Seven-enzyme in vitro cascade to (3R)-3-hydroxybutyryl-CoA

Article abstract of DOI:10.1039/C8OB02858C

Economical and environmentally-friendly routes to convert feedstock chemicals like acetate into valuable chiral products such as (R)-3-hydroxybutyrate are in demand. Here, seven enzymes (CoaA, CoaD, CoaE, ACS, BktB, PhaB, and GDH) are employed in a one-pot, in vitro, biocatalytic synthesis of (3R)-3-hydroxybutyryl-CoA, which was readily isolated. This platform generates not only chiral diketide building blocks but also desirable CoA derivatives.

Full text of DOI:10.1039/C8OB02858C

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Seven-enzyme in vitro cascade to
(3R)-3-hydroxybutyryl-CoA†
Cite this: DOI: 10.1039/c8ob02858c
Luis E. Valencia,a Zhicheng Zhang, b Alexis J. Cepedaa and
Received 15th November 2018,
Accepted 9th January 2019
a
Adrian T. Keatinge-Clay
*
DOI: 10.1039/c8ob02858c
rsc.li/obc
Economical and environmentally-friendly routes to convert feed- produce either the chiral diketide (R)-3-hydroxybutyrate or its
stock chemicals like acetate into valuable chiral products such as enantiomer (S)-3-hydroxybutyrate with the help of enzymes
(R)-3-hydroxybutyrate are in demand. Here, seven enzymes (CoaA, from the polyhydroxyalkanoate pathway.8–10 In these biosyn-
CoaD, CoaE, ACS, BktB, PhaB, and GDH) are employed in a one- thetic schemes, acetoacetyl-CoA is generated from two mole-
pot, in vitro, biocatalytic synthesis of (3R)-3-hydroxybutyryl-CoA, cules of acetyl-CoA (one CoA being recycled in the process) by
which was readily isolated. This platform generates not only chiral a biosynthetic thiolase (e.g., BktB from Cupriavidus necator,
diketide building blocks but also desirable CoA derivatives.
formerly Ralstonia eutropha), reduced by a stereoselective
reductase (e.g., PhaB from C. necator), and cleaved from CoA as
3-hydroxybutyrate by a thioesterase (e.g., TesB from E. coli).
We sought to generate the 3R-hydroxybutyryl fragment
in vitro from acetate through a multi-enzyme cascade. This
would serve as proof of principle that more complex chiral
molecules can be generated through this route utilizing PKS
KRs (Fig. 1). The primary concern was whether the unfavorable
thermodynamics of the BktB thiolase reaction (Keq = 1.1 ×
Better methods are needed to generate chiral products from
abundant feedstock chemicals. Biosynthetic pathways natu-
rally generate such compounds in vivo within the one-pot
environment of the cytosol, and synthetic biologists can often
steer the carbon flux towards a desired product. However,
manipulating such pathways in vitro using cell-free extracts
confers several advantages – the substrates and enzymes as
well as their concentrations can be controlled, molecules in
the pathway are not metabolized by organisms, and diffusion
of the substrates and products are not impeded by cellular
membranes. Indeed, multienzyme cascade reactions with cell-
free extracts are increasingly being employed in the asym-
metric synthesis of chiral alcohols, amines, and amino
acids.1,2 Impressive feats of carbon–carbon formation (even
converting CO2 into malate through a 17-enzyme system) have
been achieved.3,4
10−5
) could be overcome by exergonic reactions in the
designed pathway.11,12 Our previous studies established that
BktB operates on acyl-SNAC substrates, but how active and
stereocontrolled PhaB would be toward a truncated acyl thio-
ester was unknown.13 An acetyl-CoA synthetase (ACS,
Streptomyces coelicolor) that can ligate acetate with diverse
thiol acceptors, and a glucose dehydrogenase (GDH, Bacillus
subtilis) that oxidizes D-glucose to regenerate NADPH from
NADP+ were also employed.7
A long-term goal of ours is to employ modular polyketide
synthase (PKS) ketoreductases (KRs) that can set two stereocen-
ters during a reduction reaction in the conversion of molecules
like acetate and propionate into chiral diketide products.5,6
Some of these KRs display good stereocontrol even toward
α-alkyl, β-ketoacyl-S-N-Acetylcysteamine (SNAC) thioesters,
truncated versions of their natural substrates.7 We have been
inspired by the engineering of Escherichia coli to exclusively
We tested the pathway composed of ACS, BktB, PhaB, and
GDH with the short -SNAC handle but did not detect the
expected 3-hydroxybutyryl-SNAC product by liquid chromato-
graphy/mass spectroscopy (LC/MS). However, using the longer
handle pantetheine, prepared by reducing the abundant com-
pound pantethine with dithiothreitol (DTT), 3-hydroxybutyryl-
S-pantetheine was detected. A comparison of crystal structures
of the C. necator PhaB (the Burkholderia pseudomallei PhaB
employed in the cascade reaction is 56% identical) in the pres-
ence and absence of acetoacetyl-CoA (PDB Codes: 4N5M &
4N5L) offers an explanation for these results.14,15 The natural
substrate induces a conformational change of PhaB, including
a 4.6 Å shift of the “Clamp-lid”, into its catalytically competent
form. The interactions with PhaB extend beyond the acetoace-
aDepartment of Molecular Biosciences, The University of Texas at Austin, Austin,
TX 78712, USA. E-mail: adriankc@utexas.edu; Tel: +1-512-471-2977
bDepartment of Chemistry, The University of Texas at Austin, Austin, TX 78712, USA
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8ob02858c
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Fig. 1 Long-term goal of generating useful chiral products from feed-
stocks like acetate and propionate (* = chiral center).
tyl-SNAC and even past the acetoacetyl-pantetheine portion of
the substrate, with contacts to the diphosphate and the
adenine base. Thus, to obtain full activity and stereocontrol
from PhaB, the entire CoA handle might be necessary.
As CoA can be readily generated from pantetheine through
the action of CoaA (Staphylococcus aureus), CoaD (E. coli), and
CoaE (E. coli), these enzymes were added to the multienzyme
cascade reaction (Fig. 2).16 The seven-enzyme reaction consist-
Fig. 2 Seven-enzyme cascade reaction yielding (3R)-3-hydroxybutyryl-
CoA from pantethine, ATP, and acetate. BktB fuses acetyl groups from
two molecules of acetyl-CoA and allows one CoA to be recycled.
ing of CoaA, CoaD, CoaE, ACS, BktB, PhaB, and GDH (using (1.08 ppm, d, J = 6.3 Hz, 3H) shows the same parameters as
crude ammonium sulfate precipitates from overexpressing the methyl group of authentic (R)-3-hydroxybutyric acid.17
E. coli) was monitored in stages by LC/MS. First, the formation Eluted (3R)-3-hydroxybutyryl-CoA can be brought to neutral pH
of CoA through incubating pantethine with DTT, CoaA, CoaD, with aqueous lithium hydroxide and precipitated in 95% (v/v)
CoaE, and ATP was ensured. Next, the formation of acetyl-CoA acetone.
through the addition of ACS was checked. Finally, the for-
To determine the diastereomeric purity of (3R)-3-hydroxybu-
mation of (3R)-3-hydroxybutyryl-CoA through the addition of tyryl-CoA, the 3-hydroxybutyryl fragment was moved onto a
BktB, PhaB, GDH, NADP+, and D-glucose was observed, -SNAC handle through thiol-thioester exchange and compared
confirming that the one-pot scheme was working. The (3R)-3- with a standard synthesized from authentic (R)-3-hydroxybutyric
hydroxybutyryl-CoA product was purified by diluting the acid (Fig. 3C). The resulting 3-hydroxybutyryl-SNAC was
100 mL cascade reaction 20-fold, passing it through
a
analyzed with a chiral column (ChiralCel OC-H). It matched
Q-Sepharose ion exchange column, washing it with 50 mM the retention time of authentic (R)-3-hydroxybutyryl-SNAC.
HCl, and eluting with 100 mM HCl. Mass spectral analysis is As no (S)-3-hydroxybutyryl-SNAC was detected, the generated
consistent with (3R)-3-hydroxybutyryl-CoA, and absorbance (3R)-3-hydroxybutyryl-CoA appears to be of high diastereomeric
measurements reveal that the yield approaches 70% (120 mg) purity.
(Fig. 3A, B and ESI†). HPLC as well as 1H and 31P NMR analysis
The in vitro method described here has its limitations. It
indicate the eluted (3R)-3-hydroxybutyryl-CoA is at least 90% may not be possible to outcompete the in vivo routes to prepar-
pure. The peaks in the NMR spectrum are consistent with acyl- ing industrial quantities of (R)-3-hydroxybutyric acid or
CoAs, and the methyl group of the 3-hydroxybutyryl fragment (S)-3-hydroxybutyric acid. While none of the reagents used
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for the types of chiral products that can be obtained from such
reactions. Further development employing PKS KRs should
enable the generation of needed two-stereocenter chiral
buiding blocks from feedstock chemicals.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by NIH (GM106112) and the Welch
Foundation (F-1712). We thank Josh Beckham (UT Austin
Freshman Research Initiative Virtual Cures Stream) for the
pNIC-Bsa4-PhaB plasmid.
Fig. 3 Authentication of (3R)-3-hydroxybutyryl-CoA. (A) Negative-
mode low-resolution LC/MS. (B) High-resolution MS yielded the antici-
pated molecular formula. (C) The (R)-3-hydroxybutyryl group generated
through the cascade reaction was transferred to a -SNAC handle, and a
comparison with synthetic standards by chiral chromatography indicates
its high level of diastereomeric purity.
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