Supplementary Material
Plasmid and primer list
Table S1| Primers used in this work
Nr Name
Sequence
1
pKTS_seq_for
5’-ACCACTCCCTATCAGTGATA-3’
2
New_pSEVA_rev 5’-TACTCAGGAGAGCGTTCACC-3’
3
LibM207_IDF8_f 5’-GCAAAGGTAAANNKGGTGTGTTTC-3’
4
LibM207_IDF8_r 5’-GAAACACACCMNNTTTACCTTTGC-3’
5
LibS45_f
5’-ATAATCTGNNKGATGCCAAAAAACGTGAACTG-3‘
6
LibS45_r
5’-GTTTTTTGGCATCMNNCAGATTATGAAATTC-3‘
7
IDF10_V209H_f 5‘-GTAAAGTTGGTCATTTTCATATTGGTG-3‘
8
IDF10_V209H_r 5‘-CACCAATATGAAAATGACCAACTTTAC-3‘
9
IDF10_V244A_f 5‘-GATGGCACCATTGCGATGGAACCGTTTATG-3‘
10 IDF10_V244A_r 5’-CATAAACGGTTCCATCGCAATGGTGCCATC-3’
Degenerated codons: N = A/T/G/C; K = G/T; M = A/C
Table S2| Plasmids used in this work
Nr Name
Description
1
pAB174 pSEVA backbone with pBR322 origin of replication,
bla-resistance gene, Ptet-PT7 fusion promoter,
thermostable PcDTE-Var8, C-terminal 6His tag
2
pAB92
SEVA vector backbone, bla resistance gene, Ptet-PT7
fusion promoter, MCS, ori pBR322
3
pAB202 Derivative of pAB174, encoding mutant PcDTE IDF8
4
pAB216 Derivative of pAB202, encoding mutant PcDTE IDF9
5
pAB218 Derivative of pAB216, encoding mutant PcDTE
IDF10
6
pAB222 Derivative of pAB218, encoding mutant PcDTE
IDF10-3
7
pAB217 Derivative of pAB174, encoding PcDTE Gen3
containing mutations E214T/S45T/A172C compared
to Var8
8
pAB203 Derivative of pAB174, encoding mutant PcDTE ILS6
Reference
(Silva-Rocha et al. 2013),
(Bosshart et al., submitted)
(Silva-Rocha et al. 2013),
(Bosshart et al., submitted)
(Bosshart et al. submitted )
This work
This work
This work
This work
(Bosshart et al., submitted )
Details on site-saturation mutagenesis and screening
In short, individual clones were pre-cultured in 500 µL of LB supplemented with 100 µg mL-1 of
ampicillin at 37°C overnight before 20 µL of pre-culture was used to inoculate 1 mL of ZYM-5052
autoinduction medium (Studier 2005) in 96-well deep-well plates. Cells were grown for 16h at 30°C
before they were harvested by centrifugation and lysed by treatment with lysozyme and a freeze-thaw
cycle. Next, the crude lysate was heat-treated at 70°C for 10 min, cell debris and precipitated protein
was removed by centrifugation, and the supernatant was used for the activity assay.
For the activity assay, 20 µL of heat-treated lysate was added to 100 µL of 240 mM D-fructose in
96-well flat-bottom microplates (Greiner Bio-One) and incubated for 90 min at 30°C to allow
conversion of D-fructose to D-psicose before 120 µL of developing solution was added (50 mM TrisCl-Cl (pH 8.0), 1 mM NADH, 40 µg mL-1 KpRD) (Bosshart et al., submitted ). Reduction of Dpsicose to allitol was monitored by the concomitant oxidation of NADH at 340 nm wavelength in a
Perkin Elmer Wallac 1420 Victor platereader (Perkin Elmer). The resulting change in absorption from
NADH oxidation allowed the determination of the concentration of D-psicose using the RbtD
calibration curve recorded with known amounts of D-psicose in presence of D-fructose.
The most active clones (> 120% activity of parent from heat treated lysate) were regrown in
triplicates in 96-well plates, lysed and heat-treated as described above, and the epimerization reaction
was started as described above. After 1 h incubation 20 µL of the reaction was stopped by adding it to
145 µL of 0.1 M HCl which was followed by the addition of 135 µL of 0.1 M NaOH after 5 min.
Conversion of D-fructose to D-psicose (for IDF) or L-sorbose to L-tagatose (for ILS) was determined
by HPLC using a LC ICS-3000 system (Dionex, Olten, Switzerland) equipped with a CarboPac PA1
column (250mm x 4mm I.D.) preceded by a CarboPac PA1 guard column (50 mm x 4mm I.D.) (both
Dionex, Olten, Switzerland). Samples were eluted isocratically with 30 mM NaOH at a flow rate of
2.0 mL min-1 and detected by triple pulsed amperometry using an EC detector with a gold electrode
(all Dionex, Olten, Switzerland). Amino acid positions that showed an improvement in catalytic
activity were then targeted by single site-saturation mutagenesis.
Cost-benefit ratio of random vs. targeted libraries
Most directed evolution experiments that aim at improving certain enzymatic traits (i.e. substrate
specificity, enzymatic activity, thermostability) are still hampered by the throughput of their screens,
limiting the number of variants that can be screened to a maximum of around 104 clones. Therefore the
trend towards smaller but ‘smarter’ libraries has taken on greater significance in recent years
(Bornscheuer et al. 2012). In the present study we aimed at a side-to-side comparison between the
(screening-) cost-benefit ratios of a random mutagenesis approach (i.e. epPCR) versus a targeted
mutagenesis approach (i.e. saturation mutagenesis of residues in vicinity to the active site) for the
improvement of catalytic activity. It has been reported previously that mutations that are introduced
randomly by means or epPCR and improve enzyme activity, substrate selectivity, or enantioselectivity
are biased towards the proximity of the active site (Morley and Kazlauskas 2005; Paramesvaran et al.
2009). This is interesting insofar as mutating the entire protein generates a greater number of
mutations that are far away from the active site, simply because there are more amino acids that are far
from the active site than close ones (Morley and Kazlauskas 2005). In case of PcDTE, 48 residues
(16% out of a total of 293 residues (Ishida et al. 1997)) are located within 10Å of the center of the
active site (Bosshart et al., submitted ). Thus 84% of all residues were not targeted by saturation
mutagenesis. These 84% of residues are however very likely to be mutated by epPCR simply because
they represent the majority of residues in the protein.
From all 13 mutations that were initially discovered by screening the epPCR library, 5 were found
to be located in the dimeric interface that had previously been shown to be of central importance for
thermostability (Bosshart et al. 2013). That nearly 40% of all beneficial mutations from epPCR were
located in the dimeric interface consisting of 44 residues (i.e. 15% of all PcDTE residues) lets us
conclude that the ‘relaxation’ of the tight interface interaction is an easy evolutionary trajectory for the
enzyme to gain activity, however at the expense of thermostability.
It is worth noting that all of the 4 confirmed single beneficial mutations derived from the saturation
mutagenesis of positions located by epPCR were very distant from the active site, between 13.3Å
(E214T) and 24.7Å (A45G) of the residues Cα and the center of the active site. It is likely that the
number of clones that were screened for the epPCR library (~7500 variants) was too small to find the
(rare) mutations that are close to the active site.
Therefore we conclude that site saturation mutagenesis of residues close to the active site is
significantly more successful in finding beneficial (i.e. more active) mutants than a random
mutagenesis approach, particularly if the screening assay allows only for screening a limited number
of clones. Additionally, site-saturation mutagenesis has the advantage of excluding sites that are
already known to have adverse effects on the biocatalyst as was the case in our work, where residues
involved in the dimeric interface that has been engineered for improved thermostability could be
excluded from screening.
On the other hand, epPCR mutagenesis is able to unveil mutations that are more distant from the
active site and thus hard to predict. Therefore, epPCR and site-saturation mutagenesis can be used as
complementary approaches as shown here, to target as much of the protein sequence space as possible
but at the same time reducing the screening workload to a reasonable amount.
Supporting Tables and Figures
Table S3| Variants of PcDTE Var8 from epPCR library screening with > 1.2-fold WT activity
Variant
Sense mutation(s)
Silent mutation(s)
M_EP1
M_EP2
D173E, M207L
F187Y
M_EP3
M_EP4
M_EP5
M_EP6
Q215P
M207L
N190S, V261A
E214G
M_EP7
M_EP8
M_EP9
M_EP10
M_EP11
S45T, V261A
A172V
Q215P
W11R
N216S
E276E (GAAGAG)
G107 (GGTGGC); K204 (AAAAAG); A290
(GCAGCT)
A131 (GCAGCT)
I200 (ATTATC)
G252 (GGTGGC)
G30 (GGTGGA); A131 (GCAGCG); D176
(GATGAC)
R280 (CGTCGA)
Y73 (TATTAC); F284 (TTTTTC)
G107 (GGTGGC)
-
Improvement over
WT (-fold)
1.38
1.28
1.57
1.63
1.2
1.57
1.45
1.38
1.37
1.38
1.49
Positions F187, N190, Q215, V261, N216 were excluded from further screening as they are located in the
dimeric interface of the protein and are essential for thermostability (Bosshart et al. 2013)
Table S4| Single beneficial mutations from sites discovered in epPCR mutagenesis
Mutations from error-prone PCR
Mutation
Relative activity to Var8
Mutations from QuickChange PCR
Mutation
Relative activity to Var8
S45T
A172V
M207L
E214G
S45G
A172I
M207L
E214T
1.45
1.38
1.63
1.34
1.48
1.20
1.70
1.81
Table S5| Enzyme kinetic parameters for variants discussed in this work, determined at 25°C
Enzyme variant
WT a)
PcDTE
PcDTE Var8 b)
PcDTE IDF8 b)
PcDTE IDF9
PcDTE IDF10
PcDTE IDF10-3
PcDTE IDF10-5
PcDTE Gen3
PcDTE Var8 b)
PcDTE ILS6 b)
a)
b)
Substrate
kcat [s-1]
Km [mM]
kcat/Km [M-1 s-1]
D-fructose
D-fructose
D-fructose
D-fructose
D-fructose
D-fructose
D-fructose
D-fructose
L-sorbose
L-sorbose
23.5
4.9
42.3
31.0
37.9
44.8
22.6
16.6
0.24
3.2
40.0
45.3
473
475
530
482
171
246
54.7
63.0
588
108
89.4
65.2
71.6
92.9
132
67.6
4.4
50.8
from ref. (Bosshart et al. 2013) determined at 30°C
(Bosshart et al., submitted ), determined at 25°C
Table S6| Activation energies determined from Arrhenius Plots (Figure S2)
Enzyme variant
Substrate
Activation energy [kJ mol-1]
PcDTE WT
D-fructose
42.6
PcDTE Var8
D-fructose
59.7
PcDTE IDF10-3
D-fructose
43.6
PcDTE Var8
L-sorbose
85.2
PcDTE ILS6
L-sorbose
56.3
Table S7| Thermodynamic parameters obtained from fitting isothermal EMR experiments for
enzyme variants Var8 and IDF10-3
D-fructose
Var8
IDF10-3
Feed Concentration
Temperature
50°C
Enzyme loading
ΔGact [kJ mol-1]
ΔGinact [kJ mol-1]
kinact [s-1]
kcat [s-1]
2 mg
69.6
113.6
2.78 x 10-7
34.2
1 M D-fructose
60°C
70°C
1.3 mg
69.6
113.6
1.96 x 10-6
69.3
1.3 mg
69.6
113.6
1.15 x 10-5
125.1
50°C
2 mg
64.8
107.1
3.65 x 10-6
227.7
2 M D-fructose
60°C
70°C
1.4 mg
64.7
112.9
4.0 x 10-6
505.0
1.4 mg
64.0
116.9
6.85 x 10-6
511.0
Figure S1| Detailed view on the disassembled EMR used in this study. It consists of the main reactor
body that holds the substrate inlet capillary (1), the reactor lid with the reaction product exit capillary
(2), the magnetic stirring disk (3), the ultrafiltration membrane (4), the membrane support plate (5) and
the screws (6).
a)
b)
300
400
D-psicose [mM]
D-psicose [mM]
250
200
150
100
200
100
50°C
60°C
70°C
50
300
50°C
60°C
70°C
0
0
0
20
40
60
80
0
100
5
10
15
20
25
30
35
Time [h]
Time [h]
Figure S2| Enzyme membrane-reactor experiments at 50°C (circle), 60°C Square) and 70°C triangle).
a) PcDTE Var8 with 0.2 mL min -1 1M D-fructose (10 mM PO4, pH 7.0) and 0.25 mg mL-1 (50°C) or
0.16 mg mL-1 (60°C, 70°C) enzyme. b) PcDTE IDF10-3 with 1 mL min -1 2M D-fructose (10 mM
PO4, pH 7.0 and 0.1 mM MnCl2), 0.24 mg mL-1 (50°C) or 0.16 mg mL-1 (60°C, 70°C).
a)
b)
200
100
50°C
60°C
70°C
160
L-tagatose [mM]
L-tagatose [mM]
80
50°C
60°C
70°C
180
60
40
140
120
100
80
60
40
20
20
0
0
0
10
20
30
Time [h]
40
50
0
10
20
30
40
50
Time [h]
Figure S3 | Enzyme membrane-reactor experiments at 50°C (circle), 60°C Square) and 70°C triangle)
with 1M L-sorbose as substrate. a) PcDTE Var8 was run at 50°C (open circle), 60°C (open square)
and 70°C (open triangle) each with 0.23 mg/mL initial protein concentration in the EMR. b) PcDTE
ILS6 was run at 50°C (open circle) with 0.23 mg/mL initial protein concentration, 60°C (open square)
and 70°C (open triangle) both with 0.15 mg/ml initial protein concentration. Lines indicate linear
curve fits to the linear part of the curve. Only product concentrations below 100 mM L-tagatose (10%
conversion) were fitted to avoid significant impact of the reverse reaction to the observable decay
rate.
References
Bornscheuer UT, Huisman GW, Kazlauskas RJ, Lutz S, Moore JC, Robins K. 2012. Engineering the
third wave of biocatalysis. Nature 485(7397):185-194.
Bosshart A, Hee CS, Bechtold M, Schirmer T, Panke S., Directed divergent evolution of a
thermostable D-tagatose epimerase towards improved activity for two hexose substrates.
submitted.
Bosshart A, Panke S, Bechtold M. 2013. Systematic optimization of interface interactions increases
the thermostability of a multimeric enzyme. Angewandte Chemie, International Edition
52(37):9673-9676.
Ishida Y, Kamiya T, Itoh H, Kimura Y, Izumori K. 1997. Cloning and characterization of the Dtagatose 3-epimerase gene from Pseudomonas cichorii ST-24. Journal of Fermentation and
Bioengineering 83(6):529-534.
Morley KL, Kazlauskas RJ. 2005. Improving enzyme properties: when are closer mutations better?
Trends in Biotechnology 23(5):231-237.
Paramesvaran J, Hibbert EG, Russell AJ, Dalby PA. 2009. Distributions of enzyme residues yielding
mutants with improved substrate specificities from two different directed evolution strategies.
Protein Engineering Design & Selection 22(7):401-411.
Silva-Rocha R, Martínez-García E, Calles B, Chavarría M, Arce-Rodríguez A, de las Heras A, PáezEspino AD, Durante-Rodríguez G, Kim J, Nikel PI and others. 2013. The standard european
vector architecture (SEVA): a coherent platform for the analysis and deployment of complex
prokaryotic phenotypes. Nucleic Acids Research 41(D1):D666-D675.
Studier FW. 2005. Protein production by auto-induction in high-density shaking cultures. Protein
Expression and Purification 41(1):207-234.