Improving DBTC and Combinatorial Biosynthesis
One of the greatest challenges in synthetic biology is shortening the design, build, test cycle. Our goal was to use indigoidine as a tagging system to guide the design of novel NRPS products. NRPS are able to incorporate non canonical amino acids as well as append additional modifications, and hence were an attractive target for improving the combinatorial biosynthesis to design new peptides.
Heidelberg had strung together different NRPS modules by physically linking them together within the protein sequence, and in doing so were able to create a tripeptide verified by MS. The issue with assembling modules together through Gibson assembly is that it can be tedious and requires constant verification of DNA in order to ensure proper module expression.
We decided to explore the possibility of linking modules together through protein-protein interactions. Our system is inspired by the communication domains naturally expressed by NRPS. These domains are cleverly termed COmmunication-Mediated (COM) domains, and facilitate the unidirectional orientation of NRPS assembly lines.
COM domains have several advantages over traditional peptide linking of NRPS modules. Firstly module(s) have been cloned between COM domains that are able to be interchanged for rapid combinatorial biosynthesis.
The physical linking of whole modules often disrupts synthesis of the desired NRPS product by several orders of magnitude. Hans and Stachelhaus noted that combining modules using COM domains routinely resulted in peptide production levels of 65-80% of wild type [1]. This could often be improved up to wild type levels by directed evolution.
Lastly the ability of COM domains to assemble proteins in a specific orientation may extend far beyond NRPS production and be used for increased production of biosynthetic gene clusters designed by synthetic biologists.
References
1. Hahn, M., & Stachelhaus, T. (2004). Selective interaction between nonribosomal peptide synthetases is facilitated by short communication-mediating domains.
Proceedings of the National Academy of Sciences of the United States of America, 101(44), 15585-15590.
2. Takahashi, H., Kumagai, T., Kitani, K., Mori, M., Matoba, Y., & Sugiyama, M. (2007). Cloning and characterization of a Streptomyces single module type non-ribosomal peptide synthetase catalyzing a blue pigment synthesis.
Journal of Biological Chemistry, 282(12), 9073-9081.
3. Marahiel, M. A., & Essen, L. O. (2009). Nonribosomal peptide synthetases: mechanistic and structural aspects of essential domains.
Methods in enzymology, 458, 337-351.
