Abstract: Vitamin B₁₂（VB₁₂/Cobalamin）possesses several physiological functions and is widely used in pharmaceutical and food industries. Pseudomonas denitrificans is commonly employed in the production of VB₁₂. In order to improve the VB₁₂ productivity by a strain，the high-expression promoters were screened and then used to express the gene synthetizing VB₁₂. Via online prediction software，analyzing the promoters in promoters-included non-coding sequences that precede the genes encoding heat shock protein and molecular chaperone in P. denitrification，the non-encoding sequences of P_ibpA，P_cbpA，P_dnaJ，P_htpG，P_dnak，and P_grpE were selected and ligated to GFP report gene encoding green fluorescent protein. Then the GFP fluorescence signal value was detected by enzyme standard instrument，further for obtaining the promoters with high-expression，the expression levels of promoters in the non-coding sequences preceding the genes encoding heat shock protein and molecular chaperone were evaluated，which then was applied for the overexpression of genes in the VB₁₂ synthetic pathways . Fluorescence experimental results showed that the expression of GFP fluorescence value for the non-coding sequence with the promoter P_dnak was the highest. Subsequently，the strongest P_dnak promoter was selected to construct the recombinant P. denitrificans overexpressing gene cobA in VB₁₂ synthesis pathway，and the fermentation results revealed that the VB₁₂ yield increased by 21.5mg/L compared with the control strain. Conclusively，screening high-expression promoter for overexpressing the key gene in VB₁₂ synthesis pathway is an effective approach for improving VB₁₂ production.

Key words: Pseudomonas denitrificans, strong promoter, cobA, vitamin B₁₂, Gibson assembly