Construction of sucA-Knockout E. coli Strains and Optimization of Fermentation Conditions

Abstract:

trans-4-hydroxyproline has important applications in many fields. On the basis of the constructed< i> E. coli BL21（DE3）ΔputA strain，sucA gene was knocked out and the optimized trans-4-hydroxylase gene（hyp） was introduced. Then，the constructed plasmid pUC19-< i>ptrp2-hyp-vgb which included a tryptophan tandem promoter and Vitreoscilla hemoglobin（VHb） was imported into the recombinant strain and overexpressed. Then in the flask level，fermentation conditions and medium components were confirmed by single factor optimization and orthogonal experiment. The results showed that，compared with the original bacteria and two single knockout strains containing the same plasmid，the double knockout strain had the highest whole-cell activity，which is about 2.6 times as the original bacteria. After optimizing the fermentation conditions，trans-4-hydroxyproline was accumulated to 1.08 g/L，which increased 2.87 fold.