Enhanced protein expression and methods thereof

Abstract

The present disclosure is generally related to modified Gram positive bacterial cells producing increased amounts of one or more protein(s) of interest and modified Gram positive bacterial cells having increased genetic competency. Thus, certain embodiments of the disclosure are directed to modified Gram positive bacterial cells expressing an increased amount of a protein of interest, relative to an unmodified (parental) Gram positive bacterial cell expressing the same protein of interest, wherein the modified bacterial cell comprises at least one mutation in a rpoC gene encoding a variant RNA-polymerase (RNAP) β'-subunit polypeptide. In certain embodiments, the rpoC gene encoding the variant β′-subunit polypeptide is integrated into the chromosome of the modified cell. In other embodiments, the rpoC gene encoding the variant β′-subunit polypeptide is comprised on an extrachromosomal plasmid introduced into the modified cell. In other embodiments, the disclosure is directed to competenthost cells comprising at least one copy of a nucleic acid construct encoding a modified rpoC polypeptide comprising 90% sequence identity to SEQ ID NO: 8 and an aspartic acid to glycine substitution at position 796 of SEQ ID NO: 8, wherein the polynucleotide encoding the rpoC polypeptide is foreign to thehost cell that was non-competent prior to the introduction of the first nucleic acid construct.