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TridentSynth is a web-based retrobiosynthesis tool co-developed by researchers at Northwestern University and Lawrence Berkeley National Laboratory to design synthesis pathways to small-molecules using both biological and chemical reactions. On the chemistry side, TridentSynth uses reaction rules extracted from known synthetic organic chemistry reactions while on the biology side, TridentSynth uses reaction rules derived from monofunctional enzymes as well as from multifunctional Type I modular polyketide synthases (PKSs). By merging different synthesis routes, TridentSynth can explore a wider space of chemicals than would be possible using any one or two of these routes alone.

How this webtool works

Who should use this tool?

Any synthetic biologist, chemist, or researcher interested in designing novel pathways to valuable small molecules!

User inputs

The first step to using TridentSynth is for the user to decide which molecule they would like to synthesize. This could either be a simple commodity chemical, such as 1,4-butanediol or caprolactam, a long-chain biofuel, such as dodecane, or a structurally complex natural product, like cryptofolione. Once a target has been decided, the user can enter either the simplified molecular input linear entry system (SMILES) string of the target into the search bar on the front page of TridentSynth or draw out its molecular structure.

The next step for the user is to then decide which syntheses routes they would like to explore to this target molecule. Multifunctional type I polyketide synthase (PKS) enzymes are a crucial component of TridentSynth, as they enable the biosynthesis of large complex carbon backbones. Type I PKSs, briefly, are large modular assembly-line type enzymes comprising of smaller enzymatic domains, such as acyltransferase, ketoreductase, and enoylreductase domains that work in unison to iteratively stitch together and tailor acyl-coenzyme A (acyl-CoA) substrates, such as maloyl-CoA and methylmalonyl-CoA. For users that may be unfamiliar with PKSs, we recommend reading our previously published work for more information on how PKSs and regular, monofunctional enzymes can be used together to design novel biosynthetic pathways to key small-molecules.

Beyond PKSs, TridentSynth also allows users to design synthesis pathways using regular enzymatic as well as synthetic chemistry reactions. After deciding which target molecule to synthesize, users will have the option to decide if they would like to design pathways using either of the folllowing: (1) PKSs only, (2) PKSs + regular monofunctional enzmyes, (3) PKSs + synthetic chemistry, (4) PKSs + regular monofunctional enzymes + synthetic chemistry, (5) regular monofunctional enzmyes only, (6) synthetic chemistry, or (7) regular monofunctional enzymes + synthetic chemistry.

If a PKS-based synthesis route is chosen, TridentSynth will work in the forward direction to build towards the target molecule. This is done using two key design tools: RetroTide, which designs chimeric PKS assemblies to synthesize the carbon backbone of the target molecule, and DORAnet, which finds synthesis pathways using regular monofunctional enzymes and synthetic chemistry to tailor the PKS-generated scaffold towards the final desired target molecule.

Conversely, if a non-PKS-based synthesis route is chosen (i.e., only monofunctional enzymes or synthetic chemistry), TridentSynth will work in the reverse direction to convert the target molecule into common upstream precursors. These are a list of ~300 high-flux biological metabolites that we have curated by finding the intersection of metabolites present in various microorganisms, such as E. coli and S. cerevisiae.

Please note that TridentSynth does not take the stereochemistry of the desired target molecule into account at this time when designing pathways, however we plan to add this capability in the near future.

PKS design and PKS-based synthesis (RetroTide)

In nature, PKSs are genomically encoded within large biosynthetic gene clusters present within many bacterial genomes and are responsible for the biosynthesis of structurally complex and therapeutically relevant natural products. PKSs are an attractive biosynthetic tool for the production of small-molecules due to their unique ability to iteratively catalyze Claisen condensation reactions between simple acyl-CoA building blocks to eventually create elongated carbon scaffolds with diverse functional groups. While regular monofunctional enzymes are able to precisely catalyze single chemical transformations on a substrate's carbon backbone, they are often unable to repeatedly catalyze the formation of carbon-carbon bonds that are necessary to create these backbones.

When designing PKS-based synthesis routes, TridentSynth relies on our previously released PKS-design tool, RetroTide. In a nutshell, RetroTide designs a novel type I PKS assembly by combinatorially stitching together different PKS module designs, each of which comprises known domain architectures, with different starter and extender acyl-CoA substrates. Within RetroTide, each PKS domain (e.g., acyltransferase, ketoreductase, dehydratase) has a SMARTS-based reaction rule associated with it that describes how that domain modifies the growing polyketide chain. By iteratively applying these reaction rules in the order specified by the PKS module architecture, RetroTide can predict the final small-molecule product of a given PKS assembly.

Given a target molecule, RetroTide will attempt to get as close as possible to the target molecule. TridentSynth will output a chemical similarity score based on the maximum common substructure between the target molecule and the PKS-generated molecule that was reached with PKSs.

Post-PKS transformations (DORAnet)

Once a design for synthesizing the carbon backbone of the target molecule has been generated with PKSs/ RetroTide, TridentSynth will then use DORAnet to tailor this PKS intermediate molecule towards the final target molecule using reactions from synthetic organic chemistry and/ or regular monofunctional enzymes, depending on the user's choice. Similar to RetroTide, DORAnet relies on a SMARTS-based set of reaction rules derived from known synthetic chemistry reactions and enzymatic reactions to iteratively modify the PKS-generated intermediate towards the final target. For reaction rules encoding enzymatic reactions, DORAnet also has a list of enzyme UniProt IDs associated with each reaction rule that can be used to catalyze the reaction predicted by that rule.

If the target molecule is reached, then TridentSynth will output pathways connecting the PKS intermediate to the target. If the user has chosen to use monofunctional enzymes to tailor the PKS intermediate, then TridentSynth will also display a list of UniProt IDs that can be used to catalyze that reaction. These UniProt IDs will be hyperlinked to their respective entries on the UniProt database.

If the target molecule is not reached, however, then TridentSynth will output the most chemically similar product generated by DORAnet with respect to the target molecule.

Non-PKS synthesis (DORAnet)

As mentioned before, if users wish to design pathways to their target molecule using only regular monofunctional enzymes, and/or synthetic chemistry, but not PKSs, this is also possible with DORAnet. In order to do this, given a target molecule, TridentSynth will run in reverse and try to locate upstream precursors that are common biological metabolites typically present in high physiological concentrations within cells. To learn more about the different precursor metabolites that TridentSynth uses to find non-PKS pathways to a given target molecule, check out our page on common metabolites.

Interactive sample output

The results page surfaces structures, pathways, and enzyme annotations for downstream decision making.

To explore a fully rendered example, open the heptane PKS + biology (1 step) demo results page .

Need help?

Reach out to the TridentSynth team at [email protected] with questions, feature suggestions or bug reports. Please mention TridentSynth in the subject and body of your e-mail.

Acknowledgements and Developers