Biotechnologies in Perfume Manufacturing: Comparison
Please note this is a comparison between Version 3 by Camila Xu and Version 2 by Fabio Forlani.

The fragrance industry is increasingly turning to biotechnology to produce sustainable and high-quality fragrance ingredients. Microbial-based approaches have been found to be particularly promising, as they offer a more practical, economical and sustainable alternative to plant-based biotechnological methods for producing terpene derivatives of perfumery interest. Among the evaluated works, the heterologous expression of both terpene synthase and mevalonate pathway into Escherichia coli has shown the highest yields.

  • terpenes
  • metabolic engineering
  • bio-based ingredients

1. Introduction

Essential oils, which are aromatic and volatile liquids, are produced from plant material mainly by distillation-based methods and are usually named according to the sourcing plant species [1]. Essential oils can be described as either mixtures of fragrant substances or as mixtures of fragrant and odorless substances [2]. They have always been used for various purposes: in the past, essential oils were mainly used to treat various types of infectious diseases around the world, while nowadays at least 300 types of essential oils out of 3000 are commercially important in various types of industries, including that of fragrances, health care, cosmetics, food, beverages, agronomics, and pharmaceuticals [3]. With regard to the fragrance sector, essential oils can also be described—and to some extent classified—based on their scent [1]. In fact, essential oils that are used in perfumes are generally classified according to their volatility, which is the speed with which they diffuse in the air; based on this characteristic, each essential oil can be identified/classified into one of three “notes”: top notes, heart notes, and base notes.
From a biological point of view, these mixtures of fragrant substances within the plant are mainly composed of secondary metabolites. Secondary metabolites are historically so named to conventionally separate them from those of the energetic and biosynthetic primary metabolism. However, as their main role is to provide an evolutionary advantage to the plant which produces them, they should rather be called specialized metabolites; in fact, these metabolites have different types of biological activities, including antibacterial, antioxidant, antiviral, insecticidal, etc., and can play ecological roles, such as in fire tolerance, attracting pollinators and/or herbivores for seed dispersal, drought tolerance or plant-to-plant biosemiosis (pheromones) [1,4][1][4].
From a biochemical point of view, the scent of essential oils is associated with several unique combinations of low molecular weight (below 400 Da) volatile organic compounds [5]. In general, most of the components of essential oils derive from three main classes of compounds—terpenes, phenylpropanoids/benzenoids and fatty acid derivatives—which are often modified (oxidized, esterified, methylated, etc.) altering the volatility of such compounds in the final phases of their formation [6]. Nitrogen- and sulfur-containing compounds may also be present [7]. Amongst these three classes, terpene compounds generally constitute a major part of essential oils. In fact, monoterpenes and sesquiterpenes, a representative group of known natural volatile products [7], are classical constituents of essential oils and exhibit an extremely wide diversity of biological structures and properties. Common scented constituents of essential oil include monoterpenes, such as linalool, geraniol, myrcene, trans-β-ocimene, and limonene. Limonene is a cyclic monoterpene with citrus notes, which is often used as a top note in the production of perfumes and is nowadays mainly obtained from waste derived from orange juice production. The sesquiterpene compounds α- and β-santalol are key scented components of the sandalwood essential oil which is obtained from the heartwood and roots of mature (>25 years) oil-producing Santalaceae (Santalum genus) plants via steam distillation [8]. Patchoulol is a scented sesquiterpene compound which accounts for 30–40% of the total mass of compounds contained in patchouli oil, an essential oil commonly obtained from the leaves of Pogostemon cablin (the patchouli plant), which is widely used in the perfume industry [9].
Nowadays, most aromatic and scented compounds are produced either through chemical synthesis or extraction from natural materials (such as plant and animal sources). However, extraction from plants has some disadvantages: plant-derived materials are often subjected to fluctuations in price, annual production volumes and quality, due to factors related to seasons, geographical area of production, geopolitical problems, climate disasters and plant diseases. In addition, the price of compounds obtained from plants can increase due to limited cultivation, scarcity of the compounds of interest within the extract, high need of labor for the harvest or the depletion of natural resources [10]. Today, chemical synthesis still represents the most economical technology for the production of most aromatic and scented compounds; however, it can require unsustainable conditions (toxic catalysts, high pressure and temperature) and usually lacks adequate regio- and enantio-selectivity of the substrate, resulting in a mixture of isomers. Furthermore, the generated compounds are labeled as “artificial” or “nature-identical”, which ultimately decreases their economic value [11]. Recently, the fragrance industry has shifted towards sustainable and eco-friendly production methods and away from animal-derived raw materials. Biotechnology is gaining interest as a new means of extracting fragrance ingredients and is facilitating this transition [14][12].

2. Engineering of the Isoprenoid Pathways

Terpenes are synthesized from five-carbon precursors, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which can be derived from two alternative pathways: the mevalonate (MVA) pathway and the non-mevalonate pathway, also known as the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. While the MVA pathway produces IPP and DMAPP through a series of enzymatic reactions starting from acetyl-CoA, the MEP pathway is found in most bacteria, algae, plants (plastids), and apicomplexan protozoa (e.g., malaria parasites) and it produces IPP and DMAPP from glyceraldehyde 3-phosphate and pyruvate, using a different set of enzymatic reactions [15][13]. Due to the implication of terpene compounds in many biological functions and their economic value, the MVA and MEP pathways have been extensively studied and important regulatory mechanisms have also been clarified. The condensation of IPP and DMAPP is catalyzed by prenyltransferases, producing the linear prenyl diphosphate precursor for each class of terpenes: geranyl diphosphate (GPP), farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) for mono-, sesqui- and diterpenes, respectively. The next step involves the terpene synthases, which are part of a very large family of enzymes; they play a key role in the biosynthesis of terpenes as they catalyze various reactions (e.g., cyclization) on GPP, FPP and GGPP to form the carbon skeletons of terpene compounds and are therefore the key point for the formation of the extremely wide diversity of the final structures [10]. While many terpenes are produced directly from terpene synthases, others are formed through alterations of the initial products by oxidation, dehydrogenation, acylation and other types of reactions [6]. Finally, enzymes, such as monooxygenases of the cytochrome P450 family (P450) and oxidoreductases, are involved in further modifications and decorations of the terpene backbone, producing the thousands of naturally occurring terpenoids [10]. The knowledge accumulated to date regarding the biosynthesis of terpenes opens up various possibilities for the metabolic engineering of all phases of the entire path. In particular, it was found that a key element for the development of biocatalytic pathways is the availability of key genes which lead to the production of the target compounds, especially the genes that code for terpene synthases. Several examples of metabolic engineering for the biosynthesis of terpenes and terpenoids in microorganisms and plants demonstrate the possibilities of developing inexpensive biochemical pathways for the production of terpene compounds which are widely used in the fragrance sector. In fact, one of the most interesting areas of metabolic engineering is focused on the production of natural products in transgenic plants to improve agronomic traits, such as pest resistance and competitiveness, and to alter fragrance and flavor profiles [16][14]. In plants, two alternative approaches can be used to genetically manipulate the fragrance profile. The first is based on the introduction of foreign genes that encode for enzymes with activities that are lacking in the target plant. The second approach is based on the modulation (down or up-regulation) of the expression of one or more native genes. With the latter approach, the production of a volatile compound can be increased by up-regulating a gene in the pathway or, alternatively, by blocking the production of an unwanted volatile compound [17][15]. Other interesting methods for the synthesis of aromas and fragrances are based on the use of genetic engineering methods, microbial de novo synthesis (fermentation) or the chemical conversion of natural precursors using biological methods (enzymes or whole cells, and biocatalysis or biotransformation) [18][16], since the products obtained from this type of processes can be labeled as “natural” [11]. De novo synthesis refers to the production “from the very beginning”, that is, the synthesis of substances starting from simpler substances (sugars, amino acids, nitrogen salts and minerals, among others), which will be metabolized by microorganisms to form diverse and complex structures, generating a mixture of low concentration products [11,14][11][12]. Biocatalysis and biotransformations are processes that convert a starting material (substrate) into a desired product using a biological system. Biocatalysis uses isolated enzymes, either free or immobilized, to catalyze the reaction, while biotransformation uses whole living cells containing the necessary enzyme(s) [18][16]. The biotransformation of terpenes is of interest because it allows the production of enantiomerically pure flavors and fragrances under mild reaction conditions [19][17]. Therefore, biotechnology can help replace the natural scent and aromatic ingredients of plants such as lavender, jasmine or ylang-ylang and metabolically modified microbes can produce a variety of natural molecules ranging from patchoulol, linalool, nerolidol, valencene to sclareol through fermentation. With the help of biotechnological tools, aroma and fragrance molecules can be produced in some cases more economically and in larger quantities, overcoming many of the drawbacks associated with chemical synthesis or plant extraction [20][18].

2.1. Genetic Engineering of Plants for the Production of Terpenoids

Engineering of terpene metabolism in plants is an attractive alternative because of their elaborate biosynthetic potential and the obvious economic benefit of using photosynthesis to drive production [21][19]. Moreover, another benefit is the less expensive extraction of essential oils: the methods for their extraction, if applied on a large scale, would require little optimization and limited investments, as the methods themselves are already well known on an industrial level. Plants can be genetically engineered by means of the introduction of foreign genes or via the modulation of the expression of one or more native genes. Valid pioneering experiments performed mainly on herbaceous plants have paved the way for the genetic manipulation of the odorant trait, highlighting the potential of the expression of heterologous terpene synthase in changing the volatile profile. Interesting results have also been obtained with woody plants and mosses. For example, in 2010, Ohara et al. [22][20] engineered Eucalyptus camaldulensis, a woody plant which is widely used for the production of cellulose for the pulp and paper industries, and essential oils, by means of the expression of a heterologous synthase (the limonene synthase from Perilla frutescens, PFLS), in order to increase its limonene content. Similarly in 2014, Zhan et al. [23][21] engineered the moss Physcomitrella patens by means of two heterologous synthases, Pogostemon cablin patchoulol synthase (PTS) and Santalum album α/β-santalene synthase (STS), respectively to increase patchoulol and α- and β-santalene; the latter is the precursor of α- and β santalol. The compartmentalization of the biosynthesis of terpenes in plants was also considered in these studies. In fact, the cytosolic pathway is predominantly responsible for the generation of C15-derived terpenes, such as sterols and sesquiterpenes, whereas monoterpenes (C10), diterpenes (C20) and tetraterpenes (C40; e.g., carotenoids) are synthesized via the plastidic pathway [21][19] (Figure 1). Indeed, it has been observed that the modification of the subcellular localization of terpene synthases can lead to an increase in the production of terpenes.
Figure 1. Subcellular organization of the MVA and MEP pathways in plant cells. HMG-CoA—3-hydroxy-3-methylglutaryl coenzyme A; HMGR—3-hydroxy-3-methylglutaryl coenzyme A reductase; DMAPP—dimethylallyl diphosphate; IPP—isopentenyl diphosphate; FPP—farnesyl diphosphate; GA-3P—glyceraldehyde 3-phosphate; DXP—1-deoxy-d-xylulose 5-phosphate; DXS—1-deoxy-d-xylulose 5-phosphate synthase; DXR—1-deoxy-d-xylulose 5-phosphate reductisomerase; MEP—2-C-methyl-d-erythritol-4-phosphate; GGPP—geranylgeranyl diphosphate; GPP—geranyl diphosphate; GPS—geranyl diphosphate synthase. Dashed-line arrow indicates multiple steps.
For this reason, Zhan et al. [23][21] attempted to re-localize the synthases responsible for the production of sesquiterpenoids patchoulol and α- and β-santalene to the plastids of P. patens by adding the transit peptide of the small subunit of the RuBisCO enzyme from Arabidopsis. On the contrary, PFLS [22][20], like many other monoterpene synthases, is localized in the plastids and already has a plastid localization signal at the N-terminal. Therefore, to evaluate the different levels of expression in the cytosol, a second version of PFLS, lacking the putative signal peptide, was expressed in E. camaldulensis. These approaches showed that cytosolic expression of PFLS allows for a significantly greater (4.5-fold) accumulation of limonene than that found with the native plastid expression (Figure 2, Table 1), suggesting that the cytosolic PFLS could somehow effectively use the cytosolic GPP as a substrate.
Figure 2. Re-localization approaches of limonene synthase from Perilla frutescens. PFLS, P. frutescens limonene synthase with plastid localization signal at the N-terminal (tp). (1) Production of limonene in wild-type Eucalyptus camaldulensis. (2) Transformation of E. camaldulensis with PFLS. (3) Transformation of E. camaldulensis with modified PFLS by removal of tp. The “+” symbols indicate an increase in limonene production compared to (1).
Table 1.
Types of approaches discussed for the plant production of the selected compounds.

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