# Genetic Engineering Approaches for the Microbial Production of Vanillin
## 1 Introduction  
Vanilla flavour is widely used in the food, beverage, cosmetics, pharmaceutical, and tobacco industries. Natural vanilla flavour comprises dozens of organic compounds extracted from cured pods of different species of vanilla orchids, namely _Vanilla planifolia_, _V. pompon_, and _V. hultiensis_[1] (and references therein). The chemical composition of vanilla extracts depends on the plant species, cultivation conditions, curing and extraction methods, and storage conditions. The main constituent of vanilla flavour is vanillin (4-hydroxy-3-methoxy benzaldehyde), whose concentration can reach up to 2% \(w/w\) in cured vanilla pods [2]. Pure vanillin is widely used to boost other sweet food flavours, break down acidity, or mask the bitterness of specific food components. Furthermore, vanillin may serve as a food preservative in several dairy products, and its use in bakery significantly reduces the addition of sugar [3]. The global vanillin market was about 37,000 tonnes in 2018 and is estimated to exceed 59,000 tonnes in 2025 [4]. _Vanilla_ orchids, originally from Mexico, are currently cultivated around the tropics (in a total area of around 97,800 ha in 2018) [2]. However, vanilla beans can only meet about 1% of the annual vanillin market demand. Vanilla cultivation is labour-intensive, has a low yield of beans (around 75 kg/ha), and the extraction processes are laborious and time-consuming, making vanilla extracts very expensive. The chemical synthesis of vanillin, e.g., from guaicool and lignin, is an economical alternative. However, the lack of regioselectivity of some synthetic steps can reduce the process efficiency and increase the downstream processing costs [2]. Moreover, chemical synthesis processes require the use of hazardous chemicals (e.g., oxidising agents in alkaline conditions) and haveadverse environmental impacts. Nevertheless, chemically synthesised vanillin represents around 90% of the American market, and its price is about a hundred times lower than the one of natural extracts from _Vanilla_ beans. While, according to US food regulation, vanillin produced from a natural precursor by chemical synthesis is considered a \"natural flavour\" (FDA 101.22), by the European food regulation, the label \"natural flavour\" requires natural precursors and enzymatic or fermentative transformation processes (UE 1334/2000). \"Natural vanillin flavour\" has an intermediate price (roughly ten times more than chemically synthesised vanillin, but ten times less than natural vanilla extract), and its market share is around 9% of the global vanillin market. The increasing demand for environmentally friendly processes and for \"natural flavours\" has led to the development of biotechnological processes for the bioproduction of vanillin at the industrial level [2]. The demand for vanillin produced by biotechnological processes, also called bio-vanillin, has increased, so manufacturers are expanding their production capacity and developing new strains and production processes [5]. The biotechnological production of vanillin requires prior knowledge of the native biosynthetic pathway of vanillin in orchids and/or the de novo design of biosynthetic pathways using a retrobiosynthetic approach and expert knowledge of specific biochemical reactions [6; 7; 8; 9]. In this context, several studies have been performed to identify and characterise the enzymes and genes involved in the biosynthesis of vanillin in _Vanilla_[7; 10]. These studies will be described in detail in Section 2. Since the early 1990s, several microorganisms (hereafter defined as vanillin-producing microorganisms) were identified for their natural capacity to produce vanillin from eugenol, isoeigenol, ferulic acid, or lignin [11; 12; 13].