Scheme of A. succinogenes PCK expression cassette integration into the poxB locus using the λ Red- and Int/Xis-dependent systems. a Construction of A. succinogenes PCK expression cassette, b introduction of the PCK expression cassette into poxB locus on chromosome by λ Red-dependent recombination system using pRSFRedTER, c replacement of the poxB gene of the ES06 strain with the PCK expression cassette, and d removal of the kanamycin resistance gene from chromosome by the λ Int/Xis-dependent excision system using pRSF-P
-IX. These genetic manipulations leave a 31-bp attB
site (5′-TGAAGCCTGC TTTTTTATAC TAACTTGAGC G-3′) in the chromosome.