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Strategies for enhancing and controlling metabolic flux in engineered organisms Raghavan, Adhithi Ravi


Efforts to rejuvenate the under-exploited, but high-value, natural product space has focused on wiring nature’s biochemical reactions into microorganisms and features as a sustainable alternative to the chemical synthesis. However, for industrial relevance, the metrics of strain performance, yield, titer, and productivity, need to be improved, to achieve a better economic value. To tackle this, most metabolic engineering strategies have focused on rational deletions or overexpression of genes across metabolic pathways and given little thought to metabolic fluxes and how their channeling could affect biomass accumulation and product formation. Considering this, we propose two approaches: one for increasing the flux across metabolic pathways through the creation of fusion enzyme complexes; and the second to control flux, by tweaking the distribution of resources between biomass and product formation, through an optogenetic circuit. For the first, we have focused on increasing the flux through the non-mevalonate pathway, which is a precursor for the biosynthesis of several terpenoids. Taking inspiration from naturally occurring fusion or bifunctional enzymes of this pathway, we constructed several artificial fusions between rate limiting enzymes, by varying the catalytic domains and linkers, and tested their ability to improve flux, by enabling better substrate channeling. From our data, we found the fusion between the enzymes of IspD and IspE, with a flexible linker, outperformed the other strains especially in terms of lycopene titer. For controlling flux, we created an optogenetic circuit, which provides fine spatial control over individual cells and has advantages over chemical inducers. On exposure to red-light at 660 nm, the circuit activates T7 RNA polymerase and allocates resources between biomass accumulation and product formation. Design elements of the circuit include: plant-based phytochromes, that function as optical dimers; and yeast-based split-inteins, that can trigger a trans-splicing reaction. Using this circuit and external optical systems, we have dynamically controlled the expression of T7 RNA polymerase, which controls expression of the secondary metabolite, lycopene in our case. We expressed this circuit in bacteria and observed roughly a fivefold increase in lycopene titer with light, versus no light illumination, providing proof-of-concept of the approach.

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