Molecular Plant

Review Article

Vanillin–Bioconversion and Bioengineering of the

Most Popular Plant Flavor and Its De Novo
Biosynthesis in the Vanilla Orchid
Nethaji J. Gallage1,2,3 and Birger Lindberg Møller1,2,3,4,*
1
VILLUM Research Center for Plant Plasticity, Department of Plant and Environmental Sciences, University of Copenhagen, 40 Thorvaldsensvej, DK-1871
Frederiksberg C, Copenhagen, Denmark
2
Center for Synthetic Biology ‘‘bioSYNergy’’, Department of Plant and Environmental Sciences, University of Copenhagen, 40 Thorvaldsensvej, DK-1871
Frederiksberg C, Copenhagen, Denmark
3
Plant Biochemistry Laboratory, Department of Plant and Environmental Sciences, University of Copenhagen, 40 Thorvaldsensvej, DK-1871 Frederiksberg C,
Copenhagen, Denmark
4
Carlsberg Laboratory, 10 Gamle Carlsberg Vej, DK-1799 Copenhagen V, Denmark
*Correspondence: Birger Lindberg Møller (blm@plen.ku.dk)
http://dx.doi.org/10.1016/j.molp.2014.11.008

ABSTRACT
In recent years, biotechnology-derived production of flavors and fragrances has expanded rapidly.
The world’s most popular flavor, vanillin, is no exception. This review outlines the current state of
biotechnology-based vanillin synthesis with the use of ferulic acid, eugenol, and glucose as substrates
and bacteria, fungi, and yeasts as microbial production hosts. The de novo biosynthetic pathway of vanillin
in the vanilla orchid and the possible applied uses of this new knowledge in the biotechnology-derived and
pod-based vanillin industries are also highlighted.
Key words: metabolomics, natural products, phenylpropanoids and phenolics, molecular biology, plant biochem-
istry, synthetic biology
Gallage N.J. and Møller B.L. (2015). Vanillin–Bioconversion and Bioengineering of the Most Popular Plant Flavor
and Its De Novo Biosynthesis in the Vanilla Orchid. Mol. Plant. 8, 40–57.

INTRODUCTION possesses a pure and delicate spicy flavor, which is difficult to

In recent years, biotechnology-derived production of flavors and
fragrances has expanded rapidly. Vanillin, the worlds most pop-
ular flavor, is no exception. This review outlines the current state
of biotechnology-based vanillin synthesis with the use of ferulic
acid, eugenol, and glucose as substrates and bacteria, fungi,
and yeasts as microbial production hosts. The de novo biosyn-
thetic pathway of vanillin in the vanilla orchid and the possible
applied uses of this new knowledge in the biotechnology-
derived and pod-based vanillin industries are also highlighted.
duplicate by technological means.
Vanillin is the most characteristic flavor compound of vanilla.
Vanillin is a white crystalline powder with a pleasant, sweet, and
intense aroma, offering a vanilla-like flavor. Chemically, it is an
aromatic aldehyde belonging to the group of simple C6–C1
phenolic compounds. Structurally, it is a phenol substituted with
an aldehyde and methoxy group at specific positions (Figure 1).
Vanillin has a relatively low solubility in water at room
temperature, but is readily soluble in hot water, alcohol, and ether.

VANILLIN AND VANILLA In 2010, the annual global sales of vanillin reached more than
15 000 000 kg, with less than 1% obtained by isolation from vanilla
Vanilla is one of the most widely used flavors in the world and
pods. The production of vanilla beans and the isolation of vanillin
is applied extensively in the food, beverage, perfumery, and
from vanilla pods is a laborious and costly process (Sinha et al.,
pharmaceutical industries. Natural vanilla is a complex mixture
2008). Production of 1 kg of vanillin requires approximately
of flavors extracted from the cured pods of two different species
500 kg of vanilla pods, corresponding to the pollination of
of vanilla orchids: Vanilla planifolia and Vanilla tahitensis (Rao and
approximately 40 000 vanilla orchid flowers. The market cost of
Ravishankar, 2000). The flavor and fragrance profile of the vanilla
natural vanillin derived from vanilla pods is therefore high and
extract contains more than 200 components. Vanillin (4-hydroxy-
3-methoxybenzaldehyde) (Figure 1) is the key component, with a
concentration of 1%–2% w/w in cured vanilla pods (Sinha et al., Published by the Molecular Plant Shanghai Editorial Office in association with
2008). The vanilla extract obtained from fermented vanilla pods Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.

40 Molecular Plant 8, 40–57, January 2015 ª The Author 2015.

Vanillin–Bioconversion and Bioengineering
Figure 1. Chemical Structure of Vanillin (4-Hydroxy-3-
Methoxybenzaldehyde).
fluctuates because of the unpredictable availability of vanilla pods.
Crop yield is tightly associated with weather conditions and the
incidence of disease as well as local and international political
and economic issues. Vanillin extracted from vanilla pods has a
market price varying from around US$1200/kg to more than
US$4000/kg (Walton et al., 2003). The global supply of vanilla
pods is stagnant at about 2000 000 kg. Thus, the increasing
global demand for natural vanilla flavor can no longer be met
with pods of the vanilla orchid as the sole source. Nowadays,
less than 1% of the global production of vanillin is derived from
vanilla pods; the majority is produced synthetically using, e.g.
lignin and eugenol as starting materials (Walton et al., 2000).
The Different Routes to Vanillin
Vanillin was isolated as the main flavor constituent of vanilla pod
extracts in 1858 by Theodore Nicolas Gobley and he subse-
quently elucidated its chemical structure. Less than 20 years after
its initial isolation, synthetically produced vanillin was marketed
(Hocking, 1997). Synthetic routes to vanillin were originally
based on eugenol. Nowadays, guaiacol and lignin are the
favored starting materials (Clark, 1990). Although synthetic
vanillin is able to meet the global market and is rather cheap,
with a market price below 15 $/kg, chemical synthesis of
vanillin, like many other chemical processes, has serious
drawbacks, e.g. the use of organic solvents and hazardous
chemicals such as sodium hydroxide in vanillin purification.
Chemical synthesis of vanillin via lignin has been calculated to
be accompanied by the demand for safe removal of 160 kg of
waste per 1 kg of vanillin obtained. As a consequence,
concerns related to the negative environmental impact have
increased since the late 1970s and have resulted in closure of
all lignin-derived vanillin production in Canada and the United
Molecular Plant
States (Hocking, 1997). Nevertheless, a substantial amount of
synthetic vanillin is still derived from lignin (borregaard.com).
There has been a huge surge in the exploration of more environ-
mentally friendly biosynthetic procedures to make natural flavors.
In recent years, this quest has been spurred by a growing demand
from consumers for natural products. Many consumers equate
food quality and food safety with the natural attribute. Accordingly
and guided by novel technologies, research in bioengineering of mi-
croorganisms for flavor production is rapidly expanding (Krings and
Berger, 1998; Berger, 2007). Most biotechnological approaches
for the synthesis of vanillin are based on bioconversion of certain
natural substances such as lignin, ferulic acid, eugenol, and
isoeugenol, etc., using microorganisms such as yeasts, fungi,
and bacteria as production hosts (Lesage-Meessen et al., 1996;
Hansen et al., 2009; Di Gioia et al., 2011). Plant cells and plant
tissue cultures have also been investigated for the production of
natural vanillin but are not advanced enough to be industrially
applicable (Fu et al., 1999). The continued attempts to produce
vanillin from V. planifolia tissue cultures as well as from Capsicum
frutescens cell cultures have been hampered by slow cell growth,
low gene expression levels, and poor yields (Funk and Brodelius,
1990a, 1990b; Knorr et al., 1990; Johnson et al., 1996; Rao and
Ravishankar, 2000; Dignum et al., 2001).
Microbial transformations of natural precursors to generate vanillin
are accepted as ‘‘natural’’ according to the current European
(European Directive 88/388/CEE, JO No. L184, 22 June 1988)
and US food legislation (Krings and Berger, 1998). Although US
and EU regulatory requirements are somewhat similar, they do
have distinct differences. Thus, the definition of a ‘‘natural’’ flavor
according to the EU regulation is more stringent than the
corresponding US regulation with respect to the source material
and the manufacturing process used. The regulatory requirements
for the US and the EU ‘‘natural’’ status are quoted below:
The US requirements to qualify for a status as a ‘‘natural’’ flavor
are as follows: ‘‘A product derived from a source as a spice, fruit,
extract, oleoresin, or from a group of materials that the FDA
recognizes as ‘‘natural’’ starting materials or any product process
via distillation, extraction, roasting, heating, enzymolysis, hydro-
lysis and fermentation’’ (Sabisch and Smith, 2014).
The EU requirements to qualify for a status as a ‘‘natural’’ flavor
are as follows: ‘‘Source material must be vegetable, animal, or
microbiological and a natural substance has to be identical to
that found in nature and must be produced by a traditional food
preparation process.’’ (Sabisch and Smith, 2014).
A number of different biotechnology-derived vanillin products have
been marketed for more than a decade (Figure 2). Rhovanil,
produced by Solvay (previously known as Rhodia), was the first
commercially available fermentation-derived vanillin product and
was obtained by bioconversion of ferulic acid (rhodia.com). Ex-
turmeric vanillin is marketed by De Monchy Aromatics and is pro-
duced from curcumin (demonchyaromatics.com). Sense Capture
Vanillin is obtained by bioconversion of eugenol and marketed
by Mane (Sense Capture Vanillin by Mane, 2014). De novo
synthesized biovanillin using glucose as a precursor will be
commercialized in 2014 by Evolva and International Flavors and
Fragrances (Vanilla, 2014).
Molecular Plant 8, 40–57, January 2015 ª The Author 2015. 41
Molecular Plant
Figure 2. Different Routes to Natural Vanillin that Are Available or Soon to Be Available on the Market.
Microbial Production of Vanillin–Bioconversion
and Bioengineering
The world of microorganisms is vast and many different microbial
organisms are being used for efficient production of natural food
components (Krings and Berger, 1998; Berger, 2007). Promising
strains have been selected based on their growth properties,
production potential and tolerance to high concentrations of
both product and substrate. Microorganisms and fermentation
ingredients, which have been given GRAS status, are preferred.
GRAS is an acronym for Generally Recognized As Safe under
the regulations of the US Food and Drug Administration
(Berger, 2007).
Microorganisms that are able to metabolize a range of different
precursors into vanillin have been studied experimentally. Several
major yet common issues have challenged the successful use of
microorganisms for efficient bioconversion of putative substrates
into vanillin. Bottlenecks include (1) cytotoxicity of the flavor
products obtained and of their precursors; (2) inefficient meta-
bolic flow; (3) formation of undesired by-products; (4) costly
downstream processing methods due to the physicochemical
properties of the substrate and the product; and (5) further meta-
bolism of the desired product by the selected microorganisms.
Bioengineering tools have been employed to circumvent
these drawbacks. Bioengineering includes the use of tools such
as mutagenesis-based selection and genetic engineering for
enzyme/strain optimization and for cost-efficient downstream
processing. Microorganisms that exhibit rapid growth rates and
are amenable to molecular genetics provide suitable platforms
42 Molecular Plant 8, 40–57, January 2015 ª The Author 2015.
Vanillin–Bioconversion and Bioengineering
for biotechnological production of vanillin. The increasing knowl-
edge of enzymes that are involved in bioconversion of ferulic
acid and other substrates to vanillin as well as the identification
and characterization of genes that are encoding them offer new
opportunities for more targeted bioengineering of microorgan-
isms for vanillin production.
In the following sections, the substrates most commonly used
for vanillin synthesis employing microorganisms (bacteria, fungi,
and yeast) are presented and discussed, with emphasis on the
major issues encountered and the solutions.
PRODUCTION OF VANILLIN FROM
FERULIC ACID
Ferulic acid is the best-explored substrate for production of
vanillin. Ferulic acid is abundant in nature and shares high
structural similarity to vanillin. Free and bound ferulic acid
(4-hydroxy-3-methoxycinnamic acid) is one of the most abundant
phenylpropanoids in plants. In plants, ferulic acid is bio-
synthesized from the aromatic amino acids phenylalanine or
tyrosine (Gross and Zenk, 1969; Dewick, 1989). The two
precursor amino acids are produced via the shikimic acid
pathway (Dewick, 1989). The exact route for ferulic acid
biosynthesis in plants has not been established. When
produced from phenylalanine, the first intermediate is cinnamic
acid and the reaction is catalyzed by phenylalanine ammonia
lyase (Fritz et al., 1976). Subsequently, cinnamic acid 4-
hydroxylase (Russell and Conn, 1967; Gabriac et al., 1991;
Vanillin–Bioconversion and Bioengineering Molecular Plant

Figure 3. Possible Bioconversion Routes of Ferulic Acid to Vanillin

(A) CoA-independent conversion of ferulic acid to vanillin via a retro-aldol reaction.
(B) CoA-dependent conversion of ferulic acid to vanillin via a retro-aldol reaction.
(C) CoA-dependent b-oxidation of feruloyl-CoA into vanillic acid.
(D) Non-oxidative decarboxylation of ferulic acid to 4-vinylguaiacol. No evidence for conversion of 4-vinylguaiacol into vanillin has been obtained.
(E) Conversion of ferulic acid into vanillin involving a reducing step.

Schoch et al., 2001) catalyzes the hydroxylation of cinnamic acid
at the 4-position, resulting in the formation of p-coumaric acid. p-
Coumaric acid 3-hydroxylase (C3H) (Schoch et al., 2001)
catalyzes the hydroxylation of p-coumaric acid at the 3
position, resulting in caffeic acid formation. It is not clear
whether coenzyme A (CoA) derivatives are involved or whether
the C3-hydroxylation step proceeds, e.g. through quinate and
shikimate esters (Schoch et al., 2001). Caffeic acid could in
principle be O-methylated by an O-methyltransferase (Lam
et al., 2007) to afford ferulic acid.
Ferulic acid is an important biological and structural component
of the plant cell wall and can be found free, as homodimers, or
esterified with proteins or polysaccharides in the cell wall
(Harris and Trethewey, 2010). Ferulic acid is the precursor of
coniferyl alcohol, which provides one of the monomers for lignin
biosynthesis. In cereals, ferulic acid is esterified with arabinose,
glucose, xylose, or galactose to be integrated as part of the
pectin or hemicellulosic fraction of cell walls. These polymers
can be up to 50 000 Da in molecular mass (Harris and
Trethewey, 2010).
In complete contrast to plants, no microorganisms in nature are
known to be able to de novo synthesize ferulic acid. Neverthe-
less, a vast number of microorganisms are able to utilize ferulic
acid as their sole carbon energy source. In addition, when micro-
organisms are grown on eugenol as carbon source, ferulic acid is
formed as a transient intermediate.
Bioconversion of Ferulic Acid to Vanillin
As illustrated in Figure 3A–3E, ferulic acid degradation pathways
in microorganisms proceed with vanillin as an intermediate. It is
this inherent capability to produce vanillin that is exploited
Molecular Plant 8, 40–57, January 2015 ª The Author 2015. 43
Molecular Plant
experimentally to obtain efficient bioconversion of ferulic acid into
vanillin in microorganisms.
Five major pathways can be distinguished in microorganisms
based on the different initial reactions involved in ferulic
acid bioconversion: (1) CoA-independent retro-aldol reaction,
(2) CoA-dependent retro-aldol reaction, (3) CoA-dependent
b-oxidation, (4) non-oxidative decarboxylation, and (5) a reduc-
tive pathway (Figure 3A–3E). Some microorganisms have
developed multiple pathways for bioconversion of ferulic acid.
For example, Pseudomonas fluorescens has been reported
to metabolize ferulic acid by decarboxylation (Huang et al.,
1994), reduction (Martinez-Cuesta et al., 2005), and via a
CoA-dependent retro-aldol reaction mechanism (Gasson et al.,
1998).
The retro-aldol reaction is the reverse reaction of an aldol reaction
and is well described for fatty acid degradation (Berg et al., 2012).
The reverse aldol reaction mechanism involves elimination of
an acetate moiety from the unsaturated ferulic acid side chain,
resulting in vanillin formation. This reaction may proceed as a
CoA-dependent (Rosazza et al., 1995; Mitra et al., 1999) as well
as a CoA-independent process (Toms and Wood, 1970; Huang
et al., 1993; Jurkova and Wurst, 1993).
Toms and Wood (1970) reported the conversion of trans-ferulic
acid to vanillic acid by Pseudomonas acidovorans as early as
1970 via a CoA-independent non–b-oxidative reaction sequence.
In this study, administration of [2-14C]-labeled trans-ferulic
acid to resting cells of P. acidovorans resulted in the formation
of [2-14C]acetate and vanillic acid. A similar route for the
degradation of ferulic acid has recently been suggested to take
place in Bacillus subtilis (Gurujeyalakshmi and Mahadevan,
1987) and in Streptomyces setonii (Sutherland et al., 1983).
44 Molecular Plant 8, 40–57, January 2015 ª The Author 2015.
Vanillin–Bioconversion and Bioengineering
Figure 4. The CoA-Dependent Catabolism
of Ferulic Acid Via Vanillin in Amycolatopsis
sp. 39116.
CoA-independent non–b-oxidation of ferulic
acid in microorganisms is envisioned to
occur by initial hydration of the trans-double
bond of ferulic acid, resulting in the forma-
tion of 4-hydroxy-3-methoxyphenyl-b-hy-
droxypropionic acid as a transient inter-
mediate, which by a retro-aldol reaction
is directly converted into vanillin and
stoichiometric amounts of acetic acid
(Figure 3A). The vanillin biosynthesis
pathway in the pods of V. planifolia follows
a similar route; vanillin synthase, VpVAN,
catalyzes the direct conversion of ferulic
acid and ferulic acid glucoside to vanillin
and vanillin glucoside respectively following
a retro-aldol reaction (Gallage et al., 2014).
Ferulic acid catabolism in Delftia acido-
vorans (Plaggenborg et al., 2001),
P. fluorescens AN103 (Gasson et al., 1998;
Bennett et al., 2008), Pseudomonas sp.
strain HR199 (Overhage et al., 1999), and Amycolatopsis sp.
strain HR167 (Achterholt et al., 2000) is known to occur via
a CoA-dependent retro-aldol mechanism (Figure 3B). This
conversion is dependent on the initial activation of ferulic acid
to the corresponding CoA thioester, feruloyl-CoA. This enzymatic
activation process requires CoASH, adenosine triphosphate
(ATP), and MgCl2 as cofactors. Formation of the ferulate-CoA is
catalyzed by 4-hydroxycinnamate-CoA (4CL) ligase, also termed
ferulate-CoA ligase and feruloyl-CoA synthetase (FCS), encoded
by the gene Fcs (Narbad and Gasson, 1998; Achterholt
et al., 2000; Masai et al., 2002; Plaggenborg et al., 2003; Yang
et al., 2013).The hydration of feruloyl-CoA is catalyzed by 4-
hydroxycinnamoyl-CoA hydratase/lyase (HCHL) (EC 4.2.1.101)
(Mitra et al., 1999) or enoyl-CoA hydratase/aldolase (ECH),
encoded by the gene Ech (Huang et al., 1994; Gasson et al.,
1998; Plaggenborg et al., 2001) (Figure 4). The 4-hydroxy-3-
methoxyphenyl-b-hydroxypropionyl-CoA is formed as a transient
intermediate and subsequently converted into vanillin and acetyl-
CoA by a retro-aldol mechanism.
Ferulic acid catabolism in Pseudomonas putida (Zenk et al., 1980)
and Rhodotorularubra (Huang et al., 1993) is envisioned to
proceed via CoA-dependent b-oxidation in a similar fashion to
fatty acid catabolism. In this catabolic scheme, ferulic acid
is initially activated to its CoA thioester, feruloyl-CoA, and
converted to the intermediate 40 -hydroxy-3-methoxyphenyl-
b-hydroxypropionyl-CoA. Subsequent oxidation results in the for-
mation of 3(40 -hydroxy-3-methoxyphenyl)-3-ketopropionyl-CoA,
which, in a CoA-consuming reaction, is cleaved to form vanillyl-
CoA and acetyl-CoA. Vanillyl-CoA would then be hydrolyzed to
form vanillic acid, with concomitant release of CoA-SH (Figure 3C).
Non-oxidative decarboxylation of ferulic acid to 4-vinylguaiacol
is a competing process known to proceed rapidly and efficiently
Vanillin–Bioconversion and Bioengineering
in both bacteria, fungi, and yeast, e.g. in Bacillus coagulans
BK07 (Karmakar et al., 2000), Delftia acidovorans (Plaggenborg
et al., 2001), Enterobacter sp. Px6-4 (Li et al., 2008), Fusarium
solani (Nazareth and Mavinkurve, 1986), and Paecilomyces
variotii (Rahouti et al., 1989) (Figure 3D). This mechanism
occurs via the formation of a transient quinoid intermediate.
Formation of this type of quinoid intermediate from ferulic acid
is enzyme promoted (Bennett et al., 2008). Studies have begun
to explore the genetic basis for non-oxidative decarboxylation
of ferulic acid. In bacteria and yeast, genes encoding phenyla-
crylic acid decarboxylase (PAD1) (EC 4.1.1.-) and ferulic acid
decarboxylase (FDC1) (EC 4.1.1.-) were identified to be essential
for ferulic acid decarboxylation (Cao et al., 2010; Mukai et al.,
2010). Impaired function of either PAD1 or FDC1 resulted in
yeast unable to decarboxylate cinnamic acid–derived
compounds (Mukai et al., 2010; Gallage et al., 2014). The use
of isotope-labeled 4-vinylguaiacol demonstrated that 4-
vinylguaiacol is subsequently converted into 4-(1-hydroxy)ethyl-
guaiacol in bacteria. Biochemical evidence for further meta-
bolism of 4-vinylguaiacol or 4-(1-hydroxy)ethylguaiacol into
vanillin or other products has not been obtained (Li et al., 2008;
Gallage et al., 2014).
Chain shortening of the side chain of ferulic acid may also pro-
ceed by a reductive mechanism. The proposed reaction mecha-
nism involves initial isomerization of ferulic acid to a transient
quinoid intermediate, as described above. The quinoid intermedi-
ate is converted into 4-hydroxy-3-methoxy-phenylpropionic acid
in a reductive step and subsequently to vanillic acid (Rosazza
et al., 1995). The exact biochemical processing of 4-hydroxy-3-
methoxy-phenylpropionic acid to 2(40 -hydroxy-30 -methoxy-
phenyl)acetic acid and subsequently to vanillic acid has not
been elucidated.
The conversion of ferulic acid to vanillin is, as previously
mentioned, an intermediate step in the catabolism of ferulic
acid. Many organisms are able to rapidly catabolize the
vanillin formed into other products. Vanillin toxicity may have
spurred evolutionary selection for rapid further conversion. In
Pseudomonas and in the actinomycete Amycolatopsis sp. strain
ATCC 39116, vanillin dehydrogenase (VDH) (EC 1.2.1.67) is able
to convert vanillin to vanillic acid in a nicotinamide adenine
dinucleotide (NAD)-dependent reaction. Vanillic acid is then
catabolized to protocatechuic acid by demethylation, in which
one oxygen atom is introduced at the methyl carbon resulting
in an unstable hemiacetal, which consequently decomposes
to protocatechuic acid and formaldehyde, respectively (Priefert
et al., 1997; Fleige et al., 2013). This demethylation step
is catalyzed by vanillate O-demethylase, VANA and VANB (EC
1.14.13.82), which are encoded by the genes VanA and VanB
(Priefert et al., 1997; Plaggenborg et al., 2003). These two
genes are supposed to be organized in the same operon in
both Pseudomonas sp. strain HR199 and P. putida (Brunel and
Davison, 1988; Venturi et al., 1998).
Bioengineering of Ferulic Acid–Derived Vanillin
in Microorganisms
Increasing knowledge of the metabolic pathways for vanillin
production via ferulic acid as well as identification and character-
ization of the enzymes and genes involved in the individual steps
Molecular Plant
offer new opportunities for the bioengineering of industrial appli-
cable microorganisms for vanillin biosynthesis.
Recombinant strains of bacteria, fungi, and yeasts represent an
interesting alternative to wild-type strains, and a diverse set of
methodologies have been developed to engineer and optimize
the bioconversion of ferulic acid to vanillin.
Availability of Ferulic Acid
Agricultural plant waste constitutes cheap starting materials
for bioproduction. However, recovery of ferulic acid from plant
cell walls appears to be somewhat complicated and costly.
Nevertheless, this process has received considerable attention.
Most ferulic acid in plants is bound in the plant cell wall and re-
quires either chemical or enzymatic hydrolysis to be released.
Several studies have attempted to remove ferulic acid from plant
cell wall materials enzymatically (Lesage-Meessen et al., 1999;
Bonnin et al., 2000; Zheng et al., 2007). Feruloyl esterases (EC
3.1.1.73) are enzymes that cleave the ester bonds by which
ferulic acid is attached to the cell wall polymers and can be
isolated from a wide range of fungi, yeast, and bacteria (deVries
et al., 1997). Fungal feruloyl esterases have been proved useful
for the hydrolysis of ferulic acid from plant materials in several
in vitro and in vivo studies. Two feruloyl esterases, FaeA
and FaeB, isolated from Aspergillus niger are able to release
ferulic acid from industrial by-products such as wheat
straw, coffee pulp, apple marc, maize bran, maize fiber, etc.
(Lesage-Meessen et al., 2002; Benoit et al., 2006). Bacterial
feruloyl esterases have been used for the same purpose.
Microcapsules containing bacterial feruloyl esterase expressed
in Lactobacillus fermentum (ATCC 11976) have been shown to
release ester-bound ferulic acid from ethyl ferulate in vitro
(Bhathena et al., 2007).
Enzymatic hydrolysis of cell walls using a combination of
commercial polysaccharide-degrading enzymes and feruloyl
esterase has also been investigated (Faulds et al., 1997).
Currently, these methods are not economically feasible,
because the use of commercially available polysaccharide-
degrading enzymes is costly and would result in significantly
increased production costs of vanillin.
Ferulic acid can also be released from plant cell walls by alkaline
treatment at high temperatures (85 C–100 C). As expected
from the enzymatic treatments, this treatment causes release
of ester-bound ferulic acid from the cell walls (Higuchi et al.,
1967). A recent study of alkaline hydrolysis of corncobs reports
a high yield of hydroxycinnamic acids, mainly p-coumaric
acid and ferulic acid, for potential use in vanillin production
(Torres et al., 2009). This kind of chemical release of ferulic
acid would not be considered natural processing according to
the EU regulations, but would comply with registration as
‘‘natural’’ according to US legislation (Sabisch and Smith,
2014; Torres et al., 2009).
Today, ferulic acid used for commercial production of natural
vanillin is mostly obtained as a by-product of the production of
rice bran oil. Ferulic acid is liberated from the rice bran by
enzymatic treatment to comply with the regulations for being
Molecular Plant 8, 40–57, January 2015 ª The Author 2015. 45
Molecular Plant Vanillin–Bioconversion and Bioengineering

Substrate Microorganism Length of incubation (h) Yield (g/l) References

Ferulic acid Streptomyces setonii 23 >10 Achterholt et al., 2000
Pseudomonas putida >10 Plaggenborg et al., 2003
Streptomyces sp. V-1 55 19.2 Hua et al., 2007a
Recombinant E. coli 24 5.14 Lee et al., 2009
E. coli JM 109 6 2.52 Barghini et al., 2007
Aspergillus niger and Pycnoporus 72 2.8 Zheng et al., 2007
cinnabarinus
Eugenol Pseudomonas sp. HR199 2–200 2.6 Overhage et al., 2000
Amycolatopsis sp. HR167 32 >10 Overhage et al., 2006
Isoeugenol Bacillus fusiformis SW-B9 72 32.5 Zhao et al., 2005
Recombinant E. coli BL21 (DE3) 6 28.3 Yamada et al., 2007
P. putida 24 16.1 Yamada et al., 2007
Bacillus pumilus S-1 3.8 Hua et al., 2007b
Bacillus subtilis HS8 24 1.36 Zhang et al., 2006
Psychrobacter sp. strain CSW4 1.28 Ashengroph et al., 2012
P. chlororaphis CDAE5 24 1.2 Kasana et al., 2007
Glucose Recombinant S. cerevisiae 24–168 >0.5 Hansen et al., 2013 (WO/2013/
022881); Brochado et al., 2010

Table 1. Industrially Applicable Microorganisms that Were Bioengineered to Produce Vanillin.

classified as a natural product. The cost of naturally extracted
ferulic acid is relatively high, with a price around US$180/kg
(novorate.com). With such high costs for the starting material,
production of vanillin using this approach is very costly. Key
issues related to the use of ferulic acid from agricultural waste
materials as a starting material for production of natural vanillin
are thus related to the development of optimized and less
expensive production costs.
Toxicity
Aldehydes rarely accumulate in high concentrations in biological
systems because of their high chemical reactivity, e.g. by form-
ing Schiff bases and thereby possibly inhibiting enzymatic activ-
ity, and show toxic effects in biological systems. A major issue
for efficient biotechnology-derived production of vanillin is thus
product toxicity. High concentrations of ferulic acid are also
toxic to many microorganisms. Toxicity is manifested by inhibi-
tion of the growth of the production strain or even cell lysis
(Priefert et al., 2001). Microorganisms that have been shown
to produce vanillin at levels exceeding 1 g/l are summarized in
Table 1.
Several studies have been targeted toward isolation of bacterial
species that tolerate high concentrations of ferulic acid and
vanillin. One study was carried out to isolate strains from nature
that were resistant to high levels of ferulic acid and vanillin
and to investigate their ability to catabolize eugenol and
ferulic acid (Muheim and Lerch, 1999). Actinomycetes, such as
Amycolatopsis sp. and S. setonii, were able to accumulate
high concentrations of vanillin while at the same time exhibiting
a high tolerance toward ferulic acid.
S. setonii was able to produce vanillin, with levels reaching
6.4 g/l in shake flask experiments. The same strain has
46 Molecular Plant 8, 40–57, January 2015 ª The Author 2015.
recently been re-classified as Amycolatopsis sp. ATCC 39116
and in a later study has been shown to yield even more
vanillin, with product titers as high as 13.9 g/l and a molar
yield of 75% (Muheim and Lerch, 1999; Fleige et al., 2013).
Another Amycolatopsis species, sp. HR167, has been shown
to produce vanillin at a concentration of 11.5 g/l, with a
molar yield of 77.8% (Rabenhorst, 1996). Such strains are
thereby suitable for vanillin production on an industrial scale,
as they can tolerate both high vanillin and ferulic acid
concentrations and display a high conversion ratio of the
expensive ferulic acid substrate into vanillin. However, the
filamentous growth of actinomycetes results in highly viscous
broths, unfavorable pellet formation, and a lot of fragmentation
and lysis of the mycelium, which complicate downstream
processing.
Genes involved in ferulic acid catabolism have been heterolo-
gously expressed in Escherichia coli mutants with high vanillin
tolerance to bypass the problems related to product toxicity.
This includes the Fcs and Ech genes responsible for ferulic
acid conversion in Amycolatopsis sp. HR104 (Yoon et al.,
2005). The vanillin-resistant mutant strain was obtained following
NTG (N-methyl-N-nitro-N-nitrosoguanidine) mutagenesis. When
grown for 48 h in medium containing 2 g/l of ferulic acid as
much as 1 g/l of vanillin was obtained. To further circumvent
the inhibitory effect of vanillin, XAD-2 resin was used to bind
the vanillin formed in the medium. This increased the vanillin yield
to 5 g/l in 48 h when a five-fold increase in ferulic acid substrate
was used during incubation.
Metabolic Flow and Side Product Formation
A balanced metabolic flow in the course of conversion of sub-
strates into vanillin and prevention of formation of unwanted
side products are other important factors in the production of
Vanillin–Bioconversion and Bioengineering
vanillin. The flow of metabolites in the conversion of ferulic acid
into vanillin varies among different bacterial strains. Pseudo-
monas strains have been found to be excellent converters of
ferulic acid to vanillin (Muheim and Lerch, 1999). However, the
vanillin formed is readily oxidized into vanillic acid and/or
reduced to vanillyl alcohol. In contrast, S. setonii metabolizes
ferulic acid to vanillin as an overflow product because of a
bottleneck in the oxidation of vanillin to vanillic acid catalyzed
by vanillin dehydrogenase (VDH). When high concentrations of
ferulic acid are provided, the activity of ferulic acid–degrading
enzymes is much higher in this strain compared with VDH,
directing the metabolic flow toward accumulation of vanillin.
This makes S. setonii a good candidate for the production of
vanillin on an industrial scale.
Several approaches have been taken to impair the undesired con-
version of vanillin into vanillic acid. In Pseudomonas strains, this
was explored by mutating the vdh gene encoding VDH (Di Gioia
et al., 2011). A higher concentration of vanillin accumulated in
recombinant P. putida BO14, which was engineered to have a
defective vdh gene (Okeke and Venturi, 1999). In the case of
P. fluorescens BF13, in which expression of the vdh gene was
blocked, a complete and simultaneous loss of the ability to
metabolize ferulic acid and vanillin was observed. The effect
was reversed on introduction of multiple copies of the Fsc gene.
This gene encodes feruloyl aldehyde dehydrogenase, which
catalyzes the activation of ferulic acid to the corresponding CoA
ester, feruloyl-CoA. The recombinant P. fluorescens BF13 was
able to utilize ferulic acid and yielded up to 1.28 g/l vanillin in a
stirred tank reactor after 8 h (Di Gioia et al., 2011). A mutant of
P. fluorescens 103, with impaired VDH activity (vdh), was unable
to utilize ferulic acid in a similar manner to the wild-type
P. fluorescens BF1. It was suggested that this effect was due
to the inactivation of 4-hydroxycinnamate-CoA ligase, 4CL
(Martinez-Cuesta et al., 2005), which is also known to catalyze
the activation of ferulic acid to the corresponding CoA ester,
feruloyl-CoA. These observations indicate that inactivation of
the vdh gene affects the expression of other upstream genes
that are essential for catabolism of ferulic acid in Pseudomonas
strains; especially the reaction of ferulic acid activation to
feruloyl-CoA seems to be compromised.
The mutation-based approaches undertaken to impair undesired
oxidation of vanillin into vanillic acid are challenging because
some microorganisms possess more than one Vdh gene. vdh
mutants of Pseudomonas sp. strain HR199 and P. putida
KT2440 were able to oxidize vanillin, indicating the existence of
additional VDH-like enzymes with overlapping functional activ-
ities. Accordingly, microorganisms that lack the ability to convert
vanillin into vanillic acid when the Vdh gene is functionally
impaired are good candidates for bioengineering. For example,
the vdh-deletion mutant of Amycolatopsis sp. ATCC 39116 re-
sulted in a two to three times higher yield of vanillin than the
wild-type strain. It was demonstrated that the Amycolatopsis
sp. ATCC 39116, VDH, specifically catalyzes vanillin oxidation
without being involved in ferulic acid activation (Fleige et al.,
2013). The oxidation of vanillin to vanillic acid was strongly
reduced when Vdh gene was impaired in Amycolatopsis sp.
ATCC 39116. Consequently, Amycolatopsis sp. ATCC 39116 is
a suitable microorganism to be optimized for industrial vanillin
production by further bioengineering.
Molecular Plant
Another approach to reduce by-product formation is to use a
two-step fermentation process involving two different microbial
organisms. In one such case, ferulic acid catabolism in A. niger
and vanillin metabolism in Pycnoporus cinnabarinus were com-
bined. In the first step, ferulic acid was catabolized to vanillic
acid in high yield by the micromycete A. niger. In the second
step, the vanillic acid formed was reduced to vanillin by the
basidiomycete P. cinnabarinus. The vanillic acid and vanillin titers
obtained were 920 mg/l and 237 mg/l, respectively (Lesage-
Meessen et al., 1996).
The influence of the use of different genetic approaches to achieve
high gene expression including plasmid copy number and pro-
moters regulating genes involved in vanillin synthesis has been
studied in recombinant E. coli. When the E. coli strain JM109
was engineered with a low copy number vector pBB1 carrying
the Fcs and Ech genes isolated from P. fluorescens BF13
(Barghini et al., 2007), a final vanillin concentration of 2.52 g/l
was obtained after 6 h of incubation by sequential induction with
1.1 mM ferulic acid at resting cell conditions. To further improve
vanillin formation, an integrative vector pFR2 was constructed
carrying the Fcs and Ech genes stably integrated into the lacZ
gene of E. coli. The recombinant strain was stable and
more efficient in vanillin synthesis compared with the strain
expressing genes encoding ferulic acid catabolizing enzymes
from a low copy vector. The recombinant strain produced 6.6 kg
vanillin per 1 kg biomass in resting cells (Ruzzi et al., 1997).
PRODUCTION OF VANILLIN FROM
EUGENOL AND ISOEUGENOL
A major drawback of ferulic acid–based vanillin production in
microorganisms is the high cost of ferulic acid. Eugenol and
isoeugenol may be used as alternative substrates for vanillin for-
mation because oxidative metabolism of the two compounds by
microorganisms in nature proceeds with ferulic acid and vanillin
as intermediates. Eugenol (2-methoxy-4-(2-propenyl)-phenol) is
the principal component of clove oil prepared from the clove
tree, Syzgium aromaticum, and is isolated on an industrial scale
(Bauer et al., 2008). The market price is as low as US$5/kg for
clove oil and around US$50/kg for isolated eugenol. The
starting materials for vanillin production based on eugenol are
thus a lot cheaper for biotechnology-derived vanillin production
than ferulic acid. Eugenol is considered a GRAS compound
when used as a food additive and accordingly has developed
into a popular precursor for industrial production of vanillin using
microorganisms (fda.gov).
Bioconversion of Eugenol to Ferulic Acid and Vanillin
The oxidative catabolism of eugenol has been studied in a num-
ber of different microorganisms. In Pseudomonas, the enzymes
and the corresponding structural genes have been identified.
The catabolism of eugenol in Pseudomonas strains proceeds
sequentially via coniferyl alcohol, coniferyl aldehyde, ferulic
acid, vanillin, and vanillic acid to protocatechuic acid. Protocate-
chuic acid is further catabolized by ortho cleavage (Tadasa, 1977;
Priefert et al., 1997; Achterholt et al., 1998; Overhage et al., 1999;
Priefert et al., 1999).
Eugenol degradation in Pseudomonas involves two oxidation
steps. The initial step is the bioconversion of eugenol into
Molecular Plant 8, 40–57, January 2015 ª The Author 2015. 47
Molecular Plant
coniferyl alcohol. This step is catalyzed by the heterodimeric
enzyme eugenol hydroxylase (EC 1.14.15.-) encoded by the
genes EhyA and EhyB (Figure 5). The two subunits, EhyA and
EhyB, constitute an enzyme of the flavocytochrome c class, in
which the gene EhyA encodes the cytochrome c subunit
(Priefert et al., 1999). The two genes were characterized in
Pseudomonas sp. strain (HR199) and homologs of EhyA and
EhyB were also identified in a few other Pseudomonas strains
such as P. putida and P. fluorescens E118, which utilize
eugenol as their sole carbon energy source (Priefert et al.,
1999). Functional expression of the two genes in various
Pseudomonas strains that were unable to utilize eugenol
showed that expression of EhyA and EhyB rendered the strain
able to grow on eugenol (Priefert et al., 1999). This provided
independent confirmation that EhyA and EhyB catalyze the
initial step in oxidative eugenol degradation.
In the second oxidative step, coniferyl alcohol is oxidized to
coniferyl aldehyde. This step is catalyzed by coniferyl alcohol
dehydrogenase (ADH) (EC 1.1.1.94) encoded by the gene CalA.
Coniferyl aldehyde is subsequently converted to ferulic acid by
coniferyl aldehyde dehydrogenase (CALDH) (EC 1.2.1.68) en-
coded by the gene CalB (Achterholt et al., 1998; Plaggenborg
et al., 2006) (Figure 5). Both enzymes are dependent on NAD+
as cofactor and their structural genes CalA and CalB were
identified in Pseudomonas sp. strain (HR199). The ferulic acid
48 Molecular Plant 8, 40–57, January 2015 ª The Author 2015.
Vanillin–Bioconversion and Bioengineering
Figure 5. Eugenol Degradation Pathway in
Pseudomonas.
formed would then be catabolized to
vanillin, vanillyl alcohol, and vanillic acid, as
described above in ferulic acid metabolism.
Despite the well-characterized eugenol
degradation pathways mentioned above,
there is evidence for the existence of other
possible eugenol degradation pathways in
microorganisms.
A potential route for oxidative conversion of
eugenol into ferulic acid is via the action of
vanillyl alcohol oxidases. Vanillyl alcohol
oxidase (EC 1.1.3.7) (VAO) is a type of flavo-
protein oxidase that was first isolated from
Penicillium simplicissimum grown on ve-
ratryl alcohol (3,4-dimethoxybenzylalcohol)
(Benen et al., 1998). Enzymes of this family
are known to be active on a range of
phenolic compounds, including eugenol
and vanillyl alcohol. The enzyme has been
shown to catalyze the oxidation of eugenol
to coniferyl alcohol and vanillyl alcohol to
vanillin (Overhage et al., 2003). Genes
encoding VAO-like enzymes are known
both from bacteria, e.g. Rhodococcus
(Jin et al., 2007) and fungi (Lambert et al.,
2014). A recombinant Amycolatopsis
sp. HR167 gained the ability to utilize
eugenol following expression of the vanillyl
alcohol oxidase gene (vaoA) from P. simplicissimum together
with the coniferyl ADH (calA) and CALDH (calB) genes from
Pseudomonas sp. HR199 (Overhage et al., 2006).
The capacity for eugenol bioconversion and ferulic acid catab-
olism in Rhodococcus sp. I24 and Rhodococcus sp. PD630
shows significant differences. In contrast to Rhodococcus sp.
PD630, Rhodococcus sp. I24 can tolerate up to 2.5–3.0 mM
eugenol, implying a natural occurrence of effective eugenol
catabolism in this strain. Genomic sequence analysis of Rhodo-
coccus sp. I24 did not reveal significant sequence identity to
genes encoding known eugenol hydroxylases such as the
EhyA-B from other bacterial strains mentioned above. This
would indicate that the initial steps of degradation of eugenol
in Rhodococcus sp. I24 are catalyzed by an enzyme or several
enzymes that differ from those in pseudomonad strains. Alterna-
tively, eugenol catabolism in Rhodococcus sp. I24 may be due
to VAO activity.
In contrast to the well-described eugenol catabolism in bacteria,
eugenol degradation pathways in yeast and fungi are less inves-
tigated. The expression of the vaoA gene from P. simplicissimum
in wild-type Saccharomyces cerevisiae resulted in conversion of
administered eugenol to coniferyl alcohol. S. cerevisiae has
been reported to convert coniferyl alcohol to ferulic acid in nature
due to a dehydrogenase enzyme activity (Lambert et al., 2014).
Vanillin–Bioconversion and Bioengineering Molecular Plant
A fed-batch bioconversion process from eugenol to coniferyl eugenol- and isoeugenol-derived vanillin production encounters
alcohol by the fungus Byssochlamys fulva V107 has been re- similar drawbacks as described for ferulic acid. Consequently,
ported to yield 21.9 g/l coniferyl alcohol within 36 h with a molar only two major issues and the solutions to those are highlighted
yield of 94.6% (Furukawa et al., 1999). Likewise, bioconversion here; namely, the toxicity caused by eugenol and isoeugenol
of eugenol to 4-vinylguaiacol was observed in Fusarium solani, and the issue of side product formation.
with ferulic acid implied to be formed as an intermediate in the
conversion, although ferulic acid could not be detected in the Toxicity
medium (Nazareth and Mavinkurve, 1986).
Both eugenol and isoeugenol are synthesized in plants as antimi-
crobial compounds and are toxic at high concentrations
Ferulic acid formed via eugenol catabolism in fungi and yeast
(Karapinar, 1990; Koeduka et al., 2006). For that reason,
would be a direct substrate for the synthesis of natural vanillin.
microorganisms have also developed various degradation
S. cerevisiae does not process the ability to convert ferulic
pathways to maintain the level of eugenol and isoeugenol
acid to vanillin and is known to decarboxylate ferulic acid
below a threshold concentration to avoid toxicity and enable
efficiently to 4-vinylguacol. Nevertheless, a bioengineered
proliferation. To circumvent intoxication of the production
pathway could be envisioned using recombinant S. cerevisiae
strains used for vanillin production caused by eugenol and
mutants, in which the pad1fad1 genes encoding phenylacrylate
isoeugenol, microbial strains with high tolerance to these
decarboxylase (PAD1) and ferulate decarboxylase (FDC1)
compounds were obtained by screening and bioengineering
are disrupted, thus preventing conversion of ferulic acid
tools were employed to modify the genetic makeup of these
to 4-vinylguaiacol (Benen et al., 1998). Introduction of the
strains. One such example is the use of recombinant E. coli
gene encoding vanillin synthase (VpVAN) from the orchid
BL21(DE3) cells, which do not possess vanillin-degrading activity
V. planifolia would catalyze the conversion of ferulic acid
and express the IEM of P. putida IE27 (Yamada et al., 2008).
and ferulic acid glucoside to vanillin and vanillin glucoside,
These recombinant E. coli cells were able to efficiently produce
respectively (Gallage et al., 2014).
vanillin from isoeugenol without forming vanillic acid as a by-
product.
Bioconversion of Isoeugenol to Vanillin
Like eugenol, isoeugenol is also present in the essential oil of Side Product Formation
the clove tree (Syzygium aromaticum). Bioconversion of isoeu-
As mentioned previously, further degradation of the vanillin
genol to vanillin in both bacteria and fungi has been proposed
formed into by-products constitutes a major challenge in
to occur via an epoxide-diol pathway involving oxidation of
biotechnology-based vanillin production in microorganisms. In
the side chain of isoeugenol. Isoeugenol monooxygenase
eugenol-catabolizing bacteria, two possibilities have been inves-
(IEM) encoded by the gene Iem is the enzyme responsible for
tigated to circumvent this problem. These are based on prevent-
catalyzing the conversion of isoeugenol to vanillin. The final
ing vanillin degradation by increased pathway flux toward vanillin
bioconversion product from isoeugenol is the corresponding
formation and on continuous removal of the product, vanillin, by
substituted benzoic acid.
different product removal techniques.
Several studies report industrially viable vanillin production from
Increased production of vanillin by over-expression of IEM was
isoeugenol using bacteria and fungi (Shimoni et al., 2000, 2002;
studied in isoeugenol and eugenol metabolizing Pseudomonas
Ashengroph et al., 2010) employing strains tolerant to high
mitroreducens, which is known to contain the Iem gene and its
levels of isoeugenol (Yamada et al., 2007). P. putida IE27 and
regulatory protein IemR. IemR was suggested to be a positive
Bacillus fusiformis have been reported as efficient converters of
transcriptional regulator of Iem. It was suggested that the high
isoeugenol into vanillin (Kasana et al., 2007) with B. fusiformis
level expression and use of multiple copies of Iem and IemR re-
yielding 32.5 g/l vanillin after 72 h incubation. The P. putida
sulted in increased production of vanillin (Ryu et al., 2012) by
IE27 strain produces 16.1 g/l of vanillin following 24 h
increasing the metabolic flux toward vanillin.
incubation. The high vanillin production was obtained from
continuous addition of isoeugenol to the cultures, which helps
A few attempts have been made to study the potential advantage
to prevent further oxidation of the vanillin formed into vanillic
of using various product removal techniques to prevent vanillin
acid (Shimoni et al., 2000, 2002; Zhang et al., 2006; Kasana
degradation. These involve the use of different resins for product
et al., 2007; Yamada et al., 2007; Ashengroph et al., 2010).
removal. In cultures of P. cinnabarinus grown at bioreactor scale,
Isoeugenol bioconversion to vanillin has also been observed in
improved transformation of vanillic acid into vanillin was attemp-
the fungus, A. niger ATCC 9142 (Abraham et al., 1988). In this
ted by binding the toxic vanillin to added absorbents (Topakas
study, the yield of vanillin was considerably lower due to further
et al., 2003). The utilization of cellobiose and adsorbent resins
catabolism of vanillin into vanillyl alcohol and vanillic acid.
such as Amberlite XAD-2 and Diaion HP20 resulted in only limited
Nonetheless, currently, the yields of vanillin produced from
improvements in the product recovery process (Stentelaire et al.,
isoeugenol are higher than those obtained from eugenol (see
1998; Bonnin et al., 2000). In contrast, the use of macroporous
Table 1).
adsorbent resins like DM11 gave promising results in fed-batch
biotransformation of ferulic acid using Amycolatopsis sp. ATCC
Bioengineering of Eugenol- and Isoeugenol-Derived 39116 and S. setonii, resulting in vanillin yields of 12 g/l. High
Vanillin Production in Microorganisms concentrations of vanillin, above 10 g/l at 20 C, result in precip-
Eugenol- and isoeugenol-derived vanillin production in microor- itation of crystalline vanillin and offer a convenient method of
ganisms proceeds via ferulic acid as an intermediate. Thus, isolation.
Molecular Plant 8, 40–57, January 2015 ª The Author 2015. 49
Molecular Plant
PRODUCTION OF VANILLIN FROM
SUGARS
Plants and photosynthetic microorganisms convert energy
from sunlight into chemical energy in the form of ATP and
NADPH. The photosynthetic processes thus drive the formation
of glucose and other sugars from H2O and CO2. The abundance
and low cost of sugars make them very attractive substrates
for microbial production of chemicals like vanillin. Glucose is
the most explored sugar substrate for vanillin biosynthesis
because it is cheap and readily utilized by most microorganisms.
The cost of glucose can be as low as US$0.30/kg. It is the cheap-
est substrate used for vanillin production by bioengineering
(Hansen et al., 2009). It is also valuable as a cheap primary
energy source for the production strain. Moreover, glucose is a
more attractive substrate in comparison with ferulic acid,
eugenol, and other phenolic compounds, because it is not toxic
to microorganisms and does not pose any problem with
potential off-taste, which is known to be associated with the
bioengineering approaches to produce ‘‘natural’’ vanillin using,
e.g. eugenol or ferulic acid (Evolva, personal communication).
Bioconversion of Glucose to Vanillin
Biochemical or genetic evidence for direct bioconversion of
glucose to vanillin in any naturally occurring microorganism
has not been provided. In 1966, glucose-induced vanillin for-
mation in soils was reported (Kunc and Macura, 1966). This
50 Molecular Plant 8, 40–57, January 2015 ª The Author 2015.
Vanillin–Bioconversion and Bioengineering
Figure 6. Engineered De Novo Vanillin
Biosynthesis from Glucose in the Yeasts
S. cerevisiae and Schizosaccharomyces
pombe.
phenomenon was suggested to be due
to soil bacteria such as Pseudomonas
strains metabolizing glucose to vanillin and
vanillic acid (Ryu et al., 2012). However,
no direct evidence was provided to
demonstrate that the vanillin observed was
actually synthesized from glucose by the
Pseudomonas studied. The trace amounts
of vanillin formed most likely arose from
microbial degradation of other phenolic
compounds such as ferulic acid, eugenol,
or lignin, which would also have been
present in the soil sample.
Biotechnological Production of
Vanillin from Glucose
Despite no documented evidence for direct
bioconversion of glucose into vanillin by
microorganisms in nature, biosynthesis of
vanillin from glucose has been demon-
strated using both recombinant E. coli and
yeasts.
Li and Frost (1998) devised a route for
microbial production of vanillin from
glucose, in which de novo biosynthesis of
vanillic acid in E. coli was combined with enzymatic in vitro
conversion of vanillic acid to vanillin. The recombinant E. coli
KL7 was engineered to dehydrate 3-dehydroshikimic acid to
protocatechuic acid (3,4-dihydrobenzoic acid) by the action
of 3-dehydroshikimic dehydratase (3DSD) (EC 4.2.1.118) en-
coded by the gene AroZ from the dung mold fungus Podospora
anserina. 3-Dehydroshikimic acid is an intermediate in the shiki-
mate pathway resulting in biosynthesis of aromatic amino acids.
Protocatechuic acid was then converted to vanillic acid by a
human catechol-O-methyltransferase (COMT) (EC 2.1.1.6).
Reduction of vanillic acid to vanillin was carried out in vitro using
a cellular extract of Neurospora crassa, exhibiting the required
aromatic carboxylic acid reductase (ACAR) activity (Gross and
Zenk, 1969; Li and Frost, 1998). The use of recombinant E. coli
KL7 for vanillin production from glucose on an industrial scale
according to the outlined approach has not yet been achieved.
A main obstacle is the costly in vitro step, which is dependent
on ATP, NADPH, and Mg2+ supplementation.
Incorporation of the entire vanillin biosynthetic pathway within
a single microorganism would be much more attractive, because
it would circumvent the demand for supplementation with
expensive cofactors. In 2009, this was achieved by Hansen
et al. (2009), who demonstrated the first example of microbial
vanillin biosynthesis from glucose in the yeasts, S. cerevisiae
and Schizosaccharomyces pombe. The vanillin biosynthesis
pathways engineered into these yeasts are outlined in Figure 6.
The pathway has similarities to the pathway demonstrated by
Vanillin–Bioconversion and Bioengineering
Li and Frost in 1998, but a major difference and breakthrough is
that it encompasses the full in vivo conversion of glucose to
vanillin within a single microorganism. This was achieved by
introduction of an ACAR (EC 1.2.1.30) from Nocardia iowensis,
in combination with a phosphopantetheinyl transferase, which
in S. cerevisiae is required for proper activation of the ACAR
enzyme (Venkitasubramanian et al., 2007). The ACAR genes
catalyze the ATP- and NADPH-driven reduction of protocate-
chuic acid to protocatechuic aldehyde and of vanillic acid into
vanillin based on endogenously produced cofactors in the yeasts.
The pathway demonstrated in yeast utilizes the gene encoding
3DSD from P. anserina to catalyze the formation of protocatechuic
acid from 3-dehydroshikimate. From protocatechuic acid, the
pathway may then proceed via vanillic acid formed by O-methyl-
ation by the human COMT (EC 2.1.1.6) (Hansen et al., 2009) and
subsequent reduction to vanillin by ACAR. Alternatively,
protocatechuic aldehyde formed by reduction of protocatechuic
acid by the ACAR enzyme may be O-methylated into vanillin by
the COMT. The de novo vanillin biosynthesis platform is now in
the process of scaling up to industrial needs and will be on
market under the label ‘‘natural vanillin’’ (Vanilla, 2014).
Nonetheless, glucose-based vanillin biosynthesis pathways
constructed in recombinant E. coli and yeasts have several chal-
lenges beyond just getting the product formed. Such challenges
are related to formation of unwanted side products, balanced
efficiency of the enzymatic steps involved, and toxicity of precur-
sors or end product.
Toxicity
As pointed out previously, vanillin is toxic to living cells in high con-
centrations. Dealing with this issue is an important prerequisite for
building economically viable biotechnology-derived vanillin cell
factories. In the case of S. cerevisiae, vanillin production beyond
0.5–1 g/l was toxic to the yeast, as shown by hampered growth
and biosynthesis (Hansen et al., 2009). The natural vanillin
biosynthesis pathway in the vanilla orchid, V. planifolia, has an
elegant solution to cope with the toxicity issue by glucosylation
of vanillin to vanillin-b-d-glucoside. The same strategy was
implemented by Hansen et al. (2009), in which A. thaliana uridine
diphosphate–glucose glycosyltransferase (UGT), UGT72E2 was
employed to glycosylate vanillin to produce the less toxic
vanillin-b-glucoside as the final product. Hansen et al. reported
that extracellular concentration of vanillin-b-d-glucoside even
above 25 g/l has no effect on yeast growth (Hansen et al., 2009).
Vanillin-b-D-glucoside has higher water solubility than vanillin
and could very well also be considered to provide a sink that
can aid in directing the pathway toward vanillin synthesis.
Moreover, glycosylation of vanillin to vanillin glucoside can also
be achieved by vanillin-specific UGT, VpUGT72U1 isolated from
the vanilla pods of V. planifolia (Gallage et al., 2014).
Metabolic Flow and Side Product Formation
A major issue with developing glucose-based vanillin biosyn-
thesis pathways in bacteria and yeast is the possible inefficient
or unbalanced activities of enzymes introduced into the produc-
tion strains. Inefficiency of specific enzymes gives rise to bottle-
necks, resulting in the escape and accumulation of intermediates
and reduced amounts of the final product. The first reported route
Molecular Plant
for in vivo microbial production of vanillin from glucose resulted
in accumulation of protocatechuic acid, vanillic acid, and isova-
nillic acid, as the main products obtained (Li and Frost, 1998).
This implied a bottleneck at the methylation step, caused by
insufficient COMT enzyme activity as required to convert
protocatechuic acid into vanillic acid in E. coli KL7.
The de novo vanillin biosynthesis pathways constructed in yeast
and E. coli were based on the use of a COMT enzyme that also
catalyzed an unwanted O-methylation at the para position of
protocatechuic acid, resulting in isovanillic acid formation,
compared with the desired methylation at the meta position, giv-
ing rise to vanillic acid. Isovanillic acid may in turn be reduced
to isovanillin but not to vanillin. Isovanillin is a flavor component
and is an unwanted side product in the biotechnology-based
vanillin industry. Separation of isovanillin from the desired product
vanillin is expected to be rather complicated as vanillin and isova-
nillin share similar physicochemical properties. To circumvent this
issue, Hansen et al. screened a number of different O-methyl-
transferases (OMTs) to identify an OMT that was specific for the
methylation of the 3-hydroxy group of protocatechuic aldehyde
and not able to methylate the 4-hydroxy group. In an alternative
approach, Hansen et al. introduced site-specific mutations which
were selected based on 3-dehydroshikimic enzyme structure
prediction. By these efforts, the group managed to construct
mutant versions of the human COMT carrying either an L198Y
or an E199A mutation, which made the mutated enzyme almost
entirely specific for methylation at the meta position, thus
avoiding undesired isovanillin production as well as achieving a
higher turnover of precursors (Hansen et al., 2013).
To improve the metabolic flux toward a higher yield of vanillin in
the de novo vanillin biosynthetic pathway in yeast, mutations in
the production strains were carried out. A mutation was intro-
duced in the AROM enzyme complex (ARO1) to increase the
accumulation of 3-dehydroshikimate (Hansen et al., 2013). This
mutation resulted in increased accumulation of protocatechuic
acid and thereby redirected the metabolic flux from aromatic
amino acid synthesis to vanillin precursor production. As a
consequence, subsequent identification and isolation of a more
efficient and more specific ACAR enzyme, which was able
to catalyze the conversion of the high concentrations of
protocatechuic acid to protocatechuic aldehyde, arose as a
key factor. The group was able to isolate an efficient ACAR
gene from Neurospora crassa which, when expressed in the
yeasts efficiently catabolized the high concentrations of
protocatechuic acid. This was one of the key features for the
successful generation of recombinant S. pombe and S.
cerevisiae capable of de novo synthesizing vanillin (Hansen
et al., 2013) (Evolva, personal communication).
As described in previous sections, further degradation of vanillin
to vanillic acid and vanillyl alcohol is a major drawback in microbial
vanillin production. To circumvent this issue, S. cerevisiae ADH
enzymes became a target of modification for optimized vanillin
production in yeast. The activity of the ADH enzymes is directly
related to the concentration of dissolved oxygen, the carbon
source, and ethanol. This is because the primary function of the
ADH enzymes is to oxidize ethanol into acetaldehyde when oxida-
tion of pyruvate through the tricarboxylic acid cycle is hampered
due to lack of oxygen in the mitochondria. In total, 29 known or
Molecular Plant 8, 40–57, January 2015 ª The Author 2015. 51
Molecular Plant
hypothesized ADHs, aryl-ADHs, and related aldehyde reductases
were tested for their ability to reduce vanillin to vanillyl alcohol in
mutants of S. cerevisiae (Hansen et al., 2009). From the palette
of tested enzymes, ADH6 was recognized as the most important
gene encoding vanillin reductase activity in S. cerevisiae. The
adh6 mutants in S. cerevisiae grew normally under all growth
conditions and showed a 50% decrease in converting vanillin to
vanillyl alcohol (Hansen et al., 2009). The efforts to prevent
oxidation of vanillin into vanillic acid using genetic engineering
have had only limited success to date.
OTHER SUBSTRATES FOR MICROBIAL
VANILLIN PRODUCTION
Lignin
The suitability of lignin as a substrate for vanillin production on an
industrial scale has been investigated. Lignin is a constituent of
the plant cell wall and the second most abundant plant polymer
in the world, as well as one of the most abundant natural sources
of aromatic compounds. Although lignin is a natural polymer,
enzymatic degradation of lignin into its structural monomers is
not possible. Vanillin production from lignin on an industrial scale
is therefore based on harsh chemical oxidation. This chemically
synthesized vanillin is marketed as ‘‘synthetic vanillin’’. Several
studies have explored biological degradation of lignin using
white-rot fungi. During biological degradation of lignin, vanillin
was formed in trace amounts. However, characterization of the
enzymes that are involved in lignin degradation, resulting in
vanillin and the optimization of such pathways for industrial
biotechnology-derived vanillin production, is incomplete (Kirk
and Farrell, 1987; Priefert et al., 2001; Martinez et al., 2004).
Aromatic Amino Acids and Phenolic Stilbenes
Bioconversions of stilbenes and aromatic amino acids to vanillin
have also been studied. Stilbenes are commonly found in spruce
bark and can be oxidatively cleaved to the corresponding aro-
matic aldehydes. The cleavage reaction is catalyzed by lignostil-
bene-a,b-dioxygenases. Genes encoding stilbene-degrading
enzymes from Pseudomonas paucimobilis strain TMY 1009
have been cloned and expressed in E. coli (Priefert et al., 2001).
Phenylalanine is the main precursor for biosynthesis of phenyl-
propanoids such as flavonoids, lignin, coumarines, and stilbenes,
and some of the enzymes and genes responsible for their for-
mation are known. The metabolism of l-phenylalanine has
been studied in detail in white-rot fungi (Casey and Dobb, 1992;
Jensen et al., 1994; Krings et al., 1996). Yet, none of these
phenylpropanoids yielded considerable amounts of vanillin;
therefore, these classes of compounds are not used for
industrial-scale synthesis of vanillin at the present time.
DE NOVO BIOSYNTHESIS OF VANILLIN
IN THE VANILLA ORCHID VANILLA
PLANIFOLIA
The option to establish biotechnological production of vanillin
based on heterologous expression of the genes encoding the en-
zymes catalyzing the synthesis of vanillin in the native vanilla
orchid has not yet been tested for the simple reason that the
pathway and genes involved had not been identified in the vanilla
52 Molecular Plant 8, 40–57, January 2015 ª The Author 2015.
Vanillin–Bioconversion and Bioengineering
orchid. This situation has now changed. Gallage et al. (2014)
recently demonstrated that de novo biosynthesis of vanillin in
the orchid V. planifolia is catalyzed by a single enzyme, vanillin
synthase (VpVAN).
VpVAN catalyzes the two-carbon cleavage of ferulic acid and its
glucoside into vanillin and vanillin glucoside, respectively. The
enzyme is strictly specific to the substitution pattern at the aro-
matic ring. The substrate specificity of the enzyme was demon-
strated in vitro by coupled transcription/translation assays and
in vivo by stable expression in yeast. VpVAN was found to be
localized in the inner part of the vanilla pod and high transcript
levels were observed in single cells located a few cell layers
from the inner epidermis. Transient expression of VpVAN in
tobacco and stable expression in barley in combination with
the action of endogenous ADHs and UGTs resulted in vanillyl
alcohol glucoside formation from endogenously present ferulic
acid. Vanillyl alcohol glucoside was obtained as the final product
because vanillin is readily reduced to its alcohol and glycosylated
in plants and microorganisms. Ground ivy (Glechoma hederacea)
also produces vanillin. A gene encoding an enzyme showing
71% sequence identity to VpVAN was identified and shown by
transient expression in tobacco to be a vanillin synthase.
The conversion of ferulic acid and its glucoside into vanillin and
the corresponding glucoside is envisioned to proceed sequen-
tially by an initial hydration addition reaction followed by a CoA-
independent retro-aldol elimination reaction, resulting in the for-
mation of vanillin with concomitant release of stoichiometric
amounts of acetic acid. This reaction is similar to ferulic acid
degradation in microorganisms, as described previously.
VpVAN shows high amino acid similarity to enzymes within the
cysteine protease family (Garcia-Lorenzo et al., 2006; Gallage
et al., 2014). The cysteine protease family encompasses a large
group of enzymes with versatile physiological functions and a
broad range of substrate specificities. The cysteine proteases
identified in plants are also diverse. Plants may contain more
than 150 different cysteine proteinases, with the papain-like
cysteine proteinases as one of the major families encompassing
30–50 different members dependent on the plant species
(Garcia-Lorenzo et al., 2006). The function of the individual
cysteine proteinases is in general not well characterized; papain
(EC 3.4.22.2) from the latex of Carica papaya is one of the
exceptions (Otto and Schirmeister, 1997). In general, cysteine
proteases are expressed as pre-proteins with an N-terminal
endoplasmic reticulum (ER) - targeting signal peptide as part of
the pro-peptide domain, which comprises 130–160 amino acid
residues (Cambra et al., 2012). The pro-peptide sequence is
removed either by a processing enzyme or by autocatalytical
processing, thereby generating the mature cysteine proteinase
(Turk et al., 2012). Two putative protease cleavage sites in
VpVAN were identified after residues 61 (RFAR/RYGK) and 137
(VDGV/LPVT) (Gallage et al., 2014). In vitro transcription/
translation experiments showed no evidence of autocatalytic
processing of the VpVAN protein formed and demonstrated
that removal of the pro-peptide requires the action of a separate
processing enzyme (Gallage et al., 2014). The pro-peptide
sequence may control intracellular targeting, promote proper
folding of the mature enzyme, and/or serve to maintain the
enzyme in an inactive form in the cell to balance its function
Vanillin–Bioconversion and Bioengineering
according to physiological demands (Otto and Schirmeister,
1997). A modified VpVAN sequence in which the VpVAN pro-
peptide was replaced with the putative ER-targeting putative
signal peptide and the putative pro-peptide protease cleavage
site from the tobacco cysteine protease that showed the highest
sequence identity to VpVAN was transiently expressed in to-
bacco. The modifications resulted in the formation of increased
levels of vanillyl alcohol glucoside in comparison with parallel ex-
periments with VpVAN.
FUTURE PERSPECTIVES
Industrial application of bioengineered microorganisms for vanillin
production has gained a lot of attention not only from the flavor
and fragrance industries but also from environmental groups,
the general public, and politicians. The current and future impor-
tance of biotechnology-based vanillin production is apparent
from the vast amount of scientific papers published on this topic
and from the number of patents that have been applied for and
granted. Bioconversions aimed at industry-scale vanillin produc-
tion using microorganisms based on vanillin-like structural com-
pounds, such as eugenol, isoeugenol, and ferulic acid, have
been extensively studied in the last two decades. Recently,
vanillin production from cheaper non-toxic glucose has gained
focus. The increasing knowledge of metabolic pathways leading
to vanillin production and identification and characterization of
the enzymes and genes involved offer new opportunities for
bioengineering of industrially applicable microorganisms by sta-
ble integration of expression cassettes required for vanillin syn-
thesis. Industrially applicable microorganisms that are able to
yield more than 1 g/l of vanillin are listed in Table 1.
The available microbial bioengineering platforms for vanillin
production have a few general drawbacks, as emphasized in pre-
vious sections, and optimization of these microbial production
systems is necessary to meet the increasing demand for bio-
logically produced vanillin. Much attention has been given to
cost and yield optimization by searching for cheaper substrates,
efficient downstream processing, shorter incubation time, etc.,
with the overall aim of lowering the production cost of bio-
technologically produced vanillin in order to gain a competitive
market position in relation to chemically synthesized vanillin.
The microbial cell factories so far studied for biotechnology-
derived vanillin production are mainly based on E. coli strains
and on a few fungal and yeast strains. Nowadays, genome
sequencing of organisms may be achieved in a cost-efficient
manner and the time needed to determine complete microbial
genomes has dramatically decreased. Such datasets would
certainly guide targeted improvement of the microorganisms
that have already been studied and would provide an improved
genetic basis to engineer microorganisms that have not previ-
ously been comprehensively studied and thus not considered
as production hosts. Vanillin production in microorganisms using
bioengineering would greatly benefit from identification of micro-
organisms that are more tolerant to vanillin and to the precursors
used for its production. In addition, identification of alternative
pathways for vanillin formation that are better suited for genetic
engineering of production strains is likely to take advantage of
the fast-growing genomic databases. It should also be high-
lighted that there is a need to exploit extended use of already
approved natural GRAS microorganisms for vanillin production;
Molecular Plant
lactic acid bacteria and baker’s yeast (S. cerevisiae) are popular
choices from the consumer perspective.
An entirely new opportunity for biotechnology-based production
of natural vanillin may arise from the recent identification of the
vanillin synthase enzyme VpVAN from the vanilla orchid Vanilla
planifolia and from ground ivy (Glechoma hederacea) (Gallage
et al., 2014). As described, vanillin and vanillin glucoside
biosynthesis in the vanilla orchid proceeds with ferulic acid and
its glucoside as precursors and is catalyzed by a single enzyme
that does not require the presence of specific cofactors. If high
expression levels can be obtained in yeast production strains
and the enzyme is stable and has proper kinetic characteristics,
this may constitute an alternative to the current yeast
production systems. Similar to some of the other yeast-based
production systems, such a production route would be depen-
dent on the supply of rather costly ferulic acid as substrate.
The identification of vanillin synthase as a hydratase/lyase-type
enzyme catalyzing conversion of ferulic acid and its glucoside
into vanillin and its glucoside offers new opportunities for the
Vanilla pod–based industries. The regulation and accumulation
of vanillin glucoside in the capsules of cultivated vines in
response to abiotic and biotic environmental challenges may
now be assessed at the molecular level. Likewise, in general,
each plant species contains about 50 different papain-like
cysteine proteinases (Garcia-Lorenzo et al., 2006). They play a
role in the turnover of proteins at specific time points of plant
growth and development, e.g. in a ripening fruit. In the course
of evolution of the vanilla-producing V. planifolia orchid, natural
mutations arose in one of these cysteine proteinases, enabling
the enzyme to convert ferulic acid into vanillin. Many orchids
closely related to the vanilla orchid are present in nature but
appear not to be able to synthesize vanillin. Classic mutation
breeding to obtain vanillin production in such species may now
be initiated. This would offer a more diverse production system
less prone to diseases, and maybe the introduction of vanillin
synthesizing orchids, which would have natural pollinators in their
growth habitats and thus not require hand pollination by humans.
The discovery of the vanillin synthase enzyme has direct implica-
tions for use of vanillin in agricultural industries. Ferulic acid is a
key intermediate in lignin monomer formation in plants. In contrast
to the situation in microorganisms, ferulic acid and its glucoside are
endogenously produced in plant cells and readily available as sub-
strates for vanillin synthase. If so desired, transgenic plants with
high vanillin synthase activity may be used as production sources
for vanillin glucoside. Stable expression of VpVAN and, e.g.
AtUGT72E2 in plants would be expected to result in vanillin gluco-
side formation in varying amounts. Vanillin is an animal feeding
stimulant, and vanillin production in barley grains used for pig
feed may thus have commercial potential. As demonstrated,
molasses may be used for vanillin production based on their ferulic
acid content and following supplementation with yeast expressing
vanillin synthase but are devoid of ferulate decarboxylase activity.
The primary economic driving force for the biotechnology-
derived flavor industry is the desire to establish reliable and
economically profitable production systems that are environ-
mentally benign in comparison with the classic production
approaches based on large-scale organic chemical synthesis.
Molecular Plant 8, 40–57, January 2015 ª The Author 2015. 53
Molecular Plant
An additional driver is the fact that flavor compounds produced
from natural raw materials by microbial or enzymatic methods
in accordance with European and US legislation are labeled
as ‘‘natural’’. This type of labeling is to the benefit of the manu-
facturer, considering the current consumer trends whereby
products used in the food and flavor sector labeled ‘‘natural’’
are preferred and thus gain a higher sales price. From the
point of view of the consumers, the label ‘‘natural’’ may be
misleading, because a majority of consumers would then be
deluded into attributing the flavor compound to the plant
species known as the common original source. A proper way
to signify that a flavor compound known from nature was pro-
duced biotechnologically in microorganisms could be to add
the prefix ‘‘bio-‘‘ to the name of the flavor compound to inform
the consumer that the flavor has been produced using biotech-
nological approaches. This type of openness would offer many
long-term benefits. It would enable the consumer to clearly
distinguish between a natural extract containing a complex
mixture of flavor compounds and a biotechnologically pro-
duced product composed of one of the most important flavor
compounds of the natural extract. The biotechnologically pro-
duced flavors should thus rather be considered and promoted
as environmentally benign substitutes for identical compounds
currently obtained by large-scale chemical synthesis. Chemi-
cally synthesized flavor compounds and the introduction of
flavors not occurring in nature are in general subject to less
consumer acceptance. Such products are labeled as ‘‘nature
identical’’ and ‘‘artificial’’ (EC Flavor Directive 88/388/EEC)
(US Code of Federal Regulation 21 CFR 101.22). This has
reduced the market value of flavors produced by chemical
synthesis.
The market launch of several biotechnology-derived vanillin prod-
ucts has generated some media attention (nytimes.com) and
has awakened public suspicion and demand for government
oversight. It is important to reconsider whether or not it is
justified to label biotechnologically produced flavors such as
vanillin as ‘‘natural’’. The general public cannot be expected to
understand and adapt to definitions of flavor codes that are not
self-evident and obvious. Unclear labeling policies serve to in-
crease consumer suspicions toward biotechnological-derived
flavors and toward the entire food industry. The three main flavor
codes ‘‘natural’’, ‘‘nature identical flavor,’’ and ‘‘artificial’’ do not
properly specify and distinguish commercially available products.
This situation obviously results in confused consumers. Organi-
zations like Friends of the Earth and Greenpeace profit from this
unclear situation by establishing a communication platform to
voice their general resistance to all products obtained using
genetic engineering, even when no genetic material is present
in the commercialized product.
In conclusion, biotechnological production of vanillin using safe
substrate precursors, food-grade production organisms, and
environmentally benign and economically feasible downstream
processing is envisioned to result in a compatible and sustainable
alternative to vanillin produced by chemical synthesis.
ACKNOWLEDGMENTS
The authors have filed a patent application on the VpVAN. Birger Lindberg
Møller is a member of the Science Advisory Board of Evolva, a company
who has vested interests in vanillin.
54 Molecular Plant 8, 40–57, January 2015 ª The Author 2015.
Vanillin–Bioconversion and Bioengineering
Received: July 8, 2014
Accepted: September 15, 2014
Published: September 30, 2014
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