Advances on (+)-nootkatone microbial biosynthesis and its related enzymes

Abstract (+)-Nootkatone is an important functional sesquiterpene and is comprehensively used in pharmaceutical, cosmetic, agricultural and food flavor industries. However, (+)-nootkatone is accumulated trace amounts in plants, and the demand for industry is mainly met by chemical methods which is harmful to the environment. The oxygen-containing sesquiterpenes prepared using microbial methods can be considered as “natural.” Microbial transformation has the advantages of mild reaction conditions, high efficiency, environmental protection, and strong stereoselectivity, and has become an important method for the production of natural spices. The microbial biosynthesis of (+)-nootkatone from the main precursor (+)-valencene is summarized in this paper. Whole-cell systems of fungi, bacteria, microalgae, and plant cells have been employed. It was described that the enzymes involved in the microbial biosynthesis of (+)-nootkatone, including cytochrome p450 enzymes, laccase, lipoxygenase, and so on. More recently, the related enzymes were expressed in microbial hosts to heterologous produce (+)-nootkatone, such as Escherichia coli, Pichia pastoris, Yarrowia lipolytica, and Saccharomyces cerevisiae. Finally, the development direction of research for realizing industrialization of microbial transformation was summarized and it provided many options for future improved bioprocesses.


Introduction
Sesquiterpenoids constitute a structurally different category in terpene compounds and possess multiple biological activities, which are oxygen-containing derivatives of sesquiterpenes (Gershenzon & Dudareva, 2007). (+)-Nootkatone is a sesquiterpene compound, which belongs to bicyclic sesquiterpene ketone of Yashilane series. It was originally separated from the Nootka cypress tree and trace amount was found in grapefruit later (Macleod, 1965). Afterwards, (+)-nootkatone was found in peel oils from orange, grapefruit, lemon, mandarin, and so on (Gliszczynska et al., 2011). (+)-Nootkatone has a pleasant grapefruit flavor in orange and grapefruit juice and is used commercially as a spice or flavor ingredient (MacLeod Jr & Buigues, 1964;Sauer et al., 2003). It was reported that (+)-nootkatone tasted slightly bitter and had a low odor threshold of about 0.8 ppm in water and 30 ppm in air (Haring et al., 1972;Shaw & Wilson, 1981). (−)-Nootkatone has the higher odor threshold and less overall bioactivity, so it has little commercial interest. While (+)-nootkatone was more extensively used in the food and cosmetic industries, which providing extremely popular flavor and fragrance compounds (Wriessnegger et al., 2014).
The main production methods for terpenoids include physical extraction (Jaiswal et al., 2014), synthesis of chemical methods (Zhang et al., 2016) and biological catalytic transformation. Generally, the content of terpenoids is very low in plants and the growth of plants is easily affected by climate and environment. The method of plant extraction is easy to destroy wild plant resources seriously. At present, it mainly relies on chemical synthesis of (+)-nootkatone to meet industrial demand. Some unsafe oxidizing agents such as tert-butyl peracetate (Wilson & Shaw, 1978) or tert-butyl hydroperoxide was used to catalyze (+)-valencene to synthesize (+)-nootkatone (Salvador & Clark, 2002). Hong et al. found that hydrogen peroxide and amphiphilic molybdate ions could be used to catalyze (+)-valencene to synthetize (+)-nootkatone (Hong et al., 2016). It was also found that (+)-valencene could be oxidated by metalloporphyrins to produce (+)-nootkatone for the first time (de Melo et al., 2021). In contrast, the biocatalytic transformation method is not limited by the raw materials . The production process is environmentally friendly, sustainable and pollution free, which has obvious advantages (Gounaris, 2010). Biotransformation is a useful substitute for chemical synthesis because of its remarkable regioselectivity and enantioselectivity (Sales et al., 2018;Tai et al., 2016). It is widely used in organic synthesis, spice production, new drug development, modification, and modification of drug structure and prediction of drug metabolism model. Effective European law defined that "natural flavoring substance" as a compound "obtained by appropriate physical methods, enzymes or microbial processes from animals, plants, or microorganism" (Leonhardt & Berger, 2015). Biotransformation processes meets the Europe and the U.S. legal requirements for the "natural" properties of a flavor compound (Kolwek et al., 2018). Therefore, the use of microbial transformation to produce spices has become a hot topic.
The microbial transformation of (+)-valencene has become an increasingly important way to obtain (+)-nootkatone. Therefore, this paper focuses on the recent studies on the microbial synthesis of (+)-nootkatone. We introduce the microbial species and related enzymes involved in the microbial transformation of (+)valencene to (+)-nootkatone and the heterologous expression to synthesize (+)-nootkatone. Finally, the development direction of the industrialization of microbial transformation is summarized in order to provide reference for the future research of sesquiterpenes biotransformation.

(+)-Valencene as the Precursor
(+)-Valencene is a natural bicyclic sesquiterpene, which possesses a variety of biological activities. It was found in various citrus species, such as the sweet orange (Citrus sinensis) (Frohwitter et al., 2014;Hunter & Brogden Jr, 1965). Usually, (+)-valencene was isolated from citrus oils by steam distillation . Furthermore, previous study found that (+)valencene was biosynthesized by introducing (+)-valencene synthases in Saccharomyces cerevisiae (Paulino et al., 2020). (+)-Valencene was commercially used as an additive in drinks and flavor food because of its fruity and woody flavor. The market sales volume of (+)-valencene was about 10,000 kg every year . The enzymatic steps from (+)-valencene to (+)-nootkatone was not entirely clear in current research. It was suggested that the (+)-valencene was transformed to nootkatol through a regioselective allylic hydroxylation and nootkatol was then oxidized to (+)-nootkatone ( Fig. 1a) (Drawert et al., 1984). Both of the two steps could be catalyzed by a single multifunctional hydroxylase or oxidase and it may also be a sequential enzyme-mediated reaction ).

Biotransformation of (+)-Valencene by Microalgae and Plant Cells
There were few studies on the biotransformation of terpenoids and aromatic flavor compounds by Chlorella species recently. Microalgae are important photosynthetic microorganisms and are also used for biotransformation of terpenes. Light energy is the only source of energy for microalgae growth. Therefore, the large-scale culture of microalgae is simpler and cheaper than other microorganisms. It was reported that sesquiterpene (+)valencene was transformed to (+)-nootkatone by the green algae Chlorella fusca var. vacuolata IAMC-28 with a yield of 63% (252 mg/l) (Furusawa et al., 2005b). In contrast, Chlorella prenoidosa and Chlorella vulgaris had a better transformation effect. The yield of (+)-nootkatone exceeded 80% (320 mg/l) finally (Asakawa et al., 2013).

Enzymes Associated with Microbial Transformation of (+)-Valencene to (+)-Nootkatone
The isolation, cloning, expression, and regulation of enzyme genes have become a hot research topic. It is expected to realize the industrial and efficient production of oxygen-containing terpenes aromatic compounds through genetic engineering technology. It was reported that some enzymes played an important role in the conversion process from (+)-valencene to (+)-nootkatone and its intermediate substance (+)-nootkatol, such as cytochrome P450 enzyme, laccase, oxidase, dehydrogenase, reductase, etc. (Table 2).

CYP450s
Cytochrome P450s (CYPs) are a common enzyme in the microbial transformation. They are a kind of monooxygenated superfamily containing iron heme, which are widely distributed in bacteria, fungi and higher organisms. Cytochrome P450s (CYPs) are usually involved in a series of post-modifications of the core structure of terpenoids in a large part, resulting in the final terpenoid products with a wide range of chemical diversity (Mele et al., 2020;Xiao et al., 2019). Engineered cytochrome P450 cam and P450 BM-3 were shown to transform (+)-valencene to (+)-nootkatone (Sowden et al., 2005). The CYP109B1 from Bacillus subtilis can catalyze the oxidation of (+)-valencene to produce nootkatol

Laccase
Laccases are oxidases mainly produced by basidiomycete fungi (Calcaterra et al., 2008). It is a copper-containing polyphenol oxidase that can oxidize polyphenols, methoxy-substituted phenols, diamines, and a large number of other compounds, but it cannot oxidize tyrosine (as do the tyrosinases) (Minussi et al., 2002). (+)-Valencene and a composition having laccase activity were reacted in the presence of an oxygen source to form (+)-valencene hydroperoxide, which was degraded to generate (+)-nootkatone (Huang et al., 2001). Laccases have been found to have a lot of application functions, such as bleaching in the textile and dye industry, production of wood composite materials, and the role of bioremediation (Linke et al., 2005). The method of producing (+)-nootkatone by laccase catalyzed oxidation of (+)-valencene was shown in Fig. 1b. The catalyzed process from (+)-valencene to (+)-nootkatone was two-steps and the intermediate product was valencene 2-hydroperoxides. (+)-Nootkatone was then produced by heating or adding a chemical catalyst. Laccase isolated from the basidiomycete Funalia trogii and dyedecolorizing peroxidase (Ftr-DyP) were confirmed to converted (+)-valencene to (+)-nootkatone with the highest concentration of 1,100 mg/l (Kolwek et al., 2018).

Oxidase
Willershausen et al. proposed a cell-free enzymatic generation of (+)-nootkatone with isolated lignin peroxidase, and (+)-valencene was considered as the substrate under alkaline and high temperatures conditions (Willershausen, 1996). Krings et al. thought that some common terpene substrates, like (+)-valencene might likewise be catalyzed by dioxygenases ). Afterwards, Fraatz et al. purified the responsible oxygenase from lyophilisates of P. sapidus, which could selective and highly efficient oxidation of (+)-valencene to (+)-nootkatone. The oxygenase had 50% homologies with the putative lipoxygenases from Aspergillus fumigatus and Laccaria bicolor. According to database research, it also had 26% homology with the sequence of lipoxygenase-1 from soy bean . Later, it was found that (+)-valencene was oxidized to hydroperoxides and the homologous data of key enzymes was confirmed, indicating a kind of lipoxygenase enzyme (Fig. 1c) (Krugener et al., 2010). The oxidation was found to proceed in the highly special region, especially at C7. One of the two equal allylic positions of the ring double bond of the (+)-valencene was oxidized. There was a correlation between the activity of lipoxygenase and the production of (+)-nootkatone. The hydroperoxides [2,2 ,3,3 ] of (+)-valencene were the intermediate products of the oxidation process and the final products were β-nootkatol, α-nootkatol, and nootkatone. The gene of lipoxygenase was also cloned and expressed heterogeneously in Escherichia coli to produce (+)-nootkatone (Zelena et al., 2012). Moreover, classical genetics approach had changed and partially improved the 60% terpene transformation capability and lipoxygenase activity of Pleurotus species (Omarini et al., 2014). The dioxygenases could create catalysts in the future engineering. Some terpenes and alkenes can be catalyzed, making them the precursors of new flavor compounds.

Dehydrogenase and Reductase
In addition, there were some dehydrogenase and reductase enzymes involved in the conversion of (+)-valencene to (+)nootkatone. Cytochrome P450 monooxygenase P450 BM3 could catalyze (+)-valencene to the intermediate alcohol nootkatol, which was further oxidized to (+)-nootkatone by an alcohol dehydrogenase (ADH). Regeneration of cofactors supported by glucose dehydrogenase (GDH) can be achieved regardless of whether NADPH or NADH was used (Schulz et al., 2015). Cytochrome P450 oxidoreductases (CPR) acted as electron donors and it worked in coordination with P450s monooxygenase (Bak et al., 2011). Milhim et al. identified a novel short chain dehydrogenase (SDR) from Bacillus megaterium, which could convert (trans)-nootkatol to (+)-nootkatone for the first time (Milhim et al., 2019). Putidaredoxin reductase (PdR) was used as a coexpression enzyme in heterologous expression (Girhard et al., 2009).

Synthesis of (+)-Nootkatone by Heterologous Synthesis
In recent years, the metabolic engineering of microorganisms has made significant progress. Constructing microbial cell factories and heterologous expression of related enzymes became a great promising method for the production of (+)-nootkatone in yeast and E. coli (Fig. 2) (Table 3) (Paramasivan & Mutturi, 2017). The common precursor for terpenoid synthesis is isopentenyl diphosphate (IPP), which comes from Mevalonate pathway (MVA) or methyl-d-erythritol phosphate pathway (Maury et al., 2005). Condensation of IPP and its isomers dimethylallyl pyrophosphate (DMAPP) produce farnesyl diphosphate (FPP), which is the precursors for sesquiterpene synthesis. Currently, the plant (+)-valencene synthase was expressed in a microbial host and combined with the endogenous FPP delivery pathway and (+)valencene oxidase. This method obtained (+)-nootkatone with "natural" properties in accordance with current food legislation. In previous study, the researchers isolated a valencene synthase gene from C. sinensis, which was effective functionally expressed in E. coli and it could also be used for the subsequent production of (+)-nootkatone (Chappell & Greenhagen, 2010;Sharon-Asa et al., 2003). Afterwards, the regioselective oxidation test of (+)-valencene to (+)-nootkatone was carried out on 125 kinds of cytochrome p450 enzymes in bacteria. Studies have found that CYP109B1 of B. subtilis could catalyze the oxidation of (+)-valencene in E. coli with coexpression of PdR and putidaredoxin (Pdx) from P. putida (Girhard et al., 2009). Valencene dioxygenase (ValOx) from P. sapidus was expressed in the cytosol and periplasm of E. coli, which converted (+)-valencene to (+)nootkatone and nootkatol through intermediate hydroperoxides (Zelena et al., 2012). Various strategies used by cold shock expression, partner coexpression and mutant E. coli strains improved the yield of soluble recombinant protein.
More recently, Pichia pastoris have become one of the biochemical hosts with a wide range of microorganisms (Siripong et al., 2020). Wriessnegger et al. coexpressed the HPO and CPR in P. pastoris with the addition of (+)-valencene (Wriessnegger et al., 2014). (+)-Valencene could also be produced in the cell through the coexpression of (+)-valencene synthase, which solved the phase transfer problem of (+)-valencene. Then, it was found that the yield of (+)-nootkatone reached 208 mg/l by additional overexpression of P. pastoris ADH and truncated hydroxymethylglutaryl-CoA reductase. In addition, they also found that the overexpression of gene RAD52 had a significant positive effect on the formation of trans-nootkatol, which increased the production of trans-nootkatol (β-nootkatol) by five times compared with the original strain and conditions (Wriessnegger et al., 2016). Y. lipolytica was regarded as nonpathogenic and used as a production host for a large variety of biotechnological applications in several industrial processes classified as safe (Groenewald et al., 2014). The study achieved the heterologous production of (+)-nootkatone in Y. lipolytica by coexpressing CnVS, CYP706M1 and CPR. The final engineered strain produced (+)-nootkatone with an amount of 978.2 μg/l. This strain achieved the fusion of opCYP706M1 and opt46AtCPR1, and overexpressed the rate limiting enzymes tHMG1 and FPP synthase ERG20 in MVA pathway (Guo et al., 2018). The S. cerevisiae is a favorable host for the production of terpene compounds. It possessed the robustness of the strain, the compatibility with the existing infrastructure, and the performance of the availability of the existing genetic engineering molecular tools (Ignea et al., 2014). (+)-Nootkatone was produced by overexpressing (+)-valencene synthase and CYP71AV8 in yeast strain WAT11. The volume yields of (+)-valencene and (+)nootkatone were 1.36 and 0.04 mg/l, respectively (Cankar et al., 2011). In addition, CYP71D51v2 and P450-reductase were cloned and expressed in S. cerevisiae (Gavira et al., 2013). Another study described the coexpression of CYP706M1 and (+)-valencene synthase in yeast strain WAT11 to produce (+)-nootkatone. According to previous reports, 144 ± 10 μg/l (+)-nootkatone was produced and it had fewer intermediate products or by-products . The total terpene yield reached 31 mg/l by heterologous expression of several key enzymes in the synthesis pathway of (+)-nootkatone in S. cerevisiae w303 (Emmerstorfer et al., 2015). Related research has used S. cerevisiae as the host strain to reconstruct its metabolic pathway in vivo and biosynthesize (+)-valencene and its derivatives (+)-nootkatone. Finally, the synthesis of (+)-nootkatone reached 53.7 mg/l through resting cell transformation (Ouyang et al., 2019). Recently, a biosynthetic pathway was constructed in S. cerevisiae to produce the (+)nootkatone by overexpressing the CnVS, HPO, and ZSD1 combined with the MVA pathway engineering. The maximum yield of (+)nootkatone reached 59.78 mg/l. At the same time, it provided a solid foundation for the whole-cell production of (+)-nootkatone (Meng et al., 2020).

Conclusions and Future Perspectives
The aroma and fragrance of food will increase our sense of pleasure, arouse appetite, promote saliva secretion, and enhance the digestion and absorption of nutrients. Therefore, flavors and fragrances are an indispensable part of food. Terpenoids are the most used spices at present, and oxygen-containing sesquiterpene (+)-nootkatone not only has unique aromatic odor, but also has important pharmacological effects such as antioxidant, antibacterial, and antitumor. In terms of academic significance and application value, biotransformation has unlimited prospects in the modification and transformation of sesquiterpene (+)nootkatone. At present, researchers have conducted more research on biological transformation synthesis of natural spices. More attention should be paid on the directional transformation mechanism and regulation of (+)-valencene. The separation, cloning, expression and regulation of key enzyme genes in (+)valencene transformation and (+)-nootkatone biosynthesis have become a hot topic. It is the focus of current research for discovering new and more efficient enzymes and improving systems for expressing them heterogeneously. Gene engineering technology is used to site-directed mutation of key enzymes, change the structure of enzyme molecules, so as to improve the activity of enzyme. Gene recombination technology can also be used to obtain genetically engineered strains with high expression of key enzymes. In addition, the selection of abundant and inexpensive substrates for microbial transformation to produce spices will have more application prospects. Therefore, reducing the production costs of natural flavor (+)-nootkatone and meeting the needs of consumers and producers through bioengineering technology are a practical technology.

Author Contributions
All authors contributed to this study. XL and GF had the ideas for the article and performed the literature search and data analysis. XL wrote the manuscript. J-NR, L-LZ, and S-YP critically reviewed the manuscript.

Funding
This work was financially supported by the Fundamental Research Funds for the Central Universities (Program No. 2662020SPPY002), the National Natural Science Foundation of China (Program No. 31671824), and the Key Research and Development Program in Hubei Province (2020BBA049).