Methods for generating a glucose permease library and uses thereof

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.

1 .- 66 . (canceled) 
     
     
         67 . A host cell comprising a heterologous glucose permease 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. 
     
     
         68 . The host cell of  claim 67 , wherein the glucose permease gene is a gene that encodes a polypeptide with an amino acid sequence selected from SEQ ID NO: 13, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 9 and SEQ ID NO: 14. 
     
     
         69 . The host cell of  claim 67 , wherein the glucose permease gene is a gene with a nucleotide sequence selected from SEQ ID NO: 23, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 19 and SEQ ID NO: 24. 
     
     
         70 . The host cell of  claim 67 , further comprising a hexokinase gene functionally linked to a second promoter polynucleotide sequence, wherein the second 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. 
     
     
         71 . The host cell of  claim 70 , wherein the hexokinase gene is a gene that encodes a polypeptide sequence selected from SEQ ID NO: 15 and SEQ ID NO: 16. 
     
     
         72 . The host cell of  claim 70 , wherein the hexokinase gene is a gene with a nucleotide sequence selected from SEQ ID NO: 25 and SEQ ID NO: 26. 
     
     
         73 . 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 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 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.   
     
     
         74 . The method of  claim 73 , wherein the library of glucose permease genes comprises genes that encode polypeptide sequences of SEQ ID NO: 13, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 9, SEQ ID NO: 14 or a combination thereof. 
     
     
         75 . The method of  claim 73 , wherein the library of glucose permease genes comprises genes with a nucleotide sequence of SEQ ID NO: 23, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 19, SEQ ID NO: 24 or a combination thereof. 
     
     
         76 . The method of  claim 73 , further comprising introducing a hexokinase gene from a library of hexokinase genes, wherein each hexokinase gene from the library of hexokinase 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. 
     
     
         77 . The method of  claim 76 , wherein the library of hexokinase genes comprises genes that encode polypeptide sequences of SEQ ID NO: 15 and/or SEQ ID NO: 16. 
     
     
         78 . The method of  claim 76 , wherein the library of hexokinase genes comprises genes with nucleotide sequences of SEQ ID NO: 25 and/or SEQ ID NO: 26. 
     
     
         79 . A library of glucose permease genes, wherein each glucose permease gene in the library of glucose permease 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. 
     
     
         80 . The library of  claim 79 , wherein the library of glucose permease genes comprises genes that encode polypeptide sequences of SEQ ID NO: 13, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 9, SEQ ID NO: 14 or a combination thereof. 
     
     
         81 . The library of  claim 79 , wherein the library of glucose permease genes comprises genes with nucleotide sequences of SEQ ID NO: 23, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 19, SEQ ID NO: 24 or a combination thereof. 
     
     
         82 . The library of  claim 79 , wherein each glucose permease gene in the library of glucose permease genes is a first portion of a chimeric construct, wherein the chimeric construct comprises a second portion, wherein the second portion is a hexokinase gene. 
     
     
         83 . The library of  claim 82 , wherein the hexokinase gene 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. 
     
     
         84 . The library of  claim 83 , wherein the library of hexokinase-genes comprises genes that encode polypeptide sequences of SEQ ID NO: 15 and/or SEQ ID NO: 16. 
     
     
         85 . The library of  claim 83 , wherein the library of hexokinase genes comprises genes with nucleotide sequences of SEQ ID NO: 25 and/or SEQ ID NO: 26. 
     
     
         86 . A method of producing a biomolecule comprising introducing a glucose permease gene from the library of  claim 79  into a host cell and culturing the host cell under conditions suitable for producing the biomolecule.