Methods for generating a bacterial hemoglobin library and uses thereof

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.

1 .- 46 . (canceled) 
     
     
         47 . A host cell comprising a heterologous bacterial hemoglobin gene functionally linked to a first promoter polynucleotide sequence, wherein the first promoter polynucleotide sequence is derived from  Corynebacterium glutamicum , is less than 100 base pairs in length, is able to constitutively express genes across different growth conditions, and is able to form a ladder of promoters comprising a plurality of promoters with incrementally increasing levels of promoter activity. 
     
     
         48 . The host cell of  claim 47 , wherein the bacterial hemoglobin gene is a gene with a nucleotide sequence selected from SEQ ID NO: 12, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. 
     
     
         49 . The host cell of  claim 47 , wherein the bacterial hemoglobin gene encodes a polypeptide with an amino acid sequence selected from SEQ ID NO: 26, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34. 
     
     
         50 . The host cell of  claim 47 , wherein the host cell is  Corynebacterium glutamicum.    
     
     
         51 . A method of producing a biomolecule comprising culturing the host cell of  claim 47  under conditions suitable for producing the biomolecule. 
     
     
         52 . 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 polynucleotide sequence, wherein the promoter polynucleotide sequence is derived from  Corynebacterium glutamicum , is less than 100 base pairs in length, is able to constitutively express genes across different growth conditions, and is able to form a ladder of promoters comprising a plurality of promoters with incrementally increasing levels of promoter activity, 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.   
     
     
         53 . The method of  claim 52 , wherein the library of bacterial hemoglobin genes comprises one or more bacterial hemoglobin genes with a nucleotide sequence selected from SEQ ID NO: 12, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. 
     
     
         54 . The method of  claim 52 , wherein the library of bacterial hemoglobin genes comprises one or more bacterial hemoglobin genes that encode one or more polypeptide sequences selected from SEQ ID NO: 26, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34. 
     
     
         55 . The method of  claim 52 , wherein the library of bacterial hemoglobin genes comprises one or more bacterial hemoglobin genes from a strain, species, or sub-species of a microorganism listed in Table 2. 
     
     
         56 . The method of  claim 52 , wherein the library of bacterial hemoglobin genes comprises one or more bacterial flavohemoglobin genes from a strain, species, or sub-species of a microorganism listed in Table 2. 
     
     
         57 . The method of  claim 52 , wherein the library of bacterial hemoglobin genes comprises one or more bacterial hemoglobin genes from a strain, species, or sub-species of a microorganism listed in Table 2 and one or more bacterial flavohemoglobin genes from a strain, species, or sub-species of a microorganism listed in Table 2. 
     
     
         58 . The method of  claim 52 , wherein the host microorganism is  Corynebacterium  glutamicum. 
     
     
         59 . A library of bacterial hemoglobin genes, wherein each bacterial hemoglobin gene in the library of bacterial hemoglobin genes is functionally linked to a promoter polynucleotide sequence, wherein the promoter polynucleotide sequence is derived from  Corynebacterium glutamicum , is less than 100 base pairs in length, is able to constitutively express genes across different growth conditions, and is able to form a ladder of promoters comprising a plurality of promoters with incrementally increasing levels of promoter activity. 
     
     
         60 . The library of  claim 59 , wherein the library of bacterial hemoglobin genes comprises one or more bacterial hemoglobin genes selected from SEQ ID NO: 12, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. 
     
     
         61 . The library of  claim 59 , wherein the library of bacterial hemoglobin genes comprises one or more bacterial hemoglobin genes that encode one or more polypeptide sequences selected from SEQ ID NO: 26, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and SEQ ID NO: 34. 
     
     
         62 . The library of  claim 59 , wherein the library of bacterial hemoglobin genes comprises one or more bacterial hemoglobin genes from a strain, species, or sub-species of a microorganism listed in Table 2. 
     
     
         63 . The library of  claim 59 , wherein the library of bacterial hemoglobin genes comprises one or more bacterial flavohemoglobin genes from a strain, species, or sub-species of a microorganism listed in Table 2. 
     
     
         64 . The library of  claim 59  wherein the library of bacterial hemoglobin genes comprises one or more bacterial hemoglobin genes from a strain, species, or sub-species of a microorganism listed in Table 2 and one or more bacterial flavohemoglobins genes from a strain, species, or sub-species of a microorganism listed in Table 2. 
     
     
         65 . A method of producing a biomolecule comprising introducing a bacterial hemoglobin gene from the library of  claim 59  into a host cell and culturing the host cell under conditions suitable for producing the biomolecule. 
     
     
         66 . The method of  claim 65 , wherein the host cell is  Corynebacterium glutamicum.