To increase the menaquinone (MK) content of an Elizabethkingia meningoseptica, site-directed mutagenesis was generated to suppress 4-hydroxybenzoate octaprenyl transferase (UbiA) activity and subsequently blocked the ubiquinone (UQ) biosynthesis pathway. Fourteen conserved residues except L174 and G211 were mutated to analyze the effect of site-directed mutagenesis. The expression of UbiA in twelve mutants was decreased in both mRNA and protein levels, which resulted in the decrease of UQ concentration. Based on MenA expression level, 12 mutants were divided into two groups. Second group such as N72A, D76A, K81A, L139A, and D198A enhanced the expression of MenA, which increased MK production by 127.1%, 87.9%, 96.2%, 109.7% and 130.0% in wt-EmUbiA, respectively. In general, blocking UQ synthesis pathway for by site-directed mutagenesis of the active site of UbiA in E. meningoseptica was a promising strategy to increase MK production in E. meningoseptica.