium

Escherichia coli

P434

Escherichia coli

PAe_xerC

Pseudomonas aeruginosa

PMi_fimB

Proteus mirabilis

R721

Escherichia coli

Rci

Escherichia coli

SF6

Shigella flexneri

SLP1

Streptomyces coelicolor

IntI3

Serratia marcescens

SsrA

Methanosarcina acetivorans

SSV1

Sulfolobus sp.

T12

Streptococcus pyogenes

IntI1

Escherichia coli

Tn4430

Bacillus thuringiensis

Tn5041

Pseudomonas sp.

Tn5252

Streptococcus pneumoniae

Tn5276

Lactobacillus lactis

Tn554a

Staphylococcus aureus

Tn554b

Staphylococcus aureus

IntI2

Escherichia coli

Tn916

Entercoccus faecalis

Tuc

Lactobacillus lactis

BZo_int

Bergeyella zoohelcum

ASp_xisA

Anabaena sp.

ASp_xisC

Anabaena sp.

FLP

Saccharomyces cerevisiae

pKD1

Kluyveromyces lactis

pSB2

Zygosaccharomyces bailii

pSB3

Zygosaccharomyces bisporus

pSM1

Zygosaccharomyces fermentati

pSR1

Zygosaccharomyces rouxii

HPy_xerC

Helicobacter pylori

HPy_xerD

Helicobacter pylori

Eco_Rac

Escherichia coli

100

Eco_Qin

Escherichia coli

101

CP4-6

Escherichia coli

102

E14

Escherichia coli .

2. The system of claim 1 , wherein the first promoter is a native promoter of STE2.

3. The system of claim 1 , wherein the promoter which is activated by an α-factor receptor bound to an α-factor is a FUS1 promoter.

4. The system of claim 1 , wherein the first nucleic acid is stably integrated in a chromosome.

5. The system of claim 1 , wherein the second promoter is a constitutive promoter.

6. The system of claim 1 , wherein the second nucleic acid is stably integrated into a chromosome.

7. The system of claim 1 , wherein the second nucleic acid is an input plasmid.

8. The system of claim 1 , wherein the third nucleic acid is an output plasmid.

9. The system of claim 1 , wherein the recombinase is Escherichia coli Cre, Escherichia coli FimE, Zygosaccharomyces bailii pSB2, Zygosaccharomyces fermentati pSM1, Helicobacter pylori XerC, or Helicobacter pylori XerD.

10. The system of claim 1 , wherein the recombinase comprises a protein degradation tag.

11. The system of claim 1 , wherein the promoter which is activated by an α-factor receptor bound to an α-factor is a FUS1/2 promoter.

12. The system of claim 1 , wherein the first promoter is a first inducible promoter.

13. The system of claim 1 , wherein the first nucleic acid further comprises a MFα1 gene operatively linked to a second inducible promoter.

14. The system of claim 1 , wherein the nucleic acid encoding the x-factor receptor is operatively linked to P STE2 and/or P VAR* .

15. The system of claim 1 , wherein the second nucleic acid further comprises nucleic acid encoding BAR1 operatively linked to P TETO3 , and/or nucleic acid encoding rtTA* operatively linked to P TDH3 .

16. A genetically modified fungal cell comprising the system of claim 1 , wherein the fungal cell is a yeast cell with an endogenous α-factor.

17. The genetically modified fungal cell of claim 16 , wherein the yeast cell is a Saccharomyces cell.

18. The genetically modified fungal cell of claim 17 , wherein the Saccharomyces cell is a Saccharomyces cerevisiae cell.

19. The genetically modified fungal cell of claim 18 , wherein the Saccharomyces cerevisiae cell is a cell of the Saccharomyces cerevisiae BY4741 strain.

20. A method comprising: (a) providing a system of claim 1 , (b) introducing or expressing an α-factor to the system, and (c) expressing the GOI.

21. A method comprising: (a) providing a genetically modified fungal cell of claim 16 , (b) introducing or expressing an α-factor to the system, and (c) expressing the GOI.

Assignments (2)

CONFIRMATORY LICENSE Recorded Feb 26, 2021

From: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB

To: UNITED STATES DEPARTMENT OF ENERGY

Reel/Frame 055423/0480 →

ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Dec 11, 2020

From: ZARGAR, AMIN; LANDBERG, JENNY; KEASLING, JAY D.

To: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA

Reel/Frame 054617/0738 →

Continuity (2)

Provisional Application 62933191 · Nov 8, 2019

Related Publication 20210139923A1 · May 13, 2021

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