(620a) Model-Guided Design of a Refactored Metabolic Pathway

Synthetic biology is an emerging field that applies bottom-up engineering principles to redesigning natural biological systems for biotech applications. We present an example of these principles by completely redesigning and optimizing a seven enzyme metabolic pathway extracted from Rhodobacter sphaeroides.

Using lower cost large-scale DNA synthesis, we may now rewrite (or refactor) the DNA sequences that encode useful metabolic pathways, extracted from any sequenced host organism, and optimize them for a genetically tractable host, such as Escherichia coli. In the process, we simplify the DNA sequence to more reliably engineer and optimize the product flux. Key steps include eliminating overlapping protein coding sequences, removing extraneous transcriptional or translational regulation, improving mRNA stability, and adding unique restriction sites. We also add synthetic gene regulation to control transcription and rationally select the translation initiation rate by designing synthetic 5' UTRs for each protein coding sequence. The synthetic 5' UTRs are generated by a newly developed model of translation initiation coupled with Monte Carlo sampling (see session 15C12).

We demonstrate this systematic procedure on the crtEBICFDA genes, extracted from Rhodobacter sphaeroides. The crt metabolic pathway produces visibly colored intermediates and products, making it a good test example. The final product of the pathway is the yellow carotenoid hydroxy-spheroidene. Using HPLC purification and absorbance detection, we quantify intermediate and product formation and evaluate the effectiveness of our design procedure. The final design results in a significant improvement in accumulation of the carotenoid hydroxy-spheroidene.