Vanillin–Bioconversion and Bioengineering of the Most Popular Plant Flavor and Its De Novo Biosynthesis in the Vanilla Orchid

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 ([email protected]) 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 biochemistry, 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 In recent years, biotechnology-derived production of flavors and fragrances has expanded rapidly. Vanillin, the worlds most popular 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 biosynthetic pathway of vanillin in the vanilla orchid and the possible applied uses of this new knowledge in the biotechnologyderived and pod-based vanillin industries are also highlighted.

VANILLIN AND VANILLA Vanilla is one of the most widely used flavors in the world and is applied extensively in the food, beverage, perfumery, and pharmaceutical industries. Natural vanilla is a complex mixture of flavors extracted from the cured pods of two different species of vanilla orchids: Vanilla planifolia and Vanilla tahitensis (Rao and Ravishankar, 2000). The flavor and fragrance profile of the vanilla extract contains more than 200 components. Vanillin (4-hydroxy3-methoxybenzaldehyde) (Figure 1) is the key component, with a concentration of 1%–2% w/w in cured vanilla pods (Sinha et al., 2008). The vanilla extract obtained from fermented vanilla pods 40

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

possesses a pure and delicate spicy flavor, which is difficult to 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. In 2010, the annual global sales of vanillin reached more than 15 000 000 kg, with less than 1% obtained by isolation from vanilla pods. The production of vanilla beans and the isolation of vanillin from vanilla pods is a laborious and costly process (Sinha et al., 2008). Production of 1 kg of vanillin requires approximately 500 kg of vanilla pods, corresponding to the pollination of approximately 40 000 vanilla orchid flowers. The market cost of natural vanillin derived from vanilla pods is therefore high and

Published by the Molecular Plant Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.

Vanillin–Bioconversion and Bioengineering

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 environmentally 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 microorganisms 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).

Figure 1. Chemical Structure of Vanillin (4-Hydroxy-3Methoxybenzaldehyde).

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 subsequently 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

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, hydrolysis 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). Exturmeric vanillin is marketed by De Monchy Aromatics and is produced 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.

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Vanillin–Bioconversion and Bioengineering

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 metabolic 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 metabolism 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

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for biotechnological production of vanillin. The increasing knowledge 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 microorganisms 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 biosynthesized 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 4hydroxylase (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. pCoumaric 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. Nevertheless, a vast number of microorganisms are able to utilize ferulic acid as their sole carbon energy source. In addition, when microorganisms 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.

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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 formation of 4-hydroxy-3-methoxyphenyl-b-hydroxypropionic acid as a transient intermediate, 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).

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 reductive 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

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Ferulic acid catabolism in Delftia acidovorans (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 4hydroxycinnamoyl-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-3methoxyphenyl-b-hydroxypropionyl-CoA is formed as a transient intermediate and subsequently converted into vanillin and acetylCoA 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-methoxyphenylb-hydroxypropionyl-CoA. Subsequent oxidation results in the formation of 3(40 -hydroxy-3-methoxyphenyl)-3-ketopropionyl-CoA, which, in a CoA-consuming reaction, is cleaved to form vanillylCoA 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

Molecular Plant

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 phenylacrylic 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 4vinylguaiacol is subsequently converted into 4-(1-hydroxy)ethylguaiacol in bacteria. Biochemical evidence for further metabolism 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 proceed by a reductive mechanism. The proposed reaction mechanism involves initial isomerization of ferulic acid to a transient quinoid intermediate, as described above. The quinoid intermediate 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-3methoxy-phenylpropionic acid to 2(40 -hydroxy-30 -methoxyphenyl)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 characterization of the enzymes and genes involved in the individual steps

offer new opportunities for the bioengineering of industrial applicable 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 requires 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 polysaccharidedegrading 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.

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Molecular Plant Substrate

Microorganism

Ferulic acid

Streptomyces setonii

Vanillin–Bioconversion and Bioengineering Length of incubation (h) 23

Pseudomonas putida

Achterholt et al., 2000

>10

Plaggenborg et al., 2003

55 24

5.14

Lee et al., 2009

19.2

Hua et al., 2007a

6

2.52

Barghini et al., 2007

72

2.8

Zheng et al., 2007

Pseudomonas sp. HR199

2–200

2.6

Overhage et al., 2000

Amycolatopsis sp. HR167

32

Bacillus fusiformis SW-B9

72

32.5

Zhao et al., 2005

6

28.3

Yamada et al., 2007

24

16.1

Recombinant E. coli BL21 (DE3) P. putida Bacillus pumilus S-1 Bacillus subtilis HS8

P. chlororaphis CDAE5 Recombinant S. cerevisiae

>10

3.8 24

Overhage et al., 2006

Yamada et al., 2007 Hua et al., 2007b

1.36

Zhang et al., 2006

1.28

Ashengroph et al., 2012

24

1.2

Kasana et al., 2007

24–168

>0.5

Psychrobacter sp. strain CSW4

Glucose

>10

Recombinant E. coli E. coli JM 109

Isoeugenol

References

Streptomyces sp. V-1

Aspergillus niger and Pycnoporus cinnabarinus Eugenol

Yield (g/l)

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 forming Schiff bases and thereby possibly inhibiting enzymatic activity, 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 inhibition 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

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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 heterologously 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 substrates 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. Pseudomonas 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 conversion 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 activities. 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 resulted 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 combined. 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 (LesageMeessen et al., 1996). The influence of the use of different genetic approaches to achieve high gene expression including plasmid copy number and promoters 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 formation 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 number 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. Protocatechuic 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.

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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.

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) encoded 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

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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 flavoprotein oxidase that was first isolated from Penicillium simplicissimum grown on veratryl 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 catabolism 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 Rhodococcus 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. Alternatively, 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 investigated. 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).

Molecular Plant

Vanillin–Bioconversion and Bioengineering A fed-batch bioconversion process from eugenol to coniferyl alcohol by the fungus Byssochlamys fulva V107 has been reported to yield 21.9 g/l coniferyl alcohol within 36 h with a molar yield of 94.6% (Furukawa et al., 1999). Likewise, bioconversion of eugenol to 4-vinylguaiacol was observed in Fusarium solani, with ferulic acid implied to be formed as an intermediate in the conversion, although ferulic acid could not be detected in the medium (Nazareth and Mavinkurve, 1986). Ferulic acid formed via eugenol catabolism in fungi and yeast would be a direct substrate for the synthesis of natural vanillin. S. cerevisiae does not process the ability to convert ferulic acid to vanillin and is known to decarboxylate ferulic acid efficiently to 4-vinylguacol. Nevertheless, a bioengineered pathway could be envisioned using recombinant S. cerevisiae mutants, in which the pad1fad1 genes encoding phenylacrylate decarboxylase (PAD1) and ferulate decarboxylase (FDC1) are disrupted, thus preventing conversion of ferulic acid to 4-vinylguaiacol (Benen et al., 1998). Introduction of the gene encoding vanillin synthase (VpVAN) from the orchid V. planifolia would catalyze the conversion of ferulic acid and ferulic acid glucoside to vanillin and vanillin glucoside, respectively (Gallage et al., 2014).

Bioconversion of Isoeugenol to Vanillin Like eugenol, isoeugenol is also present in the essential oil of the clove tree (Syzygium aromaticum). Bioconversion of isoeugenol to vanillin in both bacteria and fungi has been proposed to occur via an epoxide-diol pathway involving oxidation of the side chain of isoeugenol. Isoeugenol monooxygenase (IEM) encoded by the gene Iem is the enzyme responsible for catalyzing the conversion of isoeugenol to vanillin. The final bioconversion product from isoeugenol is the corresponding substituted benzoic acid. Several studies report industrially viable vanillin production from isoeugenol using bacteria and fungi (Shimoni et al., 2000, 2002; Ashengroph et al., 2010) employing strains tolerant to high levels of isoeugenol (Yamada et al., 2007). P. putida IE27 and Bacillus fusiformis have been reported as efficient converters of isoeugenol into vanillin (Kasana et al., 2007) with B. fusiformis yielding 32.5 g/l vanillin after 72 h incubation. The P. putida IE27 strain produces 16.1 g/l of vanillin following 24 h incubation. The high vanillin production was obtained from continuous addition of isoeugenol to the cultures, which helps to prevent further oxidation of the vanillin formed into vanillic acid (Shimoni et al., 2000, 2002; Zhang et al., 2006; Kasana et al., 2007; Yamada et al., 2007; Ashengroph et al., 2010). Isoeugenol bioconversion to vanillin has also been observed in the fungus, A. niger ATCC 9142 (Abraham et al., 1988). In this study, the yield of vanillin was considerably lower due to further catabolism of vanillin into vanillyl alcohol and vanillic acid. Nonetheless, currently, the yields of vanillin produced from isoeugenol are higher than those obtained from eugenol (see Table 1).

Bioengineering of Eugenol- and Isoeugenol-Derived Vanillin Production in Microorganisms Eugenol- and isoeugenol-derived vanillin production in microorganisms proceeds via ferulic acid as an intermediate. Thus,

eugenol- and isoeugenol-derived vanillin production encounters similar drawbacks as described for ferulic acid. Consequently, only two major issues and the solutions to those are highlighted here; namely, the toxicity caused by eugenol and isoeugenol and the issue of side product formation.

Toxicity Both eugenol and isoeugenol are synthesized in plants as antimicrobial compounds and are toxic at high concentrations (Karapinar, 1990; Koeduka et al., 2006). For that reason, microorganisms have also developed various degradation pathways to maintain the level of eugenol and isoeugenol below a threshold concentration to avoid toxicity and enable proliferation. To circumvent intoxication of the production strains used for vanillin production caused by eugenol and isoeugenol, microbial strains with high tolerance to these compounds were obtained by screening and bioengineering tools were employed to modify the genetic makeup of these strains. One such example is the use of recombinant E. coli BL21(DE3) cells, which do not possess vanillin-degrading activity and express the IEM of P. putida IE27 (Yamada et al., 2008). These recombinant E. coli cells were able to efficiently produce vanillin from isoeugenol without forming vanillic acid as a byproduct.

Side Product Formation As mentioned previously, further degradation of the vanillin formed into by-products constitutes a major challenge in biotechnology-based vanillin production in microorganisms. In eugenol-catabolizing bacteria, two possibilities have been investigated to circumvent this problem. These are based on preventing vanillin degradation by increased pathway flux toward vanillin formation and on continuous removal of the product, vanillin, by different product removal techniques. Increased production of vanillin by over-expression of IEM was studied in isoeugenol and eugenol metabolizing Pseudomonas mitroreducens, which is known to contain the Iem gene and its regulatory protein IemR. IemR was suggested to be a positive transcriptional regulator of Iem. It was suggested that the high level expression and use of multiple copies of Iem and IemR resulted in increased production of vanillin (Ryu et al., 2012) by increasing the metabolic flux toward vanillin. A few attempts have been made to study the potential advantage of using various product removal techniques to prevent vanillin degradation. These involve the use of different resins for product removal. In cultures of P. cinnabarinus grown at bioreactor scale, improved transformation of vanillic acid into vanillin was attempted by binding the toxic vanillin to added absorbents (Topakas et al., 2003). The utilization of cellobiose and adsorbent resins such as Amberlite XAD-2 and Diaion HP20 resulted in only limited improvements in the product recovery process (Stentelaire et al., 1998; Bonnin et al., 2000). In contrast, the use of macroporous adsorbent resins like DM11 gave promising results in fed-batch biotransformation of ferulic acid using Amycolatopsis sp. ATCC 39116 and S. setonii, resulting in vanillin yields of 12 g/l. High concentrations of vanillin, above 10 g/l at 20 C, result in precipitation of crystalline vanillin and offer a convenient method of isolation. Molecular Plant 8, 40–57, January 2015 ª The Author 2015.

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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 demonstrated using both recombinant E. coli and yeasts.

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 cheapest 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 formation in soils was reported (Kunc and Macura, 1966). This 50

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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) encoded by the gene AroZ from the dung mold fungus Podospora anserina. 3-Dehydroshikimic acid is an intermediate in the shikimate 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 protocatechuic 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-methylation 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 challenges 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 precursors or end product.

Molecular Plant for in vivo microbial production of vanillin from glucose resulted in accumulation of protocatechuic acid, vanillic acid, and isovanillic 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, giving 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 isovanillin share similar physicochemical properties. To circumvent this issue, Hansen et al. screened a number of different O-methyltransferases (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).

Toxicity As pointed out previously, vanillin is toxic to living cells in high concentrations. 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 biosynthesis pathways in bacteria and yeast is the possible inefficient or unbalanced activities of enzymes introduced into the production strains. Inefficiency of specific enzymes gives rise to bottlenecks, resulting in the escape and accumulation of intermediates and reduced amounts of the final product. The first reported route

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 introduced 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 oxidation 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.

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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 aromatic aldehydes. The cleavage reaction is catalyzed by lignostilbene-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 phenylpropanoids such as flavonoids, lignin, coumarines, and stilbenes, and some of the enzymes and genes responsible for their formation 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 enzymes 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

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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 aromatic ring. The substrate specificity of the enzyme was demonstrated 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 sequentially by an initial hydration addition reaction followed by a CoAindependent retro-aldol elimination reaction, resulting in the formation 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 propeptide 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 tobacco. The modifications resulted in the formation of increased levels of vanillyl alcohol glucoside in comparison with parallel experiments 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 importance 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 production using microorganisms based on vanillin-like structural compounds, 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 stable integration of expression cassettes required for vanillin synthesis. 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 previous sections, and optimization of these microbial production systems is necessary to meet the increasing demand for biologically 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 biotechnologically produced vanillin in order to gain a competitive market position in relation to chemically synthesized vanillin. The microbial cell factories so far studied for biotechnologyderived 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 previously been comprehensively studied and thus not considered as production hosts. Vanillin production in microorganisms using bioengineering would greatly benefit from identification of microorganisms 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 highlighted 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 dependent 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 implications 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 substrates 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 glucoside 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 biotechnologyderived flavor industry is the desire to establish reliable and economically profitable production systems that are environmentally 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.

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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 manufacturer, 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 produced 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 biotechnological 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 produced product composed of one of the most important flavor compounds of the natural extract. The biotechnologically produced flavors should thus rather be considered and promoted as environmentally benign substitutes for identical compounds currently obtained by large-scale chemical synthesis. Chemically 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 products 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 increase 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. Organizations 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.

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Vanillin–Bioconversion and Bioengineering Received: July 8, 2014 Accepted: September 15, 2014 Published: September 30, 2014

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