An indispensable fat-soluble vitamin, menaquinone (MK) has promising applications and potential economic value for industrial production. Traditionally, MK production has been improved through the development of better strains by a repeated mutation-selection approach. However, because of its randomness and low screening efficiency from larger mutant libraries, metabolic engineering was attempted to improve strains and the fermentation process. Site-directed mutation of 4-hydroxybenzoate prenyltransferase (UbiA) D198 to Ala has been reported to increase MK content by 181% in Ellxabethkingla meningoseptica. For additional increases, several systematic metabolic engineering strategies, such as co-expression of rate-limiting genes and supplementation with substrate precursors, were employed. Co-expression of dxr and menA and supplementation with sodium pyruvate (SPY) and shikimic add (SA) led to 397% more MK content in EmUbiA (D198A) than that without SPY and SA (CK) in the wild type. Moreover, a 145% increase in the MK content in EmUbiA (D198A) was realized by the combination of co-expression of dxr, menA, and ubiE, and supplementation with SPY and SA compared to that of this combination without ubiE. Overall, MK content in E. meningoseptica increased more than 11 times by weakening the ubiquinone pathway, co-expressing dxr, menA and ubiE, and adding SPY and SA to the medium.