Journal of Biotechnology 217 (2016) 12–21
Contents lists available at ScienceDirect
Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec
Whole-cell biocatalytic production of variously substituted ␤-aryland ␤-heteroaryl-␤-amino acids
Nishanka Dilini Ratnayake a , Chelsea Theisen a,c , Tyler Walter a , Kevin D. Walker a,b,∗
a
b
c
Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA
Lake Superior State University, Sault Ste. Marie, MI 49783, USA
a r t i c l e
i n f o
Article history:
Received 25 August 2015
Received in revised form
23 September 2015
Accepted 12 October 2015
Available online 31 October 2015
Keywords:
␤-Amino acids
Whole-cell biocatalysis
Aminomutase
Batch-reaction sustainability
Chiral amine
a b s t r a c t
Biologically-active ␤-peptides and pharmaceuticals that contain key ␤-amino acids are emerging as target
therapeutics; thus, synthetic strategies to make substituted, enantiopure ␤-amino acids are increasing. Here, we use whole-cell Escherichia coli (OD600 ∼35) engineered to express a Pantoea agglomerans
phenylalanine aminomutase (PaPAM) biocatalyst. In either 5 mL, 100 mL, or 1 L of M9 minimal medium
containing ␣-phenylalanine (20 mM), the cells produced ∼1.4 mg mL−1 of ␤-phenylalanine in each volume. Representative pilot-scale 5-mL cultures, fermentation reactions converted 18 variously substituted
␣-arylalanines to their (S)-␤-aryl-␤-amino acids in vivo and were not toxic to cells at mid- to late-stage
growth. The ␤-aryl-␤-amino acids made ranged from 0.043 mg (p-nitro-␤-phenylalanine, 4% converted
yield) to 1.2 mg (m-bromo-␤-phenylalanine, 96% converted yield) over 6 h in 5 mL. The substituted ␤amino acids made herein can be used in redox and Stille-coupling reactions to make synthetic building
blocks, or as bioisosteres in drug design.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
While not abundant in nature, aromatic ␤-amino acids are found
occasionally in pharmacologically important natural products, such
as the antibiotic agent andrimid (I) from Pantoea agglomerans
(Jin et al., 2006), antifungal and insecticidal agent jasplakinolide
(II) from marine sponges (Crews et al., 1986), and aminopeptidase inhibitor bestatin (III) from Streptomyces olivoreticuli (Chen
et al., 2011; Nishizawa et al., 1977) (Table 1). In addition, pharmaceuticals such as the antiglycemic sitagliptin (Savile et al., 2010)
(IV) (Merck) and the antimitotic paclitaxel/Abraxane® (V) (Celgene Corp. and Bristol-Myers Squibb) contain moieties derived
from ␤-amino acids. A synthetically-derived agent built from (S)-␤phenylalanine is CCR-5 receptor antagonist maraviroc/Selzentry®
(VI) (Pfizer) that treats HIV infection (Haycock-Lewandowski et al.,
2008) (Table 1).
␤-Amino acids are used also as building blocks for synthetic
␤-peptide oligomers, which are not as susceptible to cleavage by
␣-peptidases, hydrolases, and metabolizing enzymes as ␣-peptides
(Frackenpohl et al., 2001; Seebach et al., 1996). ␤-Peptides can also
∗ Corresponding author at: Michigan State University, Department of Chemistry,
587 S. Shaw Lane, Room 208, East Lansing, MI 48842 USA.
E-mail address: [email protected] (K.D. Walker).
http://dx.doi.org/10.1016/j.jbiotec.2015.10.012
0168-1656/© 2015 Elsevier B.V. All rights reserved.
fold into well-defined, stable conformations in solution to form
stable quaternary structures and can form helix-turn and pleatedsheet conformations with as few as four residues in protic solutions
(Seebach et al., 1999, 1998).
Application of ␤-amino acids is further seen in the cyclic␤-tetrapeptide (VII), a mimic of the natural peptide hormone
somatostatin (regulator of endocrine and nervous system function),
that binds the human somatostatin receptors (Gademann et al.,
1999) (Table 1). In addition, a new family of nylon-3 polymers
(poly-␤-peptides) (VIII) shows significant and selective toxicity
towards the human fungal pathogen Candida albicans (Liu et al.,
2013) (Table 1).
Given the importance of ␤-amino acids as precursors of pharmaceuticals and peptidomimetics with greater in vivo stability,
several methods are described for the stereoselective chemical
synthesis of ␤-amino acids (Lelais and Seebach, 2004; Liu and
Sibi, 2002; Weiner et al., 2010). Arndt–Eistert homologation of ␣amino acids (Pinho et al., 2014; Seebach et al., 1996; Yuan and
Williams, 1997), diastereoselective (Davies et al., 2011; Davies
and Ichihara, 1991; Sewald, 1996) and enantioselective (Falborg
and Jorgensen, 1996; Sibi and Liu, 2000) conjugate addition reactions, and catalytic asymmetric hydrogenation reactions (Holz
et al., 2003; Kadyrov et al., 2005; Lubell et al., 1991) are highlighted among various methods. Oxidative stress potentially caused
by silver (Ag+ ) waste (Kim et al., 2009) and safety concerns
N.D. Ratnayake et al. / Journal of Biotechnology 217 (2016) 12–21
13
Table 1
Bioactive natural products, pharmaceuticals, and ␤-peptides based on ␤-amino acids.
associated with the volatile, carcinogenic diazomethane caution against using Arndt–Eistert homologation. Thus, alternatively
employed are catalytic asymmetric-hydrogenation reactions for
large-scale enantioselective (>95%) synthesis of ␤-amino acids. Chiral ligand design and tuning (Holz et al., 2003; Kadyrov et al.,
2005) have improved these metal-catalyzed hydrogenation routes;
however, these methods still advance through multiple steps. A
concern of multistep syntheses at commercial-scale is large-scale
chemical waste (Grayson et al., 2011). Thus, currently explored are
semisynthetic methods to integrate benign biocatalytic steps in the
synthetic pathway towards ␤-amino acids.
Currently researched are three main biocatalysts for making ␤-amino acids: lipases (Faulconbridge et al., 2000; Grayson
et al., 2011), transaminases (Rudat et al., 2012), and aminomutases
(Christenson et al., 2003; Krug and Mueller, 2009; Magarvey et al.,
2008; Walker et al., 2004; Wanninayake and Walker, 2013). Principally explored are lipases for industrial-scale enzymatic resolution
to semisynthesize ␤-amino acids (Grayson et al., 2011). Recently,
a commercially available Amano lipase PS was used to resolve a
racemic mixture of synthetically-derived ␤-aryl- and heteroaryl-␤amino acid propyl esters with excellent enantioselectivity (98–99%
ee) and moderate yields (16–50%, limited at a maximum 50% yield
by the 1:1 racemate) (Grayson et al., 2011). In addition, synthesis
of the propyl esters needed the corrosive thionyl chloride reagent,
with distillation and extraction steps before the two-phase lipase
step. Most important, after each batch reaction cycle, fresh enzyme
was added to maintain the initial turnover rate and offset the partial
inactivation of the Amano lipase PS (Grayson et al., 2011).
Aminomutase catalysis provides an alternative biocatalytic
route that involves fewer steps than the enzymatic resolution
effort. A phenylalanine aminomutase from P. agglomerans (PaPAM)
catalyzes the isomerization of (2S)-␣-phenylalanine to (3S)-␤phenylalanine (>99.9% ee) (Fig. 1) on the pathway to the antibiotic
andrimid (Magarvey et al., 2008; Ratnayake et al., 2011). PaPAM
Fig. 1. The production of ␤-phenylalanine by PaPAM catalysis.
uses several ␣-phenylalanine analogs with substituents variously
positioned on the phenyl ring (fluoro, chloro, bromo, methyl,
methoxy and nitro) and heteroaromatic analogs demonstrating an
extended substrate scope (Ratnayake et al., 2014).
Considering the high enantioselectivity, atom economy, and
expanded substrate specificity of PaPAM, herein, we evaluated the
kinetics of recombinant E. coli whole-cells harboring the papam
gene. This recombinantly-expressed enzyme was employed in an
independent, earlier biocatalysis study that examined the conversion of only the natural substrate ␣-phenylalanine (Hidesaki,
2009). The present analysis includes 21 surrogate substrates to further understand the relationship among the substrate specificity,
substituent effects, time-dependent production rates of the wholecell biocatalyst, and the in vitro-catalyzed reactions with purified
PaPAM (Ratnayake et al., 2014). In addition, this study also evaluated metabolite partitioning between the culture medium and
E. coli cells, assessed the effects of various physical parameters (e.g.,
buffer type, pH, and temperature) on the kinetics. Also explored
were amino acid toxicity and multiple batch reaction cycles to
gauge the sustainability of the whole-cell biocatalyst. Assessment
of these parameters added information on the flux parameters of
the PaPAM whole-cell biocatalyst and established a foundation for
batch-cell recycling for large-scale biosynthesis.
2. Materials and methods
2.1. Substrates, authentic standards and reagents
(S)-␣-, p-methoxy-(S)-␣-, p-nitro-(S)-␣-, and p-chloro-(R/S)␤-phenylalanine and (trimethylsilyl) diazomethane (2.0 M in
diethyl ether) were purchased from Sigma–Aldrich-Fluka (St.
Louis, MO). Racemic p-nitro-␤-phenylalanine was purchased
from Oakwood Products, Inc. (West Columbia, SC), and omethoxy-(S)-␣-, m-methoxy-(S)-␣-, o-nitro-(S)-␣-, m-nitro-(S)-␣-,
o-methoxy-(S)-␤-, m-methoxy-(S)-␤-, o-nitro-(S)-␤-, and m-nitro(S)-␤-phenylalanine were purchased from Chem-Impex International, Inc. (Wood Dale, IL). All other (S)-␣- and -␤-amino acids
were purchased from PepTech Corporation (Burlington, MA). All
chemicals were used without further purification, unless noted.
14
N.D. Ratnayake et al. / Journal of Biotechnology 217 (2016) 12–21
2.2. Bacterial strains, plasmids, and culture media
BL21(DE3) E. coli bacterial strain transformed with expression
vector pET24b(+) was used for the whole-cell biotransformations
with PaPAM. E. coli cells were grown in M9 minimal medium
[Na2 HPO4 .7H2 O (12.8 g L−1 ), KH2 PO4 (3 g L−1 ), NaCl (0.5 g L−1 ),
NH4 Cl (1 g L−1 ), MgSO4 , (2 mM), CaCl2 (0.1 mM), 100X Basal
Medium Eagle vitamins (Sigma–Aldrich, St. Louis, MO; 10 mL L−1 ),
and glucose (20%), pH 7.4] supplemented with kanamycin (Gold
Biotechnology Inc., St. Louis, MO; 50 ␮g mL−1 ). Optical density measurements of cell suspensions were obtained in 1.5 mL polystyrene
cuvettes (General Laboratory Supply, Pasadena, TX) using a UV–vis
spectrophotometer (Beckmann DU 640, Beckmann Coulter, Brea,
CA).
2.3. General instrumentation: GC/EI-MS analysis
GC–MS analysis was performed on an Agilent 6890N
gas chromatograph equipped with a capillary GC column
(30 m × 0.25 mm × 0.25 ␮M; HP-5MS; J&W Scientific) with He
as the carrier gas (flow rate, 1 mL/min). The injector port (at
250 ◦ C) was set to splitless injection mode. A 1-␮L aliquot of each
sample was injected using an Agilent 7683 auto-sampler (Agilent,
Atlanta; GA). The column temperature was increased from 50 to
110 ◦ C at 30 ◦ C/min, then increased by 10 ◦ C/min to 250 ◦ C (total
run time of 16 min), and returned to 50 ◦ C over 5 min, with a 5 min
hold. The gas chromatograph was coupled to a mass selective
detector (Agilent, 5973 inert) operated in electron impact mode
(70 eV ionization voltage). All spectra were recorded in the mass
range of 50–400 m/z.
2.4. General procedure for whole–cell biocatalytic incubations
BL21(DE3) E. coli cells (50 mL, OD600 ∼1.0) transformed to
express the papam gene from pET24b(+) were used to inoculate M9 minimal medium (1 L) supplemented with kanamycin
(50 ␮g/mL). The cells were grown at 37 ◦ C to OD600 ∼0.6, isopropyl␤-d-thiogalactopyranoside (100 ␮M) was added to induce the
expression of papam, and the cultures were incubated for 16 h
at 16 ◦ C. The next steps were performed at 4 ◦ C, unless indicated
otherwise. The cells were harvested by centrifugation at 3230 × g
(10 min), and the cell pellet was resuspended separately in three
different whole-cell feeding media (M9 minimal medium, 50 mM
phosphate buffer at pH 7, and 50 mM phosphate buffer at pH 8),
each typically adjusted separately to OD600 ∼35. For analyses at
higher biomass, the OD600 was adjusted to ∼70 and 280 in M9 minimal medium. The ␣-amino acid substrate (1 mM) was added to the
cell suspension and incubated separately at 16, 25 and 30 ◦ C. In control experiments, transformed E. coli cells were incubated without
substrate added. Additional control experiments included assays
with E. coli transformed with an empty pET-24 vector incubated
with or without the substrate. All the biotransformation feeding
assays were done in triplicate.
2.5. Derivatization and quantification of amino acids
At the end of each incubation, the reaction medium was separated from the cells by centrifuging at 3230 × g for 10 min, and
the supernatant (1 mL) from each assay was basified to pH 10 (6 M
NaOH). Internal standards m-fluoro-␤-phenylalanine, p-methyl␤-phenylalanine, and ␤-phenylalanine at 20 ␮M were added,
respectively, to quantify three sets of biosynthetic ␤-amino acids
products–Set 1: ␤-phenylalanine; o-, m-, and p-methyl-; o-, m-, and
p-methoxy-; m- and p-nitro-; m- and p-chloro-␤-phenylalanine;
Set 2: o- and p-fluoro; m-, and p-bromo-␤-phenylalanine; and
(2-thienyl)- and (3-thienyl)-␤-alanine; and Set 3: m-fluoro-␤-
phenylalanine. Each solution was treated with ethylchloroformate
(50 ␮L) for 10 min, basified again to pH 10, and reacted with a second batch of ethylchloroformate (50 ␮L) for 10 min. The solutions
were acidified to pH 2–3 (6 M HCl) and extracted with diethyl ether
(2 × 2 mL). The organic fraction was removed under vacuum, and to
the resulting residue dissolved in ethyl acetate:methanol (3:1, v/v)
(200 ␮L) was added (trimethylsilyl) diazomethane until the yellow
color persisted. The derivatized aromatic amino acids were quantified by GC/EI-MS. The peak area was converted to concentration
by solving the linear equation obtained from the standard curves
constructed with the corresponding authentic standards.
2.6. Assessing production rate of ˇ-phenylalanine by the
whole-cells in different volumes
The effect of incubation volume on whole-cell biocatalysis was
assessed by adding 20 mM of ␣-phenylalanine to a cell suspension
of E. coli (OD600 ∼35) engineered to express papam at 16 ◦ C, in 5,
100, or 1000 mL of M9 minimal medium. The cultures were incubated, respectively, in 50-mL, loosely-capped conical centrifuge
tubes (Corning Incorporated Life Sciences, Tewksbury, MA), 250mL Erlenmeyer flasks, and 2.8-L Fernbach flasks with agitation
on a shaker (225 rpm, MaxQ 5000, Thermo Scientific, Waltham,
MA) for 6 h. The cells were removed by centrifugation, and the ␤phenylalanine in the culture medium of each assay was derivatized
and quantified by GC/EI-MS.
2.7. Analysis of substrate uptake and product release by E. coli
cells
To E. coli cells expressing PaPAM incubated in M9 minimal
medium (45 mL, OD600 ∼35) was added ␣-phenylalanine (1 mM)
at 16 ◦ C. Aliquots (5 mL) were withdrawn at 0.5, 1, 2, 4, 6, and
8 h, the cells were harvested by centrifugation (3230 × g, 5 min),
and the culture medium was twice serially basified (6 M NaOH)
and treated with ethyl chloroformate. The solution was acidified
(pH 2–3, 6 M HCl), extracted with diethyl ether (2 × 2 mL), concentrated in vacuo, and the amino acids were methyl esterified with
(trimethylsilyl) diazomethane for quantification by GC/EI-MS analysis. In parallel, the cell pellet was resuspended in 50 mM sodium
phosphate buffer (pH 8.0), the cells were lysed by a Misonix XL
2020 sonicator (Misonix Inc, Farmingdale, NY) to release the soluble
amino acids, and the cellular debris was removed by centrifugation
(3230 × g, 30 min). The supernatant was decanted and the amino
acids therein were derivatized by the same reactions used for the
culture medium and analyzed by GC/EI-MS.
2.8. Analysis of enzyme release to culture medium
To measure the relative percentage of the PaPAM released in
the medium over 6 h at 16 ◦ C, a 5-mL cell suspension (OD600 ∼35)
of E. coli engineered to express papam was incubated without
␣-phenylalanine. The culture was centrifuged (3230 × g, 5 min),
and the supernatant was decanted. The cells were resuspended
in lysis buffer (5 mL of 50 mM sodium phosphate buffer (pH 8.0))
and sonicated (Misonix Inc, Farmingdale, NY) to release the soluble PaPAM. The cellular debris was removed by centrifugation
(3230 × g, 30 min), and the supernatant was decanted. To measure
the relative extracellular PaPAM activity, an aliquot (250 ␮L) of each
supernatant was incubated separately with 15 N-␣-phenylalanine
(1 mM) for 2 h at 31 ◦ C. The amino acids were derivatized and quantified by GC/EI-MS analysis.
N.D. Ratnayake et al. / Journal of Biotechnology 217 (2016) 12–21
2.9. Effect of temperature, time, and culture medium type
After expressing the papam gene in E. coli for 16 h at 16 ◦ C in
M9 minimal medium (12 × 1 L), the cultures were centrifuged, the
supernatants were decanted, and the cells from each batch were
resuspended separately in M9 minimal medium or 50 mM phosphate buffer (each at pH 7 or 8) to OD600 ∼35. ␣-Phenylalanine
(1 mM) was added to 45-mL cell suspensions in each medium, and
the feeding studies were conducted at 16, 25 and 30 ◦ C. Aliquots
(5 mL) were withdrawn from each suspension at 0.5, 1, 2, 4, 6,
8, 10, and 12 h. The supernatants were clarified by centrifugation,
decanted, and separately treated with derivatizing reagents to form
the N-(ethoxycarbonyl) ␣- and ␤-phenylalanine methyl esters, and
the cinnamic acid methyl ester, which were quantified by GC/EIMS.
2.10. Effect of substrate concentration
To a cell suspension of E. coli (OD600 ∼35), engineered to express
papam in 5 mL of M9 minimal medium was added separately 1,
5, 10, 15, 20, and 25 mM of ␣-phenylalanine. The cultures were
incubated for 6 h at 16 ◦ C. The cells were removed by centrifugation,
and the ␤-phenylalanine in the culture medium of each assay was
quantified after derivatization and analysis by GC/EI-MS.
2.11. Effect of the biocatalyst amount
The E. coli cells harboring papam were resuspended in M9 minimal medium to an OD600 of 35, 70 or 280. ␣-Phenylalanine was
added to a final concentration of 1, 5, 10, 15, 20, and 25 mM in
separate assays at each cell density, and the assays (5 mL) were
incubated for 6 h at 16 ◦ C. The culture medium was clarified by
centrifugation, and the ␤-phenylalanine in the supernatant (1 mL
aliquot) of each assay was derivatized for and quantified by GC/EIMS analysis.
2.12. Assessing the substrate scope of the biocatalytic system
␣-Phenylalanine, its analogs (ortho/meta/para-methyl, methoxy, -fluoro, -chloro, -bromo, and -nitro), and 2- and
3-thienylalanine were separately incubated in a cell suspension
of engineered E. coli (OD600 ∼35) in M9 minimal medium (5 mL)
at 16 ◦ C for 6 h. The cells were pelleted by centrifugation, and an
aliquot (1 mL) from each supernatant was separately treated with
derivatizing reagents to form the N-(ethoxycarbonyl) methyl esters
of the ␣-arylalanines and biosynthetic ␤-arylalanines, and the
methyl esters of the biosynthetic arylacrylates for quantification
by GC/EI-MS.
2.13. Assessing amino acid toxicity on whole cells at stationaryand mid-log phase
To calculate the colony forming units (CFU) of E. coli BL21(DE3)
cells at stationary-phase growth at OD600 ∼35, an aliquot (50 ␮L)
from each cell suspension incubated with ␣-phenylalanine was
diluted 108 -fold with the culture medium (in triplicate). An aliquot
(100 ␮L) of the diluted culture suspension was spread on an agar
plate supplemented with kanamycin (50 ␮g/mL) and incubated for
16 h at 37 ◦ C. The colonies on each plate were counted, and the
log(CFU mL−1 ) was calculated.
To assess the toxicity of the amino acids used in this study
on E. coli at mid-log stage growth, cells were grown at 37 ◦ C to
OD600 0.6 in 250-mL shake-flask in LB medium (100 mL) containing
kanamycin (50 ␮g/mL). Each set of two LB agar plates, supplemented with kanamycin (50 ␮g/mL), contained one (at 1 mM) of the
15
18 amino acids used in this study. An aliquot (1 mL) from the bacterial cell suspension was diluted 105 -fold with the culture medium
(in duplicate). The diluted culture suspension (100 ␮L) was spread
on each agar plate and incubated at 37 ◦ C for 18 h. The colonies on
each plate were counted, and the log(CFU mL−1 ) were calculated.
2.14. Sustainability of the biocatalytic system
E. coli cells (at OD600 ∼35) engineered to express papam were
resuspended in M9 minimal medium (5-mL), and ␣-phenylalanine
at 1 and 5 mM was added separately to different batches of cell
suspensions. The cells were incubated for 6 h at 16 ◦ C, the culture medium was clarified by centrifugation at 3230 × g (10 min).
The cell pellets were serially washed (2 × 5 mL) with M9 minimal medium and clarified by centrifugation between each wash
to remove residual substrate/product from the cells. The washed
cell pellets were resuspended in the culture medium (5 mL), a new
batch of ␣-phenylalanine substrate was added to each suspension, and the biotransformation reactions were incubated for 6 h at
16 ◦ C. The supernatant from each reaction was twice serially basified (6 M NaOH) and treated with ethyl chloroformate. The solution
was acidified (pH 2–3, 6 M HCl), extracted with diethyl ether
(2 × 2 mL), concentrated in vacuo, and treated with (trimethylsilyl)
diazomethane to methyl esterify the amino acids for quantification by GC/EI-MS. Cell harvesting, cell pellet washing, incubation,
and GC/EI-MS quantification of the synthetically derivatized ␤phenylalanine from the supernatant were repeated for 4 cycles.
3. Results and discussion
3.1. General assay conditions
E. coli (OD600 ∼35) expressing papam incubated in different volumes (5 mL, 100 mL, or 1 L) of minimal medium
containing ␣-phenylalanine (20 mM) produced ∼1.4 mg mL−1 of ␤phenylalanine in each volume. Thus, 5-mL pilot-scale whole-cell
reactions were representative of larger-scale conversion rates of
␣-arylalanines to their ␤-isomers in vivo. The host strain used for
whole-cell biocatalysis was grown in M9 minimal medium, lacking the ␣-amino acids found in rich growth media. The latter could
potentially confound the analysis of the ␤-aryl-␤-alanines made
in vivo by the aminomutase (Strom et al., 2012).
3.2. Assessment of the whole-cell biocatalytic properties of
PaPAM
Precursor biotransformations by a whole-cell organism are
designed ideally to release the products with low impedance into
the medium without cell disruption (Chen, 2007). E. coli cells
uptake aromatic amino acids through their inner membrane by
five distinct active-transporters (Chye et al., 1986; Piperno and
Oxender, 1968; Whipp and Pittard, 1977). An AroP permease
actively transports arylalanines (Chye et al., 1986), while PheP
permease transports phenylalanine (Cosgriff and Pittard, 1997; Pi
and Pittard, 1996). A permissive ATP-binding cassette type transporter, LIV-I/LS system, primarily conveys branched-chain amino
acids yet also occasionally transports phenylalanine (Koyanagi
et al., 2003). Thus, these transporters likely play a central role
in amino acid transfer into the bacteria cells used herein. In this
study, ␣-phenylalanine (benchmark substrate) entered the cells
(OD600 ∼35), and ␤-phenylalanine accumulated in the medium
(0.4 mg/5 mL) and in the cells (0.016 mg/40 mg wet wt) after 8 h
(Fig. 2). The log(CFU mL −1 ) in the E. coli samples incubated with
␣-phenylalanine at 1 mM during biocatalysis remained constant
(Fig. 2), suggesting no time-dependent accumulation of dead and/or
viable but nonculturable cells at stationary growth phase.
N.D. Ratnayake et al. / Journal of Biotechnology 217 (2016) 12–21
Relative Total Ion Count
16
11
Fig. 2. Time course and distribution of ␤-phenylalanine released to the M9 minimal
medium (pH 7.4, 5 mL) (䊏 on solid line) and remaining in the E. coli cell pellet (0.4 g)
( on dotted line) after conversion of ␣-phenylalanine (1 mM) by PaPAM whole-cell
biocatalysis at 16 ◦ C (OD600 ∼35). CFU values () for the bacterial cells at each time
point are shown; SD < 5%.
12
1.0
14.43
0.8
0.6
14.10
14.43
0.4
0.2
0.0
13
14
15
Time (min)
16
17
18
Fig. 3. Gas chromatogram profiles of the N-[(1 S)-camphanoyl] methyl
esters of authentic racemate3R– (14.10 ± 0.01 min) and 3S-␤-phenylalanine
(14.43 ± 0.01 min) (solid line), and biosynthetic ␤-phenylalanine (14.43 ± 0.01
minv) (dotted line).
Aliquots of the clarified growth medium did not convert 15 N␣- to 15 N-␤-phenylalanine, suggesting that the PaPAM enzyme did
not release to the medium from dead cells over a 6-h incubation.
Thus, biocatalysis did not occur fortuitously in the culture medium.
The labeled ␣-phenylalanine enabled distinction of de novo biosynthetic 15 N-␤-phenylalanine and unlabeled ␣- and ␤-phenylalanine
isomers made endogenously. The crude, soluble fraction isolated
from mechanically-lysed cells, containing active PaPAM enzyme,
catalyzed labeled ␤-phenylalanine from 15 N-␣-phenylalanine (see
Supplementary Fig. 1).
3.3. Enantiomeric excess of the biosynthetic (S)-ˇ-phenylalanine
The synthesis of pharmaceutically active drugs from chiral
building blocks continues the drive to develop industrially viable
methods for making enantiomerically pure compounds (Petrovic
et al., 2002). Hence, optimizing the enantiomeric excess of a
catalytic process is an important goal. To analyze the enantioselectivity of the whole-cell biocatalyst used herein, biosynthetic
␤-phenylalanine (as the reference compound) was converted to its
N-(1(S)-camphanoyl) methyl ester. The derivatized diastereomeric
␤-amino acid eluted at a retention time (14.43 min) identical
with that of authentic N-[(1S)-camphanoyl]-(3S)-␤-phenylalanine
(14.43 min) (Fig. 3), confirming the 3S-product stereochemistry
(>99.9% ee) as described in earlier in vitro assays with PaPAM
(Magarvey et al., 2008; Ratnayake et al., 2011). No biosynthetic
(3R)-␤-derivative (14.10 min) was detected.
3.4. Effect of temperature on ˇ-phenylalanine production
The activity, stability, and selectivity of a whole-cell biocatalyst are important for industrial scale biosynthesis (Zhou et al.,
2011). To examine the thermal stability of the whole-cell biocatalyst, the cell cultures were incubated at 16, 25 or 30 ◦ C.
Therefore, herein, aliquots of the supernatant from each sample
were withdrawn over time, and ␤-phenylalanine in the medium
was measured (Fig. 4). Cultures incubated at 16 ◦ C maximally
produced ␤-phenylalanine (0.092 mg mL−1 ) at 6 h, yet cultures
incubated at 25 and 30 ◦ C produced ␤-phenylalanine at ∼1.5-fold
lower maximum (∼0.065 mg mL−1 ) at 1 h (Fig. 4A). We suspect
that increased temperatures affected one or more unknown physiological parameters in E. coli at stationary phase, such as gene
Fig. 4. (A) Effect of reaction temperature on the production of ␤-phenylalanine
by engineered E. coli (OD600 ∼35) in M9 minimal medium (pH 7.4) containing ␣phenylalanine (1 mM) incubated at 16 ◦ C (䊏 on solid line), 25 ◦ C ( on dashed line),
and 30 ◦ C ( on dotted line). (B) The production of ␤-phenylalanine in M9 minimal
medium (M9 MM, pH 7.4) (䊏 on solid line), phosphate buffer (PB) at pH 7 (䊉 on short
dotted line), and pH 8 ( on dash-dotted line), each containing ␣-phenylalanine
(1 mM) incubated at 16 ◦ C.
expression (Gadgil et al., 2005), enzyme activity, substrate binding,
␣-phenylalanine flux toward post-exponential Pex protein synthesis (Kolter et al., 1993), and/or amplified phenylalanine catabolism
(Teufel et al., 2010). Thus, considering the slightly greater conversion rate at 16 ◦ C, this temperature was used to incubate the
recombinant E. coli biocatalyst for the non-natural ␣-arylalanine
substrates.
At all three temperatures, the ␤-phenylalanine decreased
markedly after the equilibrium phase beyond 8-h incubation. Analysis of the product distribution at each time point at 16 ◦ C revealed
that the concentration of cinnamic acid (a 5% by-product from
N.D. Ratnayake et al. / Journal of Biotechnology 217 (2016) 12–21
Fig. 5. Comparison of ␤-phenylalanine (䊏 dotted line) and cinnamic acid (䊉 on solid
line) production, and ␣- phenylalanine ( on dashed line) depletion by engineered
E. coli (OD600 ∼35) incubated at 16 ◦ C in M9 minimal medium (pH 7.4) containing
␣-phenylalanine (1 mM).
␣-phenylalanine during PaPAM catalysis) increased over time,
while the amount of ␣-phenylalanine rapidly decreased (Fig. 5).
An increase in cinnamic acid suggested that the occurrence of
the reverse reaction catalyzed by PaPAM (Fortin et al., 2007) is
converting ␤-phenylalanine to cinnamate as the ␤-phenylalanine
concentration increased over that of ␣-phenylalanine in the
medium (Fig. 5). ␣-Phenylalanine did not equilibrate with ␤phenylalanine in the reaction medium, likely due to its partitioning
to other metabolic pathways.
3.5. Effect of reaction medium-type on ˛- to ˇ-phenylalanine
isomerization
The influence of pH and buffer type on the stability and
activity of the whole-cell biocatalyst was evaluated in phosphate buffer (50 mM, pH 7 and 8) and M9 minimal medium (pH
7.4). The M9 minimal medium contains sources of nitrogen, carbon, phosphorous, sulfur, cations (K+ , Ca2+ , Mg2+ , and Fe2+ ), and
BME-vitamins needed for bacterial cell survival and growth. By
comparison, the phosphate buffer lacks these nutrients. The production rate of ␤-phenylalanine by the whole-cell biocatalyst
(OD600 ∼35) in M9 minimal medium was only slightly higher
(∼0.062 mg mL−1 h−1 ) than the initial rate (∼0.059 mg mL−1 h−1 )
in 50 mM phosphate buffer (pH 7 and 8) over 1 h. While ␤phenylalanine then slowly increased in minimal medium between
2 and 8 h, it decreased in phosphate buffer (pH 7 and 8) (Fig. 4B). ␣Phenylalanine depleted ∼2-fold faster in 50 mM phosphate buffer
(0.024 mg mL−1 h−1 and 0.028 mg mL−1 h−1 at pH 7 and 8, respectively), compared to when the bacteria was incubated in M9
minimal medium (0.012 mg mL−1 h−1 ) (Fig. 6). We also observed
that ␣-phenylalanine disappeared from the phosphate buffer at a
rate greater than ␤-phenylalanine was exported into the medium
over 8 h (Fig. 6). This suggested that the biocatalyst metabolized the
exogenous ␣-phenylalanine (and likely ␤-phenylalanine), directly
or via cinnamate, for viability in the nutrient-deprived phosphate
buffers. Thus, the moderately fortified M9 minimal medium was
used for other whole-cell PaPAM biocatalyst assays.
3.6. Effect of medium-type on cell viability at stationary-phase
growth
In addition, to assess viable and culturable cells in the different media, log(CFU mL−1 ) were calculated for bacteria incubated
in M9 minimal medium (pH 7.4) and phosphate buffer (pH 7)
containing ␣-phenylalanine (1 mM). The log(CFU mL−1 = 11.6) of
E. coli incubated for 4 h in minimal medium was only slightly
17
Fig. 6. Amount of ␣-phenylalanine remaining in M9 minimal medium (pH 7.4, on
dashed line), phosphate buffer at pH 7 (䊉 on solid line), and pH 8 (䊏 on dotted line)
incubated with engineered E. coli (OD600 ∼35) at 16 ◦ C. Each medium was supplied
with ␣-phenylalanine (1 mM).
higher than for cells [log(CFU mL−1 = 11.4)] incubated in phosphate
buffer (pH 7). E. coli viability in minimal medium did not change
significantly [log(CFU mL−1 ) = 11.2] in phosphate buffer without ␣phenylalanine supplementation. Thus, the exogenous amino acid
and minimal media likely provided only a small mesotrophic effect
for the bacteria, and alone does not account for the differences in
the production profiles in the various media (Fig. 4B). Nonetheless,
M9 medium was used in all incubation studies described herein.
3.7. The effect of ˛-phenylalanine concentration on cell viability
at stationary-phase growth
␣-Phenylalanine (1–25 mM) was used as the benchmark for
all the substrates tested to examine its effect on ␤-phenylalanine
production and E. coli whole-cell viability. ␤-Phenylalanine production in the whole-cells increased 0.09–1.2 mg mL−1 with increasing
substrate concentration in 6-h incubations at 16 ◦ C (Fig. 7A). The
production approached a maximum at ∼1.3 mg mL−1 for substrate
concentrations >25 mM. The production approached maximum
turnover at higher substrate concentrations either because mass
transfer to the biocatalyst was limiting or the substrate became
toxic to the cells. Therefore, substrate toxicity was analyzed by
incubating E. coli cells at stationary phase growth with increasing
substrate concentration. Calculating the log(CFU mL−1 ) of cultures
at increasing substrate concentration revealed that the cells remain
viable and culturable even at 25 mM ␣-phenylalanine (Fig. 7A).
To evaluate if the mass transfer limits could be extended, the
biocatalyst amount was increased from OD600 ∼35 to 280. The
8-fold increase in cell biomass did not significantly change the ␤phenylalanine production when the substrate was between 1 and
10 mM yet did increase the product ∼33% over the lower density
cells at 25 mM (Fig. 7B). At OD600 280 the substrate concentration
per cell is diluted ∼8-fold compared to that of cells at OD600 35, and
this dilution accordingly reduces the turnover per cell. The linear
regression fit for ␤-phenylalanine production at higher OD (Fig. 7B)
extrapolates to a production level of ∼3.2 mg mL−1 at ∼40 mM substrate, which is ∼8-fold higher than production for the OD600 35
cells incubated with 5 mM substrate. In addition, the production
of ␤-phenylalanine is projected also to reach a maximum product
level 8-fold higher (estimated at ∼10 mg mL−1 ) than in the OD600
35 reaction; thus, the biocatalyst reaction was deemed scalable for
increased ␤-amino acid production at OD600 280.
18
N.D. Ratnayake et al. / Journal of Biotechnology 217 (2016) 12–21
3.8. Substrate scope of the recombinant E. coli whole-cell
biocatalyst
Fig. 7. (A) Effect of substrate amount on the production of ␤-phenylalanine by
engineered E. coli (OD600 ∼35) incubated at 16 ◦ C in M9 minimal medium (pH 7.4)
containing various concentrations of ␣-phenylalanine (䊏 on solid line), and percent
conversion of ␣- to ␤-phenylalanine ( on dotted line) at each susbtrate concentration. log(CFU mL−1 ) values () for the bacterial cells at each susbtrate concentration
are shown; SD < 5%. (B) Comparison of PaPAM whole-cell biocalyst concentration
(OD600 ∼35 (䊏 on solid line) and 280 (䊐 on dashed line)) incubated at various
substrate concentrations.
Fig. 8. Recycling of the E. coli whole-cell biocatalyst (OD600 ∼35) for ␤phenylalanine production. The production of ␤-phenylalanine in PaPAM whole-cell
biocatalysis in M9 minimal medium (5 mL, pH 7.4) incubated with ␣-phenylalanine
(1 mM (black bars) and 5 mM (white bars)) at 16 ◦ C for each catalytic cycle. CFU values () for the bacterial cells after each reaction cycle in M9 minimal medium (pH
7.4) containing ␣-phenylalanine (5 mM) at 16 ◦ C are shown; SD < 0.05%.
Limited substrate scope is commonly a main disadvantage of
enzyme biocatalysis (Reetz, 2013). PaPAM, however, has a broad
substrate scope in vitro (Ratnayake et al., 2014), and thus was
anticipated to perform similarly in vivo. The PaPAM whole-cell
biocatalyst was probed with 21 ␣-arylalanines, including two
heteroaromatic compounds. The productive and inactive substrates were similar to those identified earlier for PaPAM in vitro
(Ratnayake et al., 2014). The transport of amino acids across the E.
coli cell membrane can depend on their steric and electronic properties (Silhavy et al., 2010) and aromatic amino acid:H+ –symporter
permeases (PheP and AroP) (Brown, 1970, 1971; Haney and
Oxender, 1992; Pi et al., 1991; Willshaw and Tristram, 1972). AroP
was shown earlier to bind p-fluoro-␣-phenylalanine and 3-(2thienyl)-␣-alanine (Brown, 1971; Willshaw and Tristram, 1972),
also used as substrates herein. These influx transporters likely
contribute significantly to the flux of the ␣-arylalanines into the
whole-cell biocatalyst in the present feeding study.
In general, the whole-cell PaPAM biocatalyst preferentially
isomerized meta- and para-substituted substrates over their
ortho-substituted isomers. The near complete conversion of five
␣-arylalanine substrates (m-bromo (96%), p-chloro (93%), p-bromo
(92%), 3-thienyl (92%), and m-methyl (90%)) (based on the 1 mM of
substrate added) by whole-cell biocatalysis to their ␤-arylalanines
suggested that efflux transporters selectively pumped these ␤amino acid products into the extracellular space over 6 h. This ∼9:1
␤:␣-arylalanine ratio greatly exceeds the intrinsic Keq values (typically ∼1.2, where the ␤:␣-arylalanine ratio is close approximately
1:1) for other amino acids in the PaPAM family(Chesters et al., 2012;
Mutatu et al., 2007). As mentioned earlier herein, the whole-cell
biocatalyst at OD600 ∼35 in both 5-mL and 1-L made ∼1.4 mg mL−1
␤-phenylalanine from ␣-phenylalanine. Thus, the production levels of ␤-amino acids from the surrogate substrates are reported
with units of “mg L−1 ” (Table 2). m-Bromo (1) accumulated in the
medium (235 mg L−1 ) more than any other substrate tested. It is
interesting to note that the in vitro catalytic rate (kcat ) of PaPAM for
m-bromo (1) coincidentally was among the fastest of the substrates
tested in an earlier study (Ratnayake et al., 2014). In addition, the
log(CFU mL−1 ) for E. coli cells at log phase growth showed that each
substrate at 1 mM was not toxic (see Supplementary Table 1).
In vitro biocatalysts are governed by cofactors (if any), physical conditions (temperature, pH, ionic strength), and the intrinsic
properties of the enzymes when reconstituted in buffer. In vivo
whole-cell biocatalysts are also dependent on other factors such as
mass transfer across cell membranes, cellular metabolism, protein
synthesis, stimulation and inactivation of microbial cell growth,
toxicity, and by-product formation (Schrewe et al., 2013). The
effects of influx and efflux permeases, in part, may have caused the
various ␣- and ␤-arylalanines to move at different rates through
the E. coli membrane. These factors, combined with other physiological pathways, are imagined to affect the production of each
␤-arylalanine by the whole-cell biocatalyst. As a result, the rankorder correlation between the in vitro catalytic rates (kcat ) and the
in vivo production rate of the whole-cell biocatalyst was largely
redistributed for the substrates tested (Table 2 and see Supplementary Fig. 2).
Thus, the intrinsic kcat of PaPAM, described in an earlier study
(Ratnayake et al., 2014), could not alone predict the turnover of the
whole-cell system for each substrate (Ratnayake et al., 2014). For
example, the intrinsic kcat values of PaPAM in vitro for m-chloro
(8) and m-bromo (1) (ranked 1st and 2nd), respectively, were followed by the natural substrate ␣-phenylalanine (11) (ranked 3rd)
as described in an earlier study (Ratnayake et al., 2014). By comparison, the product accumulation by the whole-cell biocatalyst for
N.D. Ratnayake et al. / Journal of Biotechnology 217 (2016) 12–21
19
Table 2
Production levels of PaPAM whole-cell biocatalyst for various ␣-arylalanine substrates.
Product (R=)
a
b
Production (mg L−1 )a
Yield (%)
Rankingb
Product (R=)
In vivo
In vitro
235 ± 3.6
96
1
2
224 ± 5.8
92
2
186 ± 4.2
93
162 ± 7.5
Production (mg L−1 )
Yield (%)
Rankingb
In vivo
In vitro
109 ± 9.2
56
10
16
10
96 ± 2.1
60
11
3
3
9
77 ± 2.4
43
12
17
90
4
8
74 ± 3.6
41
13
15
159 ± 0.84
87
5
14
68 ± 2.2
40
14
13
157 ± 4.6
92
6
6
49 ± 3.2
27
15
7
140 ± 4.3
72
7
4
17 ± 1.4
8
16
5
128 ± 6.1
64
8
1
13 ± 0.77
6
17
18
113 ± 1.5
62
9
12
8.5 ± 1.1
4
18
11
The concentrations of biosynthesized ␤-amino acids are reported in units of mg L−1 .
Ranking order is based on the ␤-phenylalanine production from the in vivo system and the catalytic rate (kcat ) of the in vitro system.
m-bromo (1) ranked 1st, m-chloro (8) ranked 8th, and the natural
substrate (11) ranked 11th. 11 was isomerized in vivo interestingly
slower than ten other substrates, likely because it was diverted to
other natural pathways in the E. coli cells. The whole-cell biocatalyst
did not isomerize o-chloro-, o-bromo- and o-nitro-␣-phenylalanine
(Table 2) in vivo; likely, caused by ineffective substrate binding to
PaPAM as described in the earlier in vitro study (Ratnayake et al.,
2014).
The halogenated ␤-arylalanines made herein can be used as
building blocks for bioisostere drug candidates and in Stille- and
Suzuki-type reactions to build molecules that are more elaborate
(Ahmed et al., 2015).
3.9. Sustainability of the PaPAM whole-cell biocatalytic system
Recycling of recombinant cells is a potential goal when developing a sustainable industrial process for producing fine chemicals
(Zhou et al., 2011). To test the operational stability of the recombinant E. coli whole-cell biocatalyst towards recycling, a set of
consecutive biocatalytic cycles was performed at 16 ◦ C for 30 h
using a single sample of the biocatalyst. After each 6-h reaction
cycle, the cells were recovered by centrifugation, washed with M9
minimal medium to remove residual substrate and product, and to
reduce cell aggregates (at OD600 ∼35). The cells were recovered by
centrifugation and resuspended in M9 minimal medium before the
next reaction in the series. ␤-Phenylalanine production remained
almost constant for whole-cell biocatalyst fed ␣-phenylalanine at
1 mM and 5 mM (0.075 and 0.9 mg mL−1 , respectively) in each of the
five batch reaction cycles (Fig. 8). CFU measurements suggested cell
viability was maintained even after centrifugation and cell resuspension over at least five reaction cycles (Fig. 8).
4. Conclusions
A PaPAM whole-cell biocatalyst was shown to produce several unnatural (3S)-␤-aryl-␤-amino acids at >99.9% ee, with the
highest turnover rate in M9 minimal medium at 16 ◦ C. The wholecell biocatalyst biosynthesized 18 ␤-arylalanines with moderate to
excellent converted yields (4-96%) at production levels between
8.5 and 235 mg L−1 over 6 h, respectively. More notably, E. coli cells
are reusable over at least five reaction cycles without a noticeable
loss in activity and cell viability. Using sustainable whole-cell biocatalysts are attractive over biocatalytic routes employing purified
enzymes for in vitro assays, in part, because of the potential higher
risk for enzyme denaturation during purification and incubation
with the substrate. Further, this biocatalyst offers notable advantages over conventional synthetic methods because of its excellent
enantioselectivity, broad substrate scope, single-step conversion,
20
N.D. Ratnayake et al. / Journal of Biotechnology 217 (2016) 12–21
and sustainability. In addition, the small-scale production yields
of the whole-cell biocatalyst, described here, can likely improve
by using a bioreactor, increasing the number of bacterial membrane permeases, optimizing the outer membrane permeability (Ni
and Chen, 2004), and reducing aromatic amino acid flux through
catabolic pathways in engineered E. coli.
Acknowledgements
We thank Olivia Goethe for her technical assistance. This material is based on work supported by NSF-CAREER Award 0746432
and Michigan State University AgBioResearch RA078692 (894).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jbiotec.2015.10.
012.
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