Evosight™

 

Certain proteins are extremely sensitive to mutagenesis while others accommodate a large number of mutations, hence having a bigger evolution potential. Additionally, the number of beneficial mutations may differ depending upon the characteristics that have to be engineered. EvoSight™ enables you to characterise both parameters and accelerate your protein engineering projects. 

Protéus has also developed EvoSight™ to rationalise the random mutagenesis when the screening assay is complex and not easily amenable to high-throughput. In this case, EvoSight™ allows focusing rapidly on the library with the optimal mutation load and to determine the best strategy to be applied for obtaining the expected improvement. 

  

EvoSight™ process

EvoSight™ is based on the exploitation of experimental data using a proprietary algorithm. Our EvoSight™ directed evolution service involves successive steps: 

1. Starting from your gene, Protéus produces 5 calibrated random mutagenesis libraries with increasing mutation loads 
2. The active fraction of each library is evaluated (100 clones per library tested using a simple positive / negative assay)*
3. Based on the active fraction results, Protéus determines the optimal library as well as the mimimum number of clones to be screened. 
4. The optimal library is screened, allowing the identification of improved variants 
5. The number of beneficial mutations (B factor) is determined and a strategy for further evolution is designed.

* Screening can either be performed by customer or by Protéus. See our high throughput screening services.

• Generate a set of calibrated, high quality gene libraries
• Prepare a pool of gene variants for gene shuffling
• Understand the evolution potential of your protein
• Devise an optimisation strategy
• Obtain improved versions of a single protein

• A unique, patented process
• An unprecedented insight on the protein plasticity and evolution potential
• Generation of qualified variants for shuffling
• Optimisation and acceleration of screening steps
• Rationalisation of the directed evolution process

References	Designation
DE-001	Evosight strategic study
DE-002	EvoSight library generation and analysis

Please provide:

• Your cDNA cloned in a standard vector. We strongly recommend to send purified plasmids (10 µg in 40 µl TE buffer).
• Corresponding electronic maps and sequences

Protéus can also prepare synthetic genes (wild-type or codon optimised) starting from your electronic sequences. Tags and cleavage sites may be added on your demand to ease purification and downstream processing.

 

 

EvoSight™ results are delivered in several steps:

Initial deliverables:

• A set of gene libraries with increasing mutation loads (ligation libraries) (usually 5 libraries)

After evaluation of the active fraction of each library:

• The identity of the optimal library
• The minimal number of clones to be screened

After screening of the optimal library:

• Value of the B factor
• Improved variants

Generation of five libraries: 4 weeks

The identity of the optimal library, the minimal number of clones to be screened and the value of the B factor will be transferred to you within a week following reception of your screening data (see Process).

Combination of EvoSight™ and L-Shuffling™ to achieve a 50-fold increase in the activity of a phytase

Phytases are economically significant enzymes that hydolyse phosphoesters of phytic acid and release inorganic phosphate. Phytases are used as feed additives to improve the uptake of phosphate by animals and limit the rejections in environment.

Directed evolution was applied by Protéus to improve the specific activity of a phytase. Starting with a single gene from Bacillus licheniformis, we used EvoSight™ to generate a set of calibrated mutant libraries.

After identification of the optimal library, 5000 variants were screened, of which about two thirds were found to be inactive. 273 mutants showed improved performances when compared to the wild-type enzyme.



Furthermore, EvoSight™ enabled the calculation of the B factor, ie the number of independent beneficial mutations. The value of the B factor was found to be 30.

A high B factor indicates that many different mutations can independently lead to the targeted improvement, and suggests that combining these beneficial mutations iteratively can lead to further improvement.
 
Therefore, the 73 best phytase mutants were selected as parental genes for recombination using L-Shuffling™.

After only 3 L-Shuffling™ rounds, a 50-fold increase in the activity of the phytase was achieved, demonstrating that:

• As predicted by EvoSight™, the protein had a high evolution potential
• The strategy developed thanks to EvoSight™ was successful.