This document discusses protein engineering through directed evolution. It explains that directed evolution involves randomly recombining genes from protein libraries and screening mutant proteins for improved functions. This leaves beneficial changes up to chance but generates diversity. Rational design is also used to map important protein interactions to guide evolution. The document provides an example of using directed evolution to modify a cytochrome P450 enzyme to more efficiently convert alkanes to alcohols over multiple generations. However, it notes that expressing evolved proteins in vivo and requiring expensive cofactors limit the commercial potential of this approach.

1. Protein Engineering by Directed Evolution
Ben Mair

2.  Enzymes are biological catalysts
 Vital area of biotechnology
 Can be used to create more sustainable and energy
efficient pathways to reactions
 Existing biological proteins are being engineered to
meet commercial needs
Protein Function

3. Protein Structure
Polypeptide sequence ‘folds
up’, mainly due to hydrogen
bonding and dipole
interactions
Prosthetic groups
Wikimedia, http://commons.wikimedia.org/wiki/File:Haemoglobin-3D-ribbons.png, (Accessed February 2015).

4. DNA Structure

5. Gene Splicing

6. Rational Engineering Irrational Engineering
View proteins as a sum of modular
components
View proteins as a whole
Involves changing genetic sequence
at specific points to give rise to
desired outcome in protein
Involves random recombination of
multiple homologous protein genes,
followed by screening for any
improved mutant proteins
Beneficial modification requires
great understanding of bio-
molecular interactions
Leaves beneficial changes up to
chance
Rational Vs. Irrational Protein Design

7. DNA Replication

8.  Random recombination of genes leads to good
diversity
 However large population of mutant proteins no
longer functional
 Compromise with rational design is made by mapping
important intramolecular interactions in protein
Directed Evolution

9. SCHEMA Energy of Disruption
𝐸 𝛼𝛽 =
𝑖∈𝛼 𝑗∈𝛽
𝑐𝑖𝑗 𝑃𝑖𝑗
Where c accounts for broken
interactions and P accounts for
probability of disruption
Matrix algorithm used (RASPP)
SCHEMA-RASPP technique has
enabled productive results from
55% parental homology
C. A. Voigt, C. Martinez, Z. Wang, S. L. Mayo and F. H. Arnold, Nature Struc. Bio., 2002, 9, 553-558.

10.  Cytochrome P450 from Bacillus megaterium modified
to convert aliphatic alkanes into secondary alcohols
 Turnover rate increased (× 50) over 5 generations of
homologous recombination and screening
 Active site more complementary to favourable
substrates, allowing increased productivity
Exemplar Research: Cytochrome
P450

11. Exemplar Research: Cytochrome
P450
PNAS, http://www.pnas.org/content/99/10/6725/F8.expansion.html, (Accessed February 2015).

12. Not an economical way to produce alcohols:
 Translation of gene to protein has to be done in vivo.
Protein’s modifications make it toxic to cell
(production must be regulated)
 NADPH required as electron source for reduction of
P450, which is very expensive
Exemplar Research: Cytochrome
P450

13.  Limited knowledge on existing proteins
 Difficulty scaling up enzyme reactions for commercial
potential
 Restricted access to protein and gene libraries
between institutions
Current Problems with Directed Evolution