Patents on Endophytic Fungi Related to Secondary Metabolites and Biotransformation Applications

Endophytic fungi are an important group of microorganisms and one of the least studied. They enhance their host’s resistance against abiotic stress, disease, insects, pathogens and mammalian herbivores by producing secondary metabolites with a wide spectrum of biological activity. Therefore, they could be an alternative source of secondary metabolites for applications in medicine, pharmacy and agriculture. In this review, we analyzed patents related to the production of secondary metabolites and biotransformation processes through endophytic fungi and their fields of application. We examined 245 patents (224 related to secondary metabolite production and 21 for biotransformation). The most patented fungi in the development of these applications belong to the Aspergillus, Fusarium, Trichoderma, Penicillium, and Phomopsis genera and cover uses in the biomedicine, agriculture, food, and biotechnology industries.


Introduction
The term endophyte refers to any organism (bacteria or fungi) that lives in the internal tissues of a host. This endophyte-host association is complex: it is normally driven without causing harm or apparent disease symptoms and provides benefits in survival, fitness, biodiversity, and ecosystem function for both parties by enhancing the response to environmental stress and producing the same or similar compounds that originate in the host [1][2][3]. In particular, fungal endophytes have been the focus of many studies due to their prospective promise in the production of secondary metabolites with pharmacological, agricultural, industrial, or biotechnological applications [4][5][6].
Endophytic fungi were discovered over a century ago; however, it was not until about three decades ago, with the discovery of the taxol-producing endophytic fungus Taxomyces andreanae, that they gained remarkable relevance due to the abovementioned production of active secondary metabolites [7][8][9][10].
As was the case with taxol, the process for the isolation and purification of metabolites in adequate yields remains a major concern; low yields due to the exploitation of the host for the extraction process of metabolites are also associated with environmental impacts, and new strategies such as involving the use of endophytic microorganisms instead of the host themselves have offered new niches that should be meticulously investigated and used as a base for sustainable research and development [11,12].
The present review covers patents related to the production of natural products with biomedical and agricultural applications using endophytic fungi, enabling the development of new lead compounds

Results
The description and analysis of patents was divided into two sections: those that are connected to the production of secondary metabolites and those associated with biotransformation processes. Likewise, two tables were constructed in which the main generalities of each patent are summarized.

Production of Secondary Metabolites
Early patents consisted mainly of registering the endophytic strains capable of producing specific compounds or those that represented a novel source of active metabolites (chanoclavine in EP1142986A2; resveratrol in CN1948459A; gallic acid in CN101280279A; taxol in CN101486974A) and very few applications. However, over time, patents were developed to include the registration of methods and procedures to produce and recover the compounds of interest (with a known biomedical application) or to optimize or increase their production (podophyllotoxin in US20040248265A1; taxol in CN1624103A; camptothecin in US20060134762A1; huperzine A in CN101275116A). In the last ten years, patents have been focused on using novel or enhanced fermentation processes to obtain high yields of products and provide possible applications for the metabolites (alpha-pyrone in CN110563740A; epimedins A-C in CN110511876A; differanisole A in CN109971655A; 5, 8-ergosterol epoxide in CN109971651A). The distribution of the patents in relation with the principal areas of application are illustrated in Figure 1. The production of taxol and huperzine A were considered as other application outside of their anticancer and anti-Alzheimer property respectively, due to the number of patents and economic importance.
applications. The structures of the compounds listed in Tables 1 and 2 are shown in Figure S1 (see the Supplementary Information). The principal endophytic fungi reported in this section of patents belong to the genera Aspergillus, Fusarium, Trichoderma, Penicillium, and Phomopsis with 31, 24, 18, 16, and 8 patents, respectively, and compounds such as taxol or paclitaxel, huperzine A, camptothecin, podophyllotoxin, and resveratrol. Methods for enhancing their production represented most of the registered applications. Furthermore, the diversity of compound structures demonstrates the capability of fungi to synthetize simple or very complex molecules.
Mostly, Aspergillus endophytes from plants of the genera Taxus and Torreya are described as having applications related to obtaining the highest yield of paclitaxel or its precursors, like baccatin III and cephalomannine, due to their anticancer activity. Endophytes from Huperzia serrata have been linked to the production of huperzine A and its analogs due to their anti-senile dementia and antineurodegenerative applications. Plant endophytes such as Nothapodytes nimmoniana and Camptotheca acuminata have been linked to the production of the antineoplastic agent camptothecin and some analogs. Production of the lignan-type compound podophyllotoxin has been described for several endophytes. This compound has high biomedical potential as an anticancer, antiviral, and antibacterial agent, among others, and is the precursor of the anticancer drugs etoposide and teniposide. The stilbenoid compound found on grape skin, resveratrol, could have promising therapeutic actions against obesity, type II diabetes mellitus, metabolic syndrome, cancer, autism, dementia, and Alzheimer's disease [13]. Therefore, a number of patents involving endophytes of the The principal endophytic fungi reported in this section of patents belong to the genera Aspergillus, Fusarium, Trichoderma, Penicillium, and Phomopsis with 31, 24, 18, 16, and 8 patents, respectively, and compounds such as taxol or paclitaxel, huperzine A, camptothecin, podophyllotoxin, and resveratrol. Methods for enhancing their production represented most of the registered applications. Furthermore, the diversity of compound structures demonstrates the capability of fungi to synthetize simple or very complex molecules.
Mostly, Aspergillus endophytes from plants of the genera Taxus and Torreya are described as having applications related to obtaining the highest yield of paclitaxel or its precursors, like baccatin III and cephalomannine, due to their anticancer activity. Endophytes from Huperzia serrata have been linked to the production of huperzine A and its analogs due to their anti-senile dementia and anti-neurodegenerative applications. Plant endophytes such as Nothapodytes nimmoniana and Camptotheca acuminata have been linked to the production of the antineoplastic agent camptothecin and some analogs. Production of the lignan-type compound podophyllotoxin has been described for several endophytes. This compound has high biomedical potential as an anticancer, antiviral, and antibacterial agent, among others, and is the precursor of the anticancer drugs etoposide and teniposide. The stilbenoid compound found on grape skin, resveratrol, could have promising therapeutic actions against obesity, type II diabetes mellitus, metabolic syndrome, cancer, autism, dementia, and Alzheimer's disease [13]. Therefore, a number of patents involving endophytes of the genera Cladosporium, Fusarium, Alternaria, and Penicillium for its production were registered. The demand for natural resveratrol has gained traction in various end-use industries. New source in the form of a novel endophytic fungal strain for the production of camptothecin (8) and camptothecinioids and an improved process for producing these. [26] US7070985B2

Muscodor albus Cinnamomum zeylanicum
Novel fungi and production of organic volatile antibiotics effective in the treatment of human and animal waste products. [27] CN1896232A Fusarium sp.

Ginkgo biloba
Production of plasmin.

Santalum album
Method for preparation of hexichol phenolic acid compounds (169)(170)(171) and their application in the preparation of antibacterial compounds. [202] CN109232481A Not disclosed Taxus chinensis Preparation of high-purity taxol.

Biotransformation by Endophytic Fungi
Biotechnological processes enable the production of useful molecules with a decrease in the generation of pollutants, reducing the use of solvents and reagents, minimizing the consumption of energy, and providing a way to obtain active compounds with greater specificity and efficiency. The use of endophytic fungi in biotechnological processes, such as biotransformation, is in its early stages of development and has some limitations [238]. However, there have been some reports of fungi that have been used in biotransformation [239][240][241][242]. Table 2 lists a group of patents that illustrate the efforts toward using endophytic fungi to obtain molecules of biological importance such as the ginsenosides [243] and glycyrrhetinic acid monoglucuronide [244].
Fungi from the genera Absidia, Zygorhynchus, Xylaria, and Fusarium have been patented to obtain ginsenoside Rd by the transformation of ginsenoside Rb1. Fungi from the genera Microsphaeropsis, Aspergillus, and Chaetomium have been patented for the biotransformation of glycyrrhizinic acid into glycyrrhetinic acid monoglucuronide. Production of a telomerase activator, biotransformation with endophytic fungi to obtain new/novel molecules from the saponins from natural sources and method for discovery molecules that increase telomerase enzyme activation. [265]

Discussion
The study of endophytic fungi as a source of bioactive secondary metabolites has its first beginnings in 1993 with the discovery of taxol [4], until then, the primary sources of active natural molecules were isolated mainly from plants [266]. About two decades ago, the study of endophytic fungi as producers of active molecules has been emphasized due to obtaining compounds originally produced by plants or due to the production of novel secondary metabolites [11,267], Thus, fungi from genus Aspergillus, Fusarium, Penicillium and Pestalotiopsis has been recognized as producers of anticancer compounds and having pharmaceutical potential [12,268]. It is estimated that only around 1% of the microorganisms have been cultivated, and within this groups, endophytic fungi corresponded to the least studied [269].
Through this review, we have demonstrated the wide number of endophytic fungi involved in the development of methods and techniques for the application of isolation and fermentation to obtain secondary metabolites with high potential and applications in biomedicine, agriculture, and biotechnology processes. Figure 2 shows the number of patents registered for secondary metabolites and biotransformation processes through endophytic fungi for the period from 2001 to 2019. We found 224 patents related to secondary metabolites and 21 patents related to biotransformation. Aspergillus, Fusarium, Trichoderma, Penicillium, and Phomopsis were the most representative genera for secondary metabolites.  Fusarium and Penicillium were the most commonly registered endophytic fungi genera among the 21 patents reviewed for biotransformation processes. Figure 3 shows the number of patented genera. The most notable applications patented were antimicrobial, antibacterial, anticancer, and those related to neurodegenerative diseases. For biotransformation processes, the conversion of ginsenosides and glycyrrhizinic acid were the most patentable applications due to their importance and potential in the pharmaceutical and food industries.
Tables 1 and 2 showed that the majority of the endophytic fungi were derived from plants, but we could also find patents where the host was soft corals or insects.   The global market for compounds like taxol is expected to reach USD $99 million by 2021 [270], and for resveratrol, the projected growth from 2018 to 2028 in revenue terms is 8.1% from USD $97.7 million [271]. Under the objectives of the 1992 Convention on Biological Diversity for the sustainable use of its components and the Nagoya Protocol on Access to Genetic Resources and the Fair and Equative sharing of benefits derived from the use of genetic resources [272], endophytic fungi and their derived compounds could open a new set of industries and economics in development countries with high biodiversity for the low-cost yield of high-profit molecules that can be applied in the fields discussed in this review.