An integrative expression vector for strain improvement and environmental applications of the nitrogen fixing...

An integrative expression vector, incorporating a cassette for genomic integration and expression of gene of interest into Anabaena sp. strain PCC7120 was constructed. The cassette comprised of the following elements: (a) an intergenic non-coding region from Anabaena to facilitate its genomic integration (b) a strong functional PpsbA1 promoter from Anabaena for desired gene expression and (c) neomycin phosphotransferase gene with its own promoter for the selection of transformants. The cassette was cloned in the plasmid pBluescript II SK (+), which served as a suicide vector for Anabaena. The resultant plasmid designated as pFPN, when transferred into Anabaena by electrotransformation, integrated the cassette into Anabaena genome at the defined location. The vector was evaluated by cloning, transfer, integration and expression of the native hetR gene encoding a regulator of heterocyst differentiation. Formation of multiple heterocysts and enhanced nitrogenase activity of constitutively expressing hetR transformants of Anabaena established the utility of pFPN for basic molecular biology research and strain improvement. A transgene phoN encoding a non-specific acid phosphatase from Salmonella typhi was also transferred into Anabaena using pFPN. Strong constitutive expression of PhoN from PpsbA1 facilitated easier and sensitive visual screening of PhoN for tracking of transgenic Anabaena. The vector pFPN offers a stable integrative expression system for environmental application of genetically modified Anabaena strains.