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Abstract and Figures

Vanillin is one of the most widely used flavoring agents in the world. As the annual world market demand of vanillin could not be met by natural extraction, chemical synthesis, or tissue culture technology, thus biotechnological approaches may be replacement routes to make production of bio-vanillin economically viable. This review's main focus is to highlight significant aspects of biotechnology with emphasis on the production of vanillin from eugenol, isoeugenol, lignin, ferulic acid, sugars, phenolic stilbenes, vanillic acid, aromatic amino acids, and waste residues by applying fungi, bacteria, and plant cells. Production of biovanillin using GRAS lactic acid bacteria and metabolically engineered microorganisms, genetic organization of vanillin biosynthesis operons/gene cassettes and finally the stability of biovanillin generated through various biotechnological procedures are also critically reviewed in the later sections of the review.
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Biotechnological and Molecular Approaches for Vanillin
Production: a Review
Baljinder Kaur &Debkumar Chakraborty
Received: 6 September 2012 /Accepted: 26 December 2012 /
Published online: 11 January 2013
#Springer Science+Business Media New York 2013
Abstract Vanillin is one of the most widely used flavoring agents in the world. As the
annual world market demand of vanillin could not be met by natural extraction, chemical
synthesis, or tissue culture technology, thus biotechnological approaches may be replace-
ment routes to make production of bio-vanillin economically viable. This reviews main
focus is to highlight significant aspects of biotechnology with emphasis on the production of
vanillin from eugenol, isoeugenol, lignin, ferulic acid, sugars, phenolic stilbenes, vanillic
acid, aromatic amino acids, and waste residues by applying fungi, bacteria, and plant cells.
Production of biovanillin using GRAS lactic acid bacteria and metabolically engineered
microorganisms, genetic organization of vanillin biosynthesis operons/gene cassettes and
finally the stability of biovanillin generated through various biotechnological procedures are
also critically reviewed in the later sections of the review.
Keywords Vanillin .Ferulic acid .Eugenol .Isoeugenol .Lactic acid bacteria .Genetic
organization .Metabolic engineering .Biotransformation
Vanillin (4-hydroxy-3-methoxybenzaldehyde) is one of the most widely used flavoring
agents in the world which is extracted from the orchid Vanilla planifolia,Vanilla tahitiensis,
and Vanilla pompona. It is a white needle-like crystalline powder with an intensely sweet
and very tenacious creamy vanilla-like odor. It is used as the principal flavor ingredient with
a variety of functional properties [1,2]. Pure vanillin is widely used for enhancing flavor in
food and beverage industry and as a biopreservative because of its antimicrobial and
antioxidant properties. It possesses antimutagenic activity. It is an important raw material
in pharmaceutical industries for production of drugs such as aldomet, dopamine, papaverine,
and L-DOPA and for the production of antifoaming agent. Synthetic vanillin is used to
Appl Biochem Biotechnol (2013) 169:13531372
DOI 10.1007/s12010-012-0066-1
B. Kaur (*):D. Chakraborty
Punjabi University, Patiala, Punjab, India
manufacture a number of household products, deodorants, air fresheners, floor polishes, and
herbicides [3,4].
Vanilla is widely grown in Indonesia, Madagascar, and China. Asia, Africa, and other
continents also contribute a good quantum of vanilla pods [4]. Natural vanillin extracted
from Vanilla pods cannot meet market demand alone [5,6]. Chemical synthesis is an
alternative approach as it is economical, but it leads to environmental pollution, and it also
lacks substrate selectivity that can reduce process efficiency and increase downstream
processing cost [7]. Its production is also not ideal through tissue culture techniques because
plants are slow growing and vanillin biosynthetic pathway is not very actively expressed [8].
Thus, various biotechnology-based approaches were developed for the production of van-
illin from lignin, phenolic stilbenes, isoeugenol, eugenol, ferulic acid, or aromatic amino
acids and de novo biosynthesis by applying fungi, bacteria, plant cells, or genetically
engineered microorganisms [1]. This reviews main focus is to highlight significant aspects
of biotechnology with emphasis on demonstrating potential biotransformation efficiencies of
microorganisms and drawbacks of the existing biotechnological approaches.
Biotechnology-Based Processes for Vanillin Production
Biotechnology employs the tools of genetic engineering to step up conventional methods of
manufacturing important food ingredients, drugs, and biopolymers. It broadens the range of
possible substrates for biosynthesis of food flavors, expands consumer choice, and quenches
consumer thrust for healthy, better tasting, and safe food products. Lignin, eugenol, iso-
eugenol, and ferulic acid are the major substrates for vanillin production. Vanillin production
from eugenol [9,10] and isoeugenol [1115] by different microorganisms was studied in the
last decade. Products obtained through biotechnological procedures from natural substrates
were regarded as safe and considered as natural [16]. With the approval of such products by
the FDA and European legislation, many studies focused on approaches based on biotech-
nological methods for production of flavors, fragrances, and pharmaceutical products [5,
17]. Biotechnological approaches have some advantages over other techniques, e.g., mild
reaction conditions, high substrate or product specificity which leads to only one product
isomer and eco-friendly techniques that pose less harm to the environment [1820]. That is
why tailored microbial cells or enzymes are used to get most eco-friendly and safest way of
flavor synthesis using biotransformation processes [17]. In particular, microbial biocatalysis
can be used for the production of vanillin from phenolic stilbenes, lignin, eugenol, iso-
eugenol, ferulic acid, aromatic amino acids, and glucose. Table 1enlists various micro-
organisms native as well as engineered which were used to produce vanillin from various
Bioconversion of (2-Propenyl Benzenes) to Vanillin
Eugenol is one of the most important raw materials for vanillin production and is the main
constituent of oil extracted from clove tree Syzygium aromaticum. Many studies have been
carried out to elucidate mechanism of biotransformation of eugenol to ferulic acid that
involves intermediates like eugenol epoxide, eugenol diol, coniferyl alcohol, and coniferyl
aldehyde catalyzed by EhyAB, CalA, and CalB enzymes (Fig. 1). This partial metabolic
pathway was reported in some bacterial species like Pseudomonas sp., Pseudomonas sp.
HR199, and Corynebacterium sp., but vanillin was not detected as an intermediate in
1354 Appl Biochem Biotechnol (2013) 169:13531372
Table 1 Various biotechnological approaches used for the synthesis of vanillin
Substrate Microorganisms employed Yield (g/l) References
Eugenol Pseudomonas sp. HR199 2.6 [22]
Recombinant E. coli XL1-Blue
(pSKvaomPcalAmcalB) and E. coli
0.3 [9]
Pseudomonas sp. HR199 0.3 [97]
Amycolatopsis sp. HR167(pRLE6SKvaom) [24]
Amycolatopsis sp. HR167; Rhodococcus
opacus PD630
Aspergillus niger [26]
P. resinovorans SPR1 0.24 [28]
Isoeugenol Bacillus subtilis 0.9 [30]
Pseudomonas putida I58 Trace
Arthrobacter sp. TA13 Trace
Bacillus fusiformis SW-B9 32.5 [11]
Bacillus subtilis HS8 1.36 [12]
Bacillus fusiformis CGMCC1347 8.1 [31]
Pseudomonas chlororaphis CDAE5 1.2 [13]
Bacillus pumilus S-1 3.8 [14]
Pseudomonas putida IE27 16.1 [15]
Recombinant Escherichia coli BL21(DE3) 28.3 [15]
Pseudomonas nitroreducens Jin 1 [32]
Candida galli 0.58 [33]
Psychrobacter sp. Strain CSW4 1.28 [28]
Ferulic acid Streptomyces setonii ATCC 39116 >10 [44]
Mutant Pseudomonas putida 2.247 [99]
P. cinnabarinus MUCL39533 0.16 [100]
P. cinnabarinus MUCL39533 0.584 [41]
Pseudomonas sp. 0.0085 [47]
Pseudomonas putida >10 [68]
Streptomyces halstedii GE107678 0.100.15 [48]
Escherichia coli strain JM109/pBB1 0.006 [49]
Pseudomonas fluorescens AN103 [45]
Recombinant E. coli 1.1 [77]
Aspergillus niger CGMCC0774,
Pycnoporus cinnabarinus CGMCC1115
Lactic acid bacteria Trace
Amycolatopsissp. HR167 >10 [24]
Streptomyces sp. V-1 19.2 [46]
E coli JM109 (pBB1) 2.52 [79]
Recombinant E. coli 5.14 [81]
Pseudomonas fluorescens BF13 [75]
Recombinant E. coli 6.6 kg/kg biomass [80]
Appl Biochem Biotechnol (2013) 169:13531372 1355
Pseudomonas sp. HR199 [1]. Eugenol degradation involves two oxidation reactions,
where first step involves oxidation of double bond of side chain to coniferyl alcohol,
and in the second oxidation step, it is converted into ferulic acid via coniferyl
aldehyde [1]. Ferulic acid is an important intermediate produced during hydrolysis
of eugenol which can yield vanillin through different metabolic intermediates as
illustrated in Figs. 1and 2. In most of the vanillin-producing bacterial species,
vanillin is degraded to ortho-3-carboxymuconolactone through vanillic acid and pro-
tocatechuic acid after cleavaging aromatic ring of protocatechuic acid at C
position, and it is further oxidized via β-ketoadipate pathway. Finally, metabolic
products enter into tricarboxylic acid cycle and ATP production pathway (Fig. 2).
Avdh mutant strain of Pseudomonas sp. HR199 has been constructed by inserting an
omega element containing 3-O-phosphotransferase (omega Km) and gentamycin-3-
acetyltransferase genes (omega Gm) using plasmids pSUP5011 and pBBR1MCS-5, respec-
tively [21]. Recombinant plasmids conferred resistance against kanamycin and gentamycin
along with disruption of Vdh activity of the host. Recombinant strain was able to convert
6.5 mM eugenol to 2.9 mM (0.44 g/l) vanillin within 17 h. They scaled up the process to 10 l
and recovered 2.6 g/l of vanillin from 25.3 g/l eugenol from mutant culture within 65 h of
incubation [22]. Approximately 3 years later, Ralstonia eutropha H16 was engineered by
introducing gene loci ehyAB,calA, and calB of Pseudomonas sp. HR199 using broad-host-
range vector pBBR1-JO2 [23]. cal A gene encodes a highly substrate-specific enzyme
coniferyl-alcohol dehydrogenase, and cal B gene encodes enzyme coniferyl-aldehyde dehy-
drogenase that converts coniferyl aldehyde into ferulic acid. Recombinant strain so gener-
ated was able to convert eugenol at a conversion rate of 2.9 mM/h/l into ferulic acid within
20 h of incubation with 93.8 % molar conversion. In a separate study, Overhage and
coworkers [105] constructed a recombinant strain of Escherichia coli using P
expression vector pSKvaomPcalAmcalB (consisting of vaoA gene of Penicillium simplicis-
simum CBS 170.90 and calA and calB genes of Pseudomonas sp. HR199) which success-
fully converted eugenol to coniferyl alcohol, coniferyl aldehyde finally to ferulic acid with a
molar yield of 91 % within 15 h of incubation. Ferulic acid (14.7 g/l) was recovered after
30 h of incubation (93.3 %) molar yield when this biotransformation was scaled up to 30 l
fermentation volume [105].
Table 1 (continued)
Substrate Microorganisms employed Yield (g/l) References
Staphylococcus aureus 0.045 [51]
Pycnoporous cinnabarinus 0.126 [52]
Glucose E.coli KL7/ pKL5.97A (ATCC98859)
and Neurospora crassa
Recombinant Schizosaccharomyces pombe 0.065 [56]
Recombinant S. cerevisiae 0.045 [56]
Recombinant S. cerevisiae 0.500 [57]
Vanillic acid Zygorhynchus moelleri 0.05 [99]
Micromucor isabellinus 1.96 [99]
Aspergillus fumigatus 1.09 [99]
P. cinnabarinus MUCL39533 0.767 [41]
Solid-state fermentation
using green coconut husk
Phanerochaete chrysosporium 52.5 μg/g [63]
1356 Appl Biochem Biotechnol (2013) 169:13531372
Vanillin biosynthesis route involving non beta-oxidative coenzyme A-dependent conver-
sion was established in Rhodococcus strains I24 and PD630 where ferulic acid, vanillic acid,
or coniferyl alcohols were found as major products in the biotransformation of eugenol [10].
It was observed that eugenol facilitated growth of strain I24 but not of its mutant strain
PD630. Though Rhodococcus strains I24 lack ehyAB genes whose activity is essential for
oxidative hydrolysis of eugenol to coniferyl aldehyde, participation of other oxidative
enzymes could not be ruled out (Fig. 1). Later on, the concept of metabolic engineering
was exploited for production of biovanillin in Rhodococcus strain PD630 by coexpressing
vaoA from P. simplicissimum CBS 170.90 with calAB genes from Pseudomonas sp. HR199
and the metabolically engineered strain successfully converted eugenol to ferulic acid [10].
Fig. 1 Metabolic pathways of vanillin biosynthesis
Appl Biochem Biotechnol (2013) 169:13531372 1357
Metabolic engineering was also used in vanillin-tolerant Amycolatopsis sp. HR167, and
vaoA from P. simplicissimum CBS 170.90 was cloned using expression vectorpRLE6SK-
vaom which is suitable for many Gram-positive bacteria. Intermediates like coniferyl
alcohol, coniferyl aldehyde, ferulic acid, guaiacol, and vanillic acid were detected as
excreted compounds with maximum production of coniferyl alcohol concentration of
4.7 g/l during growth on eugenol, whereas vanillin could only be detected in trace amounts
[24]. A few years later, eugenol oxidase (EUGO) of Rhodococcus sp. RHA1 was isolated
that shares 45 % amino acid sequence identity with VaoA of fungus P. simplicissimum [25].
Fig. 2 Biochemical reactions involved in the conversion of ferulic acid to vanillin, vanillin alcohol, vanillic
acid, and degradation of protocatechuic acid
1358 Appl Biochem Biotechnol (2013) 169:13531372
Homologous enzymes EUGO and VaoA are the key enzymes involved in bioconversion of
eugenol into coniferyl alcohol (Fig. 1).
Srivastava et al. for the first time established the eugenol bioconversion pathway in
fungal systems that lead to hypothesize the metabolic fate of eugenol in eukaryotic systems
[26]. They utilized reference pathway of Pseudomonas fluorescens and identified the
missing enzymes of fungal system which include CYC_ASPNG, An09g01380,
An15g01840, An01g09260, An14g 05630, An02g02820 predicted as EhyA, EhyB, CalA,
CalB, Fcs, and Ech, respectively, in Aspergillus niger for biotransformation of eugenol to
vanillin. Using in silico computational tools, PS51007, IPR003088, and PF00034 conserved
motifs were identified in EhyA enzyme of P. fluorescens that showed 58 % protein
homology coincide with cyc_aspng gene of A. niger. Similarly, EhyB of P. fluorescens
showed common evolutionary conserved motifs Pfam01565 and Pfam02913 in A.
niger with 50 % protein similarity and 34 % identity. Reconstructed pathway involves
sequential activity of eugenol hydroxylase cytochrome C subunit, eugenol hydroxylase
flavor protein subunit, coniferyl alcohol dehydrogenase, coniferyl aldehyde dehydro-
genase, feruloyl CoA synthase, and enoyl CoA hydratase/aldolase for bioconversion of
eugenol to vanillin in A. niger.
Recently, a novel metabolic pathway for conversion of eugenol to vanillin was identified
in Bacillus cereus strain PN24 [27]. It can utilize eugenol, 4-vinyl guaiacol, vanillin, vanillic
acid, and protocatechuic acid as growth substrates. Eugenol dehydrogenase and 4-vinyl
guaiacol dehydrogenase are important enzymes required for conversion of eugenol through
4-vinyl guaiacol to vanillin in B. cereus PN24. Vanillin was metabolized to protocatechuic
acid which was further degraded by a β-ketoadipate pathway as described in Fig. 2. Strain
could be explored for phenolic environmental clean-up if given optimal nutrient conditions.
More recently, another novel strain Pseudomonas resinovorans SPR1 was isolated whose
resting cells were found to convert eugenol to 0.24 g/l of vanillin with 10 % molar yield at
the end of the exponential growth phase after 30 h without further optimization [28].
Bioconversion of Isoeugenol (Propenylbenzene-1) to Vanillin
Isoeugenol is metabolized into vanillin through an epoxidediol pathway involving oxida-
tion of side chains of propenylbenzenes. The biotransformation products of 1-
propenylbenzenes differ from those obtained from 2-propenylbenzenes as these are decar-
boxylated to corresponding substituted benzoic acid. Vanillin degradation to vanillyl alcohol
and vanillic acid was reported in fungi A. niger ATCC 9142 and bacterial species Achro-
mobacter,Aeromonas,Agrobacterium,Alcaligenes,Arthrobacter sp. TA13, Bacillus,Enter-
obacter,Klebsiella,Micrococcus,Pseudomonas,Rhodobacter,Rhodococcus, and Serratia
Bacillus subtilis HS8 isolated from soil is able to produce vanillin via isoeugenol-diol and
where it undergoes non-oxidative decarboxylation that leads to production of guaiacol with
vanillic acid as an intermediate (Fig. 1). This strain could convert isoeugenol into vanillin;
with molar yield of 14.7 and 1.36 g/l, vanillin was produced after 96 h [11]. Using Bacillus
fusiformis SW-B9 60 % (v/v), isoeugenol to vanillin conversion is much faster and high
yielding as 32.5 g/l vanillin is recovered after 72 h [12]. Similarly, a very high vanillin yield
from isoeugenol using Pseudomonas putida IE27 cells has been reported, where substrate
undergoes oxidative side chain cleavage [15]. Under optimized reaction conditions, strain
IE27 showed highest vanillin-producing activity of 16.1 g/l vanillin from 150 mM isoeuge-
nol, with a molar conversion yield of 71 % at 20 °C after 24 h incubation in the presence of
Appl Biochem Biotechnol (2013) 169:13531372 1359
10 % (v/v) dimethyl sulfoxide. Authors proposed physical map of the chromosomal region
containing isoeugenol-degrading enzyme gene isoeugenol monooxygenase (icm) and its
flanking regions. The amino acid sequence of Icm enzyme shows similarity to those of
lignostilbene-α,β-dioxygenases, carotenoid monooxygenases, and 9-cis-epoxycarotenoid
dioxygenases [15].
There are few more reports where moderate levels of vanillin were generated using
biotransformation capabilities of various bacterial and fungal strains. Pseudomonas chlor-
oraphis CDAE5 was grown on 10 g/l isoeugenol, and 1.2 g/l vanillin was obtained after 24 h
reaction at 25 °C and 180 rpm [13]. In B. subtilis, vanillin recovery from isoeugenol
substrate is comparatively poor (0.610.9 g/l) due to end product toxicity [30]. In B.
fusiformis, vanillin toxicity was overcome with the addition of 12.5 g HD-8 resin in 20 ml
reaction solution at pH 7.0, 37 °C and 180 rpm converted 50 g/l isoeugenol to accumulate
8.1 g/l vanillin [31]. A strain of Bacillus pumilus S-1 capable of transforming isoeugenol to
vanillin through isoeugenol epoxide and isoeugenol-diol as intermediates was isolated and
characterized (Fig. 1). With the growing culture of B. pumilus S-1, 10 g/l isoeugenol was
converted to 3.75 g/l vanillin in 150 h, with a molar yield of 40.5 % [14]. Strains of
Arthrobacter sp. TA13 [29] and Pseudomonas nitroreducens Jin 1 [32] can produce very
low concentrations of vanillin and vanillic acid. Isolated Candida galli PGO6 can produce
vanillin and vanillic acid in concentrations of 583.2 ± 5.7 mg/l (molar yield 48 %) and
177.3±1.7 mg/l (molar yield 19 %), respectively, after 30 h of initiation of bioconversion
by this strain [33]. Recently, a halobacterium Psychrobacter sp. CSW4 was screened,
capable of converting isoeugenol to vanillin [34]. Vanillin yield was improved under resting
cell conditions with substrate optimization, and maximal vanillin concentration 1.28 g l
was achieved from 10 gl
concentration of isoeugenol after a 48-h reaction.
Bioconversion of Lignin to Vanillin
Lignin, an aromatic polymer, is one of the most abundant natural sources of aromatic
compounds. It is formed by the dehydrogenative polymerization of three cinnamyl alcohols
(monolignols) including p-coumaryl (4-hydroxycinnamyl), coniferyl (4-hydroxy-3-methox-
ycinnamyl), and sinapyl (4-hydroxy-3, 5-dimethoxycinnamyl), which are precursors of p-
hydroxyphenyl, guaiacyl, and syringyl lignin units [35].
Analytical pyrolysis has been used to investigate lignin degradation by several
white-rot fungi including Pleurotus eryngii [35] and lignin depolymerization in Pha-
nerochaete chrysosporium [36]. The enzyme lignin peroxidase, manganese peroxidase,
and laccase are responsible for lignin depolymerisation, and vanillin has been detected
only in trace amounts with other metabolites like dehydrodivanillin, vanillic acid,
coniferyl aldehyde, ferulic acid, p-hydroxycinnamyl aldehyde, p-hydroxycinnamic
acid, guaiacylglycerol-b-coniferyl ether, and guaiacylglycerol beside lignin fragments
Based upon the scientific literature, six independent lignin degradation pathways
have been identified vis-à-vis (a) β-aryl ether cleavage pathway, (b) biphenyl cata-
bolic pathway, (c) ferulate catabolic pathway, (d) tetrahydrofolate-dependent O-
demethylation pathway, (e) protocatechuate 4,5-cleavage pathway, and (f) multiple
pathways involving 3-O-methylgalate catabolism. β-aryl ether cleavage and ferulate
catabolic pathways have grabbed much interest of the microbiologists as vanillin is
found as an intermediate metabolite. β-aryl ether cleavage pathway, as reported in
Sphingomonas paucimobilis,Delftia acidovorance,andRhodococcus sp. In S.
1360 Appl Biochem Biotechnol (2013) 169:13531372
paucimobilis SYK-6, β-aryl derivatives are oxidized to α-(2-methoxyphenoxy)-β-
hydroxypropiovanillone (MPHPV) by catalytic activity of ligD encoded Cα-
dehydrogenase (LigD), and then, the ether linkage of MPHPV is reductively cleaved
to generate β-hydroxypropiovanillone and guaiacol by LigF and LigE encoded β-
etherase and ligG encoded GST, which can be converted into vanillin [37].
Bioconversion of Ferulic Acid to Vanillin
Ferulic acid (FA) is a ubiquitous plant constituent produced from the metabolism of
phenylalanine and tyrosine and is linked at different positions to arabinoxylans
through ester linkages. This phenolic component is mainly found in cell walls of
monocotyledons [38]. FA is found in abundance in corn hulls (31.0 g/kg), maize bran
(30 g/kg), sugarbeet (510 g/kg), rice endosperm cell wall (9 g/kg), wheat (6.6 g/kg),
and barley grains (1.4 g/kg). FA can be released by treatment with strong alkali or
using cinnamoyl esterases or ferulic acid esterases together with plant cell wall
glycosyl hydrolases [1,17,39,40].
Lesage-Meessen described a two-step process for production of vanillin from autoclaved
maize bran using a ferulic acid esterase (FAE) producing A. nigerI-1472 [41]. First bio-
transformation converted FA into vanillic acid. In the second step, vanillic acid was
converted into vanillin by 3-day-old Pycnoporus cinnabarinus MUCL 39533 cultures, and
767 mg/l vanillin was produced in the presence of cellobiose and XAD-2 resin.
Streptomyces sp. and Amycolatopsis sp. are more important from an industrial point of
view as their FA biotransformation efficiencies are excellent and highest among all the tested
vanillin producers [4244]. Vanillin yields in P. fluorescens AN103 were improved by
disrupting vdh gene [45]. Vanillin production was raised to 19.2 g/l in Streptomyces sp. V-
1 using adsorbent DM11 resin [46].
FA was transformed into vanillin by Pseudomonas isolate with production rate of
8.5 mg/l [47]. It was also converted to vanillin (0.100.15 g/l) and vanillic acid using
Streptomyces halstedii GE107678 [48]. Resting cells of E. coli strain JM109/pBB1 were
used for biotransformation of FA to vanillin with a yield of 0.851 mol/l at a dilution rate of
0.022 h
[49]. Enterobacter sp. Px6-4 was isolated from Vanilla roots, converted FA via
4-vinylguaiacol to vanillin [50]. Staphylococcus aureus also has an ability to consume FA
with accumulation of 45.7 mg/l vanillin on the second day of incubation [51]. However, it
was quickly degraded there by reducing vanillin yield to 9.8 mg/ml after 7 days of
incubation. In a one-step biotransformation process, P. cinnabarinus biotransformed FA
to 126 mg/l vanillin with a molar yield of 54 % under statistically optimum condition in
the presence of glucose as carbon source and corn steep liquor and ammonium chloride as
organic and inorganic nitrogen source, respectively [52].
Catabolic Pathways of FA
Based upon the scientific literature, released FA can undergo dealkylation, demethylation,
and decarboxylation to yield propionate, phenyl propionate, and 4-vinyl phenol and 4-ethyl
phenol. FA to vanillin conversions may occur through non-oxidative decarboxylation, side
chain reduction, coenzyme-A-independent deacetylation, and coenzyme-A-dependent
deacetylation. Finally, vanillic acid produced from vanillin either enters protocatechuic acid
pathway or guiacyl pathway for further degradation through TCA cycle [1,5355]as
summarized in Fig. 2.
Appl Biochem Biotechnol (2013) 169:13531372 1361
Bioconversion of Glucose or Glycoside to Vanillin
A recombinant strain of E. coli KL7 (transformed with plasmid pKL5.26A or
pKL5.97A) converted glucose into vanillic acid by shikimic acid pathway, which was
then enzymatically reduced to vanillin by aryl aldehyde dehydrogenase isolated from
Neurospora crassa. Serious drawbacks were observed in this scheme such as lack of an
in vivo step for the enzymatic reduction of vanillic acid, addition of isolated carboxylic
acid reductase, and addition of costly cofactors such as ATP, NADPH, and Mg
, and
the generation of isovanillin as a contaminating side product (Fig. 1)[1].
Using metabolically engineered Schizosaccharomyces pombe and Saccharomyces
cerevisiae, glucose was converted into vanillic acid which was further reduced to
vanillin with 92 % molar conversion within 7 h of incubation. Strains were engi-
neered by introducing three genes encoding dehydroshikimate dehydratase from the
dung mold Podospora pauciseta, aromatic carboxylic acid reductase from Nocardia,
and an O-methyltransferase from Homo sapiens into both the strains. Reduction of
vanillin to vanillyl alcohol was prevented by knockout of the host alcohol dehydro-
genase ADH6 [56]. The major drawback of this study was the glycosylation step
which implies reduction in the maximum theoretical yield. Nevertheless, this process
leads to increase in toxicity and decrease in solubility of vanillin.
In case of vanillin β-D-glucoside (or VG), extracellular concentration up to 25 g/l has
been shown not to affect growth of the strain and is thereby more suitable for commercial
production [57]. To reduce product toxicity and increase vanillin production, they
expressed glycosyltransferase of Arabidopsis thaliana in a vanillin-producing Saccharo-
myces cerevisiae strain served which was approximately fivefold improvement in free
vanillin production, compared to the previous work on de novo vanillin biosynthesis in
bakers yeast.
Bioconversion of Phenolic Stilbenes to Vanillin
Ligno stilbene-dioxygenases (isdA/isdB) extracted from P. paucimobilis strain TMY 1009
can oxidize phenolic stilbenes of spruce bark, to corresponding aromatic aldehydes (Fig. 1).
Cloning and expression of corresponding genes were carried out in E. coli. Strain was
reported to oxidize naturally occurring isorhapotin to vanillin with a molar yield of up to
70 % [1].
Bioconversion of Vanillic Acid to Vanillin
Vanillic acid is either oxidatively decarboxylated to methoxyhydroquinone or it is reduced to
vanillin and vanillyl alcohol (Figs. 1and 2). Vanillic acid decarboxylation yields vanillin in
low amounts only. This drawback can be overcome by addition of cellobiose prior to vanillic
acid supplementation that channels vanillic acid metabolism via reductive pathway, by using
high-density cultures, by using different types of bioreactors, and employing XAD-2 resin as
an adsorbant [1].
A gene encoding carboxylic acid reductase from Nocardia sp. was expressed in E.
coli BL21 (DE3) that reduced vanillic acid into vanillin. Car is the first example of a
new gene family encoding oxidoreductases with remote acyl adenylation and reductase
sites [58].
1362 Appl Biochem Biotechnol (2013) 169:13531372
Bioconversion of Aromatic Amino Acids into Vanillin
Phenylalanine ammonia lyase deaminates phenylalanine to trans-cinnamic acid, which
is the key reaction involved in flavonoid, stilbene, and lignin biosynthesis in plants.
This pathway follows with the formation of vanillin precursors like coniferyl alcohol,
ferulic acid, and coniferyl aldehyde. Several white-rot fungi and Proteus vulgaris also
possess phenylalanine ammonia lyase activity. According to P. vulgaris CMCC2840
deaminates methoxytyrosine to phenylpyruvic acid, which is then converted to vanillin
by mild caustic treatment [1](Fig.1).
Production of Biovanillin using Lactic Acid Bacteria
Phenolic compounds are directly related to sensory characteristics of foods such as
flavor, astringency, and color. Their presence in the diet is beneficial for our health
due to their chemopreventive activities against carcinogenesis and mutagenesis. Lactic
acid bacteria (LAB) are autochthonous microbiota of raw vegetables that are used in
the fermentation of food products of plant origin. However, little information is still
available on the influence of phenolic compounds on the growth and viability of LAB
species [59]. Various metabolic pathways have been described so far for the produc-
tion of vanillin from ferulic acid. But there is scanty evidence for vanillin production
using lactic acid starter culture bacteria. LABs are not able to utilize and assimilate
eugenol, isoeugenol, or vanillic acid for production of vanillin. Only a limited number
of strains like Oenococcus oeni,Lactobacillus brevis,Lactobacillus hilgardii,Lacto-
bacillus plantarum,andPediococcus damnosus are able to transform ferulic acid to
vanillin but in low yield [60]. Lactobacillus mainly produces 4-vinylguaiacol with
little amount vanillin [61]. In 2004, FAE producing human intestinal isolate Lactoba-
cillus acidophilus was characterized for the first time in context to vanillin biosyn-
thesis. These findings contribute to the understanding of malolactic fermentation in the
production of aroma compounds.
Other Substrates for Vanillin Production
Creosol and vanillyl amine can also be used for vanillin production in high yield using a
flavoprotein vanillyl alcohol oxidase (vaoA), which can act on a wide range of phenolic
compounds [2]. Vanillyl amine is initially converted to a vanillyl imine intermediate, which
is hydrolyzed non-enzymatically to vanillin, where a vaoA producing P. simplicicum strain
can be implemented (Fig. 1). This route to vanillin has biotechnological potential as the
widely available principle of red pepper; capsaicin can be hydrolyzed enzymatically to
vanillyl amine.
Vanillin Production from Waste Residue
In a study, co-culture of FA hydrolyzing A. niger CGMCC0774 and P. cinnabarinus
CGMCC1115 were used for the production of vanillin on waste residue of rice bran oil
involving vanillyl alcohol as an important intermediate. The yield of vanillin reached up to
2.8 gl
when 5 gl
of glucose and 25 g of HZ802 resin were supplemented in the
Appl Biochem Biotechnol (2013) 169:13531372 1363
bioconversion medium [62]. Similarly, sundried green coconut husk was used for production
of vanillin using basidiomycete P. chrysosporium, and 52.5 μg/g of vanillin was recovered
after 24 h of incubation [63].
Genetic Organization of Vanillin Biosynthetic Operons
Eugenol and ferulic acid catabolic operon consisted of number of genes such as ehyAB
(eugenol hydroxylase), vaoA (vanillin alcohol oxidase), calA (coniferyl alcohol dehydroge-
nase), calB (coniferyl aldehyde dehydrogenase), fcs (feruloyl CoA synthetase), ech (enoyl
CoA hydratase), aat (β-ketothiolase), vdh (vanillin dehydrogenase), vanA/vanB (vanillate-o-
demethylase), and pcaG/pcaH (protocatechueate 3,4-dioxygenase) involved in production
of coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin, vanillic acid, protocatechuic
acid, and ortho-3-carboxymuconolactone, respectively (Fig. 2). Priefert et al. for the first
time explained the organization of 6.5 kb eugenol catabolic operon containing ehyA and
ehyB encoding eugenol hydroxylase that catalyzes bioconversion of eugenol to coniferyl
alcohol by Pseudomonas sp. strain HR199 (DSM 7063). These genes are localized down-
stream to vanillin catabolism genes vanA and vanB, encoding vanillate-O-demethylase.
Operon also contains vanB gene whose product is vanillate-O-demethylase beta-subunit
which is responsible for demethylation of vanillin to protocatechuate as illustrated in Fig. 3.
fdh gene encodes for a putative formaldehyde dehydrogenase that facilitates oxidation of
Fig. 3 Genetic organization of vanillin biosynthesis cassettes/operons in various microorganisms
1364 Appl Biochem Biotechnol (2013) 169:13531372
formaldehyde to formate. A preceeding gcs, encoding putative gamma-glutamylcysteine
synthetase, helps in glutathione biosynthesis. Amino acid sequences of ehyA and ehyB
exhibited up to 29 and 55 % amino acid identity to the corresponding subunits of p-cresol
methylhydroxylase from P. putida and with amino-terminal sequences of αand βsubunits
of eugenol dehydrogenase of P. fluorescens E118. Downstream of ehyB, an open reading
frame was identified, whose deduced amino acid sequence exhibited up to 71 % identity to
azurins, representing the gene azu of the physiological electron acceptor of the eugenol
hydroxylase. Eugenol hydroxylase genes were amplified by PCR, cloned, and functionally
expressed in E. coli [64].
Genes responsible for metabolism of eugenol and isoeugenol were clustered in a 30-kb
genetic fragment consisting of 23 ORFs in P. nitroreducens Jin1 where orf 26 (iemR) and orf
27 (iem) were predicted to be involved in the conversion of isoeugenol to vanillin. The
deduced amino acid sequence of Iem of strain Jin1 showed 81.4 % sequence identity with
isoeugenol monooxygenase of P. putida IE27 transforms isoeugenol to vanillin. Its Iem was
expressed in E. coli BL21 (DE3) and recombinant so generated was able to convert
isoeugenol to vanillin. Deletion and cloning analyses indicated that the gene iemR acts as
regulatory gene located upstream of iem and is required for expression of iem in the presence
of isoeugenol [65]. On the basis of Blast P analysis, they have proposed the gene names in P.
nitroreducens jin 1ascalA,calB,aat,fcs,vdh,ech,ehyB,echA,vanB, and vanA to orfs 16,
19, 22, 23, 24, 25, 30, 32, 35, and 36, respectively [65].
The saccharomicins A and B, produced by the actinomycete Saccharothrix espanaensis,
are oligosaccharide antibiotics. Gene expression experiments were carried out in Streptomy-
ces fradiae XKS to investigate candidate genes responsible for the formation of caffeic acid
as part of the saccharomicin aglycon. Genes Sam8 (encode tyrosine ammonia lyase) and
sam5 (encode 4-coumarate 3-hydroxylase) were isolated from S. espanaensis that convert L-
tyrosine via trans-p-coumaric acid to trans-caffeic acid. Further conversion of caffeic acid to
FA took place with the help of caffeic acid-3-ortho methyltransferase (comt) gene which is
the part of the lignin biosynthesis pathway of plants, methylates -OH on C4 of the aromatic
ring. Finally, FA was converted to vanillin with the help of fcs and ech of P. fluorescens [66].
Ferulic acid catabolic genes namely fcs,ech,vdh, and aat are clustered in the genome of
Pseudomonas sp. strain HR199 and P. putida KT2440 (Fig. 3). The same arrangement of
ferulic acid-degrading genes has been identified in Gram-positive bacterium Amycolatopsis
sp. HR167. The role of structural genes fcs,ech, and vdh has been confirmed by their cloning
and expressing in E. coli fcs,ech, and vdh genes were disrupted to prove their essential
involvement of in the catabolism of FA in P. putida KT2440 [67,68].
S. paucimobilis SYK-6 degrades FA into vanillin which is further metabolized through
protocatechuate 4, 5-cleavage pathway in the presence of FerB and FerA proteins (Fig. 3).
FerB possesses 40 to 48 % amino acid identity with that of the Fch of Pseudomonas and
Amycolatopsis sp. Blast P showed 31 % identity sequence of ferA with pimeloyl-CoA
synthetase of Pseudomonas mendocina 35, a homolog of succinyl-CoA synthetase [69]. A
ferB homolog ferB2 involved in the degradation of 5,5-dehydrodivanillic acid is found
upstream of a 5-carboxyvanillic acid decarboxylase gene (ligW), and its deduced amino acid
sequence showed 49 % identity with FerB [70].
Achterholt characterized a ferulic acid catabolic operon in Amycolatopsis sp. HR167
consisting fcs of 1,476 bp, ech of 864 bp, and aat of 1,209 bp present in downstream
direction of fcs gene [44]. Orf1 of 1,626 bp encoding putative dihydroxy-acid-dehydratase
(Partial start) and Orf2 encoding putative bacterioferritin of 477 bp flanked 840 bp of ech
and 2,172 bp of fcs in upstream and orf5 encoding 1,365 bp of putative permease and orf6
encoding 404 bp of putative acyl-CoA dehydrogenase in downstream direction present in
Appl Biochem Biotechnol (2013) 169:13531372 1365
Delftia acidovorans [71] (Fig. 3). Ferulic acid catabolic operon system consisting ech,vdh,
fcs partial putative beta-kethiolase (aat) of strain P. putida was characterized [72]. Zhang and
Zhao have characterized a ferulic acid catabolic operon consisting of 831 bp of ech, 1,449 bp
of vanillin hydratase, and 1,770 bp of fcs in P. fluorescens [73]. Ech and fcs gene present in
downstream and upstream diorection of vdh gene, respectively. fcs and ech genes have been
subsequently analyzed found in Rhodococcus sp. DK17 previously described [74]. Another
ferulic acid catabolic operon system consisting fch with an overlapping transcriptional
regulator, vdh,fcs,aat, and a putative feruloyl-CoA dehydrogenase have been isolated in
P. fluorescens [75]. The gene product of ferR knockout mutant (BF1389) showed that it
negatively regulates expression of the ferulic catabolic operon in P. fluorescens BF13 [75].
Metabolic Engineering of E. coli for Production of Vanillin
Successful adoption of metabolic engineering approaches in E. coli can offer low cost and
industrially economical process for vanillin production. A recombinant strain of E. coli
engineered by cloning fcs and ech of Pseudomonas sp. HR199 catabolized FA to vanillin at a
rate of 0.022 Μm/min/ml [67]. Later on, a 4,000-bp PstI fragment of Amycolatopsis sp.
HR167 bearing 864 bp ech and 1476 bp fcs genes was cloned in pBluescript SK, and fcs
and ech were expressed in E. coli. Recombinant strains were able to transform FA to vanillin
[44]. Fcs and ech genes of Amycolatopsis sp. were expressed in metabolically engineered E.
coli to produce vanillin under control of IPTG-inducible trc promoter. Vanillin production
(1.1 g/l) was obtained with cultivation in 0.2 % (w/v) ferulate [76]. In a separate study,
Amycolatopsis sp. HR104 and D. acidovorans fcs and ech genes were introduced in
pBAD24 vector and were named pDAHEF and pDDAEF, respectively. Vanillin (450 mg/
l) was produced after 18 h of incubation through induction of fcs and ech genes from
pDAHEF, and production was optimized with the addition of 13.3 mM arabinose. Upon
induction, 500 to 580 mg/l vanillin was obtained with 0.2 % ferulic acid as an inducer in
18 h of culture [77].
In 2003, Overhage and others proposed vanillin production from eugenol using a two-
step process catalyzed by two recombinant E. coli strains. In the first step, E. coli strains
[105] XL1-Blue (pSKvaomPcalAmcalB) converted eugenol into FA with 93.3 % molar
yield, while in the second step, recombinant E. coli (pSKechE/Hfcs) converted FA to
vanillin. This process leads to the production of 4.6 g/l ferulic acid, 0.3 g/l vanillin, and
0.1 g/l vanillyl alcohol from eugenol substrate.
The isoeugenol monooxygenase gene of P. putida IE27 was introduced into E. coli BL21
(DE3) cells using expression vector pET21a, driven by T7 promoter. Vanillin (28.3 g/l) was
recovered from 230 mM isoeugenol at 20 °C after 6 h in transformed E. coli BL21 (DE3)
cultures [15].
Concentration of FA was found to influence vanillin production in E. coli DH5αtrans-
formed with plasmid pTAHEF containing fcs and ech genes of Amycolatopsis sp. HR104
[78]. The maximum production of vanillin from E. coli DH5αharboring pTAHEF was
observed to be 1.0 g/l at 2.0 g/l of FA for 48 h of incubation. Two approaches were followed
to improve the vanillin production by reducing its toxicity: (1) vanillin-resistant mutant
NTG-VR1 generation through NTG mutagenesis and (2) toxic vanillin removal from the
medium by XAD-2 resin absorption. Vanillin production was enhanced to three times at 5 g/
l of FA using NTG-VR1 when compared with its wild-type strain. After employing 50 % (w/
v) of XAD-2 resin in culture with 10 g/l of FA vanillin production of NTG-VR1 was found to
be 2.9 g/l, which was twofold higher than that obtained without using resin.
1366 Appl Biochem Biotechnol (2013) 169:13531372
Barghini engineered E. coli strain JM109 with a low-copy number vector pBB1 carrying
genes fcs and ech of P. fluorescens BF13 [79]. The final concentration of vanillin in the
production medium was 3.5 mM after 6 h incubation by sequential induction with 1.1 mM
ferulic acid. Vanillin (2.52 g/l) was obtained under resting cell conditions. Using integrative
vector pFR2, Pseudomonas genes fcs and ech were integrated into lacZ gene of E.coli. The
resultant strain was very stable and more efficiently produce vanillin than strains expressing
FA catabolic genes from a low copy vector and 6.6 kg per kg biomass vanillin was reported
using resting cells of metabolically engineered E. coli [80].
E. coli DH5α(pTAHEF-gltA) engineered with gltA (encoding citrate synthase gene
required for conversion of acetyl-CoA) yielded 1.98 g/l vanillin in 48 h of culturing on
3g/lFA.AnicdA mutant (with disrupted isocitrate dehydrogenase activity) was found
toxicity of vanillin and vanillin accumulation was enhanced by 2.6 times. The real
synergistic effect of vanillin of 5.14 g/l was produced in 24 h of the culture with molar
conversion yield of 86.6 %, which is the highest so far reported in the case of
recombinant E. coli [81].
Substrate channeling was performed by dimer formation between leucine-zippers of fcs
and ech in order to channelize feruloyl-CoA and thereby increasing vanillin production from
recombinant E. coli.E. coli harboring a plasmid pTBE-FP forming an efficient dimer of
Bait-Ech and Fcs-Prey produced 2.1 g/l vanillin at an initial FA concentration of 3 g/l for
30 h of culture, which was improved by 2.3-fold from vanillin production of 0.9 g/l of
control strain harboring pTAHEF with no leucine-zipper [82].
vdh gene of P. fluorescens BF13 strain was inactivated via targeted mutagenesis, and
results demonstrated that engineered strain BF13 accumulated vanillin with concurrent
expression of structural genes (fcs) and (ech) from a low-copy plasmid. The developed
strain produced up to 8.41 mM vanillin, which is the highest final titer of vanillin
produced by a Pseudomonas strain from agro-industrial wastes which contain ferulic
acid [83].
Vanillin Degradation
Reactive vanillin, which exhibits a toxic effect to most microorganisms, is rapidly
converted to other products as only trace amounts occur during degradation of ferulic
acid [1,84]. A complete set of genes (vdh,vanA/vanB,pcaG/pcaH), that are respon-
sible for degradation of vanillin to the ortho cleavage product 3-carboxymuconolactone,
has been identified from Pseudomonas sp. HR199 and expressed in E. coli (Fig. 2)[21,
Priefert and others characterized a genetic fragment (E230) bearing vdh,dxvanA, and
vanB genes on cosmid pVK100 based genomic library of Pseudomonas sp. strain HR199
[86]. An ORF2 has been identified upstream to vdh gene that showed 28.4 % amino acid
identity with enoyl coenzyme A hydratases. Fragment E230 was cloned in pBluescript SK
and E. coli was transformed that successfully converted vanillin to protocatechuate via
vanillate, indicating the functional expression of these genes in E. coli. Alcaligenes eutro-
phus and few Pseudomonas strains which were unable to utilize vanillin or vanillate were
also engineered with vanA,vanB, and vdh genes, and they became able to grow on these
substrates. Genes encoding vanillin dehydrogenase (vdh) and vanillate O-demethylase
(vanAB) were identified in Rhodococcus jostii RHA1 using gene disruption and enzyme
activities [87].
Appl Biochem Biotechnol (2013) 169:13531372 1367
Drawbacks of Existing Microbial Conversion Processes
There are some common observations made in the existing biotechnological procedures for
production of microbial vanillin. These observations more or less reflect drawbacks of the
existing microbial procedure, e.g., growth of filamentous actinomycetes results in highly
viscous broths, unfavorable pellet formation, and uncontrolled fragmentation and lysis of the
mycelium. This may complicate the rheology of the production processes, reduce their
productivity, and increase the downstream processing costs [88].
Attempts to prevent oxidation of vanillin by inhibition of vanillin dehydrogenase in
Pseudomonas by dithiothreitol gained limited success [39]. The vanillin yield from isoeu-
genol by microbial biotransformation is usually lower than 1 g/l [30]. Based on the analysis
of products in culture filtrate, vanillin, vanillic acid, and protocatechuic acid are implicated
as dominating intermediates of isoeugenol degradation.
Due to unspecificity of coniferyl aldehyde dehydrogenase [89], it sometimes exhibit
vanillin dehydrogenase activity that accumulated vanillin to vanillyl alcohol and vanillic
acid and results in low yields of vanillin [67]. FA intermediate products of eugenol and
isoeugenol biotransformation are degraded by p-coumaric acid decarboxylase in L. planta-
rum and to 4-vinylphenol, 4-vinylguaiacol and other by-products in the case of other LAB
species [90].
Most of the researchers paid much attention to prevent further degradation of vanillin by
optimizing bioconversion condition, metabolic engineering, and enzyme inhibition involved
in these reactions [22,91,92]. However, the vanillin yields were rarely improved. Attempts
have been made to reduce the further transformation of vanillin to vanillyl alcohol or vanillic
acid by cellobiose and adsorbent resins such as Amberlite XAD-2 and Diaion HP20 but
failed [93,94]. The most important factor in vanillin production is end product inhibition as
vanillin itself is toxic for growing cells. So, high concentration of FA in fed-batch bio-
conversions may not lead to high vanillin yield [1,95]. In some of the recombinant strains,
genetic instability can cause rapid decline in levels of vanillin production [76,77].
Biotechnological productions of vanillin have been investigated in the past using lignin,
eugenol, isoeugenol, and ferulic acid as major substrates. High chemical reactivity of the
vanillin produced and its toxicity to microbes at higher concentrations are major factors
affecting many of the existing biotechnology processes. Usually, it gets rapidly converted to
less toxic products like vanillic acid or vanillyl alcohol. Control of ferulic acid and vanillin
degradation in Pseudomonas species by gene disruption could fetch limited success since
other dehydrogenases were also present, capable of catalyzing this reaction. Hydrophobic
resins were applied to adsorb vanillin produced and to prevent its further degradation, but
this strategy also failed.
Bioconversion of eugenol to vanillin occurs at low rate using Klebsiella sp., Enterobacter,
Serratia sp. Fusarium solani, and Corynebacterium, but using vdh disruption approach,
2.6 g/l vanillin was recovered from Pseudomonas sp. HR199 [22]. Isoeugenol to vanillin
conversions occur quite reasonably in B. fusiformis SW-B9 (32.5 g/l) [11], P. putida IE27
(16.1 g/l), and recombinant E. coli BL21 (DE3) (28.3 g/l) [15], Amycolatopsis sp. [96],
Streptomyces setonii [42], P. putida [68], Streptomyces sp. V-1 (19.2 g/l) [46] have been
exploited to obtain vanillin from ferulic acid, but the substrate is very expensive that makes
the process economically unviable [1]. We should look forward for cheaper substrates
1368 Appl Biochem Biotechnol (2013) 169:13531372
available in bulk for industrial scale vanillin production that could facilitate waste utilization.
A preliminary work has already been done in this context where vanillin production was
optimized on rice bran oil [12] and green coconut husk [63].
The increasing knowledge of metabolic pathways for vanillin production as well as
the identification and characterization of the corresponding genes offers new oppor-
tunities for metabolic engineering of industrially important starter cultures for stable
expression of vanillin biosynthetic expression cassettes. Important vanillin-producing
genes ech and fcs from various organisms have been cloned in E. coli to produce
biovanillin using metabolic engineered strains and was found to have limited success.
We can exploit natural GRAS microorganism and especially belonging to the lactic
acid bacteria group for production of dairy products and process could be optimized
for in situ vanillin production in distillery, candies, chocolates, choco-eclairs, ice
creams, yogurts, desserts, etc.
Production processes are being adapted to meet increasing consumer demand for
natural and quality food additives. Production of natural vanillin through biotechno-
logical processes using safe precursors, food-grade organisms, cheaper industrial
wastes, and economical downstream processing procedures could help to increase its
importance in the flavor industry. It is also important to emphasize that industry
identifies the application of biotechnology as a component of their strategies to
address competition at the world level.
Acknowledgments The authors acknowledge UGC, New Delhi, for funding a major research project entitled
Metabolic engineering of LAB isolate for biotransformation of ferulic acid to vanillinto Dr. Baljinder Kaur
and meritorious BSR fellowship (Basic Scientific Research) to Mr. Debkumar Chakraborty.
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... The increasing demand for this molecule has propelled the search for biomass-derived pathways for vanillin production. Vanillin can be extracted from lignin, ferulic acid, glucose, vanillic acid, aromatic amino acids, isoeugenol, waste residue, and other substrates [72]. Currently, Borregaard (Sarpsborg, Norway), a Norwegian company, claims to be the only producer of biovanillin, which it produces from wood. ...
... Currently, Borregaard (Sarpsborg, Norway), a Norwegian company, claims to be the only producer of biovanillin, which it produces from wood. Table summarizes some lignocellulosic Vanillin can be extracted from lignin, ferulic acid, glucose, vanillic acid, aromatic amino acids, isoeugenol, waste residue, and other substrates [72]. Currently, Borregaard (Sarpsborg, Norway), a Norwegian company, claims to be the only producer of biovanillin, which it produces from wood. ...
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This review covers the operating conditions for extracting top value-added chemicals, such as levulinic acid, lactic acid, succinic acid, vanillic acid, 3-hydroxypropionic acid, xylitol, 2,5-furandicarboxylic acid, 5-hydroxymethyl furfural, chitosan, 2,3-butanediol, and xylo-oligosaccharides, from common lignocellulosic biomass. Operating principles of novel extraction methods, beyond pretreatments, such as Soxhlet extraction, ultrasound-assisted extraction, and enzymatic extraction, are also presented and reviewed. Post extraction, high-value biochemicals need to be isolated, which is achieved through a combination of one or more isolation and purification steps. The operating principles, as well as a review of isolation methods, such as membrane filtration and liquid–liquid extraction and purification using preparative chromatography, are also discussed.
... It is due to these properties that Vanillin is often interchangeably used with Vanilla, being the major constituent of it and responsible for flavour and aroma (Hoffman and Zapf, 2019). The application of Vanillin is not only limited to aroma and flavour but has been reported as a bio-preservative, antimutagenic agent, raw material for pharmaceutical drugs including dopamine and L-Dopa that are relevant in regulating neurological disorder (Kaur and Chakraborty, 2013). Recent studies have also reported the application of vanillin Researchers have explored tissue culture mediated Vanillin production as an alternative to conventional field cultivation but the strategy had the limitation of comparatively slow plant growth. ...
... Bacteria like Bacillus sp. and Pseudomonas sp. and fungi like Saccharomyces have been reported for their ability to convert substrate like eugenol, isoeugenol, ferulic acid and glucose into vanillin by biotransformation(Gallage and Moller, 2015). Vanillin produced using microbes is very much similar to natural Vanillin and thus it is also a safe alternative to plant derived counterpart(Kaur and Chakraborty, 2013). Several endogenous enzymes are employed in this biotransformation. ...
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Vanillin, an aromatic aldol compound and an extensively usedflavouring agent that has been used throughout the world fordecades in various sectors, primarily food industries. Lately, itsuse had also been explored in other sectors. The natural sourceof Vanillin or flavour Vanilla (whose major constituent isVanillin) is the cured pod of the Orchid plant of genus Vanilla.However, limited yield, environmental impact, and pestinfestation necessitate the need to explore other alternativesources. There are two other approaches used to produceVanillin, the major constituent of flavour Vanilla, viz. chemicalor synthetic route, and microbial biotransformation. Thesynthetic route is used to produce Vanillin in a cost-effectivemanner but is associated with the health impact on prolong useby humans. Recent advancements are focused on exploring theuse of the biotechnological route for the production of Vanillin.One of the well-explored biotechnological routes is viabiotransformation using microbes. Researchers have alsoexplored the production of Vanillin from compoundscommonly eugenol, isoeugenol, and ferulic acid, viabiotransformation, using microbial enzymes. Research isunderway to explore novel biotransformation agents andoptimization of strategies to develop a sustainable economicVanillin production strategy. Moreover, the researchers haveexpended the horizon of the application of Vanillin beyondflavour, by exploring photophysical properties and as protectiveagents against radiation-induced injury. The article highlightsthe recent advancement in Vanillin production strategies and itsapplication in different sectors. (PDF) Vanillin: A Wonder Molecule with Diverse Application and Alternative Manufacturing Strategies. Available from: [accessed Sep 07 2022].
... By employing 400 mg of the immobilized enzyme, around 50 mL was collected, achieving the highest conversion rate in the first 35 mL. In order to recover the final product, a catch-and-release system was employed using an Ambersep 900 OH resin-packed column in which the acid was entrapped, and afterwards, eluted by treatment with HCl 1.0 N. Vanillin (4-hydroxy-3-methoxybenzaldehyde) is one of the most used flavors in the world, but apart from this use, it has been also employed in the pharmaceutical and medical industries as it displays antioxidant, neuroprotective, and anticancer activities [73]. In 2017, different Deep Eutectic Solvents (DESs) were employed as cosolvents (1% v/v) in the conversion of isoeugenol (21) into vanillin (22) in a process catalyzed by Lysinibacillus fusiformis CGMCC1347 cells [74]. ...
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Asymmetric oxidation processes have constituted a valuable tool for the synthesis of active pharmaceutical ingredients (APIs), especially for the preparation of optically active sulfoxides, compounds with interesting biological properties. Classical approaches for these oxidative procedures usually require the application of non-sustainable conditions that employ hazardous reagents and solvents. In the last decades, chemists have tried to combine the preparation of valuable compounds of high yields and selectivities with the development of more sustainable protocols. To achieve this objective, greener solvents, reagents, and catalysts are employed, together with the use of novel chemical techniques such as flow catalysis or photocatalysis. The last efforts in the development of greener approaches for the preparation of APIs and their intermediates using oxidative procedure will be reviewed herein. Most of these approaches refer to biocatalytic methods, in which mild reaction conditions and reagents are employed, but other novel techniques such as photocatalysis will be described.
... Os estudos das diferentes vias metabólicas, a identificação e caracterização dos genes correspondentes à síntese da vanilina oferecem oportunidades para a engenharia metabólica de culturas importantes para a indústria. Importantes genes produtores de vanilina foram clonados em microorganismos para produzir biovanilina (Kaur Chakraborty 2013). ...
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Resumo: A vanilina está presente em diversos compostos bioativos e é o principal constituinte da baunilha, sendo utilizada durante décadas para aromatizar alimentos e bebidas. A baunilha (Vanilla planifolia) é uma planta perene e seus frutos, denominados vagens ou favas, possuem o extrato de baunilha, que confere aroma à planta. Materiais e métodos: Para possibilitar um melhor entendimento no processo de obtenção da essencial, este trabalho traz uma revisão bibliográfica referente ao processo de obtenção de forma natural, sintética e processos biotecnológicos. Lembrando que o processo de obtenção da vanilina natural dá-se por extração com etanol (60% v/v) em temperaturas brandas ou com fluidos quentes, no entanto, a toxicidade é um ponto negativo do processo de extração. O mercado de aromatizantes de baunilha é dominado pela vanilina sintética em razão da resistência ao aroma e menor preço. Existem vários métodos industriais para produzir vanilina, principalmente por síntese química a partir do guaiacol. A biotecnologia também tem contribuído para a produção de vanilina desenvolvendo uma nova rota metabólica que imita o caminho natural de plantas usando genes microbianos. Considerações: Embora seja conhecida por sua propriedade aromática, há uma gama de aplicações para a vanilina e evidências sobre suas atividades biológicas tais como: anticancerígenas, antioxidantes e antiinflamatórias. Dessa forma, a revisão aborda sobre as principais aplicações estudadas por diferentes autores que tratam sobre importância da Vanilina de forma ampla.
... Bioconversion studies have been conducted for vanillin production using ferulic acid, eugenol and isoeugenol as the major substrates (Kaur and Chakraborty 2013;Ma et al. 2022;Yamada et al. 2008), among which the ferulic acid-based route shows the highest efficiency. For example, a vanillin-tolerant bacterium Amycolatopsis sp. ...
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Vanillyl alcohol is a precursor of vanillin, which is one of the most widely used flavor compounds. Currently, vanillyl alcohol biosynthesis still encounters the problem of low efficiency. In this study, coculture engineering was adopted to improve production efficiency of vanillyl alcohol in E. coli . First, two pathways were compared for biosynthesis of the immediate precursor 3, 4-dihydroxybenzyl alcohol in monocultures, and the 3-dehydroshikimate-derived pathway showed higher efficiency than the 4-hydroxybenzoate-derived pathway. To enhance the efficiency of the last methylation step, two strategies were used, and strengthening S-adenosylmethionine (SAM) regeneration showed positive effect while strengthening SAM biosynthesis showed negative effect. Then, the optimized pathway was assembled in a single cell. However, the biosynthetic efficiency was still low, and was not significantly improved by modular optimization of pathway genes. Thus, coculturing engineering strategy was adopted. At the optimal inoculation ratio, the titer reached 328.9 mg/L. Further, gene aroE was knocked out to reduce cell growth and improve 3,4-DHBA biosynthesis of the upstream strain. As a result, the titer was improved to 559.4 mg/L in shake flasks and to 3.89 g/L in fed-batch fermentation. These are the highest reported titers of vanillyl alcohol so far. This work provides an effective strategy for sustainable production of vanillyl alcohol.
... com/ indus try-repor ts/ bio-vanil linmarket). Compounds like lignin, ferulic acid, eugenol, or isoeugenol derived from organic waste served to be the precursors for microbial production of vanillin (Fig. 5) [65]. Specific microbes like Amycolatopsis, Streptomyces, Pseudomonas, and Delftia are the promising candidates for converting ferulic acid to vanillin. ...
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Purpose of Review Despite genetic, metabolic, and enzymatic studies of xenobiotic-degrading microbe(s), bioremediation has remained inefficient at the field level. The microbial eco-physiological traits and bio-geological constraints imposed are the key hurdles. Therefore, it is imperative to comprehend how microbes survive, adapt, and overcome such constraints at the impacted niches. Furthermore, given the urgency of climate smart innovations, “microbe-driven trash-to-treasure conversion” is of the highest concern. The purposes of this review are to highlight the importance of assistive microbial responses/traits in overcoming bio-geochemical constraints for improved adaptability and to develop cleaner routes to recover value-added products during biodegradation. Recent Findings The use of specialized microbes with efficient xenobiotic/aromatic pollutant-degrading abilities, known as “bioremediation”, is a promising and eco-friendly alternative. These specialized microbes elicit a variety of cellular responses (eco-physiology traits) like chemotaxis, cell surface modifications, substrate utilization hierarchy, metabolic pathway compartmentalization, and stress management to adapt, survive, and colonize at contaminated niche as well as degrade aromatics efficiently. Recently developed system biology and metabolic engineering tools have also enabled to design-build-analyse and engineer pathways for better remediation with simultaneous recovery of value-added products, thus contributing to the circular bio-economy. Summary Efficient pollutant-degrading microbes endowed with assistive cellular traits like preferential degradation; robust stress management; compartmentalization of pathway; genetic, enzymatic, and regulonic stability; and plant growth–promoting ability are important to mitigate noxious effects of xenobiotics. Use of metabolic pathway engineering in conjunction with bioelectrochemical treatments/biorefineries having compatible consortia and efficient as well as stable enzymes will further enhance desired catalytic activities and expand the metabolic repertoire for efficient treatment-cum-resource recovery.
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Purpose Biovalorization of lignin-derived aromatic monomers such as ferulic acid (FA) has attracted considerable interest. The cold-adapted strain Paraburkholderia aromaticivorans AR20-38 converts FA to the value-added product vanillic acid (VA), without further VA degradation. The efficiency of the bioconversion of FA to VA was optimized by studying culture conditions. Methods Various cultivation parameters (agitation, temperature, FA concentration, nutrient supplementation) were assessed to increase biomass production and shorten the cultivation time, while obtaining high VA production yields. The fate of the intermediate vanillin was also studied. Lignin monomers and degradation products (FA, vanillin, VA) were quantified via UV/Vis-HPLC. Result Full bioconversion of 5 mM FA occurred over a broad temperature range of 5–30 °C. Concentrations up 30 mM FA were utilized as the sole carbon source at 20 °C. Molar VA yields (> 90%) produced from 5 to 12.5 mM FA and from 15 to 17.5 mM FA (82–87%) were not significantly different at 10 °C and 20 °C. The supplementation of the mineral medium with monosaccharides (glucose, fructose, mannose) and/or N-rich complex compounds (yeast extract, casamino acids) resulted in high biomass production, accelerated FA bioconversion, and high molar yields (96–100%). The presence of the N-rich compounds alone or in combination with glucose reduced the incubation time necessary to convert FA to VA. Vanillin, formed as an intermediate during FA degradation, was consumed and converted to VA before FA metabolization, when added in combination with FA. Vanillin bioconversion was significantly accelerated in the presence of glucose. Conclusion The variation of culture conditions improved the efficiency of the studied strain to convert FA via vanillin to VA and demonstrated remarkable FA bioconversion under varying environmental conditions, especially temperature, substrate concentration, and nutrient availability, which is of importance for potential future application.
Vanillin, an important aldehyde compound, is widely used in food and medicine industries. Natural and chemical-synthesized vanillin are facing significant challenge for the sustainable development. Here, de novo biosynthesis of vanillin in S. cerevisiae was thus designed. Through integrating sam8, CYP199A2, comt, fcs and ech into the genome of S. cerevisiae, vanillin was successfully biosynthesized with a titer of 26.3 μg/L. Preferred sam5 efficiently enriched caffeic acid pool, improving vanillin production to 50.2 μg/L in haploid engineered yeast. Expressing the mutants of ARO4 K229L and ARO7 G141S had reduced the negative feedback of tyrosine, increased the vanillin titer to 700 μg/L. Finally, the titer of vanillin was increased to 8545 μg/L through supplying 400 mg/L ferulic acid. The strategies of metabolic engineering and fermentation optimization achieved de novo biosynthesis of vanillin in S. cerevisiae, showing the feasibility of vanillin production by microbial technology.
Conference Paper
Vanillin is a valuable commodity in various industries. Therefore, its production is carried out on a large scale. The vanillin production from vanilla pods is too expensive and insufficient. In order to meet market needs, vanillin is also produced synthetically. However, due to the high consumer demand for natural or organic food production, many efforts were made to produce natural vanillin through the biotransformation process. Five Bacillus sp. bacteria were obtained from the Spent Bleaching Earth waste. They showed the ability to produce vanillin, whose biotransformation capacity was identified and analyzed by High-Performance Liquid Chromatography. Bacterial identification was performed by using 16S rRNA gene amplification, sequencing, and data analysis using the Geneious program. The DNA sequences were aligned with a database on the NCBI website using the BLAST program. The nucleotide sequences of bacterial were processed using ClustalW and Bioedit. Then, phylogenetic trees were prepared using MEGA-X for relationship analysis. The results of the identification of the five isolates were Bacillus cereus strain SBMAX30 (isolate B1), Bacillus thuringiensis strain LDC 507 (isolate B2), Bacillus thuringiensis strain LB61 (isolate B3), Bacillus cereus strain 190103- R03 (isolate B4) and Bacillus cereus strain LB87 (isolate B5). Vanillin production for 72 hours from 1% (v/v) isoeugenol for isolate B1 was 0.421 ± 0.0337 g/L vanillin (4.28%), isolate B2 was 0.420 ± 0.0679 g/L vanillin (4.28 %), isolate B3 as much as 0.315 ± 0.0983 g/L vanillin (3.20%), isolate B4 as much as 0.168 ± 0.0225 g/L vanillin (1.71%), and isolate B5 as much as 0.386 ± 0.1019 g/L vanillin (3.93%). The highest vanillin production was obtained from isolate B1(Bacillus cereus strain SBMAX30). The result highlights the potential use of bacteria as a platform to produce vanillin, although its practical use needs to be optimized.
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This review aims at providing an overview on the microbial production of vanillin, a new alternative method for the production of this important flavor of the food industry, which has the potential to become economically competitive in the next future. After a brief description of the applications of vanillin in different industrial sectors and of its physicochemical properties, we described the traditional ways of providing vanillin, specifically extraction and chemical synthesis (mainly oxidation) and compared them with the new biotechnological options, i.e., biotransformations of caffeic acid, veratraldehyde and mainly ferulic acid. In the second part of the review, emphasis has been addressed to the factors most influencing the bioproduction of vanillin, specifically the age of inoculum, pH, temperature, type of co-substrate, as well as the inhibitory effects exerted either by excess substrate or product. The final part of the work summarized the downstream processes and the related unit operations involved in the recovery of vanillin from the bioconversion medium.
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Phanerochaete chrysosporium has been found to preferentially metabolize vanillic acid using a reduction pathway which leads to vanillyl alcohol via vanillin. In order to prevent vanillin transformation into unwanted vanillyl alcohol, the ability of different adsorbents to adsorb vanillin was investigated. XAD-2 resin was shown to be very efficient and selective for vanillin. When applied to the culture medium of P. chrysosporium, the resin trapped the vanillin, which prevented the transformation of vanillin into vanillyl alcohol.
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Cellulose-rich residue was obtained by a pectinase treatment of sugar beet pulp and was enzymatically degraded to produce cellobiose. From sugar beet pulp, the hydrolysis yielded 5.3% of cellobiose and 3.8% of glucose. The cellobiose-rich fraction was added in a culture medium of the filamentous fungus Pycnoporus cinnabarinus to test it as an activator of vanillin production. Adding the sugar beet-derived cellobiose-rich fraction to a 3-day-old culture yielded a 3.3-fold increase in vanillin production when compared to a culture devoid of cellobiose, while commercial cellobiose yielded a 3.1-fold increase.© 1999 Society of Chemical Industry
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This review deals with the Vanilla plant: history; botanical description; chemistry of vanilla beans; curing of vanilla beans; commercial extraction of vanilla flavour; standard specifications and uses of vanilla flavour. The production of vanillin by both chemical and biotechnological methods is described. The biotechnological production of vanilla flavour metabolites by plant tissue/cell culture, microbial biotransformation and molecular approaches is also presented, together with a discussion on economic and safety considerations.© 2000 Society of Chemical Industry
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The present work deals with a novel bioconversion of ferulate into vanillin using resting cells of Escherichia coli strain JM109/pBB1 as a biocatalyst. Biomass recy-cling from four successive bioconversion steps demonstrated the possibility of employing the proposed resting cell system for the continuous production of vanillin. Among the test-ed immobilization supports (polyurethane, synthetic sponge and porous glass) the syn-thetic sponge proved to be the best material in terms of both vanillin formation (C v = 0.080 g l -1) and productivity (Q v = 0.019 g l -1 h -1) at the end of entrapment tests. Thus, it was used in preliminary continuous tests using a fixed-bed column with immobilized E. coli JM109/pBB1 cells. The highest vanillin yield (Y P/S = 0.851 mol mol -1) was obtained at a dilution rate of 0.022 h -1 .
The biotransformation of ferulic acid to vanillin was carried out using locally isolated bacterial cultures from manure, paddy straw and garbage samples which include Pseudomonas sp. Pseudomonas sp. was enriched with the ferulic acid thrice and subsequently named as EF1, EF2 and EF3. EF3 produced maximum vanillin (850 μg/100mg culture), followed by EF2 (842 μg/100mg culture), manure isolate MAMWR (B) (598 μg/100mg culture), EF1 (474 μg/100mg culture) and Pseudomonas sp. (230 μg/100mg culture). Garbage isolate GRMWSR (B) produced very insignificant amount (1.13 μg/100mg culture) of vanillin.
The gene loci fcs and ech, encoding feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase, respectively, are involved in the ferulic acid catabolism in Delftia acidovorans. The amino acid sequence deduced from ech exhibited 51% identity to the enoyl-CoA hydratase/aldolase from Pseudomonas sp. strain HR199, indicating that the enzyme from D. acidovorans represents a new lineage of this protein. The genes fcs and ech were expressed in Escherichia coli enabling the recombinant strain to transform ferulic acid to vanillin as revealed by photometric and HPLC analysis. An fcs deficient mutant of D. acidovorans was unable to grow on ferulic acid. The obtained data suggest that in contrast to a previous publication the biotechnologically interesting direct non-oxidative deacetylation mechanism of ferulic acid cleavage is not realized in D. acidovorans. Instead, ferulic acid degradation in D. acidovorans proceeds via a coenzyme A-dependent non-β-oxidative pathway.
This review covers the most recent literature on vanilla research. Besides the curing process, chemistry in green and cured beans, as well as studies on the biosynthetic pathways of the most important aroma compounds are discussed. Despite intensive research on the curing process, the traditional curing procedures are still widely used. The role of enzymes involved in the curing process is not fully understood.The biosynthesis of vanilla aroma compounds is still under investigation. Data obtained from plant cell cultures are not always in accordance with those from the plant. The glycosylation of the compounds in vivo is still a point of study. Alternative routes to vanillin involving microbial biotransformations are outlined.
A condensed overview is given of the potential offered by bacteria, fungi and yeasts to produce a wide range of bioflavours and fragrances. A few commercialised processes are also discussed. The advantages of microbial processes versus chemical synthesis or extraction are outlined. Both de novo fermentation processes as well as bioconversions based on adding specific precursors/intermediates to microbial cells or enzymes are illustrated via typical examples. Bottlenecks, which currently hinder a wider introduction of this flavour biotechnology, are indicated and some solutions are proposed.© 2002 Society of Chemical Industry