Screening of dissolved oxygen induced promoters in Corynebacterium crenatum and functional verification

Abstract:

Abstract:［Objective］Purpose of this work was to screen promoters which were induced by dissolved oxygen in Corynebacterium crenatum SYPA5-5． ［Methods］ Based on the genomic information of Corynebacterium，we identified target proteins which were the highest expression level of one copy of 27 known proteins from our earlier study．Based on the upstream sequence of the coding gene sequences，we amplified two promoters，named P-tkt and P-fum． The tac promoter of recombinant pDXW-8-cat and pDXW-8-gfp were replaced by the new promoters，and the recombinant plasmids were transformed into E.coli JM109 and C.crenatum SYPA5-5 respectively．At different dissolved oxygen level，we compared the function of promoters by the expression of chloramphenicol acetyltransferase (CAT) and green fluorescent protein (GFP).［Results］ Results show that we identified 2 target proteins，which were transketolase and fumarate hydratase．At the high dissolved oxygen level the CAT activity of E.coli JM109/pDXW-Ptkt-cat and C.crenatum SYPA5-5/pDXW-Ptkt-cat were 3.032 U/mg and 1.987 U/mg，which were 2.8-fold and 3.2-fold than that in the low dissolved oxygen，respectively． We got similar results by using a 5-L fermentor.［Conclusion］The P-tkt promoter induced by high dissolved oxygen was suitable for fermentation to produce amino acids and beneficial to improve ability of synthesis and metabolism of amino acids at high dissolved oxygen level.