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Controlling Metabolic Pathways Using Artificial Positive Feedback Loop in Yeast

EIB-3293

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ABSTRACT

Joint BioEnergy Institute (JBEI) scientist Dominique Loqué has developed a technology that employs in yeast cells a trait-changing strategy that he and his colleagues first applied to fine tune desirable biomass deposition in plants. Crucial to this strategy is the design of a genetic switch, or transcription factor, containing an artificial positive feedback loop (APFL) within its DNA sequence. Once inserted in yeast, the switch regulates expression of desired new traits, while the embedded feedback loop induces increased transcription of the switch itself, sustaining the production of those traits.

The APFL strategy was first employed successfully to fine tune secondary cell wall synthesis in the model plant Arabidopsis. The new JBEI technology extends this strategy with a feedback loop that works in yeast, which is a model organism for many types of fungal cells. By identifying key genes in yeast that can be controlled in this manner, the researchers have demonstrated that this technology for plants can be adapted to entirely different organisms. In yeast, it confers traits that can potentially transform fungal cell cultures into efficient factories for the production of chemicals ranging from biofuels to pharmaceuticals.

Using common genomic integration techniques, the transcription factors are inserted at strategic spots to control target metabolic pathways of yeast cells. Of particular interest to researchers is the mevalonate pathway used by yeast to produce ergosterol, a cholesterol-like molecule common in fungi. By inserting a transcription factor at roughly midpoint in the ergosterol pathway, engineers can divert this complex process at the step where it ordinarily produces small amounts of farnesyl pyrophosphate (FPP). Normally, biosynthesis of FPP is tightly regulated, and downstream enzymes from the ergosterol metabolic pathway quickly utilize it. Instead, the new transcription factor forces the yeast to overproduce FPP, a highly useful chemical that can be processed into plastics, fuels, and pharmaceuticals. With the addition of a second enzyme, this same metabolic pathway can be further altered to convert its excess FPP directly into sesquiterpenes such as bisabolene, an ingredient that can be processed into diesel and jetfuel.

More conventional research efforts to modify yeast for industrial use rely on plasmids to transfer desired genetic traits. This method is limited, however, by the instability of plasmids in yeast. As yeast multiply, selection pressure favors cells that do not carry the plasmids, making the process inherently self-limiting. JBEI’s technology does not require plasmids to reach high levels of gene expression. Instead, this method integrates relevant sets of transcription factors directly into the yeast genome, and the APFL assures that the switches will be refreshed during each new replication cycle of yeast.

           
DEVELOPMENT STAGE:  Early stage.

STATUS: Patent pending. Available for licensing or collaborative research.

REFERENCE NUMBER: EIB-3293

The Joint BioEnergy Institute (JBEI, www.jbei.org) is a scientific partnership led by the Lawrence Berkeley National Laboratory and including the Sandia National Laboratories, the University of California campuses of Berkeley and Davis, the Carnegie Institution for Science and the Lawrence Livermore National Laboratory. JBEI’s primary scientific mission is to advance the development of the next generation of biofuels.

 

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