bound cofactor.13-21 The absolute requirement for a 4-hy-
droxy aromatic substrate and the absence of exogenous
cofactors suggest a mechanism that proceeds by acid-base
chemistry via a para-quinone methide intermediate.21,22 The
enzyme-catalyzed decarboxylation and thermal decarboxyl-
ation mediated by base catalysis may both proceed by the
quinone methide intermediate.4 Except for the use of
isotopically labeled substrates to determine the reaction
stereoselectivity,22 there have been few mechanistic studies
of phenolic decarboxylase enzymes, and no protein structure
has been solved to date.
Herein, we describe optimization of the decarboxylase
biotransformation on fermentation-derived pHCA (bio-
pHCA). The approach is similar to that used by Lee et al. in
which vinylguaiacol (3-methoxy 4-vinylphenol) was pro-
duced at 9.6 g L-1 from ferulic acid in a water/hexane
mixture with ferulic decarboxylase.23 We used E. coli
recombinants for stable overexpression of PDC activity and
adopted the two-solvent, two-phase strategy in which the
product 4-VP is selectively removed from the aqueous
medium. The decarboxylation can be performed using crude
fermentation preparations of pHCA with little effect on
product yield, an advantage of enzymatic catalysis over
chemical syntheses, which exhibit less tolerance for reactant
impurities. Since the desired monomer for low branching
polymerization is acetoxystyrene (ASM), we demonstrated
its convenient preparation by a one-pot, two-stage process
where 4-VP is immediately acetylated after the aqueous
medium is drained from the two-solvent reactor.
pHCA in these studies was a tyrosine overproducing strain
expressing the Rhodotorula glutinis phenylalanine/tyrosine
ammonia lyase gene (pal).12 SDS PAGE analyses were
performed by diluting the samples in 2X sample loading
buffer (LDS, Invitrogen) to achieve a final protein concentra-
tion of 0.5 mg mL-1. Samples were placed in capped
microfuge tubes, heat denatured at 70 °C for 10 min and
then loaded (10 µg protein/lane) onto NuPage 4-12% Bis-
Tris gels (Invitrogen) using the MES buffer system for
enhanced resolution of lower molecular weight proteins. The
Mark 12 molecular weight standards (Invitrogen) were used.
Gels were run at constant voltage (200 V) for 40 min. When
SDS-PAGE gels were used for Western blots for expression
level measurements, purified L. plantarum decarboxylase
protein (1.0 to 25 ng protein per lane)11 was loaded as the
standard. When protein levels in cell-free extracts were very
high, the decarboxylase concentrations on the gels were
quantified directly by staining in Simply Blue Safe Stain
(Invitrogen) for 1.0 h. After 3 × 1.0 min washes with
distilled, deionized water, the destained gels were photo-
graphed with a FluorChem 8000 system (Alpha Innotech,
San Leandro, CA) and protein expression was quantified by
following the manufacturer’s protocols. Decarboxylation
reactions were followed using thin layer chromatography
(TLC): solid support, Merck Silica gel 60F254; mobile phase,
ethyl acetate, 100%; typical Rf values for pHCA and 4-VP
were 0.4 and 0.7, respectively. High performance liquid
chromatography (HPLC) analysis was performed on an
Agilent 1100 series liquid chromatograph (Agilent Technolo-
gies, Palo Alto, CA) with a photodiode array detector using
a Zorbax SB-C18 column (4.6 mm × 250 mm or 4.6 mm ×
150 mm rapid resolution); UV detector wavelengths, 258
and 312 nm; temperature, 40 °C; mobile phase, a gradient
combining solvent A, 0.1% trifluoroacetic acid in water, and
solvent B, 0.1% trifluoroacetic acid in acetonitrile, with 95%
solvent A/5% solvent B at start; linear gradient to 20%
solvent A and 80% solvent B over 8 min; holding at 20%
solvent A/80% solvent B for 2 min and then a linear gradient
to 95% solvent A/5% solvent B over 1 min; flow rate, 1.0
mL min-1. Typical retention times for tyrosine, pHCA, and
4-VP were 3.5, 5.3, and 7.4 min, respectively. UV/vis
spectrometry was performed on a Cary 100 Bio UV/vis
spectrophotometer (Palo Alto, CA). Extracted or purified
4-VP samples were analyzed by proton NMR (1H NMR)
spectroscopy at 500 MHz (Bruker-BioSpin, Billerica, MA).
Chemical shifts are reported in ppm (δ). The morphology
of the cells was determined using transmission electron
microscopy (TEM) (Tecnai F-20, FEI, Hillsboro, OR). The
cells were sedimented by centrifugation (10 000 × g, 20 min,
4 °C), and the paste was applied to copper planchets and
cryofixed in a Leica EM Pact (Leica, Deerfield, IL) high-
pressure freezing instrument. Sample-containing planchets
were held in liquid nitrogen and transferred to a freezer at
-85 °C for freeze substitution in 2% w/v osmium tetroxide
in anhydrous acetone for 60 h. They were then placed in a
Leica AFS automatic freeze substitution unit at -85 °C for
further processing. Cells were then ramped from -85 °C to
-30 °C (10 °C h-1), held at -30 °C for 13 h, after which
Experimental Section
General. All chemicals were reagent-grade and used as
received from either the manufacturer or distributor. Unless
otherwise noted, the biochemicals were obtained from Sigma-
Aldrich Chemical (St. Louis, MO); various dehydrated
culture media were obtained from Difco (Detroit, MI). Au-
thentic 4-VP was purchased from Lancaster Synthesis (Wind-
ham, NH). Bottled, spectroscopic grade water was obtained
from either EMD Chemicals (Gibbstown, NJ) or Aldrich
(Milwaukee, WI). Lactobacillus plantarum (ATCC14917)
and Bacillus subtilis (ATCC6633) were obtained from the
American Type Culture Collection (ATCC, Manassas, VA).
The pHCA-producing E. coli strain used to prepare bio-
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