# Biosynthesis of Camphane Volatile terpenes in _Annomum_ _villosum_ Lour: Involved Genes and Enzymes
## 1 Introduction  
_Anomum villosum (A. villosum)_ plants, belonging to the Zingiberaceae family, are robust perennial herbs that produce leafy stems that can reach heights of up to three meters fromthe creeping rhizome [1]. The cultivation of _Amomum willosum_ Lour. is a significant form of agroforestry in southern Yunnan and Guangdong, China [2]. The fruit of _A. villosum_, which called Fructus Amomi, has long been used in the Chinese diet, and is officially recognized as \"Sha Ren\" in the Pharmacopoeia of the People's Republic of China [3]. The ripe fruit of _A. villosum_ has been used in dispelling dampness, treating gastrointestinal diseases, and preventing abortion in China for hundreds of years [4; 5; 6; 7; 8]. In recent years, volatile oil and polysaccharides extracted from Fructus Amomi contribute to some important biological functions, such as anti-inflammation, antimicrobial, and antinociceptive activities [9; 10; 11; 12; 13]. The volatile oil extracted from the ripe fruits of _A. villosum_ contains bioactive compounds such as bornyl acetate (5-47%), camphor (4-17%), borneol (1.5-6%), some primary camphane volatile terpenes, etc. [14]. Among these, bornyl acetate, the key ingredient in essential oils, has long been considered as a standard for the quality of _Fructus Amomi_[15]. The quality of _A. villosum_ is usually better than that of _A. longiligulare [PERSON]_ and _A. villosum Lour. var. xanthoides [PERSON]. [PERSON]_ because of its higher content of volatile oil and bornyl acetate [16]. The demand for _A. villosum_ in the domestic market is much greater than that of the latter two, which has long left it in short supply [17]. Therefore, identifying and analyzing the key genes associated with bornyl acetate in _A. villosum_, and elucidating the accumulation rhythm of bornyl acetate in _A. villosum_ fruits, will lay a foundation for optimizing the quality of _A. villosum_. Camphane volatile terpenes are the monoterreponids and sesquiterpenoids composed of two or three isoprene units, and synthesized by the cyclization of geranyl diphosphate via the isoprenoid pathway to form the primary backbone [18; 19]. Information about the upstream biosynthesis of monoterreponid and sesquiterpenoid skeletons has been reported [20]. This coincides with the triterpen biosynthesis pathway, both via two pathways (the mexulonate [MVA] pathway and 2-C-methyl-D-erythritol-4-phosphate [MEP] pathway) to form geranyl diphosphate [21], but several enzymes were characterized in the skeleton biosynthesis of monoterreponids and sesquiterpenoids, especially the biosynthesis of camphane volatile terpenes, and little about the whole biosynthesis pathway was analyzed. Bornyl diphosphate synthase (BPPS) is an important enzyme in the synthetic pathway of Amomom medicinal terpenoids. Catalyzing geranyl diphosphate (GPP) generates bornyl diphosphate (BPP), which is the direct precursor of the medicinal substance Longnao and the indirect precursor of bornyl acetate in _A. villosum_[22]. To our knowledge, the enzymes characterized in the camphane volatile terpene biosynthesis of A. villosum were 1-deoxy-D-xylulose-5-phosphate reductio isomerase (DXR, EC: 1.1.1.267), 1-deoxy-D-xylulose-5-phosphate synthase (DXS, EC:2.2.1.7), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC:1.1.1.34), and phosphomevalonate kinase (PMK, EC:2.7.4.2) [3; 23]. Other enzymes are desired for characterization and analysis. The specific biosynthesis pathway and regulation network of this plant species should be established to meet the development of cultivation as soon as possible. Currently, research on the biosynthesis of camphene and other volatile terpenes has advanced significantly, particularly due to progress in metabolic engineering and synthetic biology [24]. Engineered E. coli and Saccharomyces cerevisiae have been used to produce camphene [25], but in low yields due to the current challenges of enzyme inefficiency, metabolic burden, and competing pathways (e.g., limonene, pinene).