Use of the lambda Red-recombineering method for genetic engineering of Pantoea ananatis

Abstract

Background: Pantoea ananatis, a member of the Enterobacteriacea family, is a new and promising subject for biotechnological research. Over recent years, impressive progress in its application to L-glutamate production has been achieved. Nevertheless, genetic and biotechnological studies of Pantoea ananatis have been impeded because of the absence of genetic tools for rapid construction of direct mutations in this bacterium. The lambda Red-recombineering technique previously developed in E. coli and used for gene inactivation in several other bacteria is a high-performance tool for rapid construction of precise genome modifications.

Results: In this study, the expression of lambda Red genes in P. ananatis was found to be highly toxic. A screening was performed to select mutants of P. ananatis that were resistant to the toxic affects of lambda Red. A mutant strain, SC17(0) was identified that grew well under conditions of simultaneous expression of lambda gam, bet, and exo genes. Using this strain, procedures for fast introduction of multiple rearrangements to the Pantoea ananatis genome based on the lambda Red-dependent integration of the PCR-generated DNA fragments with as short as 40 bp flanking homologies have been demonstrated.

Conclusion: The lambda Red-recombineering technology was successfully used for rapid generation of chromosomal modifications in the specially selected P. ananatis recipient strain. The procedure of electro-transformation with chromosomal DNA has been developed for transfer of the marked mutation between different P. ananatis strains. Combination of these techniques with lambda Int/Xis-dependent excision of selective markers significantly accelerates basic research and construction of producing strains.

Figure 1

Scheme of a construction of multiple chromosomal modifications, using the combined λ Red-Int/Xis system. A) Selective marker M flanked by attL_λ/attR_λ is used for introduction of an appropriate mutation into the chromosome by λ Red-dependent recombination. Then the marker is eliminated from the chromosome by λ Int/Xis site-specific recombination. As a result, only the 31 bp long attB_λ sequence linked to the mutation remains in the chromosome. Selective marker M can then be used in the next step of the introduction of multiple chromosomal modifications. B) The sequence of the attB_λ site. One of the six ORFs provided by this sequence does not contain stop codons. Hence, it is possible to design an "in-frame" deletion of a gene. Asterisks mark stop codons.

Figure 2

Construction of the unmarked nucleotide exchange in the P. ananatis hisD gene. A) Construction of a dsDNA fragment with appropriate mutation in the center. First, his-XhoI-1 and his-XhoI-2 oligos are annealed to each other. The nucleotide exchange of interest included in the sequence of his-XhoI-1/his-Xho2 olig is indicated by asterisks. The resulting dsDNA fragment is then used as DNA-template for PCR-amplification with his-SL and his-SR oligos. As a result a linear dsDNA fragment, harboring a XhoI restriction site and 82 bp long arms homologous to the target region of hisD gene, was obtained. B) The cassette containing dual selective/contra-selective marker is integrated into the target point of hisD gene. The constructed in vitro linear dsDNA fragment or ssDNA harboring appropriate mutation in the center is then integrated into the chromosome of this strain by the λ Red recombination system. As a result, the dual selective/contra-selective marker is eliminated from the chromosome with simultaneous introduction of the desired mutation into the hisD gene. This mutation leads to substitution of the native SmaI restriction site by the XhoI restriction site and restoration of the amino-acid sequence of HisD protein. Integrants are selected as colonies resistant to sucrose. Such colonies are subsequently tested for Cm sensitivity, ability for growth without histidine and presence of XhoI restriction site in the target chromosome point.