Technical Implementation

Our primary implementation of our technologies encompasses three main steps:

1. the creation of gene diversity libraries;
2. the selection of gene combinations that have interesting properties; and
3. the identification of the compounds encoded by these combinations.

Step 1 – Creating Gene Diversity Libraries

Step one is to create gene libraries from various organisms (plants, fungi, insects, etc.) as well as interesting biosynthetic pathways. From these libraries combinations of genes are used to synthesise artificial chromosomes. Each of these artificial chromosomes contains multiple genes. The artificial chromosomes are then introduced into yeast cells which otherwise do not contain these genes. Large populations of different (individual) yeasts are constructed in this way.

Step 2 – Selecting Promising Combinations

Step two is to see which gene combinations (i.e., which individual yeasts) have activity against a selected target. To achieve this, an assay is constructed which is used as a readout to measure whether the respective yeast cell produces an interesting small molecule. The yeasts are then individually screened against the assay using high throughput instruments (up to 109 screening events per day). Those yeasts that “hit” on the assay are selected from the rest of the population and can then either be taken directly into analytical chemistry or combined (“bred”) with other yeasts to create new combinations of the genes that conferred the initial activity – leading in turn to variations in the original compound.

A wide variety of assays can be used with this approach. Two examples are:

• A protein interaction assay that is designed to kill the yeast cell if it occurs. Those yeast cells that can make a compound that prevents the interaction will survive whilst the others will die.
• A mammalian receptor assay where activation of the receptor leads to fluorescence. The mammalian cell is placed next to the yeast cells. Those yeasts next to those mammalian cells that fluoresce are then selected.

Step 3 – Identifying the compounds responsible for the activity

The final step is to see what compounds are responsible for the activity observed in step 2. Active yeasts are taken into analytical chemistry, where they are checked to see whether a specific small molecule is the cause for the assay activity. This is done by turning the activity of the introduced biosynthetic genes on and off, and seeing which compounds correlate in their presence with the activity. Small molecules from yeasts where this correlation occurs are purified and rescreened as pure compounds. Confirmed actives are then identified and prioritised according to industry standard criteria.

Prioritised compounds can be analogued using genetic or synthetic chemistry or used as the basis for sourcing other compounds. The genes for making a given compound can also be identified and promising compounds scaled up by either synthetic chemistry or fermentation.

Scientific Paper

Article about Evolva's Technology – Yeast artificial chromosomes employed for random assembly of biosynthetic pathways and production of diverse compounds in Saccharomyces cerevisiae

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