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2 Microbial Cell Factories for the Biosynthesis of Vanillin and Its Applications
Sukumaran Karthika1, Manoj Kumar1, Santhalingam Gayathri1, Perumal Varalakshmi2, and Balasubramaniem Ashokkumar1
1 Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Madurai, TN, India
2 Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai, TN, India
2.1 Introduction
Vanillin (3‐methoxy‐4‐hydroxybenzaldehyde), a phenolic compound with organoleptic properties (Walton et al., 2003), was isolated first time from vanilla pods by Nicholas Theodore Gobley (1858). The Spanish word “vania” means pod, and “ila” means small; thus, vanilla means vine yielding small pods. The vanilla is the only edible fruit in the orchid family (Ranadive, 1994). Vanilla receives its nutrients and relies on moisture in the air like other plants’ roots connected to ground for its growth. The floret of vanilla is pale yellow‐green in color, which is 10 cm in diameter, and the bean is 5–10 inches long (Bythrow, 2005). The tropical climbing orchid, Vanilla planifolia, is the leading natural source of vanilla and to a lesser extent of Vanilla tahitiensis and Vanilla pompon. Vanilla is a sweet‐smelling and world’s second most expensive flavor compound next to the saffron. Vanillin is the foremost compound responsible for the sweet aroma of vanilla, which is mainly used as a fragrance ingredient of food preparations such as ice creams, chocolates, cakes and other milk products, and beverages (Ranadive, 1994). Moreover, vanillin serves as an intermediate in the synthesis of herbicides, antifoaming agents, or drugs such as papaverine, IL‐dopa, IL‐methyldopa, and the antimicrobial agents (Hocking, 1997). Naturally, vanilla is extracted from bean or pod‐like vanilla fruit using aqueous ethanol. Pure vanillin is a white crystalline solid, which is slightly soluble in water. Vanillin from natural sources has huge market demand due to the way of processing, cost, and bioactive properties of the product. Madagascar, Indonesia, Mexico, China, India, and Papua New Guinea are the main vanilla‐producing countries (Figure 2.1). It is also grown in a lesser extent in France and India (Kerala, Karnataka, Tamil Nadu, Assam, and Andaman Nicobar Island) (Priefert et al., 2001; Walton et al., 2003; Converti et al., 2010; Zamzuri and Abd‐Aziz, 2013; Banerjee and Chattopadhyay, 2019). Madagascar and Indonesia together are the leading producers of natural vanillin, and they have produced 5361 metric tons in the year 2018 as per Food and Agriculture Organization of the United Nations. Vanillin production from plants is a labor‐intensive process, which requires approximately 40 000 flowers to be pollinated to produce 1 kg of vanillin from 500 kg of pods. Due to low yield of natural vanillin from plants and the need of huge plant sources that grows slow in nature, vanillin extraction from plant sources is very much limited. Alternatively, vanillin is produced chemically from lignin, guaiacol, and 4‐hydroxybenzaldehyde at cheaper costs, but the low quality of the product yield and the release of hazardous contaminants in to the ecosystem by chemical synthesis make this process unlikely with regard to cost and quality. In the recent years, green synthesis of wide array of bioactive metabolites has gained much attention as an alternate to chemical synthesis in terms of high reliability, sustainability, and environment friendliness. Vanillin production through biotechnological approaches like microbes enabled biotransformation of substrates like ferulic acid and eugenol holds promise as a viable alternative and economically feasible way of obtaining vanillin. Thus, it has gained much interest in recent years due to European and US legislation already classifying the product as “natural.” This review mainly focuses on recent strategies for the vanillin production from different sources and on various strategies so far evaluated including biotechnological methods for vanillin production like biotransformation of natural precursors to vanillin using microbial cells or enzymes and on genetic manipulation by metabolic engineering. In recent years, many researchers have explained the production of vanillin from different naturally occurring sources and also reported high yield of vanillin production thus review may help in understanding past, present strategies to get better yield of vanillin to meet high demand of this flavoring compound (Vanillin) in future.
Figure 2.1 World map showing the leading countries in the production of vanillin.
2.2 Natural Sources of Vanilla and Its Production
The genus Vanilla belongs to the family Orchidaceae, which is one of the largest families of flowering plants in the world comprising 788 genera and 18 500 species. The genus itself contains over 100 species (Mabberley, 1997). V. planifolia is the only orchid that is of direct economic importance because it is the main source of the vanilla aroma. This aroma is widely used in the pharma, food, and cosmetic industry. V. planifolia has its origin in Mexico, where it was already brought by Spanish to Europe in 1520 and it became very popular. V. planifolia is still the only natural source of the vanilla aroma. In some other plants (narcissus, hyacinth, potato) traces of vanillin occur (Havkin‐Frenkel et al., 1999). Vanillin normally present in conjugated form as β‐D‐glucoside at a concentration of 1.0–2.0% of dry matter in cured vanilla pods (Westcott et al., 1993). The vanilla aroma develops in the pods or beans through a quite labor‐intensive process called curing. This process for aroma development is carried out to dry the vanilla beans and to allow chemical and enzymatic reactions to occur. Curing process aims to arrest the vegetative growth and induces the changes responsible for aroma formation. Generally, curing comprises three stages such as killing/scalding, sweating/sunning, drying, and conditioning/packaging. During curing process, conjugated glycoside molecules accumulated in the green pods are hydrolyzed enzymatically by induced β‐D‐glucosidases through drying and heating. Further packaging of processed pods by storing in closed boxes for few months facilitates various biochemical reactions such as esterification, etherification, oxidative degradation, etc. that results in desired aroma and flavor formation (Rao and Ravishankar 2000; Converti et al., 2010). Conventionally by curing process very less (1–2%) vanillin is produced naturally (Sinha et al., 2008). Moreover, natural vanillin production using plant sources is laborious, time consuming, and also expensive. With the increasing interest in producing natural vanillin and the insufficiency of plant‐derived natural vanillin production to meet the demand, alternative processes are developed to produce vanillin from a natural raw resources through many biotechnological approaches including enzyme catalyzed conversions, microbial bioconversions, the development of tissue cultures, and genetic/metabolic engineering.
2.3 Biotechnological Production of
