Combinatorial Gene Assembly and High-Throughput Assay for Increased Methyl Ketone Yields in E. Coli

The tools of synthetic biology have allowed for Escherichia coli (E. coli) to be engineered as a biocatalyst for the conversion of glucose to medium-chain (C₁₁ to C₁₅) methyl ketones. This has been accomplished by alteration of the β-oxidation pathway in E. coli by overexpression of six genes. Our interest is to optimize the pathway by enhancing desired reactions and precluding undesired reactions. This requires precise tuning of transcriptional and translational elements for each of the six genes such that they modulate methyl ketone production in a predictable manner. To accomplish this, we have employed Golden Gate cloning for combinatorial gene assembly and a Nile Red assay for high-throughput methyl ketone screening. The Nile Red assay was effective in detecting relative levels of methyl ketone production, and proteomics analyses confirmed that our assembly method was able to express our genes at the predicted levels.