Person:

Sachdeva, Gairik

This thesis presents novel synthetic biology tools and design principles usable for microbial metabolic engineering. Controlling metabolic fluxes is essential for biological manufacturing of fuels, materials, and high value chemicals. Insulating the flow of metabolites is a successful natural strategy for metabolic flux regulation. Recently, approaches using scaffolds, both in vitro and in vivo, to spatially co-localize enzymes have reported significant gains in product yields. RNA is suitable for use as an in vivo scaffold due to its diversity of structural motifs, predictability of design, and ease of expression. We used scaffolds made from RNA strands containing two sets of motifs. Polymerization domains bring individually expressed strands together as tiles to form a macromolecular assembly. Aptamers included in the sequence recruit RNA binding domains to these in vivo scaffolds. We assembled a library of eight aptamers and corresponding RNA binding domains fused to partial fragments of fluorescent proteins in E. coli. New scaffold designs could co-localize split green fluorescent protein fragments to produce activity as measured by cell-based fluorescence. The scaffolds consisted of either single bivalent RNAs or RNAs designed to polymerize in 1 or 2 dimensions. The new scaffolds were used to increase metabolic output from a two-enzyme pentadecane production pathway that contains a fatty aldehyde intermediate, as well as three and four enzymes in the succinate production pathway. Pentadecane synthesis depended on the geometry of enzymes on the scaffold, as determined through systematic reorientation of the acyl-ACP reductase fusion by rotation via addition of base pairs to its cognate RNA aptamer. Together, these data suggest that intra-cellular scaffolding of enzymatic reactions may enhance the direct channeling of a variety of chemical substrates. In the cyanobacterium S. elongatus we showed that the flow of electrons can be controlled and insulated by co-expression of a heterologous hydrogenase with different interacting ferrodoxin proteins. We used the system to demonstrate photo biological hydrogen production and hydrogen dependent cell growth. Finally, we expressed RNA scaffolds in S. elongatus using a T7 RNA polymerase system, opening doors for spatial control of metabolism in the cyanobacterium.