Methods for generating a glucose permease library and uses thereof

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.