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Review

Biotechnological Advances in Vanillin Production: From Natural Vanilla to Metabolic Engineering Platforms

by
Arnold William Tazon
,
Fatima Awwad
,
Fatma Meddeb-Mouelhi
and
Isabel Desgagné-Penix
*
Department of Chemistry, Biochemistry and Physics, Université du Québec à Trois-Rivières, Trois-Rivières, QC G8Z 4M3, Canada
*
Author to whom correspondence should be addressed.
BioChem 2024, 4(4), 323-349; https://doi.org/10.3390/biochem4040017
Submission received: 9 October 2024 / Revised: 5 November 2024 / Accepted: 25 November 2024 / Published: 27 November 2024
(This article belongs to the Special Issue Feature Papers in BioChem)
Browse Figures
Versions Notes

Abstract

:
Vanillin, an aromatic aldehyde, is one of the most popular flavors worldwide, extensively used in the food, cosmetics, pharmaceutical, and agrochemical industries. Despite its widespread use, less than 1% of the total vanillin production is natural, with the majority being synthesized chemically. While chemical synthesis can help to meet the growing demand for vanillin, a strong market trend has rapidly developed for products created from natural ingredients, including natural vanillin. Given the labor-intensive process of extracting vanillin from vanilla pods, there is a critical need for new metabolic engineering platforms to support the biotechnological production of nature-identical vanillin. This review highlights the significance of vanillin in various markets, its diverse applications, and the current state of bio-engineered production using both prokaryotic and eukaryotic biological systems. Although recent advancements have demonstrated successful vanillin production through biocatalytic approaches, our focus was to provide a current and innovative overview of vanillin bioengineering across various host systems with special consideration placed on microalgae, which are emerging as promising platforms for vanillin production through metabolic engineering. The use of these systems to support the biotechnological production of vanillin, while leveraging the photosynthetic capabilities of microalgae to capture CO2 and convert it into biomass, can significantly reduce the overall carbon footprint.

1. Introduction

In recent decades, the demand for flavor and fragrance compounds has surged, driven by the consumer preference for enhanced sensory experiences in food and other products [1]. Vanillin, in particular, has emerged as one of the most widely used flavoring agents worldwide, valued for its distinctive aroma and versatility [2,3]. Consequently, the aroma and fragrance industry has invested heavily in innovative production methods for vanillin to meet this growing demand sustainably and efficiently. Vanillin is the major organoleptic compound of vanilla aroma and is an aromatic aldehyde crystallized in the form of a white powder with a sweet taste and intense aroma [4]. The production of vanillin is possible through natural plant, biotechnological, and chemical processes [5]. However, only natural plant-extracted vanilla and vanillin produced using biotechnological platforms have been approved by the European Food Law (European Directive 88/388/EEC, OJ No. L184, 22 June 1988) and United States of America Food Law [6] as a “natural” food-grade spice [7]. Lately, a strong market trend has developed rapidly for products designed from natural ingredients [8], thus creating a strong demand for nature-identical vanillin, mainly for applications in beverages and foods with therapeutic properties. Currently, the global vanillin market is expected to grow from USD 12.73 billion in 2022 to USD 19.11 billion by 2030, with a Compound Annual Growth Rate (CAGR) of 5.2% over the forecast period between 2022 and 2030 according to the market research company SNS Insider Strategy and Stats [9] (Figure 1).
Natural vanilla and vanillin, although often used interchangeably in everyday contexts, differ significantly in their source, composition, and production methods. Natural vanilla is derived from the beans of the vanilla orchid, involving a labor-intensive process of cultivation, hand-pollination, harvesting, and curing, resulting in a complex mixture of several hundred compounds that contribute to its rich and nuanced flavor profile. In contrast, vanillin is a single chemical compound, 4-hydroxy-3-methoxybenzaldehyde, which can be produced synthetically from precursors like guaiacol, or through biotechnological methods involving microbial fermentation. This simpler composition and more straightforward production process make vanillin more affordable and widely available, albeit with a less complex flavor compared to natural vanilla. Vanillin was isolated by Theodore Nicolas Gobley in 1858 [10]. Today, it is widely coveted by industries mainly for its applications in the areas of agri-food/beverages, perfumery/cosmetology, and medicine. Approximately 60% of industrial vanillin is used in the food/beverage industry, 33% in the perfumery/cosmetics industry, and 7% in the pharmaceutical industry [11].
The production of natural vanilla from vanilla pods is a costly and laborious process; to produce a single kilogram of vanilla, it requires approximately 500 kg of vanilla pods and a manual pollination of about 40,000 flowers [12]. This generates a high cost of natural vanilla on the market, between USD 1200 and 1400 per kg [13], compared to synthetic vanillin that costs less than USD 15 per kg [6,14]. For this reason, the biotechnological or bioengineering production of vanillin is of substantial interest for higher yields and more sustainable alternatives for spice production. As vanillin is derived from aromatic amino acid metabolism, screening Vanilla planifolia plants and other natural producers of vanillin such as Glechoma hederacea, Oryza sativa, and Bruguiera gymnorhiza [4,15,16] for the genes encoding the enzymes that are responsible for vanillin biosynthesis enables the heterologous production of vanillin in other organisms and its optimization into a sustainable high-yield system (Figure 2).
The biotechnological production of vanillin is carried out by the bioconversion of various natural substrates, such as ferulic acid, eugenol, isoeugenol, lignin, and glucose, using metabolically efficient biofactories, such as fungi, yeasts, or bacteria [17]. However, these current production systems often face several challenges, including inefficient metabolism in some cases, as well as cytotoxicity of the reaction products and/or intermediates in the culture media. Additionally, the formation of by-products due to the overexpression of endogenous vanillin dehydrogenase enzymes significantly impacts yield and quality, necessitating further purification steps. This is often viewed as a challenge for manufacturers and a bottleneck for environmental sustainability [7]. However, with the recent advances in genetic engineering, CRISPR-Cas9 technology, and purification techniques, the proportion of vanillin produced from these expression systems is increasingly expanding [3,18,19,20]. In order to support the biotechnological production of high-quality, sustainable, food-grade vanillin, photosynthetic organisms such as microalgae represent an ideal biomanufacturing system. Indeed, photosynthetic microorganisms can grow in the absence of a source of organic carbon. In addition, they can sequester carbon, allowing them to limit the industrial carbon dioxide (CO2) emissions while reducing their impact in terms of greenhouse gases [21]. Several molecules of interest, including recombinant proteins such as pleiotropic antigens, and plant-specialized metabolites such as zeaxanthin, terpenoids, and growth hormones, have already been produced by this type of expression system [22,23,24]. Moreover, with the rapid advancement of metabolic engineering tools and techniques, there has been a surge in the algal production of high-value compounds. This review summarizes the importance of vanillin across various markets (food, perfumery/cosmetics, and pharmaceuticals/agrochemicals) and examines the current biological engineering production systems, including those based on bacteria, yeast, and plants. Special consideration is afforded to microalgae, which are emerging as promising platforms for supporting the biotechnological production of vanillin through metabolic engineering. The manuscript also addresses the challenges associated with implementing microalgal-based vanillin production systems. This work aims to promote the use of these systems to enhance the biotechnological production of vanillin, leveraging the photosynthetic capabilities of microalgae to capture CO2 and convert it into biomass, thereby reducing the overall carbon footprint.

2. Industrial Applications of Vanillin

2.1. Food and Beverage Industry Applications

Flavor is a sensation caused jointly by the taste and smell of a food. This is an organoleptic criterion that is regularly monitored in the agri-food industry in order to meet consumer demands [25]. The iconic and unmistakable flavor of vanillin, often described as sweet, is widely used in cold or baked confectionery, chocolate, pastries, and in the malting industry for its application to therapeutic drinks [11]. More interestingly, vanillin is often used in athletic beverage preparation to mask the bitter taste usually provided by some amino acids, such as arginine [25]. In addition to the human food applications, vanillin is applied as an additive to improve the feeding of chickens while protecting them against gastrointestinal infections [26]. In addition, the benefits of vanillin could be directly applied to the science of climate change. Ruminants (goats and dairy cows) have microorganisms (protozoa, fungi, and bacteria) in their rumen that are capable of fermenting complex carbohydrates to produce simple sugars and some atmospheric gases such as methane [27] which is known to be a potent greenhouse gas that is responsible for climate change [28]. Thus, the addition of vanillin as an ingredient in ruminant feed could improve its taste while inhibiting the growth of microorganisms, thereby limiting the release of methane into the atmosphere.

2.2. Applications in Perfume/Cosmetics

The tasty and pleasant scent of vanillin has applications in perfumery and cosmetology. Vanilla is used as a fragrance ingredient in perfumery formulations and preparations; it masks unpleasant odors and provides a very pleasant aroma [14]. Vanillin is used as a perfume fixative to preserve the appreciated fragrance in time and space. Due to its biological structure, vanillin stimulates the expression of the transcriptional factors (pOct-4, Oct-4, and Nanog) involved in the maintenance of pluripotent cells, the regeneration of epithelial stem cells in humans, and the expression of epithelial adhesive protein (E-cadherin), which helps to maintain the freshness of the body and increases its resistance against skin infections [29,30,31]. These regenerative and antimicrobial properties make vanillin a potential ingredient used in cosmetology and various applications in addition to its pleasant organoleptic properties.

2.3. Bioactivities/Pharmaceutical and Agrochemical Applications

Vanillin is used in the pharmaceutical industry as an excipient for several therapeutic drugs, such as antidepressants and antimicrobials [32,33]. This is the case for the antihypertensive drug methyl-dopamine (mixture consisting of 0.01 M of dopamine, vanillin, and N-methylacetamide, respectively) [34] used to treat hypertension, L-dopamine used to treat Parkinson’s disease, and a few anti-infectious agents such as trimethoprim [35]. In addition, numerous studies have demonstrated the pharmacological properties of vanillin, such as anticancer activity by mediating the antimutagenic potential and DNA damage [36], neuroprotective activity by attenuating biochemical impairments, learning and memory, and locomotor and motor coordination [37,38], antifungal activity through mitochondrial dysfunction [39], and, finally, antiviral and anti-inflammatory activities by neuraminidase inhibition and the modulation of the intracellular signaling pathways in THP-1 cells [40,41]. Moreover, it has recently been demonstrated that vanillin induces osteoblast-mediated bone-forming phenotypes through cell migration and F-actin polymerization [42]. Although vanillin is widely used in the food, pharmaceutical, and cosmetics industries, its application in agro-chemistry for the production of green pesticides has recently attracted significant attention from the scientific community. Indeed, the chemical structure of vanillin, which contains a hydroxyl group and a highly modifiable aldehyde function, enables the addition of a series of bioactive motifs via an ether bridge, reductive amination, and thioacetalization to generate vanillin-derived pesticides [43]. Lou, et al. [44] recently provided a comprehensive summary of the advancements in vanillin-derived pesticides, highlighting how vanillin’s unique properties are being harnessed to create more sustainable and environmentally friendly antiviral pest control solutions. These innovations contribute to the growing interest in green chemistry and sustainable agriculture, offering alternatives to the conventional synthetic pesticides that are often harmful to the environment and human health.
Due to the wide range of vanillin’s applications, farmers, industrialists, and scientists are increasingly interested in the production systems of this highly valuable molecule to ensure its timely availability in large quantities.

3. Vanillin Biosynthesis in Vanilla Plants

Vanillin, or 3-methoxy-4-hydroxybenzaldehyde (C8H8O3), is an aromatic aldehyde found in vanilla pods. Natural vanilla is a spice derived from orchids of the genus Vanilla, mainly obtained from the pods of V. planifolia, V. pompona, and V. tahitensis [7,45]. The de novo biosynthesis of vanillin through the phenylpropanoid pathway in V. planifolia was investigated by Gallage, Hansen, Kannangara, Olsen, Motawia, Jørgensen, Holme, Hebelstrup, Grisoni and Møller [4]. Vanillin biosynthesis involved several steps and enzymes, such as phenylalanine ammonia lyase (PAL; EC 4.3.1.25), cinnamate-4-hydroxylase (C4H; EC 1.14.13.11), 4-hydroxycinnamoyl-CoA ligase (4CL; EC 6.2.1.12), hydroxycinnamoyl transferase (HCT; EC 2.3.1.133), and 4-coumaroyl ester 3′-hydroxylase (C3′H; EC 1.14.13.36) [46,47]. The intermediates obtained from the catalytic reaction of these enzymes are coenzyme A derivatives as well as shikimate- and quinate esters of coumaric acid and caffeic acid [48]. Caffeic acid is a direct precursor of ferulic acid. It is methylated to ferulic acid by the enzyme caffeate O-methyltransferase (COMT; EC 2.1.1.68). Using coupled transcription/translation assays and transient expression in tobacco and yeast, a gene VpVAN encoding an enzyme called vanillin synthase (VpVAN; EC 4.1.2.41), which catalyzes the C2 side chain shortening of ferulic acid and its glucoside to vanillin or vanillin-b-D-glucoside, was identified in the chloroplasts and re-differentiated chloroplasts of V. planifolia. The discovery of this enzyme enabled Gallage, Hansen, Kannangara, Olsen, Motawia, Jørgensen, Holme, Hebelstrup, Grisoni and Møller [4] to elucidate the complete pathway of vanillin biosynthesis (Figure 3). A non-β-oxidative pathway for vanillin biosynthesis was also reported by Podstolski, et al. [49]. In this proposed pathway, 4-coumaric acid is first converted to 4-hydroxybenzaldehyde by 4-hydroxybenzaldehyde synthase (4-HBS; EC number not identified). The 3′-hydroxylation of 4-hydroxybenzaldehyde by the C3′H will then yield 3,4-dihydroxybenzaldehyde followed by the O-methylation of the 3′ hydroxyl group by an O-methyltransferase (OMT) to produce vanillin. Nevertheless, the yield of plant-based vanillin is relatively low, and the extraction and purification present several challenges [50]. In recent decades, the increased knowledge regarding the metabolic pathway and the corresponding enzymes involved in the conversion of various natural substrates, such as ferulic acid, eugenol, isoeugenol, lignin, and glucose, to bio-vanillin has provided new opportunities for bioengineering microorganisms for vanillin biosynthesis. Several authors have reported the biotechnological production of vanillin in plants, bacteria, and yeast, as summarized in Appendix A—Table A1. However, microalgae, as potential expression systems, may offer a promising alternative to support the biotechnological production of vanillin. This review will highlight the importance of these photosynthetic hosts for vanillin production, which remain underexplored compared to non-photosynthetic microorganisms despite their potential for more sustainable and efficient production.

4. Engineering Prokaryotic and Eukaryotic Expression Systems for Sustainable Vanillin Production

Metabolic engineering aims to construct a metabolic pathway of interest in host organisms for the production of high-value-added compounds, thereby enhancing the yield and efficiency for industrial-scale production [51]. Microorganisms such as bacteria and yeast are widely used in metabolic engineering projects since they can be easily genetically modified with the availability of a wide range of molecular tools. This section summarizes the recent advancements and achievements in vanillin production using bacteria, yeast, and plant tissues. It highlights the progress in optimizing the metabolic pathways, enhancing enzyme functionality, and applying innovative genetic engineering techniques to increase the vanillin yield across these biological systems. Additionally, the section addresses the current challenges in optimizing microbial-based vanillin production, such as low yield, metabolic bottlenecks, and strain stability. Potential strategies to overcome these challenges are also explored, including advanced metabolic engineering, adaptive laboratory evolution, and process optimization to improve scalability and commercial viability.

4.1. Engineering Prokaryotic Microorganisms for Vanillin Production

4.1.1. Engineering Escherichia coli

E. coli has been extensively used for the heterologous production of vanillin [52]. In particular, Torre, et al. [53] immobilized the recombinant strain M109/pBB1, which was mutated at the alcohol dehydrogenase (ADH; EC 1.1.1.1) locus and expressed the genes encoding feruloyl-CoA synthetase (FCS; EC 6.2.1.34) and enoyl-CoA hydratase/aldolase (ECH; EC 4.2.1.17) from Pseudomonas fluorescens BF13 in a reactor. This strategy resulted in the production of 0.080 g/L of vanillin from 0.200 g/L of ferulic acid in the immobilized-cell reactor. As a result, the combination of these two genes has been widely employed in E. coli expression systems. This is the case for Barghini, Di Gioia, Fava and Ruzzi [12], who were able to produce 0.53 g/L of vanillin from ferulic acid by co-expressing the same two genes in E coli strain JM109. Further progress in vanillin production using bacteria was realized eight years later by the research of Ni, et al. [54]. In fact, for the de novo production of vanillin in E. coli, this research group partially mimicked the vanillin biosynthetic pathway of the plant V. planifolia using the E. coli strain genetically engineered to overproduce L-tyrosine (Figure 4B). The carbon flux from the primary metabolism of carbohydrates and simple organic compounds, such as glycerol and xyloses, was directed to vanillin via L-tyrosine. This strategy produced 97.2, 19.3, 13.3, and 24.7 mg/L of vanillin from L-tyrosine, glucose, glycerol, and xylose, respectively [54]. The conversion of protocatechuic acid to vanillic acid is a central step in the de novo biosynthesis of vanillin [55]. A central metabolism perturbation designed to increase the flux in the de novo biosynthetic pathway of vanillic acid increased the mean of the de novo vanillate yield from 132 to 205 mg/L [55]. However, protocatechuic acid, the precursor of vanillic acid, remained the dominant biosynthetic intermediate. To maximize this step of converting protocatechuic acid to vanillic acid, Kunjapur, Hyun and Prather [55] demonstrated that supplementation with methyl donor (methionine and homocysteine) improved the vanillin acid production from protocatechuic in E. coli strain DH10B. An artificial pathway for vanillic alcohol biosynthesis from simple carbon sources was proposed by Chen, et al. [56] using the E. coli BW25113 strain. This pathway required only four heterologous enzymes, namely p-hydroxybenzoate hydroxylase (PobA), carboxylic acid reductase (CAR), caffeate O-methyltransferase (COMT), and the CAR maturation factor phosphopantetheinyl transferase (Sfp) (Figure 4A), and led to 0.24 g/L of vanillyl alcohol released into the culture medium [56]. Vanillin can be converted from isoeugenol in one step via the oxidation of the side chains by the enzyme isoeugenol monooxygenase (IEM). The isoeugenol monooxygenase gene from the bacterium Pseudomonas nitroreducens Jin1 was therefore cloned into E. coli [57]. This strain produced 38.34 g/L of vanillin from 49.2 g/L of isoeugenol. More recently, to optimize the yield of vanillyl alcohol production, Yang, et al. [58] mutated the shikimate dehydrogenase (aroE) gene in E. coli strain BW25113. In this study, gene aroE was knocked out to improve 3,4-DHBA biosynthesis of the upstream strain. Using the bioengineering E. coli BW25113 (YMC13/YMC14) co-culture strategy, this strain was able to produce 0.5 and 3.89 g/L of vanillic alcohol in shake-flask and fed-batch fermentations [58].

4.1.2. Engineering Pseudomonas sp.

Pseudomonas sp. has also been used for the production of various plants’ specialized metabolites, including vanillin. To develop an effective strain of Pseudomonas that is capable of producing vanillin with high yields, research from Di Gioia, Luziatelli, Negroni, Ficca, Fava and Ruzzi [29] suppressed, by directed mutagenesis, the gene (vdh) encoding the vanillin dehydrogenase of the Pseudomonas fluorescens BF13 strain. The inactivation associated with the simultaneous expression of the structural genes encoding feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase from a low-copy-number plasmid enabled this strain to produce up to 1.27 g/L of vanillin from ferulic acid [29]. Later, the non-pathogenic strain Pseudomonas putida KT2440 was genetically optimized to convert ferulic acid into vanillin. This genetic modification strategy aimed to enhance the efficiency of the metabolic pathway responsible for converting ferulic acid into vanillin while minimizing the formation of undesirable by-products. This was achieved by downregulating the vanillin dehydrogenase (vdh) and trans-molybdate transporter genes while enhancing the expression of feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase under the tac promoter. As a result, a yield of 1.66 g/L of vanillin was obtained, indicating the success of the genetic modifications in increasing vanillin production while minimizing by-product formation [59].

4.1.3. Engineering Corynebacterium glutamicum

C. glutamicum is a promising host for metabolic engineering studies, mostly for the industrial production of amino acids [60]. However, some publications have demonstrated the capacity of the genetically modified strain to metabolize vanillin. Chen, et al. [61] investigated the ability of the strain in culture to resist high concentrations of vanillin produced from lignocellulosic biomass. A transcriptome analysis revealed that, in addition to stimulating vanillin degradation enzymes, high concentrations of vanillin also caused differential regulation of antioxidant enzymes, secretory proteins, and the cell envelope [61]. These insights may be useful for the engineering of vanillin tolerance strains for suitable production of biofuels from lignocellulosic biomass. Thus, the conversion of ferulic acid to protocatechuic acid was enabled in C. glutamicum ATCC 21420 by cloning the vanillic acid O-demethylase (vanAB) gene of Corynebacterium efficiens NBRC 100395, which converts vanillic acid into protocatechuic acid [62]. This transformed strain was able to produce 1.06 g/L of protocatechuic acid from 3.10 g/L of ferulic acid. Also, an artificial biosynthetic pathway expressing the COMT and CAR genes from Rattus norvegicus and Nocardia iowensis, respectively, has recently been cloned into C. glutamicum [63]. The carbon flux was directed towards vanillin by the deletion of the gene NCgl0324 encoding an aromatic aldehyde reductase. The new strain PV-IYD0324 resulting in the culture produced 0.31 g/L of vanillin from endogenous 4-hydroxylbenzoic acid.

4.1.4. Engineering Pediococcus acidilactici

P. acidilactici is a Gram-positive coccus used as a host for the production of aromatic compounds such as vanillin. This bacterium has several advantages in metabolic engineering, particularly growth at high temperature, pH, and osmotic pressure values [64]. The P. acidilactici BD16 strain transformed by heat shock to express feruloyl-CoA synthetase and the enoyl-CoA hydratase/aldolase genes from Amycolatopsis sp. was cultured in the presence of ferulic acid [65]. When the culture conditions were optimized, 0.47 g/L of vanillin was recovered from 0.21 g/L of ferulic acid per mg of the recombinant cell biomass. Producing vanillin from expensive substrates such as ferulic acid has lower advantages over simple and available substrates such as glucose and industrial waste. Chakraborty, et al. [66] were able to recover 4.01 g/L of vanillin by culturing the recombinant P. acidilactici BD16 strain in the presence of rice bran.
In summary, prokaryotic microorganisms have been widely used for vanillin production by metabolic engineering. E. coli seems to be the best platform so far because few studies have mentioned the presence of vanillin degradation pathways in E. coli. Also, up to 38.3 g/L of vanillin could be produced in the bacteria in a single step using isoeugenol monooxygenase from Pseudomonas nitroreducens Jin1, as shown in Appendix A—Table A1 [57].
However, the expression of vanillin biosynthesis enzymes from plants in prokaryotic systems may, in some cases, result in poor enzyme stability or activity, requiring further optimization to improve the protein folding and expression levels. This issue can potentially be addressed by using eukaryotic expression systems, which typically possess more advanced protein-folding mechanisms and post-translational modifications (PTMs), thereby enhancing enzyme functionality and stability. Post-translational modifications such as glycosylation are essential for the optimal activity of some eukaryotic enzymes, affecting their folding, stability, and ability to interact with other molecules in the biosynthetic pathway [67,68]. When these modifications are absent or insufficient, it can lead to premature degradation of the enzymes, thereby reducing their activity and, consequently, the amount of vanillin produced.

4.2. Engineering Eukaryotic Organisms for Vanillin Production

Eukaryotic organisms, used as expression systems for vanillin production, are mainly yeasts and plant tissues.

4.2.1. Engineering Saccharomyces cerevisiae and Schizosaccharomyces pombe

S. cerevisiae is a yeast that is widely used in industry for the production of high-value-added compounds due to its alcohol tolerance, well-elucidated genome, and fermentation performance [69]. According to the Evolva project, Hansen, et al. [70] studied the ability of the yeast strains S. cerevisiae and S. pombe to produce vanillin from glucose. In this study, the vanillin biosynthetic pathway was constructed using the 3-dehydroshikimate dehydratase genes from the ascomycete Podospora pauciseta, the carboxylic acid reductase from the genus Nocardia, O-methyltransferase from Homo sapiens, and UDP-glycosyltransferase from Arabidopsis thaliana (red arrows in Figure 5A). The efficiency of this pathway was achieved by removing the alcohol dehydrogenase and the vanillin-β-D-glucosidase genes involved in vanillin and vanillin-β-glucoside degradation [71]. Therefore, 0.065 and 0.045g/L of vanillin were produced in S. cerevisiae and S. pombe, respectively, after 48 h of culture. Later, the glycosylated vanillin produced was optimized by Brochado, et al. [72] based on an in silico metabolic engineering algorithm. Indeed, this approach enables the identification of target reactions in a metabolic network whose inactivation would lead to an improvement in vanillin production. Bioinformatics analysis has thus identified pyruvate decarboxylase and glutamate dehydrogenase genes in S. cerevisiae whose suppression is thought to be associated with increased glycosyltransferase activity. The experimental analysis of four strains with two single gene deletions in these genes in the same study showed that three of the strains produced 1.5 times more glycosylated vanillin than the unmutated strains [72]. Plant tissues are mainly composed of lignin, which is a raw material residue obtained from various industrial bioprocesses. Due to its aliphatic and aromatic structures, it is a widely used substrate in the flavor industry [73]. Similarly, Shen, et al. [74] tested the ability of the EMV-8 strain of S. cerevisiae mutated with ethyl methane sulfonate to tolerate high concentrations of vanillin during the lignin biotransformation processes. The use of ethyl methane sulfonate is an engineering strategy that increases the oxidoreductase activity and antioxidant capacity of a microorganism. Such a mutation strategy enabled the EMV-8 strain to maintain a specific growth rate in the presence of 2 g/L of vanillin, in contrast to the reference strain NAN-27, which could not grow in the same conditions because of vanillin cytotoxicity [74]. To explain the redox mechanisms that allow the EMV-8 strain to tolerate high concentrations of vanillin, Liang, et al. [75] identified the different genes expressed and their implications in the mechanisms of vanillin tolerance in S. cerevisiae. Thus, the metabolism of the strain under investigation was characterized in a culture by the overexpression of the mRNA-binding aldo-keto reductase (Gcy1), its Ypr1 paralog, the peroxisome membrane receptor (Pex5), and the multiprotein complex involved in specific growth (Mbf1). However, only the Gcy1 and Ypr1 proteins showed vanillin reductase activities that are dependent on NADPH and NADH [75]. To reproduce the de novo biosynthetic pathway of vanillin from tyrosine in S. cerevisiae, Qiu, et al. [76] developed a recombinant yeast strain by cloning five heterologous enzymes, namely tyrosine ammonia lyase (TAL) from the actinomycete Saccharothrix espanaensis, the combination of three hydroxylase enzymes [(4-coumarate 3-hydroxylase (C3H) from Saccharothrix espanaensis, 4-hydroxyphenylacetate 3-hydroxylase (4HPA3H) from Pseudomonas aeruginosa, and cytochrome P450 monooxygenase (CYP199A2) from Rhodopseudomonas palustris)], caffeate O-methyltransferase (COMT) from Arabidopsis thaliana, trans-feruloyl-CoA synthetase (FCS), and enoyl-CoA hydratase/aldolase (ECH) from Streptomyces sp. (dark blue arrows in Figure 5C). By mutating the ARO4 and ARO7 genes, which encode 3-deoxy-D-arabino heptulosonate-7-phosphate synthase (DAHPS) and chorismate mutase (CM), respectively, into others to limit the negative feedback of tyrosine and increase its flux to vanillin, then by substituting the rate-limiting enzyme tyrosine ammonia lyase (sam8 gene) with 4-coumarate 3-hydroxylase (sam5 gene) to increase the caffeic acid pool, these researchers were able to produce 0.008g/L of vanillin from 0.4 g/L of ferulic acid [76]. Most microorganisms lack the ability to accumulate aldehydes due to the presence of endogenous dehydrogenases and reductases, which facilitate their rapid metabolism and prevent their accumulation in the cells. To overcome this challenge in the case of vanillin production in yeast, Mo and Yuan [20] Mo and Yuan [20] recently developed a minimal aromatic aldehyde reduction (MARE) yeast platform for engineering vanillin production from glucose. They first used a combinatorial gene deletion approach, targeting the adh6, adh7, gre3, gcy1, ydl124w, ypr1, ari1, ydr541c, and aad3 genes, which are critical for vanillin reduction and oxidation. Subsequently, they substituted the hfd1 gene with ubiC to prevent vanillin oxidation and provide the precursor for ubiquinone synthesis, thus enhancing vanillin accumulation while limiting vanillyl alcohol production. Building upon this platform, they further optimized the co-factor supply (NADPH/SAM) and implemented dual precursor synthesis pathways along with metabolic reconfiguration using a phosphoketolase pathway (green arrows in Figure 5C). Through systematic engineering, they achieved a significant milestone of producing 0.36g/L of vanillin, demonstrating the potential of the MARE yeast platform for sustainable vanillin production. Similarly, Xin, et al. [77] meticulously engineered Saccharomyces cerevisiae BY4742, which carried deletions in twenty-two genes associated with vanillin reduction and oxidation [78], to facilitate the conversion of lignin-derived monomers into vanillin. They first reconstructed two pathways separately: the CoA-independent pathway, expressing VpVAN from V. planifolia, and the CoA-dependent non-β-oxidation pathway, expressing 4CL from Petroselinum crispum and Ech from Pseudomonas putida KT2440. In addition, they fused hpaC and hpaB enzymes to enable the simultaneous hydroxylation of p-coumaric acid and p-hydroxy-benzaldehyde into caffeic acid and protocatechualdehyde, respectively. Furthermore, they regulated S-adenosylmethionine metabolism by expressing 3′-O-methyltransferase from Arabidopsis thaliana (AtCOMT), Homo sapiens (HsCOMT), and Nicotiana tabacum (NtCOMT), along with methylenetetrahydrofolate reductase from Arabidopsis thaliana. By also controlling the branch and shikimic acid pathways, the microbial cell factory, employing the CoA-dependent non-β-oxidation pathway, successfully produced 0.29g/L of vanillin from authentic lignocellulosic biomass hydrolysates [77].

4.2.2. Engineering Plant Tissues for the Production of Vanillin

Although most of the studies are limited to the production of vanillin in microorganisms, a few have investigated plant tissues. Specifically, Chee, et al. [80] genetically transformed the pepper (Capsicum frutescens) cells with the vanillin synthetase gene from Vanilla planifolia (VpVAN) by microparticle bombardment. A high-performance liquid chromatography (HPLC) analysis of the extracts from the transformant cells in a culture led to the detection of 5.7 × 10−3 g/L of vanillin, which was sixteen times higher than the control [80]. An amount of 0.54 g of vanillin per gram of fresh callus culture was also produced from rice embryonic cells transformed by Agrobacterium to express the codon optimized sequence of vanillin synthetase [81]. However, it is important to note that the enzymatic activity of this enzyme is subject to several controversies, and no kinetic parameters such as catalytic activity (Kcat) and Michaelis constant (Km) have yet been reported [82,83]. The progress of plant-tissue-culture-based vanillin synthesis has not been commercially viable and has been hindered by factors such as long culture duration, low yields, and increased by-product formation [3].

5. Current Challenges and Potential Strategies for Enhancing Microbial-Based Vanillin Production Systems

Despite the notable progress in microbial-based vanillin production, several challenges remain. One of the primary hurdles involves the low conversion efficiency and yield, largely due to the cytotoxicity of vanillin and its intermediates to host cells. This toxicity often impairs cell growth and enzyme functionality, limiting production [84]. Indeed, vanillin concentrations exceeding 0.5–1 g/L have been shown to significantly inhibit microbial growth, thereby reducing the final product [70]. Additionally, by-product formation during the biosynthesis process complicates the downstream purification, reducing the overall efficiency and increasing the production costs [50]. Another challenge is the metabolic burden on microorganisms engineered for vanillin production. Complex metabolic pathways, often involving multiple genetic modifications and CRISPR-Cas9 gene knockouts, can reduce cellular fitness, leading to lower productivity [85]. This strain on cellular resources often results in slower growth rates, decreased metabolic efficiency, and suboptimal vanillin yields, further complicating the scaling of biotechnological vanillin production for industrial applications. To address these challenges, potential strategies include metabolic pathway optimization by fine-tuning the pathways through adaptive laboratory evolution or optimizing the gene expression to enhance the flux toward vanillin biosynthesis, balancing precursor availability, co-factor regeneration, and improving enzyme activity [20,77,84,86]. Another approach is tolerance engineering, where microbial strains are engineered to increase their tolerance to vanillin and its intermediates, achieved through the overexpression of efflux pumps or modification of cellular stress response mechanisms to mitigate the cytotoxic effects of vanillin on microbial cells. Efflux pumps actively transport vanillin and its toxic intermediates out of the cell, reducing the intracellular concentrations and minimizing cellular damage [3]. Additionally, enhancing the stress response pathways can bolster the cell’s ability to tolerate and recover from vanillin-induced stress, leading to improved growth and productivity [75]. These strategies are crucial for overcoming cytotoxicity, which is one of the major challenges in microbial-based vanillin production [3,72,87]. Enzyme engineering, using rational or directed evolution-based methods, can improve catalytic efficiency and reduce the formation of unwanted by-products, thereby enhancing yield and purity [86,88]. Diversifying host systems by exploring non-traditional microbial hosts, such as photosynthetic microalgae, presents a promising alternative for vanillin production, potentially lowering costs and integrating CO2 fixation, which contributes to a more sustainable bioproduction platform.

6. Green Biology: Harnessing Microalgae as Natural Producers of Vanillin Precursors and Promising Hosts for Biotechnological Vanillin Production

Metabolically engineered photosynthetic microorganisms, such as microalgae, can offer several advantages as potential hosts for vanillin production. First, their ability to perform photosynthesis allows them to utilize CO2 as a carbon source, reducing the reliance on external feedstocks and contributing to carbon sequestration [89,90]. This makes the process more sustainable compared to non-photosynthetic microorganisms. Additionally, microalgae can produce high-value precursors and metabolites relevant to vanillin biosynthesis, potentially enhancing the overall yield. Moreover, it has been documented that the cost associated with biotechnology production in microalgae is much cheaper compared to animal cells and even lower than that of microbes and plants [91].

6.1. Microalgae as Natural Producers of Vanillin Precursors

Microalgae, with their remarkable metabolic versatility, have developed intricate biochemical pathways that enable them to produce a wide range of specialized metabolites, including valuable phenolic compounds [92]. The biosynthesis of phenolic compounds in microalgae is a complex and highly regulated process, involving several metabolic pathways that enable the production of bioactive molecules that are essential for the survival and adaptation of microalgae to their environment [93,94]. This justifies the fact that Phaeodactylum tricornutum was able to produce various vanillin precursors and metabolites, such as coumaric acid, caffeic acid, ferulic acid, protocatechuic acid, gallic acid, and vanillic acid, from in vitro cultures grown in natural seawater containing elevated levels of copper (Cu) and iron (Fe) metals [93]. The presence of these metals likely enhanced the biosynthetic pathways involved in the production of phenolic compounds, including those that serve as precursors for vanillin synthesis. This demonstrates the potential of diatoms like P. tricornutum as biotechnological platforms for producing valuable aromatic compounds such as vanillin. Moreover, Bhuvana, et al. [95], using an HPLC method, detected peaks corresponding to caffeic acid derivatives in methanolic extracts of Nannochloropsis oculata and Chlorella vulgaris. Further, Haematococcus pluvialis was fed with vanillin precursors such as ferulic acid, coniferyl aldehyde, and p-coumaric acid by Tripathi, et al. [96], resulting in the production of vanillin (0.010 g/L), vanillyl alcohol (0.003 g/L), vanillic acid (0.005 g/L), protocatechuic acid (0.001 g/L), and p-hydroxybenzoic acid (0.001 g/L) in free and immobilized cell cultures. Together, these data provide compelling evidence for the presence of the putative enzymes involved in the synthesis of vanillin, its precursors, and metabolites in microalgae, as evaluated and outlined in the following Table 1. Indeed, we identified potential homologs of vanillin biosynthetic enzymes in microalgae by comparing the sequences to those already characterized in V. planifolia (VpVAN), Streptomyces sp. V-1 (FCS and ECH), and Pseudomonas nitroreducens Jin1 (IEM). An immature VpVAN candidate displaying the cathepsin propeptide inhibitor (I29), peptidase_C1, and granulin domains was identified in the protein databases of various microalgae (Table 1). Additionally, a putative FCS similar to long-chain fatty acyl-CoA synthetases was identified in these microalgae species. This protein contains domains such as the fatty acyl-CoA synthetase domain, a dimer interface polypeptide, an acyl-activating enzyme (AAE) consensus motif, as well as AMP and CoA binding sites. Since the CoA-dependent non-β-oxidation pathway involves both FCS and ECH, a putative ECH with a crotonase-like domain, a substrate binding site, oxyanion hole (OAH)-forming residues, and a trimer interface polypeptide binding site was also found. However, for the isoeugenol monooxygenase pathway, only three of these microalgae species (C. reinhardtii, H. pluvialis, and C. vulgaris) present a putative IEM, characterized by a retinal pigment epithelial membrane protein domain. These putative proteins in microalgae could play roles similar to those in the vanillin biosynthesis pathway in V. planifolia or in bacteria. Indeed, phenolic compounds are primarily synthesized through the shikimic acid pathway, a biochemical route present in most plants and microorganisms [97,98,99]. This pathway, which leads to the formation of phenylpropanoid acid precursors like L-phenylalanine, could serve as the upstream mechanism for the biosynthesis of various vanillin precursors. The key enzyme typically initiating phenolic compound biosynthesis is phenylalanine ammonia-lyase (PAL), which catalyzes the deamination of L-phenylalanine to form cinnamic acid, a crucial precursor of vanillin. However, recent studies have shown that not all microalgal taxa possess the PAL enzyme [94]. This absence suggests that microalgae may utilize alternative pathways for phenolic compound biosynthesis, potentially opening up other routes for the production of vanillin precursors via different enzymatic mechanisms. Such alternative strategies highlight the metabolic diversity within microalgae, offering distinct advantages for engineering approaches aimed at optimizing vanillin production.

6.2. Engineering and Modulation of Robust Microalgal-Based Vanillin Production Systems

The engineering of robust microalgal-based systems for vanillin production represents a promising frontier in biotechnology, aimed at utilizing the metabolic flexibility of microalgae for sustainable vanillin biosynthesis. This development involves the integration of advanced genetic engineering techniques to optimize the metabolic pathways responsible for producing vanillin and its precursors. The key strategies include introducing or enhancing the pathways involved in the synthesis of phenylpropanoid acids such as ferulic acid, p-coumaric acid, and caffeic acid, which serve as essential precursors for vanillin production [7]. Genetic engineering techniques, including the CRISPR-Cas9 and transgenic approaches, are widely employed to modify the key enzymes and regulatory elements in the microalgal genome [102,103]. These modifications may enable more precise control of the metabolic flux toward vanillin production, minimizing the formation of unwanted by-products while maximizing efficiency.
A significant challenge in engineering microalgae for vanillin production is the efficient expression of heterologous genes within their complex cellular environment. Research efforts have been undertaken to introduce vanillin biosynthesis pathways into microalgae like P. tricornutum. In that case, the researchers attempted to propagate the full vanillin biosynthesis pathway from V. planifolia into P. tricornutum through an episomal system, incorporating multiple key enzymes required for vanillin synthesis [104]. While the pathway introduction was successful at the mRNA level, issues such as episomal instability and difficulties in protein expression hindered the actual vanillin production [105]. Indeed, episomal plasmids are commonly used in genetic engineering to introduce new pathways into host cells without altering the cell’s native genome [106,107,108]. However, these plasmids can be unstable, especially under prolonged culture conditions or selective pressures, leading to the gradual loss of the plasmid from the cell population. To overcome these challenges, researchers can explore strategies to improve the expression of heterologous genes, such as optimizing the gene integration into stable genomic locations, enhancing the promoter efficiency, and fine-tuning the expression systems [109,110]. Advances in algal synthetic biology tools, including modular plasmid systems and improved transformation techniques, are enabling more precise and efficient engineering of microalgae. Microalgae like C. reinhardtii offer a highly versatile platform for genetic manipulation, with extensive molecular tools and mutant libraries available [111,112]. Both the chloroplast and nuclear genomes of C. reinhardtii can be engineered to express vanillin biosynthesis enzymes, providing flexibility in targeting specific metabolic compartments for efficient vanillin production. Therefore, this approach enables the genetic modification of the nuclear genome (with the genes encoding enzymes that require N-glycosylation for enhanced activity) or the chloroplast genome (with the genes encoding enzymes that do not require N-glycosylation). This facilitates the reconstruction of partial vanillin biosynthesis pathways from V. planifolia or known bacterial pathways, ultimately leading to the development of industrial strains of microalgae with high vanillin yields.
Furthermore, the integration of efficient downstream processing techniques is critical to developing robust microalgal vanillin production systems. The extraction and purification of vanillin from microalgal biomass must be cost-effective and efficient to maximize the yield. Innovative approaches, such as the use of biocompatible solvents, microwave-assisted extraction, pulsed electric fields, supercritical fluids, crystallization, and membrane-based separation technologies, are currently being explored to streamline the recovery of vanillin from engineered microbial and plant-based vanillin production systems [50]. These techniques can also be adapted to microalgae, further enhancing their potential as a sustainable and scalable source of vanillin. By optimizing these extraction methods, the industrial viability of microalgal-based vanillin production could be significantly improved, ensuring high-purity vanillin at reduced processing costs and energy consumption. Ultimately, a vanillin-tolerant microalgae could be optimized for resistance to redox products, enabling the accumulation of vanillin without undergoing reduction or oxidation processes. Then, the optimized designed expression system carrying the vanillin biosynthesis pathway described in V. planifolia or the bacterial pathway can be used to engineer the nuclear and/or chloroplast genome of the tolerant microalgae. Finally, the optimized transformants are then cultivated on a large scale to produce vanillin for the cosmetics, food, and pharmaceutical industries.

7. Conclusions and Perspectives

This review has explored the potential of leveraging biotechnological advances to engineer microbial, plant, and algal systems for sustainable vanillin production. As the consumer demand for natural and eco-friendly ingredients grows, the biotechnological synthesis of vanillin offers a promising alternative to the traditional methods reliant on vanilla beans or petrochemical routes. We discussed and highlighted the production and industrial application of vanillin by deciphering the current knowledge regarding the key enzymes involved in vanillin biosynthesis. The insights gained from microbial and plant systems have already established a foundation for understanding vanillin biosynthesis. Advances in metabolic engineering have enabled the development of genetically modified microbial strains capable of efficiently producing vanillin and its precursors, although challenges such as yield optimization and pathway stability remain.
Microalgae have emerged as a promising platform for high-value molecule production owing to their metabolic diversity and capacity for large-scale cultivation. This unique biological system not only presents opportunities for vanillin biosynthesis but also offers added environmental benefits, such as carbon dioxide capture. While substantial progress has been achieved in the metabolic engineering of microalgae for the production of high-value molecules, the full potential of microalgae for vanillin production is yet to be realized. The complexity of heterologous gene expression in these systems remains a critical challenge. Researchers must continue to refine the gene integration strategies, enhance the promoter efficiency, and fine-tune the expression systems to ensure stable and high-yield production. The scaling-up of vanillin production also poses challenges. Although laboratory-level successes have demonstrated the feasibility of non-photosynthetic microorganism systems for vanillin production, scaling these systems for commercial applications will require further optimization of the growth conditions, bioreactor designs, and downstream processing methods.
Looking ahead, several key areas will drive future research into biotechnological vanillin production using microalgae. First, a deeper understanding of microalgal metabolism, particularly in the context of vanillin biosynthesis, will enable more targeted and efficient metabolic engineering. Exploring the evolutionary diversity of microalgal species could uncover novel biosynthetic pathways and enzymes that further improve the efficiency and yield of vanillin production. Second, the integration of omics technologies, including genomics, transcriptomics, proteomics, and metabolomics, will provide valuable insights into the regulation of the vanillin biosynthesis pathways and the identification of bottlenecks that limit production. Collaboration between academia and industry will be crucial to accelerating the development of scalable, cost-effective production platforms. The growing interest in bio-based vanillin from the food, cosmetics, pharmaceutical, and agrochemical industries will drive investment in research and development. By addressing the challenges of gene expression, pathway optimization, and process scalability, it is likely that microbial and algal systems will become viable alternatives for sustainable vanillin production in the near future.

Author Contributions

Conceptualization, A.W.T. and I.D.-P.; validation, A.W.T., F.A., F.M.-M. and I.D.-P.; writing—original draft preparation, A.W.T.; writing—review and editing, A.W.T., F.A., F.M.-M. and I.D.-P.; visualization, A.W.T. and F.A.; supervision, I.D.-P.; project administration, F.M.-M. and I.D-P.; funding acquisition, I.D.-P. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Université du Québec à Trois-Rivières—Annexe C program to I.D.-P.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in the study are included in the review and appendix; further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Appendix A

Table A1. Metabolic engineering of prokaryotic and eukaryotic organisms for the production of vanillin.
Table A1. Metabolic engineering of prokaryotic and eukaryotic organisms for the production of vanillin.
Engineering PlatformGenes (Origin)Bioengineering Strategies
Transformation Insertion Type Mutations
Method		Substrates (g/L)Complexity ProductVolume of Culture (mL)Fermentation Time (h)Yield
(g/L)		Ref.
Escherichia coli BL21(DE3)IEM (Pseudomonas nitroreducens Jin1)Heat shockEpisomalNone49.2 Isoeugenol2VanillinNI1638.3[57]
Escherichia coli BL21(DE3)IEM (Pseudomonas putida IE27)NIGenome integrationNone37.7 Isoeugenol2Vanillin104828.3[113]
Escherichia coli BL21 (DE3)IEM (microbial metagenome)NININone16.4 Isoeugenol2Vanillin15482.2[114]
Escherichia coli JM109fcs and ech (Pseudomonas fluorescens BF13)NININone0.64 Ferulic acid4VanillinNI60.53[12]
Escherichia coli FR13fcs and ech (Pseudomonas fluorescens BF13)NIGenome integrationNone4.5 Ferulic acid4Vanillin15244.3[115]
Escherichia coli BW25113PobA (Pseudomonas aeruginosa), CAR (Mycobacterium marinum), sfp (Bacillus subtilis), and comt (Arabidopsis thaliana)NININone1 3,4-dihydroxy benzoyl alcohol.4Vanillyl alcohol20480.24[56]
Escherichia coli K-12 MG1655CAR (Nocardia iowensis), sfp (Bacillus subtilis), comt (Homo sapiens), DSD (Bacillus thuringiensis)P1 bacteriophage transductionGenome integrationDeletion of yqhC-dkgA genesProtocatechuic acid4Vanillate50720.004[55]
Pseudomonas fluorescens BF13fcs and ech (Pseudomonas fluorescens BF13)Heat shockNIDeletion of vdh gene0.48 Ferulic acid 4Vanillin2000241.27[29]
Escherichia colital (Saccharothrix espanaensis), sam5 (actinomycete), comt (Arabidopsis thaliana), sam8 (Saccharothrix espanaensis), fcs, and ech (Streptomyces sp.)Co-transformationGenome integrationDeletion of TyrR gene L-Tyrosine6Vanillin50120.013–0.097 [54]
Escherichia coli MG1655 RARELCC (unknown prokaryote), TPADO (Comamonas sp.), DCDDH (Comamonas sp.), O-MT (Rattus norvegicus), and CAR (Nocardia iowensis).Heat shockNINoneMonomer terephthalic acid6Vanillin10240.11[116]
Escherichia coli BW25113PobA (Pseudomonas aeruginosa), CAR (Mycobacterium marinum), sfp (Bacillus subtilis), and COMT (Arabidopsis thaliana)Heat shockNIDeletion of aroE geneND6Vanillyl alcohol50
and
3000		2
and
24		3.89 and 0.55[58]
Escherichia colifcs and ech (Amycolatopsis sp.)NININoneGlucose6VanillinNI480.50[117]
Pediococcus acidilactici BD16fcs and ech (Amycolatopsis sp.)Heat shockEpisomalNone0.21 Ferulic Acid per mg of recombinant cell biomass4Vanillin1000.330.47[65]
Pediococcus acidilactici BD16fcs and ech (Pediococcus acidilactici BD16)Heat shockEpisomalNoneRice bran4Vanillin50244.01[66]
Pseudomonas putida KT2440Pp-SpuC and ATA (Chromobacterium), adh (Bacillus)ElectroporationGenome integrationNoneLignin-derived substrates4Vanillin and vanillylamine50 and
100		240.10 and 0.13[118]
Pseudomonas putida KT2440fcs and ech (Pseudomonas putida)ElectroporationGenome integrationDeletion of vdh and modABC genes1.94 Ferulic acid4Vanillin531.66[59]
Corynebacterium glutamicum ATCC 21420vanAB (Corynebacterium efficiens NBRC 100395)ElectroporationGenome integrationNone3.1 Ferulic Acid4Protocatechuic acid400241.06[62]
Corynebacterium glutamicum PV-IYΔ0324comt (Rattus norvegicus) and CAR (Nocardia iowensis)NIGenome integrationDeletion of NCgl0324 geneEndogenous 4-Hydroxylbenzoic acid6Vanillin25480.31[63]
Amycolatopsis sp. ATCC 39116fcs and ech (Amycolatopsis sp.)Direct mycelium transformationGenome integrationDeletion of vdh gene5 Glucose6Vanillin20002022.3[119]
Saccharomyces cerevisiae and Schizosaccharomyces pombe3DSD (Podospora pauciseta), ACAR (Nocardia sp.), O-MT (Homo sapiens), and UGT (Arabidopsis thaliana)Lithium acetate/polyethylene glycol/single carrier DNAGenome integrationDeletion of adh6 gene Glucose 6Vanillin5480.065 and 0.045[70]
Saccharomyces cerevisiae VAN286UGT (Arabidopsis thaliana)NINIDeletion of pdc1 and gdh1 genes Glucose6Vanillin-β-D-glucosideNI900.50[72]
Saccharomyces cerevisiaeSam8 (Actinomycete), sam5/ hpaB/ CYP199A2(Saccharothrix espanaensis/Pseudomonas aeruginosa/Rhodopseudomonas palustris), comt (Arabidopsis thaliana), fcs, and ech (Streptomyces sp.).Lithium acetate/polyethylene glycol/single carrier DNAGenome integrationDeletion of Aro4 and Aro7 genes 0.4 ferulic acid6Vanillin50960.008[76]
Saccharomyces cerevisiae S288c and CEN.PKACAR (Neurospora sp.), EntD (Escherichia coli), UGT (Arabidopsis thaliana), OMT (Homo sapiens), and 3DSD (Podospora pauciseta)Lithium acetate/polyethylene glycol/single carrier DNAGenome integrationDeletion of Bgl1 and adh6 genes 20 Glucose6Vanillin-β-D-glucoside 50452[79]
Saccharomyces cerevisiae BY4741AroZ (Podospora anserina), OMT (Homo sapiens), CAR (Segniliparus Rotundus), GapC (Clostridium acetobutylicum), Pos5c (S. cerevisiae), Sfp (Bacillus subtilis), EntD (E. coli), MetK (E. coli), hpaB (Pseudomonas aeruginosa), hpaC (Salmonella enterica), Xfpk (Bifidobacterium breve), CkPta (Clostridium kluyveri), HmaS (A. orientalis), HMO (S. coelicolor A3), and BFD (P. putida KT2440)ElectroporationCombinaison of both episomal expression and genome integrationDeletion of adh6, adh7, gre3, gcy1, ydl124w, ypr1, ari1, ydr541c, aad3, hfd1, AKR, and AL DR family genesGlucose4Vanillin101200.36[20]
Saccharomyces cerevisiae BY47424CL (Petroselinum crispum), Ech (Pseudomonas putida KT2440), VpVAN (V. planifolia), hpaB(Pseudomonas aeruginosa), hpaC (Salmonella enterica), PobA (Pseudomonas putida KT2440), COMT (Arabidopsis thaliana), METHER (Arabidopsis thaliana), and PsVAO (Penicillium simplicissimum)NIGenome integrationDeletion of adh6, adh7, bdh2, bdh1, hfd1, pdc5, aro10,
pha2, pgm1, trp2, pad1, ahfd1, AKR, and ALDR family genes		lignocellulosic biomass6Vanillin50240.29[77]
Oryza sativaVpVAN (V. planifolia)Agrobacterium-mediated transformationGenome integrationNoneEndogenous ferulic Acid 4VanillinNINI0.00054[81]
Capsicum frutescensVpVAN (V. planifolia)Microparticle bombardmentGenome integrationNoneEndogenous ferulic Acid 4VanillinNINI0.00057[80]
Abbreviations: TAL (sam 8): tyrosine ammonia lyase; C3H (sam5): 4-coumarate 3-hydroxylase; COMT: caffeate O-methyltransferase; fcs: feruloyl-CoA synthetase; ech: enoyl-CoA hydratase/aldolase; vdh: vanillin dehydrogenase; IEM: isoeugenol monooxygenase; PobA: 4-hydroxybenzoate hydroxylase; CAR: carboxyl acid reductase; Pp-SpuC: putrescine transaminase; ATA: amine transaminase; adh: alcohol dehydrogenase; sfp: phosphopantetheinyl transferase; COMT: catechol O-methyltransferase; 3DSD: 3-dehydroshikimate dehydratase; LCC: leaf–branch compost cutinase; TPADO: terephthalate 1,2-dioxygenase; DCDDH: 1,4-dicarboxylic acid dehydrogenase; OMT: O-methyltransferase; aroE: shikimate dehydrogenase; vanAB: vanillate O-demethylase; ACAR: aryl carboxylic acid reductase; UGT: UDP-glycosyltransferase; ADH6: alcohol dehydrogenase; Pdc1: pyruvate decarboxylase; gdh1: glutamate dehydrogenase; 4HPA3H (hpaB and C): 4- hydroxyphenylacetate 3-hydroxylase;CYP199A2: cytochrome P450 monooxygenase; DAHP: 3-deoxy-D-arabino heptulosonate-7-phosphate synthase; CHA: chorismate mutase; EntD: phosphopantetheine transferase; BGL1: beta-glucosidase; VpVAN: vanillin synthetase; NCgl0324: aromatic aldehyde reductase; 4CL: hydroxycinnamic acid CoA ligase; METHER: methylenetetrahydrofolate reductase; PsVAO: vanillyl alcohol oxidase; bdh1 and 2: 3-hydroxybutyrate dehydrogenase1 and 2; hfd1: fatty aldehyde deshydrogenase; pha2: prephenate dehydratase 2; pgm1: phosphoglucomutase 1; trp2: DOPAchrome tautomerase; pad1: phenylacrylic acid decarboxylase; AKR: aldo-keto reductases; ALDR: adrenoleukodystrophy-related; gcy1: glycerol 2-dehydrogenase; ari1: carbonyl reductase; ydr541c: aldehyde reductase; aad3: aryl-alcohol dehydrogenase; HpaBC: flavin-dependent monooxygenase; HMO: hydroxymandelate oxidase; BFD: benzoylformate decarboxylase. NI: not identified. The numbers 2, 4, and 6 represent the level of complexity of the heterologous pathway; 2 is less complex; 4 is moderately complex; and 6 is very complex. COMT in red is to differentiate the catechol O-methyltransferase from the caffeate O-methyltransferase.

References

  1. Spence, C. Odour hedonics and the ubiquitous appeal of vanilla. Nat. Food 2022, 3, 837–846. [Google Scholar] [CrossRef] [PubMed]
  2. Furuya, T.; Miura, M.; Kuroiwa, M.; Kino, K. High-yield production of vanillin from ferulic acid by a coenzyme-independent decarboxylase/oxygenase two-stage process. New Biotechnol. 2015, 32, 335–339. [Google Scholar] [CrossRef] [PubMed]
  3. Xu, L.; Liaqat, F.; Sun, J.; Khazi, M.I.; Xie, R.; Zhu, D. Advances in the vanillin synthesis and biotransformation: A review. Renew. Sustain. Energy Rev. 2024, 189, 113905. [Google Scholar] [CrossRef]
  4. Gallage, N.J.; Hansen, E.H.; Kannangara, R.; Olsen, C.E.; Motawia, M.S.; Jørgensen, K.; Holme, I.; Hebelstrup, K.; Grisoni, M.; Møller, B.L. Vanillin formation from ferulic acid in Vanilla planifolia is catalysed by a single enzyme. Nat. Commun. 2014, 5, 4037. [Google Scholar] [CrossRef] [PubMed]
  5. Taira, J.; Toyoshima, R.; Ameku, N.; Iguchi, A.; Tamaki, Y. Vanillin production by biotransformation of phenolic compounds in fungus, Aspergillus luchuensis. AMB Express 2018, 8, 40. [Google Scholar] [CrossRef]
  6. Krings, U.; Berger, R.G. Biotechnological production of flavours and fragrances. Appl. Microbiol. Biotechnol. 1998, 49, 1–8. [Google Scholar] [CrossRef]
  7. Gallage, N.J.; Moller, B.L. Vanillin-bioconversion and bioengineering of the most popular plant flavor and its de novo biosynthesis in the vanilla orchid. Mol. Plant 2015, 8, 40–57. [Google Scholar] [CrossRef]
  8. Omokhua-Uyi, A.G.; Van Staden, J. Natural product remedies for COVID-19: A focus on safety. S. Afr. J. Bot. 2021, 139, 386–398. [Google Scholar] [CrossRef]
  9. SNS, Vanillin Market Size, Share and Segmentation by Product Type (Natural, Synthetic Vanillin), by End-Use (Food & Beverage, Fragrance, Pharmaceutical), by Regions and Global Market Forecast 2023–2030. Microorganisms. 2022. Available online: https://www.snsinsider.com/reports/vanillin-market-2244 (accessed on 25 November 2024).
  10. Hocking, M.B. Vanillin: Synthetic Flavoring from Spent Sulfite Liquor. J. Chem. Educ. 1997, 74, 1055. [Google Scholar] [CrossRef]
  11. Zhou, N.; Thilakarathna, W.P.D.W.; He, Q.S.; Rupasinghe, H.P.V. A Review: Depolymerization of Lignin to Generate High-Value Bio-Products: Opportunities, Challenges, and Prospects. Front. Energy Res. 2022, 9, 758744. [Google Scholar] [CrossRef]
  12. Barghini, P.; Di Gioia, D.; Fava, F.; Ruzzi, M. Vanillin production using metabolically engineered Escherichia coli under non-growing conditions. Microb. Cell Fact. 2007, 6, 13. [Google Scholar] [CrossRef] [PubMed]
  13. Braga, A.; Guerreiro, C.E.; Belo, I. Generation of Flavors and Fragrances Through Biotransformation and De Novo Synthesis. Food Bioprocess. Technol. 2018, 11, 2217–2228. [Google Scholar] [CrossRef]
  14. Muheim, A.; Lerch, K. Towards a high-yield bioconversion of ferulic acid to vanillin. Appl. Microbiol. Biotechnol. 1999, 51, 456–461. [Google Scholar] [CrossRef]
  15. Chou, T.H.; Ding, H.Y.; Hung, W.J.; Liang, C.H. Antioxidative characteristics and inhibition of alpha-melanocyte-stimulating hormone-stimulated melanogenesis of vanillin and vanillic acid from Origanum vulgare. Exp. Dermatol. 2010, 19, 742–750. [Google Scholar] [CrossRef] [PubMed]
  16. Kundu, A.; Mitra, A. Flavoring extracts of Hemidesmus indicus roots and Vanilla planifolia pods exhibit in vitro acetylcholinesterase inhibitory activities. Plant Foods Hum. Nutr. 2013, 68, 247–253. [Google Scholar] [CrossRef]
  17. Ma, Q.; Liu, L.; Zhao, S.; Huang, Z.; Li, C.; Jiang, S.; Li, Q.; Gu, P. Biosynthesis of vanillin by different microorganisms: A review. World J. Microbiol. Biotechnol. 2022, 38, 40. [Google Scholar] [CrossRef]
  18. Huang, Y.; Yang, Y.; Xue, J.; Liao, Y.; Fu, X.; Zhu, C.; Li, J.; Zeng, L.; Yang, Z. Biosynthetic Pathway and Bioactivity of Vanillin, a Highly Abundant Metabolite Distributed in the Root Cortex of Tea Plants (Camellia sinensis). J. Agric. Food Chem. 2024, 72, 1660–1673. [Google Scholar] [CrossRef]
  19. Xu, L.; Liaqat, F.; Khazi, M.I.; Sun, J.; Zhu, D. Natural deep eutectic solvents-based green extraction of vanillin: Optimization, purification, and bioactivity assessment. Front. Nutr. 2024, 10, 1279552. [Google Scholar] [CrossRef]
  20. Mo, Q.; Yuan, J. Minimal aromatic aldehyde reduction (MARE) yeast platform for engineering vanillin production. Biotechnol. Biofuels Bioprod. 2024, 17, 4. [Google Scholar] [CrossRef]
  21. Jassey, V.E.J.; Hamard, S.; Lepère, C.; Céréghino, R.; Corbara, B.; Küttim, M.; Leflaive, J.; Leroy, C.; Carrias, J.-F. Photosynthetic microorganisms effectively contribute to bryophyte CO2 fixation in boreal and tropical regions. ISME Commun. 2022, 2, 64. [Google Scholar] [CrossRef]
  22. Cordero, B.F.; Couso, I.; León, R.; Rodríguez, H.; Vargas, M.A. Enhancement of carotenoids biosynthesis in Chlamydomonas reinhardtii by nuclear transformation using a phytoene synthase gene isolated from Chlorella zofingiensis. Appl. Microbiol. Biotechnol. 2011, 91, 341–351. [Google Scholar] [CrossRef] [PubMed]
  23. Shamriz, S.; Ofoghi, H. Expression of Recombinant PfCelTOS Antigen in the Chloroplast of Chlamydomonas reinhardtii and its Potential Use in Detection of Malaria. Mol. Biotechnol. 2019, 61, 102–110. [Google Scholar] [CrossRef] [PubMed]
  24. Wang, Q.; Zhuang, J.; Ni, S.; Luo, H.; Zheng, K.; Li, X.; Lan, C.; Zhao, D.; Bai, Y.; Jia, B.; et al. Overexpressing CrePAPS Polyadenylate Activity Enhances Protein Translation and Accumulation in Chlamydomonas reinhardtii. Mar. Drugs 2022, 20, 276. [Google Scholar] [CrossRef]
  25. Passariello, C.L.; Marchionni, S.; Carcuro, M.; Casali, G.; Della Pasqua, A.; Hrelia, S.; Malaguti, M.; Lorenzini, A. The Mediterranean Athlete’s Nutrition: Are Protein Supplements Necessary? Nutrients 2020, 12, 3681. [Google Scholar] [CrossRef] [PubMed]
  26. Mohammadi Gheisar, M.; Hosseindoust, A.; Kim, I.H. Evaluating the effect of microencapsulated blends of organic acids and essential oils in broiler chickens diet. J. Appl. Poult. Res. 2015, 24, 511–519. [Google Scholar] [CrossRef]
  27. Pereira, A.M.; de Lurdes Nunes Enes Dapkevicius, M.; Borba, A.E.S. Alternative pathways for hydrogen sink originated from the ruminal fermentation of carbohydrates: Which microorganisms are involved in lowering methane emission? Anim. Microbiome 2022, 4, 5. [Google Scholar] [CrossRef]
  28. Malone, S.L.; Oh, Y.; Arndt, K.A.; Burba, G.; Commane, R.; Contosta, A.R.; Goodrich, J.P.; Loescher, H.W.; Starr, G.; Varner, R.K. Gaps in network infrastructure limit our understanding of biogenic methane emissions for the United States. Biogeosciences 2022, 19, 2507–2522. [Google Scholar] [CrossRef]
  29. Di Gioia, D.; Luziatelli, F.; Negroni, A.; Ficca, A.G.; Fava, F.; Ruzzi, M. Metabolic engineering of Pseudomonas fluorescens for the production of vanillin from ferulic acid. J. Biotechnol. 2011, 156, 309–316. [Google Scholar] [CrossRef]
  30. Lee, J.; Cho, J.Y.; Lee, S.Y.; Lee, K.W.; Lee, J.; Song, J.Y. Vanillin protects human keratinocyte stem cells against ultraviolet B irradiation. Food Chem. Toxicol. 2014, 63, 30–37. [Google Scholar] [CrossRef]
  31. Loh, Y.H.; Wu, Q.; Chew, J.L.; Vega, V.B.; Zhang, W.; Chen, X.; Bourque, G.; George, J.; Leong, B.; Liu, J.; et al. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nat. Genet. 2006, 38, 431–440. [Google Scholar] [CrossRef]
  32. Paul, V.; Rai, D.C.; Ramyaa Lakshmi, T.S.; Srivastava, S.K.; Tripathi, A.D. A comprehensive review on vanillin: Its microbial synthesis, isolation and recovery. Food Biotechnol. 2021, 35, 22–49. [Google Scholar] [CrossRef]
  33. Arya, S.S.; Rookes, J.E.; Cahill, D.M.; Lenka, S.K. Vanillin: A review on the therapeutic prospects of a popular flavouring molecule. Adv. Tradit. Med. 2021, 21, 1–17. [Google Scholar] [CrossRef]
  34. Mani, A.; Ahamed, A.; Ali, D.; Alarifi, S.; Akbar, I. Dopamine-Mediated Vanillin Multicomponent Derivative Synthesis via Grindstone Method: Application of Antioxidant, Anti-Tyrosinase, and Cytotoxic Activities. Drug Des. Devel. Ther. 2021, 15, 787–802. [Google Scholar] [CrossRef] [PubMed]
  35. Olatunde, A.; Mohammed, A.; Ibrahim, M.A.; Tajuddeen, N.; Shuaibu, M.N. Vanillin: A food additive with multiple biological activities. Eur. J. Med. Chem. Rep. 2022, 5, 100055. [Google Scholar] [CrossRef]
  36. Bezerra, D.P.; Soares, A.K.; de Sousa, D.P. Overview of the Role of Vanillin on Redox Status and Cancer Development. Oxid. Med. Cell Longev. 2016, 2016, 9734816. [Google Scholar] [CrossRef]
  37. Kim, H.J.; Hwang, I.K.; Won, M.H. Vanillin, 4-hydroxybenzyl aldehyde and 4-hydroxybenzyl alcohol prevent hippocampal CA1 cell death following global ischemia. Brain Res. 2007, 1181, 130–141. [Google Scholar] [CrossRef]
  38. Yan, X.; Liu, D.F.; Zhang, X.Y.; Liu, D.; Xu, S.Y.; Chen, G.X.; Huang, B.X.; Ren, W.Z.; Wang, W.; Fu, S.P.; et al. Vanillin Protects Dopaminergic Neurons against Inflammation-Mediated Cell Death by Inhibiting ERK1/2, P38 and the NF-κB Signaling Pathway. Int. J. Mol. Sci. 2017, 18, 389. [Google Scholar] [CrossRef]
  39. Kim, J.H.; Lee, H.-O.; Cho, Y.-J.; Kim, J.; Chun, J.; Choi, J.; Lee, Y.; Jung, W.H. A vanillin derivative causes mitochondrial dysfunction and triggers oxidative stress in Cryptococcus neoformans. PLoS ONE 2014, 9, e89122. [Google Scholar] [CrossRef]
  40. Hariono, M.; Abdullah, N.; Damodaran, K.; Kamarulzaman, E.E.; Mohamed, N.; Hassan, S.S.; Shamsuddin, S.; Wahab, H.A. Potential new H1N1 neuraminidase inhibitors from ferulic acid and vanillin: Molecular modelling, synthesis and in vitro assay. Sci. Rep. 2016, 6, 38692. [Google Scholar] [CrossRef]
  41. Zhao, D.; Jiang, Y.; Sun, J.; Li, H.; Huang, M.; Sun, X.; Zhao, M. Elucidation of The Anti-Inflammatory Effect of Vanillin In Lps-Activated THP-1 Cells. J. Food Sci. 2019, 84, 1920–1928. [Google Scholar] [CrossRef]
  42. Yun, H.M.; Kim, E.; Kwon, Y.J.; Park, K.R. Vanillin Promotes Osteoblast Differentiation, Mineral Apposition, and Antioxidant Effects in Pre-Osteoblasts. Pharmaceutics 2024, 16, 485. [Google Scholar] [CrossRef] [PubMed]
  43. Gendron, D. Vanillin: A Promising Biosourced Building Block for the Preparation of Various Heterocycles. Front. Chem. 2022, 10, 949355. [Google Scholar] [CrossRef] [PubMed]
  44. Lou, M.; Li, S.; Jin, F.; Yang, T.; Song, R.; Song, B. Pesticide Engineering from Natural Vanillin: Recent Advances and a Perspective. Engineering 2024. [Google Scholar] [CrossRef]
  45. Jiang, W.; Chen, X.; Feng, Y.; Sun, J.; Jiang, Y.; Zhang, W.; Xin, F.; Jiang, M. Current Status, Challenges, and Prospects for the Biological Production of Vanillin. Fermentation 2023, 9, 389. [Google Scholar] [CrossRef]
  46. Funk, C.; Brodelius, P.E. Phenylpropanoid Metabolism in Suspension Cultures of Vanilla planifolia Andr. 1: IV. Induction of Vanillic Acid Formation. Plant Physiol. 1992, 99, 256–262. [Google Scholar] [CrossRef]
  47. Schoch, G.; Goepfert, S.; Morant, M.; Hehn, A.; Meyer, D.; Ullmann, P.; Werck-Reichhart, D. CYP98A3 from Arabidopsis thaliana is a 3’-hydroxylase of phenolic esters, a missing link in the phenylpropanoid pathway. J. Biol. Chem. 2001, 276, 36566–36574. [Google Scholar] [CrossRef]
  48. Kundu, A. Vanillin biosynthetic pathways in plants. Planta 2017, 245, 1069–1078. [Google Scholar] [CrossRef]
  49. Podstolski, A.; Havkin-Frenkel, D.; Malinowski, J.; Blount, J.W.; Kourteva, G.; Dixon, R.A. Unusual 4-hydroxybenzaldehyde synthase activity from tissue cultures of the vanilla orchid Vanilla planifolia. Phytochemistry 2002, 61, 611–620. [Google Scholar] [CrossRef]
  50. Liaqat, F.; Xu, L.; Khazi, M.I.; Ali, S.; Rahman, M.U.; Zhu, D. Extraction, purification, and applications of vanillin: A review of recent advances and challenges. Ind. Crops Prod. 2023, 204, 117372. [Google Scholar] [CrossRef]
  51. Zhao, M.L.; Cai, W.S.; Zheng, S.Q.; Zhao, J.L.; Zhang, J.L.; Huang, Y.; Hu, Z.L.; Jia, B. Metabolic Engineering of the Isopentenol Utilization Pathway Enhanced the Production of Terpenoids in Chlamydomonas reinhardtii. Mar. Drugs 2022, 20, 577. [Google Scholar] [CrossRef]
  52. Converti, A.; Aliakbarian, B.; Domínguez, J.M.; Bustos Vázquez, G.; Perego, P. Microbial production of biovanillin. Braz. J. Microbiol. 2010, 41, 519–530. [Google Scholar] [CrossRef] [PubMed]
  53. Torre, P.; De Faveri, D.; Perego, P.; Ruzzi, M.; Barghini, P.; Gandolfi, R.; Converti, A. Bioconversion of ferulate into vanillin by Escherichia coli strain JM109/pBB1 in an immobilized-cell reactor. Ann. Microbiol. 2004, 54, 517–527. [Google Scholar]
  54. Ni, J.; Tao, F.; Du, H.; Xu, P. Mimicking a natural pathway for de novo biosynthesis: Natural vanillin production from accessible carbon sources. Sci. Rep. 2015, 5, 13670. [Google Scholar] [CrossRef] [PubMed]
  55. Kunjapur, A.M.; Hyun, J.C.; Prather, K.L. Deregulation of S-adenosylmethionine biosynthesis and regeneration improves methylation in the E. coli de novo vanillin biosynthesis pathway. Microb. Cell Fact. 2016, 15, 61. [Google Scholar] [CrossRef] [PubMed]
  56. Chen, Z.; Shen, X.; Wang, J.; Wang, J.; Zhang, R.; Rey, J.F.; Yuan, Q.; Yan, Y. Establishing an Artificial Pathway for De Novo Biosynthesis of Vanillyl Alcohol in Escherichia coli. ACS Synth. Biol. 2017, 6, 1784–1792. [Google Scholar] [CrossRef]
  57. Wang, Q.; Wu, X.; Lu, X.; He, Y.; Ma, B.; Xu, Y. Efficient Biosynthesis of Vanillin from Isoeugenol by Recombinant Isoeugenol Monooxygenase from Pseudomonas nitroreducens Jin1. Appl. Biochem. Biotechnol. 2021, 193, 1116–1128. [Google Scholar] [CrossRef]
  58. Yang, M.; Meng, H.; Li, X.; Wang, J.; Shen, X.; Sun, X.; Yuan, Q. Coculture engineering for efficient production of vanillyl alcohol in Escherichia coli. aBIOTECH 2022, 3, 292–300. [Google Scholar] [CrossRef]
  59. Graf, N.; Altenbuchner, J. Genetic engineering of Pseudomonas putida KT2440 for rapid and high-yield production of vanillin from ferulic acid. Appl. Microbiol. Biotechnol. 2014, 98, 137–149. [Google Scholar] [CrossRef]
  60. Park, S.H.; Kim, H.U.; Kim, T.Y.; Park, J.S.; Kim, S.-S.; Lee, S.Y. Metabolic engineering of Corynebacterium glutamicum for L-arginine production. Nat. Commun. 2014, 5, 4618. [Google Scholar] [CrossRef]
  61. Chen, C.; Pan, J.; Yang, X.; Guo, C.; Ding, W.; Si, M.; Zhang, Y.; Shen, X.; Wang, Y. Global Transcriptomic Analysis of the Response of Corynebacterium glutamicum to Vanillin. PLoS ONE 2016, 11, e0164955. [Google Scholar] [CrossRef]
  62. Okai, N.; Masuda, T.; Takeshima, Y.; Tanaka, K.; Yoshida, K.I.; Miyamoto, M.; Ogino, C.; Kondo, A. Biotransformation of ferulic acid to protocatechuic acid by Corynebacterium glutamicum ATCC 21420 engineered to express vanillate O-demethylase. AMB Express 2017, 7, 130. [Google Scholar] [CrossRef]
  63. Kim, H.S.; Choi, J.A.; Kim, B.Y.; Ferrer, L.; Choi, J.M.; Wendisch, V.F.; Lee, J.H. Engineered Corynebacterium glutamicum as the Platform for the Production of Aromatic Aldehydes. Front. Bioeng. Biotechnol. 2022, 10, 880277. [Google Scholar] [CrossRef] [PubMed]
  64. Mora, D.; Fortina, M.G.; Parini, C.; Manachini, P.L. Identification of Pediococcus acidilactici and Pediococcus pentosaceus based on 16S rRNA and ldhD gene-targeted multiplex PCR analysis. FEMS Microbiol. Lett. 1997, 151, 231–236. [Google Scholar] [CrossRef] [PubMed]
  65. Kaur, B.; Chakraborty, D.; Kumar, B. Metabolic engineering of Pediococcus acidilactici BD16 for production of vanillin through ferulic acid catabolic pathway and process optimization using response surface methodology. Appl. Microbiol. Biotechnol. 2014, 98, 8539–8551. [Google Scholar] [CrossRef] [PubMed]
  66. Chakraborty, D.; Selvam, A.; Kaur, B.; Wong, J.W.C.; Karthikeyan, O.P. Application of recombinant Pediococcus acidilactici BD16 (fcs+/ech+) for bioconversion of agrowaste to vanillin. Appl. Microbiol. Biotechnol. 2017, 101, 5615–5626. [Google Scholar] [CrossRef]
  67. Scheper, A.F.; Schofield, J.; Bohara, R.; Ritter, T.; Pandit, A. Understanding glycosylation: Regulation through the metabolic flux of precursor pathways. Biotechnol. Adv. 2023, 67, 108184. [Google Scholar] [CrossRef]
  68. Bryan, L.; Clynes, M.; Meleady, P. The emerging role of cellular post-translational modifications in modulating growth and productivity of recombinant Chinese hamster ovary cells. Biotechnol. Adv. 2021, 49, 107757. [Google Scholar] [CrossRef]
  69. Liu, Z.L.; Ma, M.; Song, M. Evolutionarily engineered ethanologenic yeast detoxifies lignocellulosic biomass conversion inhibitors by reprogrammed pathways. Mol. Genet. Genom. 2009, 282, 233–244. [Google Scholar]
  70. Hansen, E.H.; Møller, B.L.; Kock, G.R.; Bünner, C.M.; Kristensen, C.; Jensen, O.R.; Okkels, F.T.; Olsen, C.E.; Motawia, M.S.; Hansen, J. De novo biosynthesis of vanillin in fission yeast (Schizosaccharomyces pombe) and baker’s yeast (Saccharomyces cerevisiae). Appl. Env. Microbiol. 2009, 75, 2765–2774. [Google Scholar] [CrossRef]
  71. Moehs, C.P.; Allen, P.V.; Friedman, M.; Belknap, W.R. Cloning and expression of solanidine UDP-glucose glucosyltransferase from potato. Plant J. 1997, 11, 227–236. [Google Scholar] [CrossRef]
  72. Brochado, A.R.; Matos, C.; Møller, B.L.; Hansen, J.; Mortensen, U.H.; Patil, K.R. Improved vanillin production in baker’s yeast through in silico design. Microb. Cell Factories 2010, 9, 84. [Google Scholar] [CrossRef] [PubMed]
  73. Zamzuri, N.A.A.; Abd-Aziz, S. Biovanillin from agro wastes as an alternative food flavour. J. Sci. Food Agric. 2013, 93, 429–438. [Google Scholar] [CrossRef] [PubMed]
  74. Shen, Y.; Li, H.; Wang, X.; Zhang, X.; Hou, J.; Wang, L.; Gao, N.; Bao, X. High vanillin tolerance of an evolved Saccharomyces cerevisiae strain owing to its enhanced vanillin reduction and antioxidative capacity. J. Ind. Microbiol. Biotechnol. 2014, 41, 1637–1645. [Google Scholar] [CrossRef] [PubMed]
  75. Liang, Z.; Wang, X.; Bao, X.; Wei, T.; Hou, J.; Liu, W.; Shen, Y. Newly identified genes contribute to vanillin tolerance in Saccharomyces cerevisiae. Microb. Biotechnol. 2021, 14, 503–516. [Google Scholar] [CrossRef] [PubMed]
  76. Qiu, D.; Wang, M.; Zhou, C.; Zhao, J.; Zhang, G. De novo biosynthesis of vanillin in engineered Saccharomyces cerevisiae. Chem. Eng. Sci. 2022, 263, 118049. [Google Scholar] [CrossRef]
  77. Xin, X.; Zhang, R.-K.; Liu, S.-C.; He, Z.-J.; Liu, R.-Y.; Lan, H.-N.; Liu, Z.-H.; Li, B.-Z.; Yuan, Y.-J. Engineering yeast to convert lignocellulose into vanillin. Chem. Eng. J. 2024, 485, 149815. [Google Scholar] [CrossRef]
  78. Chen, R.; Gao, J.; Yu, W.; Chen, X.; Zhai, X.; Chen, Y.; Zhang, L.; Zhou, Y.J. Engineering cofactor supply and recycling to drive phenolic acid biosynthesis in yeast. Nat. Chem. Biol. 2022, 18, 520–529. [Google Scholar] [CrossRef]
  79. Strucko, T.; Magdenoska, O.; Mortensen, U.H. Benchmarking two commonly used Saccharomyces cerevisiae strains for heterologous vanillin-β-glucoside production. Metab. Eng. Commun. 2015, 2, 99–108. [Google Scholar] [CrossRef]
  80. Chee, M.J.; Lycett, G.W.; Khoo, T.J.; Chin, C.F. Bioengineering of the Plant Culture of Capsicum frutescens with Vanillin Synthase Gene for the Production of Vanillin. Mol. Biotechnol. 2017, 59, 1–8. [Google Scholar] [CrossRef]
  81. Arya, S.S.; Mahto, B.K.; Sengar, M.S.; Rookes, J.E.; Cahill, D.M.; Lenka, S.K. Metabolic Engineering of Rice Cells with Vanillin Synthase Gene (VpVAN) to Produce Vanillin. Mol. Biotechnol. 2022, 64, 861–872. [Google Scholar] [CrossRef]
  82. Diamond, A.; Barnabé, S.; Desgagné-Penix, I. Is a spice missing from the recipe? The intra-cellular localization of vanillin biosynthesis needs further investigations. Plant Biol. 2022, 25, 3–7. [Google Scholar] [CrossRef] [PubMed]
  83. Yang, H.; Barros-Rios, J.; Kourteva, G.; Rao, X.; Chen, F.; Shen, H.; Liu, C.; Podstolski, A.; Belanger, F.; Havkin-Frenkel, D.; et al. A re-evaluation of the final step of vanillin biosynthesis in the orchid Vanilla planifolia. Phytochemistry 2017, 139, 33–46. [Google Scholar] [CrossRef] [PubMed]
  84. Liu, Y.; Sun, L.; Huo, Y.-X.; Guo, S. Strategies for improving the production of bio-based vanillin. Microb. Cell Factories 2023, 22, 147. [Google Scholar] [CrossRef] [PubMed]
  85. Mao, J.; Zhang, H.; Chen, Y.; Wei, L.; Liu, J.; Nielsen, J.; Chen, Y.; Xu, N. Relieving metabolic burden to improve robustness and bioproduction by industrial microorganisms. Biotechnol. Adv. 2024, 74, 108401. [Google Scholar] [CrossRef]
  86. Chen, Q.; Jiang, Y.; Kang, Z.; Cheng, J.; Xiong, X.; Hu, C.Y.; Meng, Y. Engineering a Feruloyl–Coenzyme A Synthase for Bioconversion of Phenylpropanoid Acids into High-Value Aromatic Aldehydes. J. Agric. Food Chem. 2022, 70, 9948–9960. [Google Scholar] [CrossRef]
  87. Pattrick, C.A.; Webb, J.P.; Green, J.; Chaudhuri, R.R.; Collins, M.O.; Kelly, D.J. Proteomic Profiling, Transcription Factor Modeling, and Genomics of Evolved Tolerant Strains Elucidate Mechanisms of Vanillin Toxicity in Escherichia coli. mSystems 2019, 4. [Google Scholar] [CrossRef]
  88. Fujimaki, S.; Sakamoto, S.; Shimada, S.; Kino, K.; Furuya, T. Engineering a coenzyme-independent dioxygenase for one-step production of vanillin from ferulic acid. Appl. Environ. Microbiol. 2024, 90, e0023324. [Google Scholar] [CrossRef]
  89. Zeng, X.; Danquah, M.K.; Chen, X.D.; Lu, Y. Microalgae bioengineering: From CO2 fixation to biofuel production. Renew. Sustain. Energy Rev. 2011, 15, 3252–3260. [Google Scholar] [CrossRef]
  90. Kselíková, V.; Singh, A.; Bialevich, V.; Čížková, M.; Bišová, K. Improving microalgae for biotechnology—From genetics to synthetic biology—Moving forward but not there yet. Biotechnol. Adv. 2022, 58, 107885. [Google Scholar] [CrossRef]
  91. Hammerschmidt, N.; Tscheliessnig, A.; Sommer, R.; Helk, B.; Jungbauer, A. Economics of recombinant antibody production processes at various scales: Industry-standard compared to continuous precipitation. Biotechnol. J. 2014, 9, 766–775. [Google Scholar] [CrossRef]
  92. Cichonski, J.; Chrzanowski, G. Microalgae as a Source of Valuable Phenolic Compounds and Carotenoids. Molecules 2022, 27, 8852. [Google Scholar] [CrossRef] [PubMed]
  93. Rico, M.; López, A.; Santana-Casiano, J.M.; Gonzàlez, A.G.; Gonzàlez-Dàvila, M. Variability of the phenolic profile in the diatom Phaeodactylum tricornutum growing under copper and iron stress. Limnol. Oceanogr. 2013, 58, 144–152. [Google Scholar] [CrossRef]
  94. Del Mondo, A.; Sansone, C.; Brunet, C. Insights into the biosynthesis pathway of phenolic compounds in microalgae. Comput. Struct. Biotechnol. J. 2022, 20, 1901–1913. [Google Scholar] [CrossRef] [PubMed]
  95. Bhuvana, P.; Sangeetha, P.; Anuradha, V.; Ali, M.S. Spectral characterization of bioactive compounds from microalgae: N. oculata and C. vulgaris. Biocatal. Agric. Biotechnol. 2019, 19, 101094. [Google Scholar] [CrossRef]
  96. Tripathi, U.; Ramachandra Rao, S.; Ravishankar, G.A. Biotransformation of phenylpropanoid compounds to vanilla flavor metabolites in cultures of Haematococcus pluvialis. Process Biochem. 2002, 38, 419–426. [Google Scholar] [CrossRef]
  97. Liyanage, N.S.; Awwad, F.; Gonçalves dos Santos, K.C.; Jayawardena, T.U.; Mérindol, N.; Desgagné-Penix, I. Navigating Amaryllidaceae alkaloids: Bridging gaps and charting biosynthetic territories. J. Exp. Bot. 2024, erae187. [Google Scholar] [CrossRef]
  98. Desgagné-Penix, I. Biosynthesis of alkaloids in Amaryllidaceae plants: A review. Phytochem. Rev. 2021, 20, 409–431. [Google Scholar] [CrossRef]
  99. Tzin, V.; Galili, G. New Insights into the Shikimate and Aromatic Amino Acids Biosynthesis Pathways in Plants. Mol. Plant 2010, 3, 956–972. [Google Scholar] [CrossRef]
  100. Yang, W.; Tang, H.; Ni, J.; Wu, Q.; Hua, D.; Tao, F.; Xu, P. Characterization of two Streptomyces enzymes that convert ferulic acid to vanillin. PLoS ONE 2013, 8, e67339. [Google Scholar] [CrossRef]
  101. Ryu, J.-Y.; Seo, J.; Park, S.; Ahn, J.-H.; Chong, Y.; Sadowsky, M.J.; Hur, H.-G. Characterization of an Isoeugenol Monooxygenase (Iem) from Pseudomonas nitroreducens Jin1 That Transforms Isoeugenol to Vanillin. Biosci. Biotechnol. Biochem. 2013, 77, 289–294. [Google Scholar] [CrossRef]
  102. Jeong, B.-r.; Jang, J.; Jin, E. Genome engineering via gene editing technologies in microalgae. Bioresour. Technol. 2023, 373, 128701. [Google Scholar] [CrossRef] [PubMed]
  103. Patel, V.K.; Das, A.; Kumari, R.; Kajla, S. Recent progress and challenges in CRISPR-Cas9 engineered algae and cyanobacteria. Algal Res. 2023, 71, 103068. [Google Scholar] [CrossRef]
  104. Slattery, S.S.; Diamond, A.; Wang, H.; Therrien, J.A.; Lant, J.T.; Jazey, T.; Lee, K.; Klassen, Z.; Desgagné-Penix, I.; Karas, B.J.; et al. An Expanded Plasmid-Based Genetic Toolbox Enables Cas9 Genome Editing and Stable Maintenance of Synthetic Pathways in Phaeodactylum tricornutum. ACS Synth. Biol. 2018, 7, 328–338. [Google Scholar] [CrossRef]
  105. Diamond, A.; Diaz-Garza, A.M.; Li, J.; Slattery, S.S.; Merindol, N.; Fantino, E.; Meddeb-Mouelhi, F.; Karas, B.J.; Barnabé, S.; Desgagné-Penix, I. Instability of extrachromosomal DNA transformed into the diatom Phaeodactylum tricornutum. Algal Res. 2023, 70, 102998. [Google Scholar] [CrossRef]
  106. Roig-Merino, A.; Urban, M.; Bozza, M.; Peterson, J.D.; Bullen, L.; Büchler-Schäff, M.; Stäble, S.; van der Hoeven, F.; Müller-Decker, K.; McKay, T.R.; et al. An episomal DNA vector platform for the persistent genetic modification of pluripotent stem cells and their differentiated progeny. Stem Cell Rep. 2022, 17, 143–158. [Google Scholar] [CrossRef]
  107. Wade-Martins, R.; James, M.R.; Frampton, J. Long-term stability of large insert genomic DNA episomal shuttle vectors in human cells. Nucleic Acids Res. 1999, 27, 1674–1682. [Google Scholar] [CrossRef]
  108. Chen, Y.; Partow, S.; Scalcinati, G.; Siewers, V.; Nielsen, J. Enhancing the copy number of episomal plasmids in Saccharomyces cerevisiae for improved protein production. FEMS Yeast Res. 2012, 12, 598–607. [Google Scholar] [CrossRef]
  109. Suttangkakul, A.; Sirikhachornkit, A.; Juntawong, P.; Puangtame, W.; Chomtong, T.; Srifa, S.; Sathitnaitham, S.; Dumrongthawatchai, W.; Jariyachawalid, K.; Vuttipongchaikij, S. Evaluation of strategies for improving the transgene expression in an oleaginous microalga Scenedesmus acutus. BMC Biotechnol. 2019, 19, 4. [Google Scholar] [CrossRef]
  110. Cagney, M.H.; O’Neill, E.C. Strategies for producing high value small molecules in microalgae. Plant Physiol. Biochem. 2024, 214, 108942. [Google Scholar] [CrossRef]
  111. Einhaus, A.; Baier, T.; Kruse, O. Molecular design of microalgae as sustainable cell factories. Trends Biotechnol. 2024, 42, 728–738. [Google Scholar] [CrossRef]
  112. Naduthodi, M.I.S.; Claassens, N.J.; D’Adamo, S.; van der Oost, J.; Barbosa, M.J. Synthetic Biology Approaches To Enhance Microalgal Productivity. Trends Biotechnol. 2021, 39, 1019–1036. [Google Scholar] [CrossRef] [PubMed]
  113. Yamada, M.; Okada, Y.; Yoshida, T.; Nagasawa, T. Vanillin production using Escherichia coli cells over-expressing isoeugenol monooxygenase of Pseudomonas putida. Biotechnol. Lett. 2008, 30, 665–670. [Google Scholar] [CrossRef]
  114. Zhao, L.; Xie, Y.; Chen, L.; Xu, X.; Zhao, C.X.; Cheng, F. Efficient biotransformation of isoeugenol to vanillin in recombinant strains of Escherichia coli by using engineered isoeugenol monooxygenase and sol-gel chitosan membrane. Process Biochem. 2018, 71, 76–81. [Google Scholar] [CrossRef]
  115. Luziatelli, F.; Brunetti, L.; Ficca, A.G.; Ruzzi, M. Maximizing the Efficiency of Vanillin Production by Biocatalyst Enhancement and Process Optimization. Front. Bioeng. Biotechnol. 2019, 7, 279. [Google Scholar] [CrossRef] [PubMed]
  116. Sadler, J.C.; Wallace, S. Microbial synthesis of vanillin from waste poly(ethylene terephthalate). Green Chem. 2021, 23, 4665–4672. [Google Scholar] [CrossRef] [PubMed]
  117. Yoon, S.-H.; Li, C.; Kim, J.-E.; Lee, S.-H.; Yoon, J.-Y.; Choi, M.-S.; Seo, W.-T.; Yang, J.-K.; Kim, J.-Y.; Kim, S.-W. Production of Vanillin by Metabolically Engineered Escherichia coli. Biotechnol. Lett. 2005, 27, 1829–1832. [Google Scholar] [CrossRef]
  118. Manfrão-Netto, J.H.C.; Lund, F.; Muratovska, N.; Larsson, E.M.; Parachin, N.S.; Carlquist, M. Metabolic engineering of Pseudomonas putida for production of vanillylamine from lignin-derived substrates. Microb. Biotechnol. 2021, 14, 2448–2462. [Google Scholar] [CrossRef]
  119. Fleige, C.; Meyer, F.; Steinbüchel, A. Metabolic Engineering of the Actinomycete Amycolatopsis sp. Strain ATCC 39116 towards Enhanced Production of Natural Vanillin. Appl. Environ. Microbiol. 2016, 82, 3410–3419. [Google Scholar] [CrossRef]
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Figure 1. Vanillin market size reported by SNS Insider in 2022 [9].
Figure 1. Vanillin market size reported by SNS Insider in 2022 [9].
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Figure 2. Current and future approaches for biologically engineered hosts for the production of vanillin. The black arrows indicate current approaches, while red arrows represent future approaches.
Figure 2. Current and future approaches for biologically engineered hosts for the production of vanillin. The black arrows indicate current approaches, while red arrows represent future approaches.
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Figure 3. Vanillin and vanillin-β-D-glucoside biosynthesis pathways in V. planifolia pods. L-phenylalanine from the shikimate pathway will undergo a series of reactions to yield ferulic acid, which will subsequently be cleaved into vanillin by the enzyme vanillin synthetase according to Gallage, Hansen, Kannangara, Olsen, Motawia, Jørgensen, Holme, Hebelstrup, Grisoni and Møller [4]. As present in red according to Podstolski, Havkin-Frenkel, Malinowski, Blount, Kourteva and Dixon [49], 4-coumaric acid can be directly converted to 4-hydroxybenzaldehyde followed by 3′ hydroxylation and O-methylation to yield vanillin. Abbreviations: COMT: caffeate O-methyltransferase; PAL: phenylalanine ammonia lyase; OMT: O-methyltransferase; 4HBS: 4-hydroxybenzaldehyde synthase; C4H: cinnamate 4-hydroxylase; 4CL: 4-hydroxycinnamoyl-CoA ligase; HCT: hydroxycinnamoyl transferase; C3′H: coumaroyl ester 3′-hydroxylase; and VpVAN: V. planifolia vanillin synthase.
Figure 3. Vanillin and vanillin-β-D-glucoside biosynthesis pathways in V. planifolia pods. L-phenylalanine from the shikimate pathway will undergo a series of reactions to yield ferulic acid, which will subsequently be cleaved into vanillin by the enzyme vanillin synthetase according to Gallage, Hansen, Kannangara, Olsen, Motawia, Jørgensen, Holme, Hebelstrup, Grisoni and Møller [4]. As present in red according to Podstolski, Havkin-Frenkel, Malinowski, Blount, Kourteva and Dixon [49], 4-coumaric acid can be directly converted to 4-hydroxybenzaldehyde followed by 3′ hydroxylation and O-methylation to yield vanillin. Abbreviations: COMT: caffeate O-methyltransferase; PAL: phenylalanine ammonia lyase; OMT: O-methyltransferase; 4HBS: 4-hydroxybenzaldehyde synthase; C4H: cinnamate 4-hydroxylase; 4CL: 4-hydroxycinnamoyl-CoA ligase; HCT: hydroxycinnamoyl transferase; C3′H: coumaroyl ester 3′-hydroxylase; and VpVAN: V. planifolia vanillin synthase.
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Figure 4. Engineered biosynthetic pathways for the de novo heterologous production of vanillin in E. coli. (A,B) represent the different metabolic pathways proposed by Chen, Shen, Wang, Wang, Zhang, Rey, Yuan and Yan [56] and Ni, Tao, Du and Xu [54]. (A) The arrows with black annotation in Chen’s proposed pathway indicate the native pathway in E. coli, and the arrows blue annotation indicate the heterologous steps that have been added. (B) Pathway proposed by Ni, Tao, Du and Xu [54]; the green arrows contain three non-bacterial enzymes involved in the artificial pathway from tyrosine to ferulic acid, and the last black arrows of this pathway represent steps catalyzed by two bacterial enzymes involved in the conversion of ferulic acid to vanillin. Abbreviations. PEP: phosphoenolpyruvate; E4P: D-erythrose 4-phosphate; DAHP: 3-deoxy-D-arabinoheptulosonate 7-phosphate; 4-HPP: 4-hydroxyphenylpyruvate; L-Tyr: L-tyrosine; pCA: coumaric acid; CA: caffeic acid; FA: ferulic acid; FC: feruloyl-CoA; HMH-CoA: 4-hydroxy-3-methoxyphenyl-β-hydroxypropionyl-CoA; 3-DHS: 3-dehydroshikimic acid; Chim: chorismate; 4-HBA: 4-hydroxybenzoic acid; 3,4-DHBA: 3,4-dihydroxybenzoic acid; 3,4-DHBAad: 3,4-dihydroxybenzaldehyde; 3,4-DHBAal: 3,4-dihydroxybenzyl alcohol; VA: vanillyl alcohol; PEPS: phosphoenolpyruvate synthase; TKT: transketolase; AroG: 2-dehydro-3-deoxyphosphoheptonate aldolase; AroL: shikimate kinase II; UbiC: chorismate lyase; PobA: p-hydroxybenzoate hydroxylase; CAR: carboxylic acid reductase; ADH: alcohol dehydrogenases; COMT: caffeate O-methyltransferase; VAO: vanillyl alcohol oxidase; AT: aminotransferase; fbr-CM/PDH: fbr-chorismate mutase/prephenate dehydrogenase; TAL: tyrosine ammonia lyase; C3H: 4-coumarate 3-hydroxylase; FCS: trans-feruloyl-CoA synthetase; and ECH: enoyl-CoA hydratase/aldolase.
Figure 4. Engineered biosynthetic pathways for the de novo heterologous production of vanillin in E. coli. (A,B) represent the different metabolic pathways proposed by Chen, Shen, Wang, Wang, Zhang, Rey, Yuan and Yan [56] and Ni, Tao, Du and Xu [54]. (A) The arrows with black annotation in Chen’s proposed pathway indicate the native pathway in E. coli, and the arrows blue annotation indicate the heterologous steps that have been added. (B) Pathway proposed by Ni, Tao, Du and Xu [54]; the green arrows contain three non-bacterial enzymes involved in the artificial pathway from tyrosine to ferulic acid, and the last black arrows of this pathway represent steps catalyzed by two bacterial enzymes involved in the conversion of ferulic acid to vanillin. Abbreviations. PEP: phosphoenolpyruvate; E4P: D-erythrose 4-phosphate; DAHP: 3-deoxy-D-arabinoheptulosonate 7-phosphate; 4-HPP: 4-hydroxyphenylpyruvate; L-Tyr: L-tyrosine; pCA: coumaric acid; CA: caffeic acid; FA: ferulic acid; FC: feruloyl-CoA; HMH-CoA: 4-hydroxy-3-methoxyphenyl-β-hydroxypropionyl-CoA; 3-DHS: 3-dehydroshikimic acid; Chim: chorismate; 4-HBA: 4-hydroxybenzoic acid; 3,4-DHBA: 3,4-dihydroxybenzoic acid; 3,4-DHBAad: 3,4-dihydroxybenzaldehyde; 3,4-DHBAal: 3,4-dihydroxybenzyl alcohol; VA: vanillyl alcohol; PEPS: phosphoenolpyruvate synthase; TKT: transketolase; AroG: 2-dehydro-3-deoxyphosphoheptonate aldolase; AroL: shikimate kinase II; UbiC: chorismate lyase; PobA: p-hydroxybenzoate hydroxylase; CAR: carboxylic acid reductase; ADH: alcohol dehydrogenases; COMT: caffeate O-methyltransferase; VAO: vanillyl alcohol oxidase; AT: aminotransferase; fbr-CM/PDH: fbr-chorismate mutase/prephenate dehydrogenase; TAL: tyrosine ammonia lyase; C3H: 4-coumarate 3-hydroxylase; FCS: trans-feruloyl-CoA synthetase; and ECH: enoyl-CoA hydratase/aldolase.
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Figure 5. Biosynthesis pathways for vanillin and glycosylated vanillin production in Saccharomyces cerevisiae yeast [20,70,76,79]. (A) (Red arrows) represent the pathway proposed by Strucko, Magdenoska and Mortensen [79] and Hansen, Møller, Kock, Bünner, Kristensen, Jensen, Okkels, Olsen, Motawia and Hansen [70], while (B) (dark blue arrows) and (C) (green arrows) represent the pathways proposed by Qiu et al. (2022) and Mo and Yuan [20], respectively. Abbreviations. 3DHS: 3-dehydroshikimic acid; E4P: erythrose-4-phosphate; PCA: protocatechuic acid; L-Tyr: L-tyrosine; p-CA: p-coumaric acid; CA: caffeic acid; FA: ferulic acid; FC: feruloyl-CoA; 3,4-DHBAad: 3,4-dihydroxybenzaldehyde; PAL: protocatechuic aldehyde and VAC: vanillic acid; HPP: hydroxyphenylpyruvate; HMA: hydroxymandelate; DHMA: 3,4-dihydroxymandelate; DHGA: 3,4-dihydroxyphenylglyoxylate; 3-DSD: 3-dehydroshikimate dehydratase; ACAR: aromatic carboxylic acid reductase; EntD: phosphopantetheine transferase; HsOMT: O-methyltransferase (homo sapiens); UGT: UDP-glycosyltransferase; TAL: tyrosine ammonia-lyase; C3H: 4-coumarate 3-hydroxylase; 4HPA3H: 4-hydroxyphenylacetate-3-hydroxylase; COMT: caffeate O-methyltransferase; FCS: trans-feruloyl-CoA synthetase; ECH: enoyl-CoA hydratase/aldolase; HmaS: hydroxymandelate synthase; HpaBC: flavin-dependent monooxygenase; HMO: hydroxymandelate oxidase; BFD: benzoylformate decarboxylase; and VDH: vanillin dehydrogenase. The red cross materializes the suppression of genes involved in vanillin oxidation and reduction.
Figure 5. Biosynthesis pathways for vanillin and glycosylated vanillin production in Saccharomyces cerevisiae yeast [20,70,76,79]. (A) (Red arrows) represent the pathway proposed by Strucko, Magdenoska and Mortensen [79] and Hansen, Møller, Kock, Bünner, Kristensen, Jensen, Okkels, Olsen, Motawia and Hansen [70], while (B) (dark blue arrows) and (C) (green arrows) represent the pathways proposed by Qiu et al. (2022) and Mo and Yuan [20], respectively. Abbreviations. 3DHS: 3-dehydroshikimic acid; E4P: erythrose-4-phosphate; PCA: protocatechuic acid; L-Tyr: L-tyrosine; p-CA: p-coumaric acid; CA: caffeic acid; FA: ferulic acid; FC: feruloyl-CoA; 3,4-DHBAad: 3,4-dihydroxybenzaldehyde; PAL: protocatechuic aldehyde and VAC: vanillic acid; HPP: hydroxyphenylpyruvate; HMA: hydroxymandelate; DHMA: 3,4-dihydroxymandelate; DHGA: 3,4-dihydroxyphenylglyoxylate; 3-DSD: 3-dehydroshikimate dehydratase; ACAR: aromatic carboxylic acid reductase; EntD: phosphopantetheine transferase; HsOMT: O-methyltransferase (homo sapiens); UGT: UDP-glycosyltransferase; TAL: tyrosine ammonia-lyase; C3H: 4-coumarate 3-hydroxylase; 4HPA3H: 4-hydroxyphenylacetate-3-hydroxylase; COMT: caffeate O-methyltransferase; FCS: trans-feruloyl-CoA synthetase; ECH: enoyl-CoA hydratase/aldolase; HmaS: hydroxymandelate synthase; HpaBC: flavin-dependent monooxygenase; HMO: hydroxymandelate oxidase; BFD: benzoylformate decarboxylase; and VDH: vanillin dehydrogenase. The red cross materializes the suppression of genes involved in vanillin oxidation and reduction.
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Table 1. Putative vanillin biosynthesis and degradation enzymes identified in microalgae.
Table 1. Putative vanillin biosynthesis and degradation enzymes identified in microalgae.
Homologues of Vanillin biosynthesis EnzymesConserved
Domains
Database		FunctionComponentProcessTarget Biochemical Mechanisms
Putative Vanillin synthetase214853,
425470,
239068,
197621.		Enables cysteine-type endopeptidase activityExtracellular space and lysosomeInvolved in protein catabolic processRetro-aldol-type reaction
[4]
Putative Feruloyl-CoA synthetase341284;Enables acyl-activating enzyme activityNICatalyze the ATP-dependent acylation of fatty acids in a two-step reactionCoA-dependent non-β-oxidation reaction
[100]
Putative Enoyl-CoA Aldolase/hydratase474030
119339		Enables enoyl-CoA hydratase activityMitochondrion Involved in fatty acid beta-oxidationCoA-dependent non-β-oxidation reaction
[100]
Putative Isoeugenol Monooxygenase442887Enables carotenoid dioxygenase activityNIInvolved in carotene catabolic processOxidative cleavage
[101]
A BlastP was performed in NCBI using the vanillin synthase from Vanilla planifolia (GenBank: AKG47593.1); feruloyl-CoA synthetase from Streptomyces sp. V-1 (GenBank: AGR34008.1); enoyl-CoA aldolase/hydratase from Streptomyces sp. V-1 (GenBank: AGR34009.1), and isoeugenol monooxygenase from Pseudomonas nitroreducens Jin1 (GenBank: ACP17973.1) as queries in the protein databases of four microalgae species (C. reinhardtii, P. tricornutum, H. pluvialis, and C. vulgaris). Homologous enzymes sharing the same active domain were found in these microalgae species. CDD: Conserved Domains Database; NI: not identified.
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Tazon, A.W.; Awwad, F.; Meddeb-Mouelhi, F.; Desgagné-Penix, I. Biotechnological Advances in Vanillin Production: From Natural Vanilla to Metabolic Engineering Platforms. BioChem 2024, 4, 323-349. https://doi.org/10.3390/biochem4040017

AMA Style

Tazon AW, Awwad F, Meddeb-Mouelhi F, Desgagné-Penix I. Biotechnological Advances in Vanillin Production: From Natural Vanilla to Metabolic Engineering Platforms. BioChem. 2024; 4(4):323-349. https://doi.org/10.3390/biochem4040017

Chicago/Turabian Style

Tazon, Arnold William, Fatima Awwad, Fatma Meddeb-Mouelhi, and Isabel Desgagné-Penix. 2024. "Biotechnological Advances in Vanillin Production: From Natural Vanilla to Metabolic Engineering Platforms" BioChem 4, no. 4: 323-349. https://doi.org/10.3390/biochem4040017

APA Style

Tazon, A. W., Awwad, F., Meddeb-Mouelhi, F., & Desgagné-Penix, I. (2024). Biotechnological Advances in Vanillin Production: From Natural Vanilla to Metabolic Engineering Platforms. BioChem, 4(4), 323-349. https://doi.org/10.3390/biochem4040017

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