20. Protein space:
VASTLY (Very much more than astronomically) large
Mostly empty
sequences20450
20 natural amino acids
EVOLUTION IS HARD!
~ 450 amino acids long
21. Directed evolution is a molecular optimization on a fitness
landscape, where fitness is performance, defined by the user.
Fitness
22. Evolution works because…
?
…the regions that life
has discovered and
explored are rich in
function.
And, at least in some of its
many dimensions, the
fitness landscape is smooth.
This allows adaptation, one
step (one mutation) at a
time.
28. Sit
Relativeproductivity
Directed enzyme evolution
Savile et al. Science, 2010, 329, 5989
Replace all that with a single enzyme step!
Round of Evolution
1
10
100
1000
10000
100000
1 2 3 4 5 6 7 8 9 10 11 12
100000
10000
1000
100
10
1
Improved
70,000 fold
Started here
29. Sit
Relativeproductivity
Solvent waste
reduced 60%
Increased yield
from starting
materials
Directed enzyme evolution
Replace all that with a single enzyme step!
Round of Evolution
1
10
100
1000
10000
100000
1 2 3 4 5 6 7 8 9 10 11 12
100000
10000
1000
100
10
1
Improved
70,000 fold Toxic heavy metals
eliminated
Savile et al. Science, 2010, 329, 5989
30. Sit
Replace all that with a single enzyme step!
FDA Approval in 2012
AWARD
Thomas Edison
Patent Award
Toxic heavy metals
eliminated
Solvent waste
reduced 60%
Increased yield
from starting
materials
Savile et al. Science, 2010, 329, 5989
PRESIDENTIAL
31.
32. We need better cellulases to break
down biomass and generate sugars
for microbial chemicals and fuel
production.
35. Sexual recombination populates fitness peaks
(conservative of structure and function while making
large jumps in sequence)
Parent proteins,
found in nature
36. Chimeric progeny
can be better than
their parents.
Sexual recombination populates fitness peaks
(conservative of structure and function while making
large jumps in sequence)
37. We used recombination and random mutations to make
thermostable cellulases that hydrolyze more biomass at
elevated temperatures
0,0
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
0,9
1,0
35 45 55 65 75 85 95
Cellobiose(mM)
Temperature (ºC)
3C6P
HJPlus
HICel6
HJCel6
38. Enzymes (and other proteins) are highly ‘evolvable’
Proteins can adapt via simple uphill walks
New functions by changing tiny fractions of
(<2 %) of the sequence
Scary fact: Many beneficial
mutations are far from the
active site.
Directed evolution can optimize enzyme function
in real time, circumventing our profound ignorance
of how sequence encodes function.
39. How can we create whole new enzymes to expand the
chemistry of the biological world?
(catalyze reactions not known in nature)
40. How can an inherently conservative process like
this create novelty?
At any given time, nature takes the most probable paths
(the fewest mutational ‘steps’).
45. “Novelty” is often already there.
Access to new features relies on some (even very small) functional
overlap that can be drawn out and optimized during evolution.
Native reaction New reaction
48. Second most
abundant element
in the Earth’s crust
Si
14
28.0855
Silicon
...the literature is void of examples of biologically
synthesized, either in vivo or in vitro,
silicon-carbon bonds.
Silicon 2009, 1, 147.
49. N
H
Si
OH
R
R
catalysts / ligands
SiR3
reagents for cross-coupling
Si
18
F-acceptors for
PET imaging
Si(i-Pr)3
Si(i-Pr)3
materials for organic LED
biological probes
and drug leads
O
N
H
H2N
Me3Si O
OH
18F
RR
3
52. Physically possible molecules…
Relevant to biology
Freed from the constraints of biological
function, we can explore the physically
possible proteins to find new capabilities.
53. Bacterial enzymes that mimic human drug metabolism so that
we can test the toxicity of new drug candidates