What Is Biosynthesis of Vanillin?

Vanillin is the main component of vanilla, also known as vanillin, vanillin, vanillin aldehyde, etc. The chemical name is 4-hydroxy-3-methoxybenzaldehyde (4-hydroxy-3-methoxybenzaldehyde), relative molecular mass 152.15, white to light yellow crystalline powder or needle crystal. Vanillin exists in the plant in free form and as glucosides, accounting for 2% to 3% of the dry weight of vanilla beans. Pure vanillin has a strong milky aroma and no odor. Vanillin has a wide range of uses, in addition to being used as a perfume and flavoring agent, it is also an important raw material and intermediate in the pharmaceutical industry, and can also be used as an electroplating polish, plant growth promoter, ripening agent and so on.

Vanillin is the largest synthetic spice in the world, with an annual consumption of about 12,000 t, and its demand is still increasing at a rate of 10% per year. The production of natural vanillin extracted and prepared from vanilla is low, only about 20~50 t per year, and the remaining consumption gap is filled by chemical synthesis method, which has many unavoidable drawbacks, such as single aroma, easy to be adulterated, etc., but people's demand for natural vanillin is always on the rise.

The demand for natural vanillin is always on the rise. Since the cultivation of vanilla is restricted by climatic and geographical conditions, and the processing of its aroma is complicated, it is difficult for the production of vanillin to meet the market demand. Biological methods can synthesize natural vanillin [1], and have the advantages of less pollution, cleaner production and safety. At present, the reported biological methods include microbial fermentation, plant cell culture and enzyme method.

1 Microbial Fermentation

In 1977, Tadasa K isolated a strain of Corynebacterium sp. that could convert eugenol into vanillin [2], which opened up a new way to prepare vanillin biologically. Subsequently, many bacteria and molds were found to be able to convert eugenol, isoeugenol, ferulic acid, glucose and other compounds into vanillin, and vanillin could be synthesized by microbial fermentation using a variety of substrates.

1 . 1 Eugenol or Isoeugenol as Substrate

Eugenol and its isomer isoeugenol, mainly derived from the volatile oil of clove basil of Labiatae, the chemical name of eugenol is 4-propenyl-2-methoxyphenol [2-methoxy-4-(2- propenyl)-phenol], and the chemical name of isoeugenol is 4-propenyl-2-methoxyphenol [2-methoxy-4-(1- propenyl)- phenol]. The chemical name of isoeugenol is 4-propenyl-2-methoxyphenol [2-methoxy-4-(1-propenyl)-phenol], both of which can be chemically or biologically converted to vanillin. In addition to Corynebacterium sp., Serratia sp., Klebsiella sp., Enterobacter sp., and some fungi in the class of Hemiptera are also capable of converting eugenol into vanillin [3, 4]. Moreover, Serratia marcescens, Klebsiella marcescens, and Enterobacter marcescens can convert isoeugenol into vanillin. Bacillus subtilis and Rhodococcus subtilis are also capable of converting isoeugenol into vanillin.

rhodochrous) also possesses the ability to convert isoeugenol into vanillin [5, 6].

The conversion of eugenol to vanillin by fungal strains of the class Hemiptera did not produce high yields, with a maximum of 0.0271 g/L [4]. Bacillus subtilis B2 strain can use isoeugenol as the only carbon source and convert it into vanillin with a yield of 0.61 g/L and a molar yield (in terms of isoeugenol) of 12.4% [5]; when B. purpurea MTCC 289 strain uses isoeugenol as the substrate, under the optimal culture conditions in the laboratory, the molar yield of vanillin (in terms of isoeugenol) can be as high as 58% [6].

Although Serratia marcescens strain DSM 30126 was also utilized, the results varied greatly depending on the substrate [3]. The molar yield of vanillin (in terms of eugenol) was only 0.1% with a yield of 0.018 g/L, while the yield of vanillin (in terms of isoeugenol) was 20.5% with a yield of 3.8 g/L. In contrast, the yield of vanillin obtained by using isoeugenol as the substrate was 20.5%, and the yield of vanillin obtained by using isoeugenol as the substrate was 20.5%. In contrast, the yield of vanillin obtained with isoeugenol as substrate was higher than that with eugenol as substrate, which was also the result of the authors' fermentation with Serratia marcescens strain AB 90027. Isoeugenol is a more suitable substrate than eugenol for the production of vanillin by microbial fermentation.

1 . 2 Ferulic Acid as Substrate

Ferulic acid, chemically known as 4-hydroxy-3-methoxycinnamic acid, is widely found in agricultural by-products such as corn gluten and grain bran. There are two ways to produce vanillin by fermentation using ferulic acid as a substrate: one requires the action of a single bacterium, while the other requires the combined action of multiple strains of bacteria.

The single strains of fungi used for the production of vanillin by fermentation have been reported [4, 7, 8], including certain fungi of the order Bacillus coagulans, and soil filamentous fungi (Amycolatopsis sP.). Only 0.0552 g/L of vanillin was obtained by using a fungal strain of the order Hemiptera [4]. In contrast, vanillin was produced from 4-vinylguaiacol by Bacillus coagulans strain BK 07, which uses ferulic acid as the sole carbon source. This strain can metabolize more than 95% of ferulic acid within 7 h, and the main product is 4-vinylguaiacol, which is the fastest metabolism process of ferulic acid reported so far [7]. In the patented method invented by Rabenhorst J et al. [8], a soil filamentous bacterium, DSM 9992 strain, was used to convert ferulic acid into vanillin with a yield as high as 11.5 g/L. The mechanism of this process is not yet reported.

The "two-step bioconversion method" proposed by Lesage-Meessen L et al [1] is a typical example of the production of vanillin by the combined action of multiple strains, which utilizes Aspergillus niger and pycnoporus cinnabarinus to complete the whole conversion process. This method utilizes the combination of Aspergillus niger and Pycnoporus cinnabarinus. Firstly, Aspergillus niger converted ferulic acid into vanillic acid with a yield of 0.92 g/L and a molar yield of 88% in terms of ferulic acid, and then reduced vanillic acid to vanillin with a yield of 0.237 g/L and a molar yield of 22% in terms of vanillic acid by Pycnoporus cinnabarinus. Recently, they [9] changed ferulic acid to corn bran as the only carbon source, and created a "two-step bioconversion method" to obtain vanillin crystals without any purification measures.

According to the study [10, 11], during the reduction of vanillic acid to vanillin by M. verticillioides, the yield of vanillin will be reduced due to the generation of the by-product methoxyhydroquinone; when the mass concentration of vanillin in the medium exceeds 1.0 g/L, it will be very toxic to M. verticillioides, which will slow down the growth of M. verticillioides, and at the same time the large amount of proliferation of this strain is not conducive to the production of vanillin. The addition of cellobiose to this system significantly reduced the production of hydroxymethoxyquinone and increased the vanillin yield by 3.3 times to 0.725 g/L. The addition of appropriate amount of XAD-2 resin adsorbed vanillin could regulate the mass concentration of vanillin in the medium and the growth rate of the strain, and the use of mechanically stirred bioreactor could greatly improve the mass transfer effect, and the yield of vanillin could reach 1.575 g/L. The results showed that the vanillin production in this system was not favorable to the production of vanillin, and it could be used in the production of vanillin.

1 . 3 Glucose as a Substrate

Glucose can be obtained from starch hydrolysis, which is a sufficient raw material with low cost, but there is only one case of glucose biotransformation as a substrate to produce vanillin [12]. Firstly, glucose was converted to vanillic acid by Escherichia coli recombinants (Escherichia coli KL7/PKL5. 26A or KL7/PKL5. 97A), and then vanillic acid was reduced by aromatic aldehyde dehydrogenase isolated from Neurospora crassa to produce vanillin. In order to make the glucose-substrate bioconversion method an industrially applicable technology for the production of natural vanillin, the stable expression of recombinants in E. coli must be solved.

In summary, although glucose is inexpensive, how to realize the stable expression of recombinants of E. coli is the key to practical application; from the viewpoint of raw material resources and the current status of biotechnology in China, the bioconversion of isoeugenol and ferulic acid as the substrate for the production of vanillin has more promising prospects for development.

2 Plant Cell Culture Method

The development of modern bioengineering technology has led to the idea and practice of using cell culture to produce vanillin. The first one was the artificial culture of vanilla planolia cells, which utilized the property of exocytosis of vanillin by the cells, but the yield per unit volume was not high, only about 0.01 g/L [13]. Recently, new methods have been found to convert specific additives into vanillin, such as capsicum 'rutescens cells' [14-16].

These cell cultures and their production of vanillin are affected by factors such as additives and cell growth environment [17]. Cao Mengde et al. [13] found that the addition of different types and concentrations of phytohormones did not have a significant effect on cell growth, but had a significant effect on the production of vanillin, and the combination of naphthalene acetic acid and 6-benzyl adenine resulted in the production of more vanillin. The formation of vanillin is negatively correlated with cell growth, i.e., slow cell growth favors the formation of the target product, therefore, a two-step culture method can be considered for the cultivation of vanillin cells and production of vanillin [18].

In addition to vanillin-producing plant cells in suspension culture, there are also shrubby pepper cells and Haematococcus pluoialis cells that can convert additives into vanillin [14-16]. Due to the disadvantages of cell suspension culture, such as unfixed cell position, unfavorable cell organization, and difficult to control the culture conditions, the yield of the target product vanillin is not high, which can be improved by cell immobilization culture, so that the yield of vanillin can be increased.

As mentioned earlier, shrubby chili cells can convert some specific additives, such as isoeugenol, protocatechuic aldehyde, and caffeic acid, into vanillin. Table 1 shows the maximum production of vanillin and its occurrence in relation to the additives during the immobilization of shrubby chili cells [14, 15].

It can be seen that isoeugenol is the best of the three additives, and it is more effective if β-cyclodextrin is added to improve the solubility of isoeugenol and reduce its toxicity to the cells, or if Aspergillus niger mycelium is added as an inducer. Similar to shrubby pepper cells, R. rainbowii cells can also convert ferulic acid or turpentine into vanillin [16]. The maximum yield of vanillin was 0.0106 g/L with the addition of ferulic acid (at a concentration of 1.0 mmol/L) and 0.0205 g/L with the addition of turpentine aldehyde (at a concentration of 1.0 mmol/L) during the solidification process. In conclusion, the efficiencies of the existing plant cell cultures for vanillin production are not high, and they are still far from industrialization.

3 Enzymatic

All biometabolic reactions are enzymatic, and although the mechanism of action of the various methods described above is not yet clear, it can be determined that they are produced by the action of one or more enzymes. If these enzymes can be isolated and utilized in enzymatic reactions, vanillin can be produced more directly.

van den Heuvel Robert H H et al. [19] from the University of Wageningen, Netherlands, found that vanillin can be produced by vanillyl alcohol oxidase (VAO) in two ways. One is the production of vanillin from wood tar alcohol via vanillyl alcohol in the presence of VAO. In the other case, vanillylamine is converted by VAO under alkaline conditions to an intermediate product that can be directly hydrolyzed to vanillin. Since red pepper and capsaicin can be enzymatically degraded to produce vanillin, the latter route provides a richer source of raw material and makes it more competitive. A patent [20] reported that the enzyme named chirazyme L  20] reported that a lipase named chirazyme L-2, c-f, c2 lyo was able to convert isoeugenol and pine aldehyde to vanillin with mass fractions of 30.4% and 83.1%, respectively, and that this lipase could also be isolated from Candida antarctic.

Because of the advantages of high efficiency, specialization, mild reaction conditions, easy purification of products, low energy consumption, low pollution, simple operation and easy control, the digital reaction should have incomparable superiority. However, how to make use of enzyme theory, chemical engineering and modern biotechnology to modify the existing enzymes, study their immobilization technology and develop a suitable multi-enzyme reactor will be the main task for the production of vanillin by enzyme method.

4 Conclusion

The production of vanillin by biological method is getting more and more attention, and many famous vanillin producing companies in the world, such as Schniken (Italy), Boreai (Norway) and Monsanto (USA), have strengthened the research on the production of vanillin by biological method, in order to occupy a dominant position in the spice market in the future. China is still in the initial stage in this aspect, at present, there are only the research of Cao Mengde of Huazhong University of Science and Technology on the suspension culture of vanillin cells, and the research of Yao Risheng of Hefei University of Technology on the production of vanillin by bio-oxidation method in conjunction with Xiamen Yongquan Group Company Limited.

Therefore, it is necessary to take advantage of the combination of the methods of production, learning, research, and governmental funding, and to intensify the research of cooperation, so as to make our country occupy a place in the market of biosynthesized vanillin. Therefore, it is necessary to increase the cooperative research with the help of the combination of industry, academia, research and government funding, so that China can occupy a place in the biosynthetic vanillin market.

Although the research on the non-chemical synthesis of vanillin has made a lot of substantial progress, the following problems still need to be solved in order to realize the industrial production: obtaining excellent engineering strains; improving the plant cell culture technology; researching on the technology of enzyme isolation, purification and immobilization, as well as the development of multi-enzyme reactors. According to the current state of the art, the cost of producing vanillin by plant cell culture will be as high as 15,000 US dollars/kg, while the cost of extracting vanillin from vanilla beans by traditional methods is 4,000 US dollars/kg, and the cost of using microbial fermentation is about 1,000 US dollars/kg [17]. Therefore, the research and development of microbial fermentation of vanillin using isoeugenol and ferulic acid as substrates will be more promising for the production of vanillin in China.

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