Carboxylic Acid Reductase 2015

459
2.5.2 Carboxylic Acid Reductase
A. S. Lamm, P. Venkitasubramanian, and J. P. N. Rosazza
General Introduction
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Carboxylic acids are ubiquitous in natural systems and are an important functional group
in a myriad of organic compounds. They are present in free acids such as acetic, pyruvic,
or citric acid, or in the derivated forms of esters, anhydrides, lactones, amides, and lac-
tams. Many carboxylic acids form stable salts with reasonably high water solubility. As a
group they are present in many value-added chemicals or synthons for natural biosynthe-
sis, as well as having uses in the pharmaceutical, agricultural, and food industries. How-
ever, such a critical moiety typically represents the end of the oxidation process from a
primary alcohol to an aldehyde and then to a carboxylic acid. The aldehyde and alcohol
functionalities are also well sought after and the ability to interconvert between all three
functionalities is highly desired. Consequently, several well-known chemical synthetic
methods to manipulate this oxidation–reduction process have been developed. Most re-
ductive synthetic methods employ expensive reducing agents, typically metallic in nature
(a catalyst accompanied by hydrogen under high temperature and pressure), or the use of
hydride ions. These conditions routinely result in very expensive, extremely hazardous,
and potentially wasteful production operations.
Over the years, specific microorganisms and their intrinsic enzymes have been
shown to have the ability to perform biocatalytic oxidative reactions (oxidases), and,
more importantly, some entities can perform the more difficult reductive reaction (reduc-
tases). These discoveries are significant because they allow for these reactions to occur
under the mildest of conditions. Desired products are formed via specific binding reac-
tions of organic substrates and are highly regio- and stereospecific. These selectivities
and specificities are often realized because of rigid interactions occurring between en-
zyme active sites and substrate molecules. Of course, this creates an opportunity for bet-
ter production operations that require lower input costs because of safer, more sustain-
able processes and which give potentially greater yields. The advantages of using a carbox-
ylic acid reductase (CAR) versus a chemical synthesis are given in Table 1. The points men-
tioned in Table 1 help to illustrate the novel green chemistry approach of carboxylic acid
reductase to synthesis, isomeric resolution, and biofuel production.
Table 1 Comparison of Carboxylic Acid Reductase Based Reduction Versus Synthetic

Chemical Reduction
Carboxylic Acid Reductase (CAR) Synthetic Chemical Reduction

regio- and stereospecific products reactions usually lack specificity
resolution of isomeric mixtures possible due to no differentiation of isomeric mixtures
reaction specificity
no unwanted side products side-product formation
no protecting groups required protecting groups required for certain
functionalities
mild temperature, pressure, and pH requires high temperature, pressure,
and pH
can differentiate substrates no discrimination
for references see p 477

460 Biocatalysis 2.5 Carbonyl Reduction
Table 1 (cont.)
Carboxylic Acid Reductase (CAR) Synthetic Chemical Reduction
no hazardous conditions or chemicals required requires hazardous conditions and
chemicals, and expensive metal cata-
lysts
no organic solvents required requires organic solvents
reaction mechanism not affected by cations and feedstock should be purified to prolong
anions present in the feedstock catalyst lifetime
value-added aldehydes or alcohols are easily gen- more selective and expensive catalyst
erated for purification, production, or synthetic composition required for selective ca-
applications talysis
Several of these carboxylic acid reducing biocatalysts have been mentioned in the litera-
ture.[1–12] The most recent and comprehensive of these references[13] gives suitable cover-
age to carboxylate reductions by fungi, bacteria (aerobic and anaerobic), and plants, as
well as by isolated and overexpressed enzyme systems. Some of these organisms are uti-
lized in their whole-cell form and give the corresponding alcohols from the carboxylic
acid substrate. Such organisms include Actinomyces,[14] Clostridium thermoaceticum,[15] As-
pergillus niger,[16,17] Corynespora melonis,[16] Coriolus,[16] Neurospora,[18] Glomerella cingula-
ta,[19,20] Gloeosporium laeticolor,[20] and Nocardia[21] species.
2.5.2.1 Isolation and Purification of Carboxylic Acid Reductase
2.5.2.1.1 Carboxylic Acid Reductase from Nocardia iowensis DSM 45197
Only the carboxylic acid reductase (CAR) isolated from Nocardia iowensis DSM 45197T,
NRRL 5646T, NRRL B-24671T, UI 122540T (formerly known as Nocardia species NRRL
5646) has been characterized sufficiently for academic and commercial applications.[22]
As reported by Rosazza and co-workers, N. iowensis efficiently reduces aryl carboxylic
acids to aldehydes and alcohols; however, it is noted that the substrate specificity for re-
ducing carboxylic acids with this organism appears to be significantly different to that re-
ported previously for the enzyme.[23–25] To further investigate this enzyme, these workers
have devised a purification protocol for obtaining pure, active, and relatively stable car-
boxylic acid reductase.
This purification has enabled the analysis of large amounts of pure, active carboxylic
acid reductase for the elucidation of its properties and its catalytic mechanism. Purified
carboxylic acid reductase is shown to require various factors for catalytic activity (ATP,
Mg2+, and NADPH). Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-
PAGE) and gel filtration chromatography indicate a molecular mass of 128 000 Da, similar
to that for the carboxylic acid reductase from Nocardia asteroides[23] and that from Neuro-
spora crassa.[23,24,26] Apparent Km values [where Km is the Michaelis constant (the substrate
concentration at which the enzyme reaction rate is half of the maximum velocity of the
reaction)] for N. iowensis carboxylic acid reductase (for benzoate, ATP, and NADPH) are
more than 1000-fold higher than those reported for the enzyme from N. asteroides.[23]
The Km of benzoate for N. crassa carboxylic acid reductase is 63 M and its activity is inhib-
ited by 300 M benzoate.[26] No inhibition is observed for the enzyme from N. iowensis,
even when benzoate concentrations are increased to 2 mM. This suggests that the N. io-
wensis carboxylic acid reductase has different catalytic properties to those reported earli-
er.[23,25] An illustrative experimental procedure for the preparation of N. iowensis DSM
45197 cells, enzyme extraction and purification, and assay involving the reduction of so-
dium benzoate to give benzaldehyde (1, Scheme 1) is described below.[27]
2.5.2 Carboxylic Acid Reductase 461
Scheme 1 Reduction of Benzoate Using the Carboxylic Acid Reductase

from Nocardia iowensis DSM 45197[27]
CAR from Nocardia iowensis DSM 45197

O ATP, NADPH, MgCl2 O
Tris-HCl buffer
Ph ONa Ph H
1
Benzaldehyde (1); Typical Procedure for the Preparation, Purification, and Assay of the
CAR from Nocardia iowensis DSM 45197:[27]
Preparation of N. iowensis cells: N. iowensis DSM 45197 is available from the ATCC (Ameri-
can Type Culture Collection), NRRL (US Agricultural Research Service Culture Collection),
and the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen). It is grown
and maintained on slants of Sabouraud dextrose agar or sporulating agar (ATCC no.
5 medium). Shaken-flask cultures were grown by a standard two-stage incubation proto-
col[4] in sterile medium (200 mL) held in stainless-steel capped 1-L DeLong culture flasks.
The medium contained (w/v) 2% glucose, 0.5% yeast extract, 0.5% soybean flour, 0.5% NaCl,
and 0.5% K2HPO4 in distilled H2O, adjusted to pH 7.2 with 6 M HCl and then autoclaved at
121 8C for 20 min. Cultures were incubated by shaking at 250 rpm at 28 8C on Gyrotory
shakers. A 10% inoculum derived from a 72 h old first-stage culture was used to initiate
the second-stage culture, which was incubated as before. After 24 h of incubation in the
second stage, BzOH was added to a concentration of 5 mg • mL–1 (44.6 mM) as an inducer
for enzyme synthesis. The culture was harvested 24 h after addition of benzoate, and the
cells were collected from the medium by centrifugation at 8000 gravities for 20 min and
washed twice with 0.9% (w/v) NaCl. Cell pellets were stored at –38 8C until needed. Typical
wet cell yields by this cultivation process were approximately 25 g • L–1.[28]
Preparation of cell extracts: A cell pellet (25 g; wet weight) was suspended in cold 50
mM Tris-HCl buffer (pH 7.5; 150 mL) containing 1 mM dithiothreitol (DTT), 0.1 mM
BnSO2F, 1 mM edta, and 10% (v/v) glycerol. The cell suspension was disrupted with a Soni-
fier Cell Disrupter 350 at 250 W with a 20% intermittent duty cycle for 10 min. Cell debris
was removed by centrifugation at 100 000 gravities for 40 min at 4 8C. The 100 000 gravity
supernatant was used directly for subsequent enzyme purification steps, which were all
conducted at 4 8C.
Purification of wild-type carboxylic acid reductase from N. iowensis: The crude 100 000
gravity supernatant (170 mL; approx. 500 mg of protein) was applied to a Mono-Q column
(2 20 cm) pre-equilibrated with 50 mM Tris-HCl (pH 7.5) containing 1 mM dithiothreitol
(DTT), 1 mM edta, 0.1 mM BnSO2F, and 10% (v/v) glycerol. The column was washed with
starting buffer (60 mL) before the enzyme was eluted in starting buffer (400 mL) with a
0–1 M linear NaCl gradient while 5-mL fractions were collected. The active fractions (frac-
tions 22 to 28) were combined and concentrated to a volume of 15 mL in an Amicon con-
centrator (PM-30 membrane). This preparation was diluted with 20 mM Tris-HCl buffer
(pH 7.5; 200 mL) containing 1 mM dithiothreitol (DTT), 10 mM MgCl2, and 10% (v/v) glycer-
ol and concentrated to a volume of 50 mL. This material was loaded onto a Reactive Green
19 agarose[29,30] column (2 10 cm) pre-equilibrated with 20 mM Tris-HCl buffer (pH 7.5)
containing 1 mM dithiothreitol (DTT), 10 mM MgCl2, and 10% (v/v) glycerol. The column
was then washed with starting buffer (30 mL) and the enzyme was eluted with the Tris-
HCl buffer (100 mL) with a combined linear gradient of 0 to 2 mM edta and 0 to 0.5 mM
NADP+ (200 L) while 2.5-mL fractions were collected. The active fractions (fractions 9 to
14) were combined, concentrated with an Amicon concentrator (PM-30 membrane) to a
volume of 5 mL, and diluted to a volume of 50 mL with 5 mM phosphate buffer (pH 6.8)
containing 1 mM dithiothreitol (DTT), 5 mM MgCl2, and 10% (v/v) glycerol. This prepara-

tion was then loaded onto a hydroxyapatite column (0.8 5 cm) equilibrated with 5 mM
phosphate buffer (pH 6.8). The column was then washed with starting buffer (20 mL) and
the enzyme was eluted with a linear gradient of 5 to 80 mM phosphate buffer (80 mL).
Enzyme assay: A standard aryl aldehyde oxidoreductase assay soln (final volume:
0.7 mL) was prepared containing 0.15 mM NADPH, 1 mM ATP, 5 mM NaOBz, 10 mM
MgCl2, and the enzyme (0.01–0.3 U) in 50 mM Tris-HCl buffer. The reference cuvette con-
tained all components except enzyme. Reaction was initiated by adding the enzyme, and
the process was monitored by recording the absorption decrease at 340 nm at 25 8C with a
spectrophotometer. One unit of the aryl aldehyde oxidoreductase was defined as the
amount of enzyme that catalyzed the reduction of NaOBz to benzaldehyde (1) at a rate
of 1 mmol • min–1 under standard assay conditions.
Protein assay: The concentration of protein was measured by the Bradford protein mi-
croassay using bovine serum albumin as the standard.
2.5.2.1.2 Recombinant Carboxylic Acid Reductase from E. coli
Carboxylic acid reductase is produced in Nocardia iowensis, an organism that is relatively

difficult to grow and which produces limited quantities of the required enzyme. There-
fore, Rosazza and co-workers have sought to clone and express the enzyme in a common
expression host such as E. coli. The results of this are well documented and will not be de-
tailed here.[28,31] In brief, to express recombinant carboxylic acid reductase (rCAR), a
100-mL culture of E. coli [BL21(DE3) or BL21-CodonPlus(DE3)-RP] harboring pHAT305 plas-
mid is grown overnight in LB–ampicillin medium.[31] This culture is diluted 20-fold with
fresh medium and then incubated at 170 rpm on a rotary shaker at 37 8C until an optical
density of 0.6 is reached at 600 nm. At this point, 1 mM isopropyl -d-1-thiogalactopyran-
oside (IPTG) is added and the induced culture is incubated for a further 4.5 hours. Lysate
from E. coli BL21(DE3) and BL21-CodonPlus(DE3)-RP cells carrying pHAT305 has moderate
carboxylic acid reductase activity, and a typical 1 L culture of E. coli BL21-CodonPlus(DE3)-
RP cells carrying pHAT305 yields approximately 50–70 mg of purified enzyme. However,
recombinant carboxylic acid reductase (rCAR) from E. coli BL21-CodonPlus(DE3)-RP/
pHAT305 shows a lower specific activity than that of wild-type carboxylic acid reductase
purified from N. iowensis.[31] Compared to wild-type Nocardia, the E. coli based expression
of the carboxylic acid reductase biocatalyst can be scaled up with relative ease and pro-
duces fewer side products than Nocardia. Although the pure enzyme itself can be used in
small-scale reactions, whole E. coli cells containing carboxylic acid reductase and the nat-
ural cofactors (ATP, NADPH) are easily used to reduce a wide range of carboxylic acids,
conceivably on any scale. To obtain the most active form of rCAR, E. coli bearing the plas-
mid pPV2.85 is recommended (see Section 2.5.2.4.2).
2.5.2.2 Mechanism of Carboxylic Acid Reduction
The mechanism of carboxylic acid reductase (CAR) is presented here for a deeper under-
standing of the process. It has been discovered that carboxylic acid reductase produced in
wild-type Nocardia is actually a holo-enzyme produced by post-translational phospho-
pantetheinylation of apo-carboxylic acid reductase (apo-CAR) to afford maximal enzyme
activity.[28] This post-translational modification is performed by a phosphopantetheine
transferase (PPTase)[32,33] within Nocardia iowensis in the presence of coenzyme A (Scheme
2).
Scheme 2 Phosphopantetheinylation Catalyzed by Phosphopantetheine Transferase[28,32]
NH2
OH N
H H O O N
N N
HS O P O P O N N
O O O O O
O OH
O P O
O
coenzyme A
OH
N
H
O OH
apo-enzyme H H O
N N
PPTase HS O P O
O O O
HN
O
holo-enzyme
This 4¢-phosphopantetheine prosthetic group in active carboxylic acid reductase serves as

a “swinging arm” that can react with acylated adenosine monophosphate (AMP) inter-
mediates. The initial binding of both the carboxylic acid substrate (in this case, benzoic
acid) and adenosine triphosphate (ATP) at the carboxylic acid reductase N-terminus to
form an acyl adenylate is a crucial step (Scheme 3). The cysteine residue of the phospho-
pantetheinyl moiety of the holo-carboxylic acid reductase (holo-CAR) reacts with benzoyl-
functionalized adenosine monophosphate to form a benzoyl sulfide that is covalently at-
tached to the enzyme. The sulfide tethered to the enzyme is brought into the proximity of
the C-terminal domain where hydride attack from NADPH reduces the sulfide, yielding
the free aldehyde [in this case, benzaldehyde (1)], NADP+, and a free phospho-
pantetheinylsulfhydryl group for another catalytic cycle.[28] A simplified view of this
cycle is shown in Scheme 4.

Scheme 3 Catalytic Process of the holo-Carboxylic Acid Reductase Reduction of Benzoic

Acid to Benzaldehyde[28]
NH2
N
O N
O O P O N N
OH O
Ph
HO OH
OH
H H O
N N
HS O P O
O O O
phosphopantetheine
adenylating domain attachment site reduction domain
N C
O OH
H H O
N N
Ph S O P O
O O O
phosphopantetheine
N C
OH O
O H H
N N
O P O S Ph
O O O
phosphopantetheine
N C
NADPH
OH
H H O O
N N
HS O P O + + NADP+
O O O Ph H
1
phosphopantetheine
N C
Scheme 4 Simplified Mechanism of Carboxylic Acid Reductase Based Reduction[28]
O
ATP
O
Ph OH
Ph OH HS-CAR PPi
O
NPT
CAR HS-CAR
Ph O AMP
HS-CAR
coenzyme A
AMP
O
O
Ph H O
1 Ph H
Ph S CAR
HS-CAR
NADP+ NADPH
NPT = Nocardia phosphopantetheine transferase; PPi = pyrophosphate; HS-CAR = phosphopantetheinylated holo-CAR
2.5.2.3 Substrate Scope with Carboxylic Acid Reductase
Reduction of carboxylic acids can be accomplished by growing cultures of wild-type No-

cardia iowensis or heterologously expressed CAR in E. coli, as well as by purified enzymes.
The reduction of a range of aromatic carboxylic acids and a variety of aliphatic acids has
been examined. The range of oxidoreductase activities of crude N. iowensis cell-free extract
(CFE) with different substrates is summarized in Scheme 5.[23,24,26,27,34] Benzoic acids substi-
tuted with halogens, methyl, methoxy, hydroxy, acetyl, nitro, benzoyl, phenyl, and phen-
oxy groups have been examined, as well as aromatic systems containing two and three
rings (naphthalene and fluorene, respectively) and various heterocyclic aromatic
acids.[29] The 3-substituted benzoic acids (bromo, chloro, hydroxy, methoxy) are the best
substrates within their respective aryl carboxylic acid series (but not the fluoro- and meth-
yl-substituted species). The range of aromatic substrates for carboxylic acid reductase is
similar to that observed elsewhere.[25] In general, 2-substituted benzoic acids are the poor-
est substrates of those compared. As mentioned above, the cell-free extract (CFE) from N.
iowensis also efficiently reduces naphthoic acids, but only reduces indole-3-, and indole-5-
carboxylic acids. In addition, cell-free extracts reduce furoic and nicotinic acid, as well as
cinnamic, phenylacetic, phenylmalonic, phenylsuccinic, and 2-phenylpropanoic acids, al-
beit at slower rates than benzoic acid itself.[27] Of all the compounds examined, the best
substrates are benzoic acid, 3-bromobenzoic acid, 3-chlorobenzoic acid, 4-fluorobenzoic
acid, 4-methylbenzoic acid, 3-methoxybenzoic acid, and 2-naphthoic acid. However,
nitro-substituted benzoic acids are not reduced at all. Relative rates of substrate reduction
are determined by measuring the optical density (OD) at 340 nm as NADPH is oxidized to
NADP+ during the reduction reaction.

Scheme 5 Substrate Specificity of Carboxylic Acid Reductase Obtained from Dif-

ferent Sources[23,24,26,27,34]
O O
carboxylic acid reductase
Ar1 OH Ar1 H
Ar1 Enzyme Source Relative Activitya Ref

[27]
Ph Nocardia iowensis 100
[23,34]
Ph Nocardia asteroides JCM 3106 100
[24,26]
Ph Neurospora crassa 100
[27]
2-HOC6H4 Nocardia iowensis 0
[23,34]
2-HOC6H4 Nocardia asteroides JCM 3106 0
[27]
[23,34]
[24,26]
3-HOC6H4 Neurospora crassa 24
[27]
[23,34]
[24,26]
4-HOC6H4 Neurospora crassa 11
[27]
2-ClC6H4 Nocardia iowensis 3
[23,34]
2-ClC6H4 Nocardia asteroides JCM 3106 0
[27]
[23,34]
[27]
[23,34]
[27]
2-Tol Nocardia iowensis 6
[23,34]
2-Tol Nocardia asteroides JCM 3106 6
[27]
[23,34]
[27]
[23,34]
[27]
2-MeOC6H4 Nocardia iowensis 3
[27]
[24,26]
3-MeOC6H4 Neurospora crassa 53
[27]
[27]
2-O2NC6H4 Nocardia iowensis 0
[23,34]
2-O2NC6H4 Nocardia asteroides JCM 3106 0
[27]
[23,34]
[27]
[23,34]
a
Relative activity in relation to the reaction of benzoate (Ar1 = Ph).
Recombinant carboxylic acid reductase (rCAR) also reduces a number of natural mono-,
di-, and tricarboxylic acids (Table 2).[28,35] Rates of reduction of d-tartaric acid, Æ-ketoglutar-
ic acid, cis- and trans-aconitic acid, as well as citric and malic acids are higher than that for
benzoic acid. Regiospecificity in carboxylic acid reduction remains to be established for
citric, tartaric, and malic acids; however, recombinant carboxylic acid reductase is d-tar-
taric acid specific, offering the possibility of a method of resolving tartaric acid racemates.
Pyruvic acid, isocitric acid, fumaric acid, and maleic acid are not substrates, indicating
that carboxy moieties adjacent to C(sp2) centers might be less susceptible to reduction
with recombinant enzyme. Relative rates of substrate reduction are again determined by
measuring the optical density (OD) at 340 nm as NADPH is oxidized to NADP+ during the
reduction reaction.
Table 2 Substrate Specificity of Recombinant Carboxylic Acid Reductase[28,35]
O O
rCAR from E. coli
R1 OH R1 H
Substrate Relative Activitya with rCAR from E. coli Ref
O
[28,35]
100
Ph OH
OH O
HO 120 [28,35]
OH
O OH
OH O
HO 0 [28,35]
OH
O OH
OH 50 [28,35]
OH
rac
O
[28,35]
OH
50
OH
O
OH 0 [28,35]
O O
[28,35]
HO OH 160
O
O O
HO 80 [28,35]
OH
O
O OH
O O [28,35]
130
HO OH

Table 2 (cont.)
Substrate Relative Activitya with rCAR from E. coli Ref
O OH
O
[28,35]
HO 130
O OH
O OH
O O [28,35]
80
HO OH
O HO O
[28,35]
40
HO OH
O OH
O O
[28,35]
120
HO OH
OH
O OH
O O
[28,35]
0
HO OH
OH
O
HO 0 [28,35]
OH
O
HO O
O [28,35]
0
OH
O
HO
OH 105 [28,35]
O OH
rac
O
HO 105 [28,35]
OH
O OH
O
HO 130 [28,35]
OH
O OH
a
Relative activity in relation to the reaction of benzoate.
Ibuprofen is an attractive substrate candidate for investigation because of its overall im-
portance as a widely used drug, and the opportunity to examine the possible stereoselec-
tive properties of the carboxylic acid reductase reaction with cultures of N. iowensis (see
Section 2.5.2.4.1). The biocatalytic reduction of vanillic acid to the commercially valuable
product vanillin (see Section 2.5.2.4.2) is a useful illustration of the ease of use and effi-
ciency of the recombinant carboxylic acid reductase E. coli reduction system.[35] Further-
more, the broad substrate specificity of carboxylic acid reductase enables the wide appli-
cation of this biocatalyst to other important applications, such as enantiomeric resolution
and the reduction of many other natural and synthetic carboxylic acids.
2.5.2.4 Applications of Carboxylic Acid Reductase
Chemists continuously explore means for improving chemical processes, enabling them
to become both economically viable and environmentally sustainable. This involves selec-
tive catalysis, using more viable sources of raw material, and modifying synthetic proce-
dures.[36] The introduction of biological systems in traditional chemical synthesis provides
the added advantages of catalyst versatility, regio-, chemo-, and enantioselectivity, and ca-
talysis at ambient temperature and pressure.[37] Highly selective biocatalytic reductions of
carboxylic acids open new avenues for producing value-added chemicals that are current-
ly derived either from petroleum feedstock or by chemical synthetic means. These range
from vanillin to chiral aldehydes and alcohols obtained by kinetic resolution, and find use
in the pharmaceutical, food, and agricultural industries. Several examples are described
in the following sections.
2.5.2.4.1 Synthesis of Ibuprofen
The 2-arylpropanoic acid derivative ibuprofen (2) is a potent oral nonsteroidal anti-inflam-
matory, antipyretic, and analgesic agent.[38–40] Pharmacological activities of ibuprofen re-
side almost exclusively in its S-enantiomer (S)-2.[39] With growing cultures of Nocardia io-
wensis, two major metabolites of racemic ibuprofen are formed; these have been isolated
and identified by spectral methods as ibuprofenol 3 and the corresponding acetate deriv-
ative 4 (Scheme 6).[21] An unoptimized preparative-scale reaction conducted with racemic
ibuprofen (rac-2) has been performed.[21] This reaction was stopped at 24 hours when
analysis (TLC) indicated that approximately 50% of the substrate had been converted
into other products. Unreacted, recovered ibuprofen is largely (+)-(S)-ibuprofen [(S)-2],
which is obtained with an enantiomeric excess of 47.9% as determined by measurement
of the optical rotation. The enantiomeric excess of the (–)-(R)-ibuprofenol (R)-3 obtained
from the biotransformation is determined to be 61.2% by spectroscopic analysis (NMR)
of the (+)-(S)-2-acetoxymandelic acid derived ester 5. This finding shows that whole-cell
N. iowensis reactions are (–)-(R)-selective with racemic ibuprofen, and offer the possibility
that other racemic carboxylic acids can be resolved.
Scheme 6 Biocatalytic Reduction of Ibuprofen[21]
OH OH OAc
N. iowensis
+
O
Bui Bui Bui
rac-2 3 4

OH CAR from
OH OH
N. iowensis
+
O 50% conversion O
Bui Bui Bui
rac-2
(S)-2 47.9% ee (R)-3
Ph
O
OAc
O
Bui
5 61.2% ee
While it may appear to be disadvantageous for whole cells to reduce carboxylic acids to
aldehydes and alcohols, and to further esterify them to the corresponding acetates, such a
combination of reactions may be highly desirable in, for example, the conversion of car-
boxy substrates into esters of value or fragrances and flavors.
2.5.2.4.2 Synthesis of Vanillin
Natural vanilla, obtained from the dried pods of the orchid Vanilla planifolia, accounts for
only <1% of the world flavor market (global demand 18 000 metric tons/year) and the re-
mainder is supplemented by chemical synthesis of vanillin (4-hydroxy-3-methoxybenzal-
dehyde) from guaiacol (2-methoxyphenol).[41] Vanillin has been produced by microbial
transformation from natural substrates, including phenolic stilbenes,[42,43] eugenol,[44–46]
and ferulic acid.[47,48] In whole-cell Nocardia biotransformation reactions, decarboxylation
of vanillic acid (6) to give guaiacol (7) is the major complicating pathway.[49,50] The identi-
fication of guaiacol and vanillyl alcohol (9) as metabolites confirms that Nocardia iowensis
possesses two different metabolic pathways for the biotransformation of vanillic acid.
These pathways are (i) decarboxylation to guaiacol and (ii) reduction to vanillin (8) and
its subsequent reduction to vanillyl alcohol (9) (Scheme 7).[49] With purified Nocardia car-
boxylic acid reductase, the adenosine triphosphate and NADPH dependent reduction of
vanillic acid is quantitative, yielding only vanillin and no complicating byproducts.[51]
Scheme 7 Biotransformation of Vanillic Acid To Give Guaiacol, Vanillin, and Vanillyl Alcohol
Using Whole-Cell Nocardia iowensis[49]
HO
MeO
O 7
HO
OH
MeO ATP O AMP
6 arylaldehyde oxidoreductase
(EC 1.2.1.30) NADPH
− PPi
OMe
OH
O NADPH
HO HO
H OH
MeO MeO
8 9
PPi = pyrophosphate
The biocatalytic reduction of vanillic acid to the commercially valuable vanillin is used to
illustrate the ease and efficiency of the recombinant carboxylic acid reductase (rCAR) E.
coli reduction system.[35] Carboxylic acid reductase does not reduce aldehydes to alcohols;
however, the reduction of vanillin to vanillyl alcohol is made possible by the endogenous
aldehyde reductase/dehydrogenase present in E. coli. The relative concentrations of these
metabolites in reduction with enzyme expressed by various E. coli strains have been plot-
ted over the course of the reaction, and these results are displayed in Figure 1. In graphs
A–C the biocatalyst concentration is 30 g • L–1 while for graph D an XAD-2 resin [5 g(resin) •
g(substrate)–1] is added for product removal and a biocatalyst concentration of 120 g • L–1 is
used. In this latter example, most of the vanillic acid (6) is reduced by E. coli expressed car-
boxylic acid reductase with in situ product removal in 2 hours to give vanillin (8; 80%) and
vanillyl alcohol (9; 20%).
Figure 1 Reduction of Vanillic Acid to Vanillin and Vanillyl Alcohol Using Recombinant Car-
boxylic Acid Reductase Expressed in Various E. coli Strains[35]a
5 catalyst:
metabolite concentration (g•L−1)
E. coli BL21-CodonPlus(DE3)-RP/pHAT305
4
3
(A)
2
0
0 2 4 6 8 10
time (h)
= vanillic acid (6); = vanillin (8); = vanillyl alcohol (9)

5 catalyst:

E. coli BL21-CodonPlus(DE3)-RP/pPV2.83
4
3
(B)
2
0
0 2 4 6 8 10
time (h)
5
catalyst:
4
3
(C)
2
0 2 4 6 8 10
time (h)
5
catalyst:
4 with XAD-2 resin
3
(D)
2
0
0 2 4 6 8 10
time (h)
a
Reprinted from (Venkitasubramanian; Daniels; Das; Lamm; Rosazza, Enzyme and Microbial
Technology, Vol. 42), Copyright (2008), p 130 with permission from Elsevier.
E. coli BL21-CodonPlus(DE3)-RP/pHAT305 (Placcar) expressing CAR (Graph A) reduces 50% of

vanillic acid to vanillin in 10 hours. Coexpression of CAR and NPT (Nocardia phospho-
panthetine transferase) in E. coli BL21-CodonPlus(DE3)-RP/pPV2.83 (Placcar Placnpt) results
in a purified recombinant carboxylic acid reductase with improved specific activity of
2.2 U • mg–1 of protein. Furthermore, resting cells of this enzyme reduce 90% of vanillic
acid to vanillin in 6 hours. Enhanced in vivo cofactor NADPH regeneration by glucose de-
hydrogenase (GDH) is accomplished using E. coli BL21-CodonPlus(DE3)-RP/pPV2.85 (Placcar
Placnpt Placgdh), which expresses CAR, NPT, and GDH (Graph C). Resting-cell reactions using
E. coli BL21-CodonPlus(DE3)-RP/pPV2.85 with in situ product removal by XAD-2 resin effi-
ciently reduce 5 g • L–1 of vanillic acid and benzoic acid within 2 hours (Graph D).[35] This
concept has been further exemplified by constructing a true de novo biosynthetic path-
way for vanillin production from glucose in Schizosaccharomyces pombe as well as in bak-
ers yeast (Saccharomyces cerevisiae).[52] Productivities are 65 and 45 mg • L–1 after introduc-
tion of three and four heterologous genes, respectively. The engineered pathways involve
incorporation of 3-dehydroshikimate dehydratase from the dung mold Podospora paucise-
ta, an aromatic carboxylic acid reductase (ACAR) from a bacterium of the Nocardia genus,
and an O-methyltransferase from Homo sapiens. In S. cerevisiae, the aromatic carboxylic
acid reductase enzyme requires activation by phosphopantetheinylation, and this is
achieved by coexpression of a Corynebacterium glutamicum phosphopantetheine transfer-
ase.
Vanillin (8) and Vanillyl Alcohol (9); Typical Procedure for Reduction of Vanillic Acid
using a Recombinant Carboxylic Acid Reductase (rCAR):[35]
Initial culture growth: Crystals from a frozen glycerol stock of E. coli BL21-CodonPlus(DE3)-
RP/pPV2.85 were streaked onto LB agar plates with ampicillin (100 g • mL–1) to obtain sin-
gle colonies, which were in turn inoculated into LB medium (20 mL containing 100 g •
mL–1 ampicillin) in 125-mL stainless steel capped DeLong flasks. The cultures were incu-
bated with shaking at 250 rpm on a rotary shaker at 37 8C, and a 1% inoculum derived
from 8-h stage I cultures was used to initiate fresh LB cultures (200 mL) with antibiotics
in a 1-L DeLong flask. These cultures were incubated at 37 8C for 16 h with shaking at
250 rpm. Carboxylic acid reductase containing E. coli cells were then pelleted by centrifu-
gation at 5000 gravities for 6 min at 4 8C.
Whole-cell carboxylic acid reduction: The carboxylic acid reductase containing E. coli
cells were resuspended in 0.9% (w/v) NaCl (200 mL) and pelleted once again by centrifuga-
tion at 5000 gravities for 6 min at 4 8C. A sodium vanillate stock soln (50 mg • mL–1) was pre-
pared by dissolving equimolar amounts of vanillic acid (6) and NaHCO3 in 0.1 M Na2HPO4
(pH 7) [a 50-mL mixture thus contained glucose (0.4%), wet E. coli cells (1.5 g), and sodium
vanillate (200 mg) in 0.1 M Na2HPO4 (pH 7)]. The resulting mixture was incubated at 28 8C
with shaking at 220 rpm and 1-mL samples were withdrawn at various time intervals for
analysis.
Analytical methods: Standard solns were prepared by dissolving weighed amounts of
compounds in a 1:1 (v/v) mixture of 0.1 M Na2HPO4 (pH 7) and MeCN. Aliquots (0.5 mL) of
the biotransformation samples were mixed with MeCN (0.5 mL) and the resulting mix-
tures were vortexed for 30 s. After standing at rt for 30 min, samples were microcentri-
fuged at 20 000 gravities for 3 min and the supernatants were filtered through 0.22 m
poly(vinylidene difluoride) syringe filters. Then, a sample (1–2 L) was analyzed by HPLC
(Econosil C18, 5 m column; MeCN/H2O/HCO2H 20:80:1; flow rate: 0.4 mL • min–1). HPLC
retention volumes and detection wavelengths for standards were as follows: vanillyl alco-
hol (9) 1.7 mL, 277 nm; vanillic acid (6) 2.5 mL, 260 nm; vanillin (8) 4.1 mL, 284 nm. TLC
analysis (silica gel, CH2Cl2/MeCN/HCO2H 75:25:1) was conducted by careful spotting of
standard compounds (10–20 g) and bioconversion samples (30 L) followed by develop-
ment and visualization with a UV lamp (254 nm) and/or spraying with a phosphomolybdic
acid/95% EtOH spray [30% (w/v)] followed by gentle heating. Rf values of standards were as
follows: vanillyl alcohol (9) 0.8; vanillic acid (6) 0.5; vanillin (8) 0.4. Other suitable aromat-
ic carboxylic substrates for this procedure include 3-chlorobenzoic acid, 4-chlorobenzoic
acid, and 3-(4-hydroxy-3-methoxyphenyl)propenoic acid (ferulic acid).
2.5.2.4.3 Reduction of Ferulic Acid
Ferulic acid [10; 3-(4-hydroxy-3-methoxyphenyl)propenoic acid] is an extremely abundant

plant product available from corn kernel hulls, which are themselves obtained from wet
milling.[53] The major product obtained by microbial oxidation of ferulic acid by strains of
Bacillus,[54] Pseudomonas,[55,56] Polyporus,[57] Rhodotorula,[58] and Streptomyces[59] is vanillic acid

(6). Given that vanillic acid is itself a precursor for the biocatalytic synthesis of vanillin (8)
using an arylaldehyde oxidoreductase, one can envision using this abundant, readily
available material in a simple two-step/one-pot process for the preparation of vanillin
from ferulic acid (Scheme 8).[31,53] In addition, in vitro treatment of ferulic acid with re-
combinant carboxylic acid reductase (rCAR) affords smooth reduction to coniferyl alde-
hyde (11),[49] while incubation of ferulic acid with growing cultures of E. coli BL21-
CodonPlus(DE3)-RP/pHAT305 expressing carboxylic acid reductase leads to the produc-
tion of coniferyl alcohol (12). The current market price of coniferyl aldehyde is approxi-
mately $8000/lb, while coniferyl alcohol is obtainable for about $159 000/lb. Such reduc-
tions of the carboxy moiety of ferulic acid to its aldehyde and alcohol are not observed
when ferulic acid is incubated with resting cells of Nocardia iowensis.[58,53]
Scheme 8 Reduction of Ferulic Acid To Give Value-Added Chemicals[31,53]
HO OH
MeO
10
microbial purified rCAR or

oxidation E. coli BL21-CodonPlus(DE3)-RP/pHAT305
O
HO HO O
OH
MeO MeO H
6 11
purified rCAR or
O
HO HO
H
MeO MeO OH
8 12
HO
OH
MeO
9
2.5.2.4.4 Synthesis of 3-Hydroxytyrosol
Olive oil and wine contain 3-hydroxytyrosol (3-HT), a phenolic compound with strong an-
tioxidant properties that is associated with a number of beneficial effects on human
health.[60] In addition to the pronounced effects of 3-hydroxytyrosol itself, the chemical
structure is found as a building block in various pharmaceutically active compounds,
and it can be used as a precursor for their synthesis. For example, 3-hydroxytyrosol is a
building block in vernakalant (Brinavess), a drug used for the maintenance of the sinus
rhythm of the heart. The production of 3-hydroxytyrosol is achieved through reduction
of (3,4-dihydroxyphenyl)acetic acid. The reduction is performed using E. coli BL21(DE3)
cells overexpressing carboxylic acid reductase from Nocardia and phosphopantetheinyl
transferase from E. coli.[60]
2.5.2.4.5 Reduction of Fatty Acids
Fatty alcohols are an important class of molecules in the detergent/surfactant industry.

These molecules can either be produced from natural fats and oils or from petroleum-de-
rived alkenes. Copper chromite reduction of the methyl esters of fatty acids or hydroge-
nation of free fatty acids yields saturated fatty alcohols. The oxo process[36] using ethylene
and syngas, or Ziegler chemistry using triethylaluminum with ethylene are typically em-
ployed by the surfactant industry to obtain fatty alcohols in bulk quantities (typically lin-
ear saturated alcohols). Fatty alcohols find application in cosmetics and toiletries as well
as in the pharmaceutical and plastics industries. The fatty alcohols can be further derivat-
ized as ethoxylates, sulfonates, phosphates, or amine oxides, which are mostly used in the
detergent industry. Fatty alcohols and aldehydes play important roles in the metabolic
pathways of a number of organisms because they can generate fatty acids along with for-
mation of the reduced forms of enzyme cofactors in a reaction involving large favorable
free-energy changes. The reduction of free fatty acids to aldehydes using recombinant car-
boxylic acid reductase (rCAR) was first exemplified by the conversion of oleic acid into
oleyl aldehyde.[61] A carboxylic acid reductase (CAR) from Mycobacterium marinum converts
a wide range of aliphatic fatty acids (C6-C18) into the corresponding aldehydes.[62] Togeth-
er with the broad substrate specificity of an aldehyde reductase or an aldehyde decarbon-
ylase, the catalytic conversion of fatty acids into fatty alcohols (C8-C16) or fatty alkanes
(C7-C15) has been reconstituted in vitro. This concept is applied in vivo, in combination
with a chain-length specific thioesterase, to engineer E. coli BL21(DE3) strains that are ca-
pable of synthesizing fatty alcohols and alkanes. The carboxylic acid reductase enzyme
activity has been verified in vitro via the oxidation of NADPH in the presence of benzoic
acid. It is found to have a selective preference for NADPH over NADH with apparent Km
values of 362 € 13 M, 48 € 8 M, and 115 € 13 M for benzoic acid, NADPH, and ATP, re-
spectively, and a Vmax of 2.32 € 0.1 mol • mg–1 (carboxylic acid reductase). Optimal activity
is observed at pH 7.5 with in vitro half-lives of 73, 70, and 48 hours at 26, 30, and 37 8C,
respectively, indicating it to be a relatively stable enzyme.[62] The analyses and bioconver-
sions are conducted using a methodology similar to that described previously.
2.5.2.4.6 Hydrogenation of Fatty Acids
The double cofactor dependency of carboxylic acid reductases on ATP and NADPH poses
additional challenges related to in situ regeneration of the catalytically active cofactors.
This issue has been beautifully exemplified using Pyrococcus furiosus as a microbial cata-
lyst and employing hydrogen gas to maintain the redox balance.[63,64] P. furiosus contains
suitable enzymes to couple hydrogen oxidation [catalyzed by hydrogenases (Hases)] to the
reduction of carboxylic acids catalyzed by carboxylic acid reductases [alternatively known
as aldehyde oxidoreductases (AOR)]. Whole-cell reduction of carboxylic acids is achieved

in the presence of hydrogen gas or syngas, and, in comparison with metal-based hydroge-
nation, biocatalytic hydrogenation can convert hexanoic acid into its corresponding alco-
hol in high yields with a high turnover frequency of 389 h–1 (Scheme 9).
Scheme 9 Reduction of Carboxylic Acids by Pyrococcus furiosus Cells[63,64]
H2
Hase Hase
reduction oxidation NADPH NADP+
O O
AOR ADH
R1 OH R1 H R1 OH
Hase = hydrogenase; AOR = aldehyde oxidoreductase; ADH = alcohol dehydrogenase

R1 = (E)-CH=CHPh, (CH2)2Ph, alkyl
2.5.2.5 Conclusions
The direct reduction of free carboxylic acids to value-added aldehydes and alcohols has
wide-ranging applications in the chemical, pharmaceutical, and food industries. Chemi-
cal reduction of free carboxylic acids uses metal alloy catalysts under extreme reaction
conditions that lead to unwanted side reactions,[17–19,29] such as the cracking of aliphatic
carboxylic acids or ring hydrogenation of aromatic carboxylic acids. Furthermore, the
classical hydride reduction of carboxylic acids requires initial protection of the starting
acid prior to carbonyl group reduction.[36] Biocatalytic reduction of free carboxylic acids
is advantageous because it is carboxylic acid group specific and requires no cumbersome
protecting groups. Furthermore, the carboxylic acid substrates are water soluble, reac-
tions are carried out at ambient temperature and physiological pH, and the reactions are
enantioselective.[27,65]
The efficient expression of carboxylic acid reductase (CAR) in E. coli can provide an
avenue for generating a range of value-added aldehydes of importance in the pharmaceu-
tical, food, and agricultural industries. In addition to aromatic acids, recombinant carbox-
ylic acid reductase (rCAR) can efficiently reduce aliphatic carboxylic acids, leading to a
route to aliphatic alcohols and polyols that can be used as chiral synthons as well as pre-
cursors in polymer synthesis. Using directed evolution and other related techniques, mu-
tants of carboxylic acid reductase can be generated that may be more efficient in reducing
aliphatic carboxylic acids. The challenges of cofactor recycling can be addressed by incor-
porating an engineered hydrogenase with hydrogen gas as a source of electrons. Current-
ly, several aliphatic acids are derived from the fermentation of carbohydrate feedstock,
and, using metabolic and reaction engineering, one can envision engineered strains ex-
pressing recombinant carboxylic acid reductase that efficiently produce these polyols
and aliphatic alcohols from the same feedstock.
References 477
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2.5.2 Carboxylic Acid Reductase

A. S. Lamm, P. Venkitasubramanian, and J. P. N. Rosazza

Table 1 Comparison of Carboxylic Acid Reductase Based Reduction Versus Synthetic

Carboxylic Acid Reductase (CAR) Synthetic Chemical Reduction

for references see p 477

2.5.2.1 Isolation and Purification of Carboxylic Acid Reductase

2.5.2.1.1 Carboxylic Acid Reductase from Nocardia iowensis DSM 45197

Scheme 1 Reduction of Benzoate Using the Carboxylic Acid Reductase

CAR from Nocardia iowensis DSM 45197

for references see p 477

2.5.2.1.2 Recombinant Carboxylic Acid Reductase from E. coli

Carboxylic acid reductase is produced in Nocardia iowensis, an organism that is relatively

2.5.2.2 Mechanism of Carboxylic Acid Reduction

Scheme 2 Phosphopantetheinylation Catalyzed by Phosphopantetheine Transferase[28,32]

This 4¢-phosphopantetheine prosthetic group in active carboxylic acid reductase serves as

for references see p 477

Scheme 3 Catalytic Process of the holo-Carboxylic Acid Reductase Reduction of Benzoic

Scheme 4 Simplified Mechanism of Carboxylic Acid Reductase Based Reduction[28]

NPT = Nocardia phosphopantetheine transferase; PPi = pyrophosphate; HS-CAR = phosphopantetheinylated holo-CAR

2.5.2.3 Substrate Scope with Carboxylic Acid Reductase

Reduction of carboxylic acids can be accomplished by growing cultures of wild-type No-

for references see p 477

Scheme 5 Substrate Specificity of Carboxylic Acid Reductase Obtained from Dif-

Ar1 Enzyme Source Relative Activitya Ref

Table 2 Substrate Specificity of Recombinant Carboxylic Acid Reductase[28,35]

Substrate Relative Activitya with rCAR from E. coli Ref

for references see p 477

2.5.2.4 Applications of Carboxylic Acid Reductase

2.5.2.4.1 Synthesis of Ibuprofen

Scheme 6 Biocatalytic Reduction of Ibuprofen[21]

for references see p 477

2.5.2.4.2 Synthesis of Vanillin

for references see p 477

metabolite concentration (g•L−1)

E. coli BL21-CodonPlus(DE3)-RP/pHAT305 (Placcar) expressing CAR (Graph A) reduces 50% of

2.5.2.4.3 Reduction of Ferulic Acid

Ferulic acid [10; 3-(4-hydroxy-3-methoxyphenyl)propenoic acid] is an extremely abundant

for references see p 477

microbial purified rCAR or

2.5.2.4.4 Synthesis of 3-Hydroxytyrosol

2.5.2.4.5 Reduction of Fatty Acids

Fatty alcohols are an important class of molecules in the detergent/surfactant industry.

2.5.2.4.6 Hydrogenation of Fatty Acids

for references see p 477

Scheme 9 Reduction of Carboxylic Acids by Pyrococcus furiosus Cells[63,64]

Hase = hydrogenase; AOR = aldehyde oxidoreductase; ADH = alcohol dehydrogenase

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