Systems and methods for enhancing gene expression
View Patent ↗The present invention provides for a system for increasing the production of a compound using an artificial positive feedback loop (APFL). In some embodiments, the system diverts a compound produced in a first metabolic pathway into a second metabolic pathway in order to produce a compound of interest.
1. A genetically modified fungal host cell, comprising: (a) a first polynucleotide encoding a fungal transcription factor operably linked to a first promoter that is native to the transcription factor, (b) a second polynucleotide encoding a first biosynthetic enzyme operably linked to a second promoter that is induced or activated by the transcription factor, wherein the first biosynthetic enzyme catalyzes a first reaction which produces a first compound from a second compound, and (c) a third polynucleotide encoding an expression cassette inserted between a polynucleotide encoding a second biosynthetic enzyme and a native promoter of the second biosynthetic enzyme, wherein the expression cassette comprises a polynucleotide encoding the fungal transcription factor, a third promoter that is induced at a level lower than the induction of the first promoter and the second promoter, and a terminator located between the polynucleotide encoding the fungal transcription factor and the third promoter;
such that the native promoter of the second biosynthetic enzyme is operatively linked to the polynucleotide encoding the fungal transcription factor, and the third promoter is operatively linked to the polynucleotide encoding the second biosynthetic enzyme; wherein the transcription factor is Upc2, Ecm22, Upc2G888D, or Ecm22G790D and the second biosynthetic enzyme catalyzes a second reaction that is downstream of the first reaction in a metabolic pathway.
2. The genetically modified fungal host cell of claim 1 , wherein any two or all of the first polynucleotide, second nucleotide, and third polynucleotide are of the same polynucleotide molecule.
3. The genetically modified fungal host cell of claim 1 , wherein the third promoter is a met3 or met25 promoter.
4. The genetically modified fungal host cell of claim 1 , wherein the fungal host cell is a yeast host cell.
5. The genetically modified fungal host cell of claim 1 , wherein the genetically modified fungal host cell overproduces the first compound.
6. The genetically modified fungal host cell of claim 5 , wherein the first biosynthetic enzyme is ERG19, the second biosynthetic enzyme is ERG9, and the first compound is farnesyl diphosphate (FPP).
7. The genetically modified fungal host cell of claim 6 , wherein the transcription factor is Upc2 or Upc2G888D.
8. A method of producing a first compound, the method comprising:
(a) providing a genetically modified fungal host cell of claim 1 , and
(b) culturing the host cell under conditions in which the transcription factor is expressed;
such that the first compound is produced.
9. The method of claim 8 , wherein any two or all of the first polynucleotide, second nucleotide, and third polynucleotide are the same polynucleotide.
10. The method of claim 8 , wherein the second promoter that is induced or activated by the transcription factor is a erg9 promoter, or functional variant thereof.
11. The method of claim 8 , wherein the third promoter that is induced at a level lower than the induction of the first promoter and the second promoter is a the met3 or met25 promoter, or functional variant thereof.
12. The method of claim 8 , wherein the providing step (a) comprises introducing one or more of the first polynucleotide, second polynucleotide, and third polynucleotide into the host cell.
13. The method of claim 8 , wherein the fungal host cell is a yeast host cell.