Knock-in/Knock-out (KIKO) vectors for rapid integration of large DNA sequences, including whole metabolic pathways, onto the Escherichia coli chromosome at well-characterised loci

Suriana Sabri,Lars K Nielsen,Jennifer A Steen,Mareike Bongers,Claudia E Vickers

doi:10.1186/1475-2859-12-60

Abstract

BackgroundMetabolic engineering projects often require integration of multiple genes in order to control the desired phenotype. However, this often requires iterative rounds of engineering because many current insertion approaches are limited by the size of the DNA that can be transferred onto the chromosome. Consequently, construction of highly engineered strains is very time-consuming. A lack of well-characterised insertion loci is also problematic.ResultsA series of knock-in/knock-out (KIKO) vectors was constructed for integration of large DNA sequences onto the E. coli chromosome at well-defined loci. The KIKO plasmids target three nonessential genes/operons as insertion sites: arsB (an arsenite transporter); lacZ (β-galactosidase); and rbsA-rbsR (a ribose metabolism operon). Two homologous ‘arms’ target each insertion locus; insertion is mediated by λ Red recombinase through these arms. Between the arms is a multiple cloning site for the introduction of exogenous sequences and an antibiotic resistance marker (either chloramphenicol or kanamycin) for selection of positive recombinants. The resistance marker can subsequently be removed by flippase-mediated recombination. The insertion cassette is flanked by hairpin loops to isolate it from the effects of external transcription at the integration locus. To characterize each target locus, a xylanase reporter gene (xynA) was integrated onto the chromosomes of E. coli strains W and K-12 using the KIKO vectors. Expression levels varied between loci, with the arsB locus consistently showing the highest level of expression. To demonstrate the simultaneous use of all three loci in one strain, xynA, green fluorescent protein (gfp) and a sucrose catabolic operon (cscAKB) were introduced into lacZ, arsB and rbsAR respectively, and shown to be functional.ConclusionsThe KIKO plasmids are a useful tool for efficient integration of large DNA fragments (including multiple genes and pathways) into E. coli. Chromosomal insertion provides stable expression without the need for continuous antibiotic selection. Three non-essential loci have been characterised as insertion loci; combinatorial insertion at all three loci can be performed in one strain. The largest insertion at a single site described here was 5.4 kb; we have used this method in other studies to insert a total of 7.3 kb at one locus and 11.3 kb across two loci. These vectors are particularly useful for integration of multigene cassettes for metabolic engineering applications.

Highlights

Metabolic engineering projects often require integration of multiple genes in order to control the desired phenotype
The rbs operon is incomplete and non-functional in E. coli W, an industrially relevant sucrose utilizing strain [2], the target sequences for homologous recombination are conserved
The lacZ locus is often subjected to modification in laboratory strains, and has been successfully used as a site for integration of sucrose utilization genes in E. coli K-12, C and B [46]

Summary

Introduction

Metabolic engineering projects often require integration of multiple genes in order to control the desired phenotype. This often requires iterative rounds of engineering because many current insertion approaches are limited by the size of the DNA that can be transferred onto the chromosome. While relatively large fragments can be introduced on plasmids, plasmid-based expression has a number of disadvantages These include the metabolic burden placed on the cell for plasmid maintenance and expression of gene products from multiple copies, the potential for internal rearrangements, segregational instability, fluctuations in copy number and resulting effects on interpretation of data, and the burden placed on cell in presence of antibiotic or other selective agents required for plasmid maintenance [6,7,8,9,10,11]. High copy-number plasmids are known to exert a much stronger metabolic drain than low copy-number vectors [13], and for this and associated reasons, low copy-number vectors are often found to be superior for engineering and other applications [11,14,15]

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Results

Conclusion