Methods for generating a bacterial hemoglobin library and uses thereof
AssigneeZymergen Inc.
InventorsShawn Manchester, Alexander Neckelmann
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Abstract
The present disclosure describes methods for generating microbial strains expressing a heterologous bacterial hemoglobin gene that produce biomolecules of interest. In aspects, the disclosure provides novel bacterial strains, which express a heterologous bacterial hemoglobin 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 hemoglobin genes using a promoter ladder comprising a plurality of promoters derived from Corynebacterium glutamicum.
Claims30
A host cell comprising a heterologous bacterial hemoglobin gene functionally linked to a first promoter polynucleotide, wherein the first promoter polynucleotide comprises a 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.
-7. (canceled)
-13. (canceled)
A method for generating a microorganism capable of increased production of a biomolecule, the method comprising:
- a) genetically modifying a host microorganism, wherein the modifying comprises introducing a bacterial hemoglobin gene from a library of bacterial hemoglobin genes into the genome of the host microorganism, wherein each bacterial hemoglobin gene from the library of bacterial hemoglobin 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 bacterial hemoglobin 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 bacterial hemoglobin gene from the library of bacterial hemoglobin genes;
- c) contacting each strain of the plurality of strains of the host microorganism with a carbon source under fermentative conditions;
- and
- d) selecting each strain of the host microorganism that produces an increased amount of a biomolecule as compared to the amount of the biomolecule produced from a control microorganism, wherein the control microorganism does not express a bacterial hemoglobin gene from the library of bacterial hemoglobin genes.
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(canceled)
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-28. (canceled)
A library of bacterial hemoglobin genes, wherein each bacterial hemoglobin gene in the library of bacterial hemoglobin 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)
(canceled)
-42. (canceled)
-46. (canceled)
Dependencies are inferred from the claim text.