Post on 11-Aug-2019
Protein Engineering
for Enzyme Catalysis with Microgels
Lehrstuhl für Biotechnologie, RWTH Aachen University und DWI-Leibniz Institut für Interaktive Materialien
Prof. Dr. Ulrich Schwaneberg
Monschau SFB Mikrogele Summer School 2018, 11.7.2018
Microgels as Versatile Containers for
Immobilisation of Enzymes
Motivation: Enhance performance of enzymes
- Avoid deactivations e.g. in organic solvents
- Avoid degradation e.g. proteolytic digest
- Recovery in processes for reuse
- Efficient diffusion
- General applicable
- High compound loads possible
4
Polymer-Biomacromolecule
Conjugates
-fast reactions in water
-no toxic by-products
-formation of stable covalent bonds
-orthogonal reactions
Sortase-Mediated Surface Functionalization of Stimuli-Responsive Microgels
5Internal manuscript No. 188 Gau‡, Mate‡, Zou, Oppermann, Töpel, Jakob, Schwaneberg* and Pich* . Biomacromolecules. 2017;18,2789–2798.
Sortase-mediated conjugation of eGFP on the surface of PVCL/GMA
AFM images and confocal images of (a) PVCL/GMA-LPETG-microgels (no sortase) and (b) PVCL/GMA-LPETG-eGFP hybrid (withsortase ligation)
Conclusions:
• SrtA-mediated ligation enabled the efficient andcontrollable decoration of the PVCL microgel surfacewith the model protein GGG-eGFP
• Sortase-mediated ligation is a very promising andpowerful tool for the surface functionalization ofmicrogels with biomolecules which opens upnumerous possibilities to develop microgels withtailored properties for biomedical and otherapplications
Acknowledgements: EG, AT and AP thank Volkswagen Foundation and DFG Collaborative Research Center 985 “Functional Microgels and Microgel Systems” for financial support. ZZ gratefully acknowledges the Chinese Scholarship Council (CSC) for his PhD fellowship.
Elisabeth Gau Diana M. Mate
Facile approach for encapsulation of enzymes in nanogels
Tunable Enzymatic Activity and Enhanced Stability of Cellulase Immobilized in Biohybrid Nanogels
6167 Peng, H., Rübsam, K., Jakob, F., Schwaneberg, U., Pich, A. BioMacromolecules, 2016
Synthetic Procedure To Obtain Biohybrid Nanogels via Cross-Linking in w/o emulsion
poly(N-vinylpyrrolidone-co-N-methacryloxysuccinimide)
How is the biocatalytic activity and stability of trapped protein
influenced by the chemical structure of nanogel network?
7167 Peng, H., Rübsam, K., Jakob, F., Schwaneberg, U., Pich, A. BioMacromolecules, 2016
Tunable Enzymatic Activity and Enhanced Stability of Cellulase Immobilized in Biohybrid Nanogels
CD spectra of cellulase biohybridnanogels CNG4, CNG6, CNG8, and CNG10
Secondary structures of the immobilized cellulase is altered with increase of crosslink
density
Cellulase nanogels show decreased activity compared to free cellulase
Cryo-FESEM of biohybrid nanogels CNG4
Specific activities of biohybrid nanogels CNGn (n = 4, 6, 8, and 10)
Residual activities of free cellulase, CNG4, CNG6, CNG8, and CNG10, incubated with organic solvents
Cellulase nanogels demonstrate compared to free cellulase
significantly improved stability
organic solvents chaotropic agents storage stability
Biohybrid nanogels with varied degree of crosslinking density (CNG4, CNG6, CNG8, and CNG10) were generated
Reversible Deactivation of Enzymes (Cellulase) by Redox-Responsive Nanogel Carriers
8165 Peng, H., Rübsam, K., Jakob, F., Pazdzior, P., Schwaneberg, U., Pich, A. Macromol. Rapid Commun., 2016
Highly efficient enzyme encapsulation and reversible modulation of enzyme activity by novel redox-responsive polymeric nanogels
Figure 1. Enzyme encapsulation/deactivation in polymeric nanogels followed by release/activation under reducing conditions. DDT: dithiothreitol, PMT: pentaerythritol tetrakis (3-mercaptopropionate), EED: 2,2′-(ethylenedioxy)diethanethiol
co-polymer
self-assembly
add cross-linker
add DTT
cross-linker
proteins
Aggregate formation (trapping of enzyme)
Protein encapsulated nanogels (inactive)
Protein encapsulated nanogels (active)
9165 Peng, H., Rübsam, K., Jakob, F., Pazdzior, P., Schwaneberg, U., Pich, A. Macromol. Rapid Commun., 2016
Reduced activity of cellulase, when it is encapsulated in the
nanogels
Cellulase activity is rapidly recovered in DTT solution
Reversible Deactivation of Enzymes by Redox-Responsive Nanogel Carriers
Ligh
t sc
atte
rin
g in
ten
sity
RH/nm
DLS meassurement of PNG1 degradability in presence of 10 mM DTT
Degradation of generated protein nanogels by addition of
DTT
Particle size remains the same when no DTT was
supplemented
PNG – protein nanogel; NG - nanogel
Cellulase activity is determined by substrate diffusion process
in nonreductive conditions
Increased acitivity of PNG incubated with DTT can be
attributed to release of cellulase from nanogel
Incubation timeSpec
ific
nan
oge
l act
ivit
y [U
/mg]
Determination of protein nanogel cellulase activity (4-MUC assay) w/o 10 mM DDT
FRET: Chromophore release from 2 differently loaded nanogels; upon addtion
of DTT emmision intensity shifts immediately
Wavelength [nm]
No
rmal
ized
em
mis
sio
n
inte
nsi
ty
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Protein Engineering: Focused vs Random mutagenesis
Shivange, A., Marienhagen, J., Schenk, A and Schwaneberg, U. (2009). Advances in Generating Functional Diversity, Curr. Opin. Biotechnol., 13, 19-25.
Localized & rationally addressable properties• Activity / Selectivity• Substrate profile• Thermal resistance
• ?pH profile?
Non-understood properties• Organic solvent• Ionic liquid• pH stability• Molar substrate/product
concentrations….
Mutagenesis methods for diversity generation in directed evolution
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Why is focused mutagenesis necessary?
● Methods for ideal diversity generation: 100 aa: 20100= 1.267*10124 variants
● 1.000.000 variants screening: represents in [%] of sequence space
<0.0000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000001 %
● Peptide with 6 aa= 20*20*20*20*20*20= 64 000 000 variants
Screening limit of state of the art technologies
● Key technologies are diversity generation and high throughput screening
● Traditional directed evolution:● Low mutagenesis frequency: 1 to 5 mutations per 1000 bp usually 1 to 3 amino acid exchanges● Small library size sampled: 1000-3000 clones
● Three to six iterative cycles often >6 amino acid substitutions in most beneficial variants
● Usually no molecular understanding of improved property
● Time requirements usually 1 to 2 years including screening development
14
Directed protein evolution
15
Classification of random mutagenesis methods
Wong, T. S., Zhurina, D. and Schwaneberg, U. (2006). The diversity challenge in directed protein evolution, Combinatorial Chemistry and High Throughput Screening, 9, 271-289.
Limitations of epPCR
Polymerase bias Only one nt of a codon is mutated
Organisation of the genetic code
63 of 100 mutations areA to G or T to C mutations
150 of 380 possible amino acid exchanges
1.67 codons per “aromatic” amino acid
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Consequences of bias on the protein levelExample: Subtilisin from Bacillus lentus (1GCI), epPCR (balanced dNTPs + Mn2+)
From: ● Schenk, A., Wong, T. S., Roccatano, D., Hauer, B. and Schwaneberg, U. (2006). SeSaM (Sequence Saturation
Mutagenesis): Eine Methode zur Sättigungsmutagenese eines Genes, BIOspektrum, 3 , 277-279.● Wong, T. S., Roccatano, D., Zacharias, M. and Schwaneberg, U. (2006). A statistical analysis of current random
mutagenesis methods for directed protein evolution, J. Mol. Biol. 355, 858-871 (cover page).
● Mutagenic hot spots and barely
mutated regions
● Low diversity and preference
for aa-substitutions to chemically
similar ones
13
MAP 3D
Verma,R., Schwaneberg, U., and Roccatano, D. (2012). MAP2.03D: A sequence/structure based server for protein engineering. Synth. Biol., 1, 139-150.
BSLA lipase (181 aas): State of the art random mutagenesis by epPCR
• epPCR library with low mutations frequency (3.1 per kb) sequencing of 1000 mutations
• epPCR library with high mutations frequency (11.7 per kb) sequencing of 1000 mutations
How many of the 181 amino acid positions are found to improve ionic liquid resistance BMIM-Cl?
15 positions epPCR-low18 positions epPCR-high
Zhao J, Kardashliev T, Joëlle Ruff A, Bocola M, Schwaneberg U. (2014). Lessons from diversity of directed evolution experiments by an analysis
of 3,000 mutations. Biotechnol Bioeng, 111(12), 2380-2389.
epPCR-high
epPCR-low
What do you find?
0-4 amino acid substitutions of 19 possible substitutions
15
What potential for improvement is obtainable?
Library of BSLA variants is generated in which
• every variants habors one amino acid exchange
• the FULL natural diversity is covered (20*181= 3620 variants)
19
Case study BSLA lipase (181 aas): focused mutagenesis (with KE Jaeger)
• Saturation mutagenesis library of each position• Extensive sequencing • All 341 missing substitutions were manually
prepared via site directed mutagenesis• Screening of eight properties• Sequencing of the best 20 clones per position to
gain a molecular understanding of interactions
For eight properties we can have the information on how many positions contribute to property improvement and what improvement is obtainable with ONE amino acid exchange
How many of the 181 amino acid positions contribute to improve ionic liquid resistance BMIM-Cl?
104 positions>50 %
Bacillus subtilis BSLA181 amino acids = 181 tripletts
64 x 181 = 11,584 mutant genes
19 x 181 = 3,440 variant proteins
VJ Frauenkron-Machedjou, A Fulton, L Zhu, C Anker, M Bocola, KE Jaeger and U Schwaneberg, ChemBioChem, 2015,
16(6):937-945
20
Similar trends for other properties: Organic solvent resistance: out 181 positions
No. of beneficial positionsDMSO Dioxane TFE
SSM 107 75 74
epPCR-low 24 11 14
epPCR-high 29 13 19
104 positions40 to 59 % of positions
contribute again!
Location of beneficial positions for dioxaneexposed buried
WT all positions 71% 29%
SSM 81% 19%
epPCR-low 82% 18%
epPCR- high 92% 8%
a) SSM TFE
Strong preference forcharged and aromatic
substitutions
Exposed positionspreferred
21
26
Number of substitutions
Markel*, U., Zhu*, L., Frauenkron-Machedjou, V. J., Zhao, J., Bocola, M., Davari, M. D., Jaeger, K.-E., Schwaneberg, U.
(2017). Are Directed Evolution Approaches Efficient in Exploring Nature’s Potential to Stabilize a Lipase in Organic
Cosolvents? Catalysts, 7, 142
27
Coverage of amino acid substitutions patterns for P450 BM3 heme domain
error-prone PCR SeSaM-TV SeSaM-TV-III
See: www.sesam-biotech.com
Main conclusions
III. A higher throughput and smarter evolution strategies are required!
II.Libraries with one amino acid substitution per variant thatcovers all natural diversity are a prerequiste to understandgeneral principles to stabilize enzymes
I. State of the art directed evolution experiments identify only a small fraction of beneficial positions (<20 %)
22
Key: Balance throughput and time requirement (5 to 6 aas)
Thursday, July 26, 2018
29
Time efficient protein engineeringKey: Balance throughput and time requirement (5 to 6 aas)
in Concepts: Knowledge gaining directed evolution: KnowVolution
Cheng, F, Zhu, L, Schwaneberg, U (2015) Directed evolution 2.0: improving and deciphering enzyme properties, ChemComm, 51(48):9760-72.
State of the art
32
Focused Mutagenesis
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
1 AS 2 AS 3 AS 4 AS 5 AS
20 400 8000 160000
3200000
Pro
tein
var
ian
ts
Fragment Amplification Primer Name Primer Sequence (5’-3’)
A2-Vector-A1 E31Fw
E31Rv
ctagtgcttcagCGTAAGGGGCAAG
gataaccactcgMNNCAAAGTGTAACCCGTC
B T77Fw
T77Rv
cgagtggttatcTTGAGCCGCCATG
catagaagccgccCATCAGMNNCACTAATTGCGC
C K139Fw
K139Rv
ggcggcttctatgGTGATTATTTCCG
gaaacagtggatcAACCTTMNNCAAATCAGCCTG
D G187Fw
G187Rv
gatccactgtttcACCCCGTCGAAG
cagtgaaattgagAATCTCMNNCATCTGGGCAAATG
E V298Fw
V298Rv
ctcaatttcactgCTTCCCCCTATTGC
ctgaagcactagCGCCGTMNNAATCTGTTGCAAC
C/D/E/A2-Vector-A1 K139Fw
D52Rv
ggcggcttctatgGTGATTATTTCCG
ccacttatccggTGTGACMNNATTCATTAACTCTG
B’ D77Fw’
T77Rv’
ccggataagtggCCTCAATGGCCGGTAC
catagaagccgccCATCAGMNNCACTAATTGCGC
Colony PCR Forward primer
Reverse primer
TAATACGACTCACTATAGGG
TCCAAAAGAAGTCGAGTGG
1
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OmniChange: Focused mutant library generation (EU patent granted)
Vector A1A2 EDB C
Vector A1 A2EDB C Vector
D52Rv T77Rv K139Rv G187Rv V298Rv
Vector A1A2 EDB C
Amplification by PCR, DpnI digestion and pufication
Cleavage (6 mM I2/EtOH; 5 min at 70 C)
Hybridization (5 min at 20 C)
Transformation
V
V
V
V
V
V
START
START
START
STOP
STOP
STOP
* * * * *
* * *
* * * * *
D52FwT77Fw K139Fw G187Fw V298Fw
*
*
**
*
*
* **
*
*
* **
*
*
Step 1
Step 3
Step 2
Step 4
*
Position G31 T77 K139 G187 V298 1 aag aag aaa aat aat 2 aag aat aag aag aat 3 aat acg aag aag aat
4 aat acg aag aag aat 5 acg act aat acg act 6 acg agt acg acg act 7 acg agt acg act agt
8 acg agt agg act atg 9 acg atg agg act att
10 acg att agg act cag
11 acg cag agg agg cag 12 act cag agt agt cat 13 atg cat agt agt cat 14 cat cat agt atg ccg
15 ccg ccg agt att cct 16 ccg ccg atg cag cct 17 ccg ccg att cag cct 18 ccg cgg att cag ctg
19 ccg cgg cag cat ctg 20 cct ctg cag ccg ctt 21 cct ctg ccg cct ctt 22 cct ctg ccg cgg gag
23 cct ctt ccg cgt gag 24 cct gat cct ctg gag 25 cct gcg ctg ctg gag
26 cct gcg ctt ctg gat 27 cgg gcg gag ctt gcg 28 ctg gcg gag gag gct 29 ctg gct gtg gag ggg
30 ctg ggg gcg gat ggg 31 gag ggt gcg gat ggt 32 gag ggt gcg gct ggt 33 gat gtg gct gct gtg
34 gat gtg gct ggc gtg 35 gcg gtt ggg ggt gtt 36 gct tag tat ggt tag 37 gtt tag tat gtg tat
38 tag tat tcg gtt tcg 39 tat tat tcg tct tct 40 tat tcg tcg tgg tgg
41 tat tcg tcg tgg tgg 42 tat tcg tct ttg tgt Dennig, A., Shivange, A.V., Marienhagen, J., and Schwaneberg, U. (2011). OmniChange: The Sequence
Independent Method for Simultaneous Site-Saturation of Five Codons, PLoS ONE 6(10): 26222.bt.
No gel extraction stepNo final PCR applification stepNo additional sequencesNo limitation by aa-positions
3 200 000 variants generated in one afternoon
35
iVDT-Basistechnologie-Platform High throughput screening by flow cytometry
BDInflux
• Analyzing of 200.000 and sorting of 70.000 events per s-1
( running at a few thousands per s-1)
• Sorting of 10.000.0000 events per hour
• Analysis and sorting based on fluorescence
• Enrichment in the active enzyme population as abenchmark
Enables novel directed evolution strategies withhigh mutational loads for efficient identification of beneficial amino acid positions
Best use as PRESCREENING system: sorting of ~2000 beneficial variants in MTPs/agar plates
36
BMBF Basistechnologie Project iVDT:
Highlights
• Coupled enzymatic reaction (phytase – glucose oxidase)
• E. coli cells expressing active phytase form a fluorescent hydrogel aroundStrategy
Scanning force
microscopy
Confocal
microscopy
E. coli cells
phytase (-)
E. coli cells
phytase (+)
Phytase activity
before sorting
Phytase
activity after
sorting
Activity distribution
Whole cell: Fluorescent hydrogel-based FACS platform for hydrolytic enzymes
37
A fluorescent hydrogel-based FACS screening platform for hydrolytic enzymes
Pitzler, C., Wirtz, G., Vojcic, L., Hiltl, S., Böker, A., Martinez, R.,
Schwaneberg, U., Chem Biol. (2014) 21 (12):1733-42
BMBF Basistechnologie Project iVDT
Proof of concept for phytases:
Fur-Shell technology was advanced for three additional hydrolases: cellulase, esterase, and lipase
Simple handing
High throughput screening toolbox for directed hydrolase evolution
Esterase variant with 7-fold increase in kcat and 2-fold reduced KM
Traditional directed evolution yield often 1.5-2.5-fold improvement per round
Toolbox for hydrolases
Lülsdorf, N., Pitzler, C. Biggel, M., Martinez, R., Vojcic, L. and
Schwaneberg, U. A flow cytometer-based whole cell screening toolbox
for directed hydrolase evolution through fluorescent hydrogels, Chem.
Commun., 2015, 51(41):8679-82.
39
Part I – Optimization of in vitro cellulase production in emulsions
Amount of
DNA
25°C lin. DNA
(30°C)
4 h
pIX3.0RMT7
vector°C
Substrate
concentration
0.328 µM DNA
0.656 µM DNA
0.656 µM
DNA
0 mg/ml BSA 1 mg/ml BSA
2 mg/ml BSA
Additives e.g.
BSA
1 mg/ml
BSA
0.46 mM FDC
Substrate
concentration
40
Validation of the uHTS-IVC platform for directed cellulase evolution
- Screening of >1,2 Mio. events- MTP analysis of 528 variants with 4-MUC assay revealed 33 cellulase variants improved
activity compared to parent- Most promising variants were selected for rescreening- Best identified variant CelA2-H288F-M1 (N273D/N468S) was purified and kinetically
characterized
Characterization in MTP format revealed the 13-fold improved cellulase variant M1 Flow cytometer-based uHTS-IVC technology platform successfully validated
kcat
[min-1]KM
[µM]kcat/KM
[min-1 µM-1]
Specific activity[U mg-1]
Amino acid substitutions
CelA2-WT 0.11 (± 0.02) 48.37 (± 24.30) 0.002 16.57 (± 3.13) -
CelA2-H288F 0.50 (± 0.02) 8.95 (± 1.62) 0.056 72.62 (± 3.21) H288F
CelA2-H288F-M1 1.52 (± 0.04) 9.66 (± 1.19) 0.157 220.6 (± 6.71) N273D/H288F/N468S
Körfer, G., Pitzler, C., Vojcic, L., Martinez, R., Schwaneberg, U. (2016) In vitro flow cytometry-based screening platform for cellulase engineering,
Scientific Reports, 6, 1-12. DOI: 10.1038/srep26128.
41
iVDTv2:Cellulase evolution: OmniChange library using in vitro flow cytometer platform
Georgette
Körfer
-In vitro expression in (w/o/w) emulsion and screening of 36,757,972 events by flow cytometer-Sorting of 395,229 events
Reanalysis of sorted sample revealed 30-fold enrichment of active fraction
Variantskcat
[min-1]KM
[µM]
kcat/KM
[min-1
µM-1]
Specific activity[U mg-1]
Amino acid substitution
CelA2-WT 0.11 (± 0.01) 22.53 (± 4.78) 0.005 16.27 (± 1.04) -
CelA2-H288F 0.58 (± 0.02) 7.66 (± 1.12) 0.075 83.87 (± 2.89) H288F
CelA2-H288F-M3 4.65 (± 0.26) 51.19 (± 7.55) 0.091 674.50 (± 38.17) H288F/H524Q
M3 shows 41-fold higher specific activity compared to CelA2-WT Combinatorial effect between F288 and Q524 (N524, M524) successfully identified
Kinetic characterization:
Summary
KnowVolution a strategy to balance throughput and time requirement (5 to 6 aas)
Barely exploit the potential of protein sequence space
Main technical limitation: throughput screening technologies
Microgels enable to the converge the worlds of biocatalysis and chemical catalysis