Efficient Transformation of the Cephamycin C Producer Nocardia lactamdurans and Development of Shuttle and Promoter-Probe Cloning Vectors

摘要

A high transformation efficiency (1 × 105 to 7 × 105 transformants per μg of DNA) of Nocardia lactamdurans LC411 was obtained by direct treatment of mycelium with polyethylene glycol 1000 and cesium chloride. A variety of vectors from Streptomyces lividans, Brevibacterium lactofermentum, Rhodococcus fascians, and a Nocardia (Amycolatopsis) sp. were tested; transformants could be obtained only with vectors derived from an endogenous plasmid of the Amycolatopsis sp. strain DSM 43387. Vectors carrying the kanamycin resistance gene (kan) as a selective marker were constructed. The transformation procedure has been optimized by using one of these vectors (pULVK1) and studying the influence of the age of the culture, concentrations of cesium chloride and polyethylene glycol, amount of plasmid DNA, and nutrient supplementations of the growth medium. Versatile shuttle cloning vectors (pULVK2 and pULVK3) have been developed by subcloning the pBluescript KS(+) multiple cloning site or a synthetic polylinker containing several unique restriction sites (EcoRV, DraI, BamHI, SstI, EcoRI, and HindIII). A second marker, the apramycin resistance gene (amr) has been added to the vectors (pULVK2A), allowing insertional inactivation of one of the markers while using the second one for selection. An alternative marker, the amy gene of Streptomyces griseus (pULAM2), which is easily detected by the release of extracellular amylase in transformants of N. lactamdurans carrying this vector, has been added. Two promoter-probe plasmids, pULVK4 and pULVK5, have been constructed, with the promoterless xylE gene as a reporter, for utilization in N. lactamdurans.