A novel plasmid vector designed for chromosomal gene integration and expression: Use for developing a genetically stable Escherichia coli melanin production strain

Recombinant Escherichia coli strains for the production of valuable products are usually generated by transformation with plasmid expression vectors. However, in spite of their usefulness, common problems associated with plasmid use include segregrational and structural instability as well as undesired copy-number effects. A viable alternative to plasmid use is chromosomal gene integration. With the purpose of facilitating the process of stable strain generation, a novel chromosomal integration vector was developed and tested. We describe the construction and use of novel expression vector pLoxGentrc that contains the strong trc promoter (Ptrc), a multiple cloning site, the T1 and T2 rrnB terminator sequences, the lacIq gene and the aacC1 gene conferring gentamicin resistance flanked by two loxP sites. As a demonstration of utility, melanin-producing strains of E. coli were generated employing this vector. Melanin is a polymer synthesized by the enzyme tyrosinase using l-tyrosine as substrate. The melA gene encoding a tyrosinase from Rhizobium etli was ligated to pLoxGentrc to generate pLoxGentrcmelA. This plasmid was transformed into E. coli W3110 to generate a melanin-producing strain. A region from this plasmid including PtrcmelA, T1 and T2 rrnB and the aacC1 gene was amplified by PCR employing primers with 45 b regions of homology to the lacZ gene. The PCR product was electroporated into strain W3110 that expressed the λ-Red enzymes. From this experiment, strain W3110PtrcmelA, was obtained having the melA gene inserted in the lacZ locus. Fermentor cultures with strain W3110/pLoxGentrcmelA grown in the presence and absence of gentamicin as well as W3110PtrcmelA without antibiotic revealed that the latter displays high genetic stability as well as the highest melanin titer. Vector pLoxGentrc should be useful during strain generation processes, enabling direct comparison of plasmid and chromosome-based production systems.