ANAEROBICALLY CONTROLLED EXPRESSION SYSTEM DERIVED FROM THE ARCDABC OPERON OF PSEUDOMONAS-AERUGINOSA - APPLICATION TO LIPASE PRODUCTION

The anaerobically inducible arcDABC operon encodes the enzymes of the arginine deiminase pathway in Pseudomonas aeruginosa, Upon induction, the arcAB mRNAs and proteins reach high intracellular levels, because of a strong anaerobically controlled promoter and mRNA processing in a rcD, leading to stable downstream transcripts, We explored the usefuln ess of this system for the construction of expression vectors. The lac Z gene of Escherichia coli was expressed to the highest levels when fu sed close to the arc promoter, Insertion of lacZ further downstream in to arcA or arcB did not stabilize the intrinsically unstable lacZ mRNA , On the contrary, lacZ mRNA appeared to be a vulnerable endonuclease target destabilizing arcAB mRNAs in the 5'-to-3' direction in P. aerug inosa, The native are promoter was modified for optimal expression in the -10 sequence and in the -40 region, Which is a binding site for th e anaerobic regulator ANR. In P. aeruginosa grown either anaerobically or with oxygen limitation in unshaken cultures, this promoter was str onger than the induced tac promoter, The P. aeruginosa lipAH genes, wh ich encode extracellular lipase and lipase foldase, respectively, were fused directly to the modified are promoter in an IncQ vector plasmid . Semianaerobic static cultures of P. aeruginosa PAO1 carrying this re combinant plasmid overproduced extracellular lipase 30-fold during sta tionary phase compared with the production by strain PAO1 without the plasmid, Severe oxygen limitation, in contrast, resulted in poor lipas e productivity despite effective induction of the ANR-dependent promot er, suggesting that secretion of active lipase is blocked by the absen ce of oxygen, In conclusion, the modified are promoter is useful for d riving the expression of cloned genes in P. aeruginosa during oxygen-l imited growth and stationary phase.