Regulated expression systems for the development of whole-cell biocatalysts expressing oxidative enzymes in a sequential manner

This work reports the prepn. of two recombinant strains each contg. two enzymic activities mutually expressed through regulated systems for prodn. of functionalized epoxides in one-pot reactions. One strain was Pseudomonas putida PaW340, contg. the gene coding for styrene monooxygenase (SMO) from Pseudomonas fluorescens ST under the auto-inducing Ptou promoter and the TouR regulator of Pseudomonas sp. OX1 and the gene coding for naphthalene dihydrodiol dehydrogenase (NDDH) from P. fluorescens N3 under the Ptac promoter inducible by IPTG. The second strain was Escherichia coli JM109, in which the expression of SMO was under the control of the Pnah promoter and the NahR regulator of P. fluorescens N3 inducible by salicylate, while the gene expressing NDDH was under the control of the Plac promoter inducible by IPTG. SMO and NDDH activities were tested in bioconversion expts. using cinnamyl alc. as ref. substrate. The application that we selected is one example of the sequential use of the two enzymic activities which require a temporal control of the expression of both genes.