High gene expression yeast strain
OwnerKyle Schneider, Catherine L. Foune +3inventors
InventorsKyle Schneider, Catherine L. Foune, Lin Bao, Robert Venable, Robert M. Kennedy
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Abstract
New insecticidal proteins, nucleotides, peptides, their expression in plants, methods of producing the peptides, new processes, production techniques, new peptides, new formulations, new organisms, and a process which increases the insecticidal peptide production yield from yeast expression systems. The present invention is also related to novel cell culture methods and conditions that can be used to express heterologous polypeptides, along with new transgenic yeast strains.
Claims45
A method of expressing one or more heterologous Cysteine-Rich Bioactive Peptides (CRBPs), the method comprising:
- culturing a modified yeast cell in the presence of a sugar alcohol, wherein the modified yeast cell is a Kluyveromyces lactis cell or a Kluyveromyces marxianus cell;
- wherein the modified yeast cell comprises:
- (i) a polynucleotide encoding a CRBP;
- and (ii) at least one modification rendering an endogenous Ga180 gene or an endogenous Ga180 activity at least partially inoperable or partially inactive.
A method of producing one or more Cysteine-Rich Bioactive Peptides (CRBPs) in a yeast cell, wherein the yeast cell is selected from a Kluyveromyces lactis cell or a Kluyveromyces marxianus cell, the method comprising:
- a. preparing a vector comprising a first expression cassette flanked by a site-specific integration (SSI) sequence, wherein the first expression cassette contains a polynucleotide encoding a CRBP, or complementary nucleotide sequence thereof, and wherein the site-specific integration (SSI) sequence is operable to knock-out Ga180 or ndt80;
- b. introducing the vector into the yeast cell, wherein the vector permits integration of the first expression cassette into the yeast genome by knocking-out Ga180 or ndt80, and knocking-in said first expression cassette;
- and
- c. growing the yeast cell in a growth medium containing a sugar alcohol, under fermentation conditions operable to enable expression of the CRBP and secretion into the growth medium.
A modified Kluyveromyces lactis or Kluyveromyces marxianus yeast strain comprising:
- a. at least one genome modification rendering an endogenous Ga180 gene or an endogenous Ga180 activity at least partially inoperable or partially inactive;
- and
- b. a heterologous first expression cassette comprising a a polynucleotide encoding a CRBP, or a complementary nucleotide sequence thereof, wherein the ratio of the CRBP produced by the modified Kluyveromyces lactis or Kluyveromyces marxianus yeast strain grown in a medium containing a sugar alcohol, relative to the yeast strain grown in a medium not containing sugar alcohol, is at least from about 2:1 to about 10,000:1.
Dependencies are inferred from the claim text.