From 2007.igem.org

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Diauxie Project

Dosimeter Project

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What is Diauxie?

Bacterial cells in growth phase preferentially metabolize glucose before utilizing any other sugar. This is because glucose is the most efficient sugar for growth. Only when glucose is no longer available for the cell's use does it switch to the metabolization of other sugars like lactose or xylose. When the cell switches from glucose to another sugar there is a lag period during which the cell produces the alternate, sugar specific metabolization proteins. This delay is called diauxic lag.

Our Goal

It is of our interest to selectively eliminate the preferential processing of glucose before xylose. Xylose is a common lignocellulose sugar found in plant digests, and is therefore a ubiquitous energy source the can be used in the production of compounds such as ethanol. By eliminating xylose diauxie, bacteria such as E. coli could be made to produce ethanol much more efficiently because of the absence of diauxic lag.

How to eliminate Diauxie

Our approach to eliminate xylose diauxie was to remove glucose's transcriptional control over xylose catabolism without significantly altering wildtype metabolism in other pathways. The xylose operon is under regulation of the xylose sensing protein XylR and the energy availability indicator CRP.CRP activates xylose machinery only when it is bound to cAMP, which is prevalent in the cell when glucose levels are low. CRP-cAMP along with XlyR stabilizes RNA polymerase, activating transcription. We took two separate approaches to altering the expressed levels of xylose machinery.

CRP*

One approach devised was to express a mutant of CRP in the presence of xylose that acts like CRP-cAMP even in the absence of cAMP (and therefore the presence of glucose). By expression this protein at higher levels than regular CRP, the expression of xylose catabolism machinery is activated.

Promoter Region

Another approach was to strengthen the promoter region in order to abolish the need for CRP-cAMP stabilization while maintaining XylR control. In effect, this makes the promoter insensitive to glucose levels but still sensitive to xylose levels. The relatively poor promoter of xylose operator is incrementally changed to resemble the consensus promoter until XylR alone is sufficient to stabilize RNA polymerase.