A simple and reliable method to conduct and monitor expression cassette integration into the Escherichia coli chromosome

Abstract

We report a method for the integration of expression cassettes into the Escherichia coli chromosome using rare and dispensable sugar degradation gene loci as sites for integration. Clones carrying successfully recombined DNA fragments in the chromosome are easily screened using a solid differential medium containing the respective sugar compound. As an example for the heterologous expression of a complex natural product biosynthesis pathway, we show the stepwise chromosomal integration of the zeaxanthin biosynthesis pathway from Pantoea ananatis into E. coli.

Synthesis of fucosylated lacto-N-tetraose using whole-cell biotransformation

“…For the intracellular synthesis of fucosylated LNTs, the previously described E. coli strain LJ-AY (E. coli LJ110, lacZYA::lgtA-FRT fucIK::lacY-FRT), capable of synthesizing lacto-N-triose II (LNT II), was used as parent strain. 27 By using chromosomal integration technique, 33,34 the Bacteroides fragilis fkp gene (encoding for an L-fucose kinase and L-fucose-1-phosphate guanylyltransferase) 38 was inserted into E. coli LJ-AY, resulting in strain LJ-AYF-cat and strain LJ-AYF (after removal of an antibiotic resistance cassette). In subsequent analysis of intracellular GDP-L-fucose formation by these strains in a manner described before, 13 we found an intracellular level of GDP-L-fucose of about 5 mg per g cellular dry weight, ensuring sufficient substrate supply for the recombinant fucosyltransferase reactions.…”

“…antibiotics, the stable, chromosomal integration of recombinant gene expression cassettes can be favorable in terms of process efficiency and product safety. 34 Thus, the gene futC was chromosomally integrated under control of a tac-promoter, which has the advantage that only a single inductor was necessary during synthesis. The resulting strain, E. coli LJ-AYFOC-cat, was cultivated in the same manner as the plasmid carrying strain E. coli LJ-AYFO pCAW55, but without the addition of ampicillin and L-rhamnose.…”

High versus low level expression of the lycopene biosynthesis genes from Pantoea ananatis in Escherichia coli

“…The expression of each gene is controlled by an IPTG- inducible tac-promoter. The chromosomal insertion of the heterologous expression cassettes were conducted by a method described recently (Albermann et al 2010). This method is based on the k-Redrecombineering technique (Datsenko and Wanner 2000;Zhang et al 1998).…”

“…Takara PrimeStar DNA polymerase (Lonza, Belgium) was used for amplification. The chromosomal integration of recombinant crt-gene expression cassettes and elimination of antibiotic resistants gene cassettes using plasmid pCP20 (Cherepanov and Wackernagel 1995) were conduct as described by Albermann et al (2010).…”

Construction of an Escherichia coli mutant producing monophosphoryl lipid A

“…In addition, we also developed a rapid and efficient method to integrate heterologous genes in the chromosome of E. coli by combining the Red recombination system (Sharan et al 2009;Albermann et al 2010;De Mey et al 2010) and the blue-white screen. Heterologous genes are usually expressed in E. coli using plasmids as the vector.…”