Methods for generating a glucose permease library and uses thereof
AssigneeZymergen Inc.
InventorsShawn Manchester, Jeffrey Mellin
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Abstract
The present disclosure describes methods for generating microbial strains expressing a heterologous bacterial glucose permease gene that produce biomolecules of interest. In aspects, the disclosure provides novel bacterial strains, which express a heterologous bacterial glucose permease gene whose expression is controlled by a native Corynebacterium glutamicum promoter or a mutant promoter derived therefrom. Also provided herein are methods for producing a library of bacterial glucose permease genes using a promoter ladder comprising a plurality of promoters derived from Corynebacterium glutamicum.
Claims30
A host cell comprising a heterologous glucose permease gene functionally linked to a first promoter polynucleotide, wherein the first promoter polynucleotide comprises a nucleotide sequence selected from SEQ ID NO:
- 1, SEQ ID NO:
- 2, SEQ ID NO:
- 3, SEQ ID NO:
- 4, SEQ ID NO:
- 5, SEQ ID NO:
- 6, SEQ ID NO:
- 7, and SEQ ID NO:
- 8.
(canceled)
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A method for generating a microorganism capable of increased production of a biomolecule from glucose, the method comprising:
- a) genetically modifying a host microorganism, wherein the modifying comprises introducing a glucose permease gene from a library of glucose permease genes into the genome of the host microorganism, wherein each glucose permease gene from the library of glucose permease genes is functionally linked to a promoter comprising a nucleotide sequence selected from SEQ ID NO:
- 1, SEQ ID NO:
- 2, SEQ ID NO:
- 3, SEQ ID NO:
- 4, SEQ ID NO:
- 5, SEQ ID NO:
- 6, SEQ ID NO:
- 7, and SEQ ID NO:
- 8 and wherein the modification generates a strain of the host microorganism expressing the glucose permease gene;
- b) repeating step a) for a plurality of rounds until a plurality of strains of the host microorganism are generated, wherein each strain of the plurality of strains of the host microorganism expresses a separate glucose permease gene from the library of glucose permease genes;
- c) contacting each strain of the plurality of strains of the host microorganism with a carbon source comprising glucose under fermentative conditions;
- and
- d) selecting each strain of the host microorganism that produces an increased amount of a biomolecule from glucose as compared to the amount of the biomolecule produce from glucose from a control microorganism, wherein the control microorganism does not express a glucose permease gene from the library of glucose permease genes.
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A library of glucose permease genes, wherein each glucose permease gene in the library of glucose permease genes is functionally linked to a promoter comprising a nucleotide sequence selected from SEQ ID NO:
- 1, SEQ ID NO:
- 2, SEQ ID NO:
- 3, SEQ ID NO:
- 4, SEQ ID NO:
- 5, SEQ ID NO:
- 6, SEQ ID NO:
- 7, and SEQ ID NO:
- 8.
(canceled)
-46. (canceled)
-50. (canceled)
-66. (canceled)
Dependencies are inferred from the claim text.