High-throughput screening for terpene-synthase-cyclization activity and directed evolution of a terpene synthase

Article abstract of DOI:10.1002/anie.201301362

An easy assay: A synthetic substrate enables a colorimetric screen for terpene synthase cyclization activity, thereby facilitating the engineering of these enzymes. By using directed evolution, the thermostability of a sesquiterpene synthase was increased without the loss of other properties. The technique also enabled rapid optimization of conditions for expression and stabilization in lysate of another terpene synthase. PPO=diphosphate. Copyright

Full text of DOI:10.1002/anie.201301362

Angewandte
Chemie
DOI: 10.1002/anie.201301362
Directed Evolution
High-Throughput Screening for Terpene-Synthase-Cyclization Activity
and Directed Evolution of a Terpene Synthase**
Ryan Lauchli, Kersten S. Rabe, Karolina Z. Kalbarczyk, Amulya Tata, Thomas Heel,
Rebekah Z. Kitto, and Frances H. Arnold*
The development of high-throughput assays can be extremely
challenging, yet is essential for many applications in drug
discovery and enzyme engineering.[1] Directed evolution has
proven to be a reliable method for optimizing the perfor-
mance of enzymes in a variety of applications,[2] but it requires
an appropriate high-throughput assay for screening mutant
libraries. Terpene synthases catalyze the key cyclization step
in the biosynthesis of terpenoids, which are by far the largest
class of natural compound and are highly valued as medicines,
materials, fuels, and chemicals.[3] Although metabolic engi-
neering efforts have improved access to some terpenoids by
production in microbial hosts, the terpene synthase enzymes
responsible for the cyclization of linear isoprenoid diphos-
phates into cyclic terpenoids have proven difficult to engi-
neer.[4] The cyclizations proceed through complex carbocation
bond-forming reactions and migrations, and are terminated
by either elimination or aqueous quenching of carbocationic
intermediates. The complexity of these processes renders
rational engineering of terpene synthases nearly impossible.
Furthermore, mutagenic studies are reliant upon tedious GC–
MS analysis of products, and thus, such studies have explored
only small selections of amino acid mutations. The use of
directed evolution, therefore, has been difficult because no
easy high-throughput assay for productive cyclization activity
has been described.
a modified substrate containing a vinyl methyl ether func-
tionality and a coupled enzyme assay to detect the cyclization
byproduct, methanol. We have used this screen to increase the
thermostability of one terpene synthase by directed evolu-
tion, and to optimize growth and reaction conditions for
a second enzyme.
Several nonnatural isoprenoids were shown to serve as
substrates for terpene synthases.[5] Notably, Virgil and Corey
demonstrated that lanosterol synthase could utilize vinyl
ether 1 (Scheme 1) as a substrate in place of squalene oxide.[5a]
Scheme 1. Terpene synthase substrates containing vinyl ether function-
alities. PPO=diphosphate.
In subsequent work by Jin and Coates,[5b] vinyl methyl ether
containing isoprenoid diphosphate 2 was used as a substrate
for a diterpene synthase. This vinyl methyl ether, however,
hydrolyzed rapidly in neutral aqueous conditions. Inspired by
these precedents, we hypothesized that a stable version of
a vinyl methyl ether containing substrate might react in the
presence of a terpene synthase to generate methanol as a side
product. Thus, we designed substrate 3, which was expected to
be less nucleophilic, and therefore less susceptible to hydrol-
ysis, than 2. Substrate 3 was synthesized in four steps from
a known compound[6] and was produced as a mixture of cis
and trans isomers. Substrate 3 was stable for at least 10 days at
room temperature as a 10 mm stock solution in aqueous
NH4HCO3 (25 mm).
Synthetic substrate 3 has nearly the same length and
general shape as farnesyl diphosphate (FPP), the natural
substrate for sesquiterpene synthases. Thus, we tested sesqui-
terpene synthases BcBOT2 and SSCG_02150 for activity on 3
in small-scale reactions and used gas chromatography for
product analysis.[7] BcBOT2, from the agriculturally impor-
tant powdery mildew Botrytis cinerea, produces presilphiper-
folan-8b-ol (PSP), a key intermediate in the biosynthesis of
the fungusꢀ virulence factor.[7a] SSCG_02150 is a (À)-d-
cadinene synthase from Streptomyces clavuligerus.[7b] To our
satisfaction, these two enzymes generated identical products
A screen for cyclization activity that provides a colori-
metric read-out and can be used in cell lysate would expedite
the engineering of terpene synthases by directed evolution
and would facilitate the optimization of conditions for
soluble, active expression and purification of terpene syn-
thases. We have developed a high-throughput screen that uses
[*] Dr. R. Lauchli, Dr. K. S. Rabe, K. Z. Kalbarczyk, A. Tata, Dr. T. Heel,
R. Z. Kitto, Prof. Dr. F. H. Arnold
Division of Chemistry and Chemical Engineering
California Institute of Technology
1200 California Blvd. MC 210-41, Pasadena, CA 91125 (USA)
E-mail: frances@cheme.caltech.edu
[**] We are grateful to David Cane, Scott Virgil, Robert M. Coates, and
David Christianson for their pioneering studies, invaluable advice,
encouragement, and materials. We thank Indira Wu, Chris Farwell,
and Jack Zhang for assistance. R.L. acknowledges the support of
NIH fellowship F32GM095061. The content is solely the responsi-
bility of the authors and does not necessarily represent the official
views of the NIH. K.S.R. thanks the Deutscher Akademischer
Austauschdienst (DAAD) for a postdoctoral fellowship. K.Z.K. and
R.Z.K. acknowledge the support of Summer Undergraduate
Research Fellowships from the California Institute of Technology.
T.H. was funded by the FWF grant number: J3327-B21.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201301362.
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from 3. According to GC, the product was a mixture of two
compounds, which we determined was the result of a thermal
Cope rearrangement (caused by the injection port of the GC
at 2508C, see the Supporting Information) of a single product
believed to be 7.[8] The terpene-synthase-catalyzed reaction
that generates 7 is shown in Scheme 2. Upon cyclization of
substrate 3, the vinyl methyl ether moiety generates a stabi-
lized carbocation 5, which in turn is quenched by water to
generate hemiacetal 6. Hemiacetal 6 is unstable and decom-
poses to the aldehyde 7, releasing one equivalent of methanol
in the process.
starting points for the evolution of new functions. Thermo-
stabilization enables enzymes to operate at higher temper-
atures, with concomitant faster reaction rates, and allows
longer catalyst lifetimes and lower catalyst loadings.[11] Weiss
and co-workers undertook the thermostabilization of the
plant sesquiterpene synthase, tobacco epi-aristolochene syn-
thase (TEAS), by using a computational method, based on
the crystal structure of the enzyme, to introduce specific
stabilizing interactions.[12] Though the authors were able to
improve thermostability, their mutant enzyme was poorly
active, insolubly expressed, and the specificity of the product
mixture was lost to a large degree (significant side products
were formed).
We undertook a directed evolution approach to creating
a thermostable terpene synthase with native catalytic activity.
A library of about 2800 BcBOT2 mutants was created by
error-prone PCR (epPCR) with an average of three base-pair
mutations per gene. This library was heat-treated for 10 min
at 458C, and the screen was used to identify mutants that
retained activity. Three thermostable mutants were identified.
The most improved variant, 19B7, had lost its three C-
terminal amino acids and had the mutation K85R. The
locations of these mutations within the folded protein
structure are not known, because no crystal structure of
BcBOT2 has been published. The mutations in 19B7 are
stabilizing yet do not noticeably change expression, activity,
or the specificity for the product (see the Supporting
Information). The product analysis from GC-scale reactions
using the native substrate, FPP, showed only traces of side
product formation. Expression was maintained as well. The
T50 was increased from 428C to 478C. (T50 is the incubation
temperature at which 50% of productivity on 3 remained
after 10 min of heat treatment.)
We then confirmed that productivity on the native
substrate, FPP, correlated with productivity on 3, as measured
by using the screen, for methanol production. The reaction of
purified 19B7 on FPP was followed by GC for 20 min, and the
amount of cyclization product PSP was quantified at multiple
time points. The reaction of 19B7 with 3 was also monitored
over 20 min under the same conditions. Productivity data with
3 were obtained at time points by terminating the terpene
synthase reactions with ethylenediaminetetraacetic acid
(EDTA), and subsequently determining the quantities of
methanol released by using the AOX coupled enzyme assay.
As shown in Figure 1, the 19B7-mediated production of
methanol from 3 closely follows the cyclization of FPP to
make PSP. Importantly, this correlation allows one to use the
screen to rapidly estimate the effects of conditions on enzyme
productivity instead of using GC-based measurements.
Another round of directed evolution performed on
variant 19B7 (again by using random mutagenesis with
a mutation rate of 3 bp mutations per gene) increased the
T50 value to about 548C in variant 9D6 (screening ca. 1800
mutants). Variant 9D6 had the mutation H383R, and again
product specificity was maintained with the native substrate,
FPP, as measured by GC. Expression and activity were also
similar to that of BcBOT2. The thermostabilities of 9D6 and
19B7 are shown compared to parent BcBOT2 in Figure 2. In
only two rounds of directed evolution and without the
Scheme 2. Generation of methanol upon terpene-synthase-catalyzed
reaction on synthetic substrate 3.
After having shown that these sesquiterpene synthases are
able to use 3 as a substrate, we adapted the reaction of 3 for
high-throughput screening by using an established coupled
enzyme assay for methanol.[9d] Substrate 3 and terpene
synthase enzymes were allowed to react for 1 min to 24 h in
microtiter plates. After the incubation time was reached,
alcohol oxidase (AOX), which converts methanol to formal-
dehyde in the presence of molecular oxygen, was added to the
microtiter wells.[9] After 10 min of incubation with AOX,
Purpald was added to react with the formaldehyde to
generate a blue-purple color in wells with active enzyme
(Scheme 3).[10] The absorbance, and corresponding concen-
Scheme 3. Enzyme-coupled assay for methanol quantification.
tration of methanol, was read by a plate reader at 550 nm.
Under the conditions used here, concentrations of about 20–
250 mm of methanol were found to represent a reasonable
working range for the assay, as confirmed by conducting this
sequence of AOX and Purpald treatment with methanol
standards (see the Supporting Information). Notably, this
screen works well with both purified enzyme and in crude
E. coli lysate, and it has the advantage that dithiothreitol
(DTT) can be used to stabilize proteins without interfering
with the screen response.
Next, the screen was applied to the thermostabilization of
terpene synthase BcBOT2 by directed evolution. Thermo-
stable enzymes are much easier to use and are also better
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for 1 h, and the AOX-Purpald screen was used to determine
which condition(s) gave active enzymes. As shown in Table 1,
the screen rapidly revealed that expression in TB was superior
to that in LB. Furthermore, expression at 258C appeared to be
Table 1: Absorbance values of the screen for various expression
conditions.
LB[a] 188C TB 188C LB 258C TB 258C
Blank no additive[b]
Blank + betaine
Blank + sucrose
SSCG_02150 no additive 0.20
SSCG_02150 + betaine
SSCG_02150 + sucrose
0.20
0.18
0.22
0.17
0.20
0.24
0.27
0.38
0.38
0.17
0.17
0.22
0.16
0.24
0.22
0.17
0.18
0.24
0.20
0.40
0.22
0.32
0.26
Figure 1. Correlation of methanol release from 3 (measured by using
the screen) with presilphiperfolan-8b-ol (PSP) generation from FPP as
measured by GC. Reactions were run with the thermostable BcBOT2
variant 19B7 (100 nm) at 308C using 0.20 mm of substrate. Back-
ground signal was subtracted from the response.
[a] TB is terrific broth with kanamycin sulfate (50 mgLÀ1), LB is Luria
broth with kanamycin sulfate (50 mgLÀ1). [b] Blanks are reported as an
average of 2 wells; reactions with SSCG_02150 were reported as an
average of 8 wells.
slightly better than at 188C. Notably, no activity was seen for
SSCG_02150 in the absence of stabilizing additives, and
betaine proved to be the best additive for stabilization.
Sucrose appeared to give a slight background response,
whereas betaine produced no response above background.
In summary, we have shown that synthetic substrate 3 is
effective for screening the cyclization reactions catalyzed by
two sesquiterpene synthase enzymes. By using this screen we
were able to increase the thermostability of terpene synthase
BcBOT2 by 128C. No structural information was required for
this work, and the screen response of thermostabilized mutant
19B7 was predictive for activity on the native substrate FPP.
The screen also proved to be a powerful tool for optimizing
the expression and buffer conditions for a second sesquiter-
pene synthase, SSCG_02150. The successful application of
this high-throughput screen using synthetic substrate 3 to two
distinct enzyme optimization problems suggests that this
approach may be generally useful. Optimization challenges
for terpene synthases including overcoming product (inor-
ganic diphosphate) inhibition, increasing activity on native
substrates or analogues, altering cofactor (divalent metal
cation) preference, and tolerance to organic solvents are all
problems that are not obviously solved by rational methods,
but may be solved by directed evolution using this screening
system.
Figure 2. Methanol release, measured by using the screen, of BcBOT2
and mutants after heat treatment for 10 min at the indicated temper-
atures and subsequent incubation at room temperature for 1 h with 3.
Error bars indicate standard deviation of at least 3 repeats.
advantage of structural knowledge, the thermostability of
BcBOT2 was increased by about 128C with no loss in other
desirable properties. Because thermostable proteins are more
robust to mutation, 9D6 is an excellent starting point for
further functional evolution.
This screening system can also facilitate optimization of
expression, purification, and reaction conditions for terpene
synthases. Optimization of these properties can be a tedious
process, because terpene synthases tend to be difficult to
express and work with. These enzymes frequently form
inclusion bodies or are expressed in inactive form.[13] Even
when soluble expression is achieved, purification and reaction
buffer conditions must be optimized to keep these sensitive
enzymes soluble and active.
We induced protein production in cultures of E. coli
containing the SSCG_02150 gene in Luria broth (LB) and
terrific broth (TB) at both 18 and 258C. The resulting cells
were lysed with either no buffer additive or with 10% of
either sucrose or betaine. The lysates were then treated with 3
Experimental Section
Procedure for the assay: A microtiter plate with cell lysate (100 mL
per well) is treated with substrate 3 (100 mL per well of 0.50 mm
solution) in standard buffer (50 mm PIPES, 10 mm MgCl2, 100 mm
NaCl, 2 mm DTT, pH 7.5). This solution is made immediately prior to
use from a stock solution of substrate 3 (10 mm) in 25 mm aqueous
NH4HCO3 (This stock solution is stable for at least 3 months at
À208C). The reactions are incubated at room temperature for 1–2 h,
and then a solution (10 mL per well) of dilute alcohol oxidase is added
(50 mL of AOX stock solution from MP Biomedicals dissolved in
950 mL of cold 0.1m potassium phosphate buffer, pH 8.0). The
microtiter plate is shaken at room temp for 10 min at 600 rpm on
an orbital platform shaker. Optionally, 16 mL of EDTA (0.5m, pH 8.0)
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can be added to each well to stop the terpene synthase reaction at any
point in the process, with shaking for 1 min to mix the reaction
contents. Purpald solution (Aldrich, 351 mg dissolved in 15 mL of 2n
NaOH, 50 mL per well) is added and shaking is resumed for 20–
30 min. The plates are briefly centrifuged at 3000–5000 rpm to
remove bubbles, and the absorbances are read with a plate reader at
550 nm. Background absorbance is typically 0.2 or below, with active
enzymes sometimes giving absorbances exceeding 1.0.
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Published online: March 26, 2013
Keywords: directed evolution · high-throughput screening ·
.
protein engineering · terpenes · thermostability
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