In depth

Introduction

 

Site-specific recombinases are nature's word processing cut and paste functions for DNA. Recombinase proteins recognize specific short (30-50 bp) sequences in DNA, bring two of these sites together, cut both sites, swap partner half sites and then rejoin the DNA in the new configuration.

recomb

recm-1.jpg

 

 

Each recombination site has a distinct left and right half, and the recombinase always joins the left half of one site to the right half of the other. If two sites are in the same left to right orientation in a DNA molecule, recombination deletes the sequence between the two sites, producing an excised circular DNA molecule containing a single recombination site, and leaving just a recombination site behind in the original molecule.

 

recm-2.jpg

 

In the reverse of this reaction, a circular molecule with a single recombination site can integrate into a site in another DNA molecule. The integrated DNA is then flanked by two recombination sites.

 

Integrons

 

Integrons are naturally occurring bacterial systems that use site-specific recombination for storing, expressing and shuffling gene cassettes that might be useful for their host under certain circumstances. Integrons consist of a recombinase gene and a recombination site attI (for integron attachment site) into which circular gene cassettes containing an attC (for cassette attachment site) can be integrated. There is also a strong promoter upstream of attI, from which the gene cassettes are transcribed into mRNA. Cassettes can be deleted from the integron by recombination between pairs of attC sites, or by recombination between an attC site and attI. Excised cassettes are efficiently integrated into the integron by recombination between the cassette attC site and attI. The order of genes in an integron can therefore be changed by excision followed by reintegration of the excised cassette(s) into attI.

 

 

integron-1.jpg

 

Integrons were first found in bacteria with multiple drug resistant markers. These integrons contain as many as five or six different antibiotic and disinfectant resistance gene cassettes. More recently superintegrons, containing as many as 100 different gene cassettes, have been found on the chromosomes of bacterial species including Vibrionaceae and Pseudomonads. The superintegron gene cassettes encode a variety of different biochemical functions that may help their host survive in certain ecological niches. It is thought that only those gene cassettes integrated close to attI and the constitutive promoter, and the small number of cassettes that contain their own promoter, are expressed at any one time.

 

The sythetic integron

 

syntegron-1.jpg

Many complex chemicals with therapeutic or other commercial applications are too difficult or expensive to synthesise chemically and may be produced at only low levels by their natural host. Genes encoding complex metabolic pathways can be engineered into bacteria allowing the production of the desired products. However, the yield of the desired product in engineered bacteria is rarely optimal. Sub-optimal expression levels of the different enzymes in the pathway can lead to low yields, build up of toxic intermediates, or excessive metabolic load. Our aim is to use synthetic integrons to explore all possible levels of expression of the different genes in a metabolic pathway to optimise metabolic flow through the pathway and increase product yields. All of the genes for the metabolic pathway are assembled into integron like structures containing gene "cassettes" downstream of a strong promoter. Each gene is separated from the next by a recombination site, and cassettes can contain regulatory sequences as well as structural genes for the enzymes in the pathway. Expression of the recombinase protein randomizes the gene order, leading to different expression levels of all genes in the pathway, depending on their position relative to the promoter and any regulatory cassettes. The optimal gene arrangement is then selected, using a screen for increased production of the desired product. Alternatively, selective pressure is put on bacterial cultures to increase metabolic flow through the pathway in a directed evolution approach.