of 21
Genetically programmed chiral organoborane synthesis
S. B. Jennifer Kan
,
Xiongyi Huang
,
Yosephine Gumulya
,
Kai Chen
, and
Frances H.
Arnold
*
Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 East
California Boulevard, MC 210-41, Pasadena, CA 91125, United States
Summary paragraph
Recent advances in enzyme engineering and design have expanded nature’s catalytic repertoire to
functions that are new to biology
1
3
. Yet only a subset of these engineered enzymes can function
in living systems
4
7
. Finding enzymatic pathways that forge chemical bonds not found in biology
is particularly difficult in the cellular environment, as this hinges on the discovery not only of new
enzyme activities but also reagents that are simultaneously sufficiently reactive for the desired
transformation and stable
in vivo
. Here we report the discovery, evolution, and generalisation of a
fully genetically-encoded platform for producing chiral organoboranes in bacteria.
Escherichia
coli
harbouring wild-type cytochrome
c
from
Rhodothermus marinus
8
(
Rma
cyt
c
) were found to
form carbon–boron bonds in the presence of borane-Lewis base complexes, through carbene
insertion into B–H bonds. Directed evolution of
Rma
cyt
c
in the bacterial catalyst provided access
to 16 novel chiral organoboranes. The catalyst is suitable for gram scale biosynthesis, offering up
to 15300 turnovers, 6100 h
–1
turnover frequency, 99:1 enantiomeric ratio (e.r.), and 100%
chemoselectivity. The enantio-preference of the biocatalyst could also be switched to provide
either enantiomer of the organoborane products. Evolved in the context of whole-cell catalysts, the
proteins were more active in the whole-cell system than in purified forms. This study establishes a
DNA-encoded and readily engineered bacterial platform for borylation; engineering can be
accomplished at a pace which rivals the development of chemical synthetic methods, with the
ability to achieve turnovers that are two orders of magnitude (over 400-fold) greater than that of
known chiral catalysts for the same class of transformation
9
11
. This tunable method for
manipulating boron in cells opens a whole new world of boron chemistry in living systems.
Boron-containing natural products are synthesised in the soil by the myxobacterium
Sorangium cellulosum
as antibiotics against Gram-positive bacteria
12
. In the sea, these
molecules give the Jurassic red alga
Solenopora jurassica
its distinct pink colouration
13
; they
Reprints and permissions information is available at
www.nature.com/reprints
.
Correspondence and requests for materials should be addressed to F.H.A. (frances@cheme.caltech.edu).
These authors contributed equally to this work.
Supplementary Information is available in the online version of the paper.
Author Contributions
S.B.J.K. and X.H. designed the research with guidance from F.H.A. S.B.J.K., X.H., Y.G., K.C. performed the
experiments and analysed the data. S.B.J.K., X.H. and F.H.A. wrote the manuscript with input from all authors.
The authors declare no competing financial interests.
Readers are welcome to comment on the online version of the paper.
A provisional patent application has been filed through the California Institute of Technology based on the results presented here.
HHS Public Access
Author manuscript
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. Author manuscript; available in PMC 2018 December 07.
Published in final edited form as:
Nature
. 2017 December 07; 552(7683): 132–136. doi:10.1038/nature24996.
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are also produced by the bioluminescent bacterium
Vibrio harveyi
for cell-cell
communications
14
(Extended Data Fig. 1). To prepare boron-containing biomolecules, living
organisms produce small molecules that spontaneously react with boric acid available in the
environment
15
,
16
. While this non-enzymatic method for capturing boron is sufficient for an
organism’s survival, it is limited to a substrate’s inherent affinity towards boric acid, and
lacks tunability and generality for synthetic biology applications. Moreover, organisms that
produce organoboranes (compounds that contain carbon–boron bonds) are unknown.
We envisioned that enzyme-catalyzed borylation could provide living organisms the ability
to produce boron-containing products tailored to our needs. Such an enzyme is not known in
nature, but we hypothesised that existing natural proteins might be repurposed and
engineered to perform this task. In the past, we and others have exploited the promiscuity of
natural and engineered haem proteins for various non-natural reactions
4
,
6
,
7
,
17
. The resulting
enzymes are fully genetically-encoded and carry out their synthetic functions in their
bacterial expression hosts. Here, we focused on introducing boron motifs to organic
molecules enantioselectively, as this would generate boron-containing carbon-stereocentres,
which are important structural features in functional organoboranes such as the FDA-
approved chemotherapeutics Velcade
®
and Ninlaro
®
18
. They are also versatile precursors for
chemical derivatisation through stereospecific carbon–boron to carbon–carbon/carbon–
heteroatom bond conversion
19
21
.
Though boron reagents applicable for carbon–boron bond formation in water are
known
22
,
23
, their biocompatibility, cell permeability, stability, and reactivity in living
systems, where biomolecules, nucleic acids, and metal ions abound, are uncertain.
Nevertheless, since boron reagents designed for
in vivo
chemical biology applications are
precedented
24
26
, we reasoned that reagents suitable for biological borylation could be
found. We identified borane-Lewis base complexes as potential candidates due to their
aqueous stability and reactivity towards carbenoid B–H insertion
9
11
,
27
(Extended Data Fig.
2), a mechanistic pathway we believed might be adapted for use in the biological
environment due to its orthogonality to living systems’ existing biochemistry.
We first set out to assess whether biological organoborane production might be feasible in a
bacterial cell. When
E. coli
BL21(DE3) cells harbouring wild-type cytochrome
c
from
Rhodothermus marinus
, a Gram-negative, thermohalophilic bacterium from submarine hot
springs in Iceland
8
(
Rma
cyt
c
), were incubated with
N
-heterocyclic carbene borane
28
,
29
(NHC-borane)
1
and ethyl 2-diazopropanoate (Me-EDA)
2
in neutral buffer (M9-N minimal
medium, pH 7.4) at room temperature,
in vivo
production of organoborane
3
was observed,
with 120 turnovers (calculated with respect to the concentration of
Rma
cyt
c
expressed in
E.
coli
; Fig. 1a, b) and an e.r. of 85:15 (
R
/
S
isomer = 6; Fig. 1c). Since the pET22b/pEC86
expression system translocates
Rma
cyt
c
to the
E. coli
periplasm for post-translational
maturation (during which the haem cofactor is covalently ligated to the cyt
c
apoprotein)
30
,
we assumed that borylation takes places in the periplasmic compartment. In the absence of
Rma
cyt
c
,
E. coli
yielded only a trace amount of borylation product with very low
stereoselectivity (Extended Data Table 1). Both substrates and the organoborane product
were stable under these conditions. The haem cofactor alone could also promote the
borylation reaction, although with no stereoselectivity. Other cytochrome
c
proteins,
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cytochromes P450, and globins also demonstrated carbon–boron bond forming ability, but
their selectivities were unsatisfactory (Extended Data Table 1).
To improve the performance of this whole-cell catalyst, we subjected the wild-type
Rma
cyt
c
(which we refer to as BOR
WT
hereafter) to site-saturation mutagenesis, sequentially
targeting active-site amino acid residues M100, V75 and M103, which are closest to the
haem iron in BOR
WT
(within 7Å, Fig. 1d). Each single-site site-saturation mutagenesis
library was cloned using the 22c-trick method
31
, screened as whole-cell catalysts in 96-well
plates for improved borylation enantioselectivity, and the best variant was used to parent the
next round of mutation and screening. With a single mutation M100D replacing the distal
axial ligand, the first-generation biocatalyst exhibited 16-fold improvement in turnover over
the wild-type (1850 TTN, Fig. 1b), with 88:12 e.r. (
R
/
S
isomer = 7; Fig. 1c). The M100D
mutation also substantially improved carbene transfer reactivity for Si–H insertion catalyzed
by
Rma
cyt c
6
. This improvement in catalytic performance is likely due to removal of the
axial ligand from the haem iron, which opens a site primed for iron carbenoid formation and
subsequent product formation
32
. Two subsequent rounds of mutagenesis and screening led
to variant BOR
R1
(V75R M100D M103T), which exhibited a turnover of 2490 and an e.r. of
97.5:2.5 (
R
/
S
isomer = 39). This genetically programmed biological function is readily
scalable from analytic to mmol scale – with 0.5 mmol substrates, BOR
R1
produced
organoborane
3
in 97.5:2.5 e.r. and 75% isolated yield (3000 TTN). The absolute
configuration of product
3
was unambiguously assigned to be
R
by X-ray crystallography.
With an excellent borylating bacterium in hand, the properties and potential of the system
were assessed. We characterised the initial rates of
in vivo
borylation and found that
screening for improved enantioselectivity also led to an overall rate enhancement: whole-cell
BOR
R1
is 15 times faster than BOR
WT
, with a turnover frequency of 6100 h
–1
. Interestingly,
as purified protein or in cell lysate, both BOR
R1
and BOR
WT
are orders of magnitude slower
(Fig. 1e). When isolated BOR
R1
protein and whole-cell BOR
R1
were preincubated with
Me-EDA
2
before the borylation reaction, the isolated protein retained only ~50% of its
activity, whereas whole-cell BOR
R1
retained >90% activity (Fig. 1f). NHC-borane
1
and
organoborane product
3
did not inactivate the enzyme. Me-EDA likely inactivates BOR
R1
through carbene transfer to the haem cofactor and/or nucleophilic side chains of the protein,
a mechanism we previously studied in detail for a cytochrome P450-based carbene
transferase
33
. The intact periplasm apparently protects BOR
R1
from inactivation by Me-
EDA, and carbene transfer to yield the organoborane product is generally faster than protein
inactivation pathway(s) under those conditions. Similar observations have been reported for
other protein-based carbene transfer reaction systems.
7
,
34
Analysis of colony-forming units
shows that
in vivo
organoborane production does not dramatically reduce the viability of the
E. coli
(Extended Data Fig. 3).
We next explored the scope of boron reagents that could function in the cellular
environment. Ten boron reagents were tested under turnover-optimised conditions: though
the size, solubility and lipophilicity of these reagents varied, all were found to permeate the
cell membrane and give the desired products in excellent selectivities and turnovers (Fig.
2a). Various substitutions on the NHC nitrogen are tolerated (
3
to
10
). The reaction is
chemoselective in the presence of terminal olefins (
5
), which could function as a reaction
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handle suitable for downstream biological or bio-orthogonal derivatisation. Sterically more
demanding tetra- and penta-substituted NHCs are also accepted (
7
to
10
). Beside imidazole-
based boron reagents, triazolylidene borane and picoline borane could also be used for
in
vivo
borylation, yielding products
11
and
12
in 1070 TTN and 2440 TTN, respectively, with
uniformly high selectivities (96:4 e.r.). On gram scale,
in vivo
borylation produced 740 mg
of picoline organoborane
12
with 2910 TTN, 96:4 e.r. and 42% isolated yield (64% based on
recovered starting material, Fig. 2b). The absolute configuration of
12
was assigned to be
R
by X-ray crystallography. When substrates were added portion-wise at regular time intervals
to
E. coli
expressing BOR
R1
(we tested the sequential addition of up to eight equivalents of
substrates over a period of 12 hours, Fig. 2c; Extended Data Table 2), organoborane
3
was
produced with 10400 turnovers (50% yield, 96:4 e.r.), whereas organoborane
9
was obtained
with 15300 turnovers (73% yield, 96:4 e.r.). No significant loss in activity or
enantioselectivity was observed, demonstrating the potential of this bacterial catalyst for
biosynthesis and incorporation into natural or engineered metabolic pathways.
Systematic modification of the diazo ester substituents from Et to Me,
i
-Pr or Bn revealed
that the borylation ability of BOR
R1
is not limited to Me-EDA (
3
,
13
to
15
, Fig. 2d). The
protein’s relative insensitivity to steric bulk of the ester might indicate that in the putative
iron carbenoid intermediate this moiety is solvent-exposed rather than embedded within the
active site. By re-randomising the 103 position in BOR
R1
, a residue we believe might
modulate loop dynamics for improved binding of this substrate, the borylation turnover of
15
improved (from 2560 to 4200 TTN) using V75R M100D M103D (BOR
R2
, Fig. 3a).
From the same site-saturation library, a borylation catalyst for trifluoromethyl-substituted
diazo ester (CF
3
-EDA) was also discovered (V75R M100D M103F, BOR
R3
). Acceptor/
acceptor diazo reagents such as CF
3
-EDA are less reactive towards carbenoid formation due
to their electron-deficient nature and have not been employed before this for enzymatic
carbene-transfer reactions. The present system tolerates this class of substrates and yielded
product
16
with 95:5 e.r. and 1560 TTN.
To further broaden the generality of this borylation platform, we re-examined the
evolutionary landscape from BOR
WT
to BOR
R1
to search for promiscuous mutants that
might unlock new reactivities. Double mutant V75P M100D (BOR
P*
) stood out as highly
productive but poorly selective (69:31 e.r.) for Me-EDA borylation in the M100D V75X
site-saturation library. As proline-mediated helix kinks are known to induce structural and
dynamic changes to proteins, we asked whether the V75P mutation might provide access to
a unique reaction space. Ethyl 2-diazophenylacetate (Ph-EDA) is a bulky donor/acceptor
diazo reagent inactive towards BOR
WT
, but when added to
E. coli
harbouring BOR
P*
with
NHC-borane
1
, Ph-EDA was transformed to organoborane
17
in 100 TTN and 75:25 e.r.
(Fig. 3a). By accumulating three additional loop mutations though directed evolution
(M99Y, T101A and M103F; Extended Data Table 3), BOR
P*
evolved into a synthetically
useful catalyst (BOR
P1
) for the borylation of Ph-EDA, supporting 340 turnovers with an e.r.
of 94:6.
BOR
P*
also allows us to move beyond diazo ester-based substrates and apply bacterial
production to a different class of chiral organoboranes: though inactive towards BOR
WT
,
CF
3
-substituted (diazomethyl)benzene (CF
3
-DMB) reacted with NHC-borane
1
in the
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presence of BOR
P*
to yield organoborane
(
R
)-18
in vivo
with 74 turnovers and modest
selectivity (79:21 e.r.). We enhanced this through three cysteine mutations at Y71, M89 and
M99 (BOR
P2
; Extended Data Table 3) to produce organoborane
(
R
)-18
in 96:4 e.r. and 1010
TTN. Through X-ray crystallography, the absolute configuration of
(
R
)-18
was
unambiguously assigned as
R
.
Finally, we asked whether the stereochemical preference of biological borylation could be
switched. Towards this end, examination of the M100D V75X site-saturation library for
CF
3
-DMB borylation led us to identify a variant (V75G M100D; BOR
G*
) having an inverted
stereochemical preference to BOR
P*
in the carbon–boron bond-forming step (31:69 e.r. for
R
/
S
isomer; 340 TTN). The selectivity of BOR
G*
was further tuned through mutations
M89F, T98V, M99L, T101L and M103F (BOR
G1
; Extended Data Table 3) to yield
organoborane
(
S
)-18
with 90:10 e.r. and 1120 TTN.
Chiral
α
-trifluoromethylated organoboranes are useful synthetic building blocks that
combine the unique properties of fluorinated motifs with the versatile synthetic applications
of organoboranes
35
; however, methods for their asymmetric preparation are rare
11
,
36
. Our
ability to biosynthesise both enantiomers of these molecules may have applications in
pharmaceutical and agrochemical synthesis. For example, product
(
R
)-18
was converted to
pinacol boronate
19
with retention of the stereogenic carbon centre (Fig. 3b). Through well-
established stereospecific transformations
19
21
, pinacol boronates can be diversified into a
broad array of chiral compounds. We demonstrated the transformation of
19
to alcohol
20,
a
motif found in compounds useful for the treatment of cancer
37
and neurodegenerative
diseases
38
, and the Mattheson homologation-oxidation product
21
, both of which were
obtained with good stereocontrol.
In conclusion, we present a platform for biological borylation, which can be tuned and
configured through DNA manipulation. Microorganisms are powerful alternatives to
chemical methods for producing pharmaceuticals, agrochemicals, materials, and fuels. They
are available by fermentation at large scale and low cost, and their genetically-encoded
synthetic prowess can be systematically modified and optimised. Borylation chemistry can
now be added to biology’s vast synthetic repertoire.
Methods
Detailed experimental methods are available in the Supplementary Information.
Materials
Plasmid pET22b(+) was used as a cloning vector, and cloning was performed using Gibson
assembly
39
. The cytochrome
c
maturation plasmid pEC86
30
was used as part of a two-
plasmid system to express prokaryotic cytochrome
c
proteins. Cells were grown using Luria-
Bertani medium or HyperBroth (AthenaES) with 100 μg/mL ampicillin and 20 μg/mL
chloramphenicol (LB
amp/chlor
or HB
amp/chlor
). Cells without the pEC86 plasmid were grown
with 100 μg/mL ampicillin (LB
amp
or HB
amp
). Electrocompetent
Escherichia coli
cells were
prepared following the protocol of Sambrook
et al.
40
. T5 exonuclease, Phusion polymerase,
and
Taq
ligase were purchased from New England Biolabs (NEB, Ipswich, MA). M9-N
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minimal medium (abbreviated as M9-N buffer; pH 7.4) was used as a buffering system for
whole cells, lysates, and purified proteins, unless otherwise specified. M9-N buffer was used
without a carbon source; it contains 47.7 mM Na
2
HPO
4
, 22.0 mM KH
2
PO
4
, 8.6 mM NaCl,
2.0 mM MgSO
4
, and 0.1 mM CaCl
2
.
Plasmid construction
All variants described in this paper were cloned and expressed using the pET22b(+) vector
(Novagen). The gene encoding
Rma
cyt
c
(UNIPROT ID B3FQS5) was obtained as a single
gBlock (IDT), codon-optimized for
E. coli
, and cloned using Gibson assembly
39
into
pET22b(+) (Novagen) between restriction sites
Nde
I and
Xho
I in frame with an
N
-terminal
pelB leader sequence (to ensure periplasmic localization and proper maturation;
MKYLLPTAAAGLLLLAAQPAMA) and a
C
-terminal 6xHis-tag. This plasmid was co-
transformed with the cytochrome
c
maturation plasmid pEC86 into
E. cloni
®
EXPRESS
BL21(DE3) cells (Lucigen).
Cytochrome
c
expression and purification
Purified cytochrome
c
proteins were prepared as follows. One litre HB
amp/chlor
in a 4 L flask
was inoculated with an overnight culture (20 mL, LB
amp/chlor
) of recombinant
E. cloni
®
EXPRESS BL21(DE3) cells containing a pET22b(+) plasmid encoding the cytochrome
c
variant, and the pEC86 plasmid. The culture was shaken at 37 °C and 200 rpm (no humidity
control) until the OD
600
was 0.7 (approximately 3 hours). The culture was placed on ice for
30 minutes, and isopropyl
β
-
D
-1-thiogalactopyranoside (IPTG) and 5-aminolevulinic acid
(ALA) were added to final concentrations of 20 μM and 200 μM, respectively. The incubator
temperature was reduced to 20 °C, and the culture was allowed to shake for 22 hours at 200
rpm. Cells were harvested by centrifugation (4 °C, 15 min, 4,000xg), and the cell pellet was
stored at −20 °C until further use (at least 24 hours). The cell pellet was resuspended in
buffer containing 100 mM NaCl, 20 mM imidazole, and 20 mM Tris-HCl buffer (pH 7.5 at
25 °C) and cells were lysed by sonication (2 minutes, 2 seconds on, 2 seconds off, 40% duty
cycle; Qsonica Q500 sonicator). Cell debris was removed by centrifugation for 20 min
(5000xg, 4 °C). Supernatant was sterile filtered through a 0.45 μm cellulose acetate filter and
purified using a 1 mL Ni-NTA column (HisTrap HP, GE Healthcare, Piscataway, NJ) using
an AKTA purifier FPLC system (GE healthcare). The cytochrome
c
protein was eluted from
the column by running a gradient from 20 to 500 mM imidazole over 10 column volumes.
The purity of the collected cytochrome
c
fractions was analysed using sodium dodecyl
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Pure fractions were pooled and
concentrated using a 3 kDa molecular weight cut-off centrifugal filter and dialyzed overnight
into 0.05 M phosphate buffer (pH = 7.5) using 3 kDa molecular weight cut-off dialysis
tubing. The dialyzed protein was concentrated again, flash-frozen on dry ice, and stored at
−20 °C. The concentration of cytochrome
c
was determined in triplicate using the
hemochrome assay described below.
Cytochrome P450 and globin expression and purification
Purified P450s and globins were prepared differently from the cytochrome
c
proteins, and
described as follows. One litre HB
amp
in a 4 L flask was inoculated with an overnight
culture (20 mL, LB
amp
) of recombinant
E. cloni
®
EXPRESS BL21(DE3) cells containing a
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pET22b(+) plasmid encoding the P450 or globin variant. The culture was shaken at 37 °C
and 200 rpm (no humidity control) until the OD
600
was 0.7 (approximately 3 hours). The
culture was placed on ice for 30 minutes, and IPTG and ALA were added to final
concentrations of 0.5 mM and 1 mM, respectively. The incubator temperature was reduced
to 20 °C, and the culture was allowed to shake for 20 hours at 200 rpm. Cells were harvested
by centrifugation (4 °C, 15 min, 4,000xg), and the cell pellet was stored at −20 °C until
further use (at least 24 hours). The cell pellet was resuspended in buffer containing 100 mM
NaCl, 20 mM imidazole, and 20 mM Tris-HCl buffer (pH 7.5 at 25 °C). Hemin (30 mg/mL,
0.1 M NaOH; Frontier Scientific) was added to the resuspended cells such that 1 mg of
hemin was added for every 1 gram of cell pellet. Cells were lysed by sonication (2 minutes,
1 seconds on, 2 seconds off, 40% duty cycle; Qsonica Q500 sonicator). Cell debris was
removed by centrifugation for 20 min (27,000xg, 4 °C). Supernatant was sterile filtered
through a 0.45 μm cellulose acetate filter, and purified using a 1 mL Ni-NTA column
(HisTrap HP, GE Healthcare, Piscataway, NJ) using an AKTA purifier FPLC system (GE
healthcare). The P450 and globin proteins were eluted from the column by running a
gradient from 20 to 500 mM imidazole over 10 column volumes. The purity of the collected
protein fractions was analysed using SDS-PAGE. Pure fractions were pooled and
concentrated using a 10 kDa molecular weight cut-off centrifugal filter and buffer-
exchanged with 0.1 M phosphate buffer (pH = 8.0). The purified protein was flash-frozen on
dry ice and stored at −20 °C. P450 and globin concentrations were determined in triplicate
using published extinction coefficients and the hemochrome assay described below.
Hemochrome assay
A solution of sodium dithionite (10 mg/mL) was prepared in M9-N buffer. Separately, a
solution of 1 M NaOH (0.4 mL) was mixed with pyridine (1 mL), followed by centrifugation
(10,000xg, 30 seconds) to separate the excess aqueous layer gave a pyridine-NaOH solution.
To a cuvette containing 700 μL protein solution (purified protein or heat-treated lysate) in
M9-N buffer, 50 μL of dithionite solution and 250 μL pyridine-NaOH solution were added.
The cuvette was sealed with Parafilm, and the UV-Vis spectrum was recorded immediately.
Cytochrome
c
concentration was determined using
ε
550–535
= 22.1 mM
−1
cm
−1.
41
Protein
concentrations determined by the hemochrome assay were in agreement with that
determined by the bicinchoninic acid (BCA) assay (Thermo Fisher) using bovine serum
albumin (BSA) for standard curve preparation.
Mutagenesis library construction
Cytochrome
c
site-saturation mutagenesis libraries were generated using a modified version
of the 22-codon site-saturation method
31
. For each site-saturation library, oligonucleotides
were ordered such that the coding strand contained the degenerate codon NDT, VHG or
TGG. The reverse complements of these primers were also ordered. The three forward
primers were mixed together in a 12:9:1 ratio, (NDT:VHG:TGG) and the three reverse
primers were mixed similarly. Two PCRs were performed, pairing the mixture of forward
primers with a pET22b(+) internal reverse primer, and the mixture of reverse primers with a
pET22b(+) internal forward primer. The two PCR products were gel purified, ligated
together using Gibson assembly
39
, and transformed into
E. cloni
®
EXPRESS BL21(DE3)
cells.
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Mutagenesis library screening in whole cells
Single colonies were picked with toothpicks off of LB
amp/chlor
agar plates, and grown in
deep-well (2 mL) 96-well plates containing LB
amp/chlor
(400 μL) at 37 °C, 250 rpm shaking,
and 80% relative humidity overnight. After 16 hours, 30 μL aliquots of these overnight
cultures were transferred to deep-well 96-well plates containing HB
amp/chlor
(1 mL) using a
12-channel EDP3-Plus 5–50 μL pipette (Rainin). Glycerol stocks of the libraries were
prepared by mixing cells in LB
amp/chlor
(100 μL) with 50% v/v glycerol (100 μL). Glycerol
stocks were stored at −78 °C in 96-well microplates. Growth plates were allowed to shake
for 3 hours at 37 °C, 250 rpm shaking, and 80% relative humidity. The plates were then
placed on ice for 30 min. Cultures were induced by adding 10 μL of a solution, prepared in
sterile deionized water, containing 2 mM IPTG and 20 mM ALA. The incubator temperature
was reduced to 20 °C, and the induced cultures were allowed to shake for 20 hours (250
rpm, no humidity control). Cells were pelleted (4,000xg, 5 min, 4 °C), resuspended in 380
μL M9-N buffer, and the plates containing the cell suspensions were transferred to an
anaerobic chamber. To deep-well plates of cell suspensions were added NHC-borane
substrate (10 μL per well, 400 mM in MeCN) and diazo reagent (10 μL per well, 400 mM in
MeCN). The plates were sealed with aluminium sealing tape, removed from the anaerobic
chamber, and shaken at 500 rpm for 6 h (24 h for reactions with Ph-EDA or CF
3
-DMB due
to their lower aqueous solubility). After quenching with hexanes/ethyl acetate (4:6 v/v, 0.6
mL), internal standard was added (20 μL of 20 mM 1,2,3-trimethoxybenzene in toluene).
The plates were then sealed with sealing mats and shaken vigorously to thoroughly mix the
organic and aqueous layers. The plates were centrifuged (4,000xg, 5 min) and the organic
layer (200 μL) was transferred to autosampler vials with vial inserts for gas
chromatography-mass spectrometry (GC-MS) or chiral high performance liquid
chromatography (HPLC)/supercritical fluid chromatography (SFC) analysis. Hits from
library screening were confirmed by small-scale biocatalytic reactions.
Cell lysate preparation
Cell lysates were prepared as follow:
E. coli
cells expressing
Rma
cyt
c
variant were pelleted
(4,000xg, 5 min, 4 °C), resuspended in M9-N buffer and adjusted to the appropriate OD
600
.
Cells were lysed by sonication (2 minutes, 1 seconds on, 2 seconds off, 40% duty cycle;
Qsonica Q500 sonicator), aliquoted into 2 mL microcentrifuge tubes, and the cell debris was
removed by centrifugation for 10 min (14,000xg, 4 °C). The supernatant was sterile filtered
through a 0.45 μm cellulose acetate filter, and the concentration of cytochrome
c
protein
lysate was determined using the hemochrome assay. Using this protocol, the protein
concentrations we typically observed for OD
600
= 15 lysates are in the 8 – 15 μM range for
wild-type
Rma
cyt
c
and 1 – 10 μM for other
Rma
cyt
c
variants.
Small-scale whole-cell bioconversion
In an anaerobic chamber, NHC-borane (10 μL, 400 mM in MeCN) and diazo reagent (10 μL,
400 mM in MeCN) were added to
E. coli
harbouring
Rma
cyt
c
variant (380 μL, adjusted to
the appropriate OD
600
) in a 2 mL crimp vial. The vial was crimp-sealed, removed from the
anaerobic chamber, and shaken at 500 rpm at room temperature for 6 h (24 h for reactions
with Ph-EDA or CF
3
-DMB). At the end of the reaction, the crimp vial was opened and the
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reaction was quenched with hexanes/ethyl acetate (4:6 v/v, 0.6 mL), followed by the
addition of internal standard (20 μL of 20 mM 1,2,3-trimethoxybenzene in toluene). The
reaction mixture was transferred to a microcentrifuge tube, vortexed (10 seconds, 3 times),
then centrifuged (14,000xg, 5 min) to completely separate the organic and aqueous layers
(the vortex-centrifugation step was repeated if complete phase separation was not achieved).
The organic layer (200 μL) was removed for GC-MS and chiral SFC/HPLC analysis. All
biocatalytic reactions reported were performed in replicates (duplicates to quadruplicates)
from at least two biological replicates. The total turnover numbers (TTNs) reported are
calculated with respect to
Rma
cyt
c
expressed in
E. coli
and represent the total number of
turnovers obtained from the catalyst under the stated reaction conditions. For reactions using
OD
600
= 15
E. coli
cells, the catalyst loadings are 0.0001 – 0.0015 mol% of enzymes with
respect to the limiting reagent in the reaction. The g
borylation product
/g
dry cell weight
ratios
ranged from ~0.05 (wild-type) to ~2 (engineered variant).
Cell viability assay
The colony forming units (cfu) of whole-cell reactions (+ borylation) and controls without
borylation reagents (− borylation) were determined with biological replicates according to
the following procedures. Six 2 mL screw cap vials containing 380 μL suspension of
E. coli
harbouring BOR
R1
(OD
600
= 15) were transferred to an anaerobic chamber. To three of these
vials were added NHC-borane
1
(10 μL, 400 mM in MeCN) and Me-EDA
2
(10 μL, 400
mM in MeCN). These vials were capped and shaken at 500 rpm in the anaerobic chamber (+
borylation). The remaining three vials were capped and shaken in the absence of reagents
1
and
2
(− borylation). After 2.5 hours, all six vials were removed from the anaerobic
chamber. Aliquots of cell suspension were removed the vials and subjected to serial dilution
to obtain stock solutions of 10
6
, 10
7
, and 10
8
-fold dilution. 50 μL of each stock solution was
plated on LB
amp/chlor
agar plates and incubate at 37 °C overnight. The cfu of the cell
suspensions were calculated based on the colony counts of 10
7
-dilution plate. The cfu for
each vial are shown in Extended Data Fig. 2.
Biosynthesis of organoboranes 9 and 3 via serial substrate addition
Twelve 2 mL screw cap vials containing 400 μL suspension of cells harbouring
Rma
cyt
c
BOR
R1
(OD
600
= 15) and 100 μL of glucose (250 mM) were transferred to an anaerobic
chamber. The twelve vials were grouped into four group sets to determine the yield, TTN,
and e.r. for reactions involving the stepwise addition of 2, 4, 6 or 8 equivalents of reagents.
Each equivalent is 2.5 μL solution of NHC-BH
3
substrate in MeCN (2 M) and 2.5 μL Me-
EDA solution in MeCN (2 M). The time interval between each equivalent was 75 minutes.
All four group sets were shaken at 480 rpm in the anaerobic chamber until the completion of
the addition and reaction for the last group set. The vials were then removed from the
anaerobic chamber and quenched with 1 mL of 4:6 hexanes/ethyl acetate and 100 μL
internal standard (1,2,3-trimethoxybenzene, 20 mM in toluene). The reaction mixture was
transferred to a microcentrifuge tube, vortexed (10 seconds, 3 times), then centrifuged
(14,000xg, 5 min) to completely separate the organic and aqueous layers (the vortex-
centrifugation step was repeated if complete phase separation was not achieved). The
organic layer was removed. Another 1 mL of 4:6 hexanes/ethyl acetate and 100 μL internal
standard were added for a second round of extraction and the organic solutions of two
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rounds of extraction were combined. 300 μL of the extract was taken for GC-MS and chiral
HPLC analysis to determine the yield, TTN, and e.r..
Data Availability
Data supporting the findings of this study are available within the paper and its
Supplementary Information, or are available from the corresponding author upon reasonable
request. Crystallographic coordinates and structure factors have been deposited with the
Cambridge Crystallographic Data Centre (CCDC) under accession codes 1572198 for
organoborane
3
, 1572200 for organoborane
18
, and 1572201 for organoborane
12
.
Extended Data
Extended Data Figure 1.
Examples of boron-containing natural products
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Extended Data Figure 2. Summary of known catalytic systems for metal-carbenoid insertion
reactions of boranes
a
, Rh
2
(esp)
2
-catalysed borylation of diazo esters with NHC-boranes.
27
b
, Cu(MeCN)
4
PF
6
-
catalysed borylation of diazo esters with phosphine-borane.
9
c
, [Rh(C
2
H
4
)
2
Cl]
2
-catalysed
borylation of diazo esters with amine-borane adducts.
10
d
, Cu(MeCN)
4
PF
6
-catalysed
borylation of CF
3
-substituted (diazomethyl)benzene with phosphine-borane.
11
e
, Rh
2
(
R
-
BTPCP)
4
-catalysed borylation using alkynes as carbene precursors.
42
f
, I
2
-catalysed
borylation of diazo esters with NHC-boranes.
43
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Extended Data Figure 3. Effect of biological borylation on E. coli cell viability
Cell viability assay was performed in biological triplicate, see Methods section for
experimental protocol.
Extended Data Table 1
Preliminary borylation experiments with haem and haem proteins using NHC-borane (1) and
Me-EDA (2) as substrates
Catalyst
TTN
e.r.
None
0
N/A
Haemin
80 ± 5
0
Haemin + BSA
170 ± 10
54:46
E. coli
cell background
trace
55:45
R. marinus
cyt
c
120 ± 20
85:15
H. thermophilus
cyt
c
140 ± 10
55:45
P. ferrireducens
protoglobin Y60V
NR
-
P411 CIS
trace
n.d.
BM3 P450 wild-type
NR
-
BM3 Hstar
trace
n.d.
N/A – not applicable; NR – no product was detected; n.d. – not determined; Experiments with cytochromes
c
, globin, or
cytochromes P450 were performed using
E. coli
harbouring the corresponding protein (OD
600
= 15). Reactions were
performed in biological triplicate. TTNs reported represent mean values averaged over three biological replicates, and the
error bars indicate one standard deviation. Within instrument detection limit, variability in e.r. was not observed. Unreacted
starting materials were observed at the end of all reactions and no attempt was made to optimize these reactions.
Experiments with hemin were performed using 100 μM hemin, 10 mM NHC-borane
1
, 10 mM Me-EDA
2
, 10 mM
Na
2
S
2
O
4
. Experiments with hemin and BSA were performed using 100 μM hemin in the presence of BSA (0.75 mg/mL)
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instead. Experiments to determine
E. coli
cell background reaction was performed with
E. cloni
®
EXPRESS BL21(DE3)
cells containing a pET22b(+) plasmid encoding halohydrin dehalogenase (HHDH) from
Agrobacterium tumefaciens
(UNIPROT ID Q93D82) instead of
Rma
cyt
c. A. tumefaciens
HHDH is inactive towards NHC-borane
1
and Me-EDA
2
.
P411 CIS
4
and BM3 Hstar
44
are previously reported engineered BM3 P450 variants.
Extended Data Table 2
Biosynthesis of organoboranes 3 and 9 via serial substrate addition
total equiv. of reagents
biological replicate 1
biological replicate 2
yield%
TTN
e.r.
yield%
TTN
e.r.
2
42
2270
97.5 : 2.5
40
2130
97.5 : 2.5
4
43
4560
97:3
37
3840
97 : 3
6
57
9000
96.5 : 3.5
43
6800
96.5 : 3.5
8
50
10500
96.5 : 3.5
48
10300
96.5 : 3.5
total equiv. of reagents
biological replicate 1
biological replicate 2
yield%
TTN
e.r.
yield%
TTN
e.r.
2
63
3300
97.5 : 2.5
59
3100
97.5 : 2.5
4
67
7000
97 : 3
55
5800
97 : 3
6
71
11100
96.5 : 3.5
69
10900
96.5 : 3.5
8
75
15800
96 : 4
72
14800
96 : 4
Experiments performed in biological duplicate. See Methods section for experimental protocol.
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Extended Data Table 3
Directed evolution of whole cell
Rma
cyt
c
for improved enantioselectivity in the
biosynthesis of organoboranes 17, (
R
)-18 and (
S
)-18
mutations
e.r. of 17
M100D V75P
75 : 25
M100D V75P M99Y
81 : 19
M100D V75P M99Y T101A
89 : 11
M100D V75P M99Y T101A M103F
94 : 6
mutations
e.r. of (
R
)-18
M100D V75P
76 : 24
M100D V75P M89C
90 : 10
M100D V75P M89C Y71C
94 : 6
M100D V75P M89C Y71C M99C
96 :4
mutations
e.r. of (
S
)-18
M100D V75G
73 : 27
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mutations
e.r. of (
S
)-18
M100D V75G M89F M103F
78 : 22
M100D V75G M89F M103F T101L
86: 14
M100D V75G M89F M103F T101L M99L
88 : 12
M100D V75G M89F M103F T101L M99L T98V
90 : 10
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
This work was supported in part by the National Science Foundation, Office of Chemical, Bioengineering,
Environmental and Transport Systems SusChEM Initiative (grant CBET-1403077), and the Gordon and Betty
Moore Foundation through Grant GBMF2809 to the Caltech Programmable Molecular Technology Initiative. X.H.
is supported by a Ruth L. Kirschstein NIH Postdoctoral Fellowship (F32GM125231). We thank O. F. Brandenberg,
S. Brinkmann-Chen, T. Hashimoto, R. D. Lewis, and D. K. Romney for discussions and/or comments on the
manuscript, and N. W. Goldberg and A. Zutshi for experimental assistance. We are grateful to S. Virgil (Caltech
Center for Catalysis and Chemical Synthesis), N. Torian (Caltech Mass Spectrometry Laboratory), M. K. Takase
and L. Henling (Caltech X-ray Crystallography Facility) for analytical support; and H. Gray for providing the
pEC86 plasmid.
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