Engineering aldehyde-based metabolic pathways to novel small molecules

Metabolic pathways leading to aromatic and aliphatic aldehydes

Aldehydes serve many useful purposes as a reactive functional group but do not naturally accumulate in common microbes. Instead, molecules containing aldehyde functional groups are rapidly converted into their corresponding alcohols. We previously engineered an E. coli strain capable of accumulation of a broad range of aromatic and aliphatic aldehydes. In initial demonstrations, we used this strain to overproduce aldehydes and also to redirect carbon flux towards other biochemical classes via aldehyde intermediates. We are only beginning to explore the tip of the iceberg of possibilities for the aldehyde platform in small-molecule biosynthesis or chemical biology. Development of these pathways could unlock new routes for production of value-added biochemicals or site-specifically modified proteins.

Developing more selective tools for nsAA incorporation/discrimination

Cartoon depicting quality control (kindly touched up by Dr. Jeff Ting)

Proteins are incredible polymeric nano-machines and the molecular workhorses of cells. In biotechnology, they play important roles as catalysts (enzymes) or binders (antibodies, receptors, etc.). Researchers in the chemical biology community have pioneered tools to incorporate non-standard amino acids, which are amino acids beyond the standard twenty. These molecules contain functional groups that are useful for diverse purposes. However, incorporation tools are not very selective and often lead to heterogeneity in protein synthesis. It is also very cumbersome to identify what fraction of target proteins correctly incorporated the desired non-standard amino acids. Our goal is to develop novel strategies to accurately evaluate non-standard amino acid incorporation and to use these strategies to engineer more selective incorporation tools. These approaches could achieve higher yield and purity of therapeutics, including antibody-fragment drug conjugates.

Exploring novel functional groups in biocatalysis

Cartoon and stick representation of the ClpS binding pocket (green protein with red residues) bound to a Phe N-end peptide (yellow) (PDB ID: 3O2B)

Catalysis is at the heart of what defines chemical and biomolecular engineers - it is unique from other engineering disciplines such as mechanical or petroleum engineering. Biocatalytic processes use enzymes or whole-cells under environmentally friendly conditions, often harness renewable inputs, and frequently achieve high specificity. Nature has generated a diverse array of enzymatic reactions using the natural twenty amino acids, and directed evolution approaches have made incredible strides in broadening natural reactivities. We seek to elucidate the role that non-standard amino acids may play in achieving finer modifications in enzymatic active sites of certain well-characterized or promiscuous enzymes. The development of such design rules may aid improvement of catalytic efficiency or substrate specificity and could add to the toolbox for metabolic engineering.