The Endophytes: A New Resource for Vulnerable Plant Bioactive Compounds

Abstract

Plant-associated microorganisms that live symbiotically in the plant body without causing disease symptoms are called endophytic microorganisms. Endophytes, including bacteria and fungi, can enhance the growth of the host plant and increase its resistance to pests, phytopathogens, and environmental stresses. In addition, endophytes can regulate the synthesis of plant secondary metabolites. Endophytes are a new reservoir for the discovery and production of valuable active substances. Some endophytic secondary metabolites are the same as host plants, such as paclitaxel. This finding has increased the importance of endophytes because the production of effective substances on an industrial scale in microorganisms is easier than in plants and has lower environmental costs. Therefore, endophytes need more attention in the pharmaceutical industry.

1. Introduction

The rapid growth of human societies has increased the need to improve health standards and intensify food production. On the other hand, the emergence of drug resistance in pathogens and pests has become an increasing need to promote the search for new pharmaceutical and agricultural sources. Medicinal plants have been a valuable source of bioactive substances for a long time; however, environmental considerations, labor-intensive, high cost, and time-consuming have limited the use of these plant resources. On the other hand, the production of plant material in cell cultures faces technical challenges. The production of effective plant substances entered a new age with the discovery of the endophytic fungus Taxomyces andreanea in the yew, which could produce bioactive such as its host. Microorganisms are an attractive source of new biomaterials; also, they have the potential to increase the production of existing valuable materials. Plant-associated microorganisms called endophytes live in symbiosis with the tissues of their host plants. Many microorganisms, such as fungi, bacteria, and actinomycetes, have been discovered in endophytic relationships with plants [1].

The endophytes live asymptomatically in mutual association with plants. The endophytic lifestyle of microbes plays an important role in maintaining the health of plants by providing nutrients and defending plants against abiotic and abiotic stresses [2]. In addition, endophytes can produce many bioactive. Some of these substances are similar to the profile of the host plant’s bioactive, which has increased the hope for cost-effective and environmentally friendly production. In the pharmaceutical and agricultural industries, bioactive compounds are known for their many applications. During the last two decades, endophytes have been recognized as important sources of bioactive compounds. Also, the proportion of new structures produced by endophyte isolates (51%) is significantly higher than that of soil isolates (38%), which has made endophytes one of the main natural product screening programs [3].

2. Endophytes

Microorganisms colonize many living plants in nature, and the degree of this microbial colonization varies by plant species. If the host plant tissue remains stable during this colonization, the relationship may vary from latent pathogenesis to mutual symbiosis. These microorganisms may be epiphytes, endophytes, or latent pathogens. Endophyte refers to microorganisms that are found under normal conditions in the tissues of living plants, without causing apparent diseases or visible symptoms of disease [4]. Endophytes are ubiquitous and spend a significant part of their life cycle without causing negative or obvious symptoms in the living tissues of the host plant. The word endophyte was first coined in 1866, where “endo” means “inside” and “phyte” means plant. They are mostly located in internal tissues such as roots, stems, leaves, flowers, and seeds. Endophytes may be transmitted horizontally or vertically [2], and some may even be seed-borne and passed on to the next generation [4]. A large community of endophytes lives inside the tissues of any plant. The diversity of endophytes is influenced by the host plant and its characteristics, including genotype, tissue, growth stage (age), and health status [5].

Endophytes have been isolated from all different parts of the plant. More than 200 genera from 16 bacterial phyla have been documented to be associated with endophytes [6]. It is also estimated that out of about 1.5 million species of fungi, one million of them are endophytic [7].

2.1 Endophytes: Plant interaction

Endophytes can provide benefits to their host plants. They mediate abiotic and biotic stress tolerance, reduce water consumption, and defend against pests and phytopathogens [8]. This interaction is controlled by endophyte and plant genes. The endophytic relationship is a novel and cost-effective plant-microbe evolutionary relationship that is driven by location and not defined by function [9]. Endophytic microbes are chemical synthesizers inside plants [10]. The imperceptible association of endophytes with the plant enables them to evolve [9]. It is the coevolution between endophytes and their host plant that determines the production of bioactive compounds. These compounds often play a role in the plant-microbe interaction in different ways and can bring different fitness benefits to the host plant [11, 12].

Plant compounds can be of plant origin or derived from endophytes or even can be produced by both. In the latter case, the endophyte may be involved in the entire pathway, but another scenario may be that only parts of the biosynthesis originate from the endophyte. In plant-endophyte interactions, significant changes appear in the secondary metabolism of symbionts, and these changes can be as a result of (i) induction of host metabolism by endophyte, (ii) induction of endophyte metabolism by the host, (iii) host and endophyte share part of a specific pathway, (iv) the host metabolizes endophyte products, and (v) the endophyte can metabolize host secondary compounds. [13]. Endophytes isolated from medicinal plants can produce bioactive metabolites and play a vital role in inducing secondary metabolite production by host plants [5, 14].

3. Secondary metabolites

Endophytes play a critical role in enhancing plant growth and are also known for their ability to produce bioactive with biotechnological applications. The use of herbal medicines is common in developing countries and up to 80% of people use this medicine. This traditional medicine has a long history. Medicinal plants are known for their rich sources of natural products. They are very valuable for disease prevention and treatment [15]. Endophytes communicate with their host plant through metabolic interactions [1, 16], which enable them to produce signaling molecules with interesting biological activities. In addition, the coevolution of endophytes with the host plant enables them to mimic the biological properties of the host and produce similar bioactive compounds [16].

Endophytes synthesize various bioactive compounds. However, compounds that have shown anticancer properties have attracted more attention, and in the meantime, the discovery of paclitaxel production by endophytic fungi has been a turning point in endophyte research.

3.1 Paclitaxel (Taxol)

Paclitaxel, with the brand name Taxol, is a terpenoid that was mainly obtained from the tissues of the yew plant; due to its amazing properties in binding to microtubules and inhibiting the division spindle, it is used in the treatment of various types of cancer, especially breast and ovarian cancer. It has been used a lot. However, extraction from plant sources due to the slow growth of the plant, the difficulty of purifying paclitaxel, and also its low amount in the plant tissues did not meet the needs of the market. Therefore, several methods, such as chemical synthesis, were also developed and commercialized. The scientists were also looking for alternative sources until the ability to synthesize it in the endophytic fungi of the host plant was discovered.

The discovery of Taxomyces andreanea from the Pacific yew, Taxus brevifolia, was undoubtedly a turning point in the field of bioprospecting for endophytes. This endophytic fungus demonstrated the ability to synthesize paclitaxel in the culture broth same as its host plant [17]. Microbial production of paclitaxel is very important for the development of the first billion-dollar anticancer drug business [17, 18]. Since this important discovery, several other endophytic fungi and bacteria showing paclitaxel production from yew and other plant species have been discovered (Table 1), including Alternaria, Bartalinia, Fusarium, Lasiodiplodia, Metarhizium, Monochaetia, Pestalotiopsis, Penicillium, Phoma, and Spomatoichoanthermium [13, 81, 82, 83]. The efficiency of paclitaxel among these fungal species varies [from nanograms to milligrams per liter], and their productivity is often lost during several generations of cultivation in laboratory conditions [84]. Microbial production of paclitaxel by endophytes has been observed mostly in fungal isolates. However, there are limited reports of the production of paclitaxel and some of its precursors by several strains of endophytic bacteria, such as Erwinia taxi, Micromonospora sp., Streptomyces sp., Kitasatospora sp., Bacillus cereus, B. megaterium, Sphingomonas ssp. taxi, B. subtilis, Pantoea sp., and Curtobacterium sp. [85]. Also, the discovery of paclitaxel-producing bacteria symbiotic with marine macroalgae Sargassum polycystum and Acanthaphora specifera showed that the search for endophytic sources of paclitaxel should not be limited to plants and terrestrials [68].

Secondary metabolite	Host	Endophyte	Yield (μg/L)	Ref
Paclitaxel (Taxol)	Taxus brevifolia	Taxomyces andreanae	0.02–0.05	[17]
	T. wallichiana	Pestalotiopsis microspora	0.06–0.07	[19]
	Taxodium distichum	Pestalotiopsis microspora Cp-4	0.05–1.49	[20]
	Taxus cuspidata	Alternaria sp. Ja-69	0.16	[19]
	Taxus baccata	Fusarium lateritium Tbp-9	0.13
	T. baccata	Monochaetia sp. Tbp-2	0.10
	T. baccata	Pestalotia bicilia Tbx-2	1.08
	T. cuspidata	Pestalotiopsis microspora Ja-73	0.27
	T. wallachiana	Pestalotiopsis microspora Ne-32	0.5
	T. sumatrana	Pithomyces sp. P-96	0.095
	T. canadensis	Erwinia taxi*	2.5–15	[21]
	Wollemia nobilis	Pestalotiopsis guepinii W-1f-2	0.49	[22]
	Torreya grandifolia	Periconia sp. No. 2026	0.03–0.83	[23]
	Ginkgo biloba	Alternaria sp.	0.12–0.26	[24]
	T. baccata	Kitasatospora sp. *	120	[25]
	T. baccata	Penicillium sp.	111
	T. canadensis, T. brevifolia, T. hunnewelliana, T. baccata, T. cuspidata	Bacillus cereus ssp. taxi, Bacillus megaterium ssp. taxi, Pantoea sp., Bacillus cereus, Bacillus subtilis ssp. taxi, Bacillus megaterium, Curtobacterium sp., Sphingomonas ssp. taxi*	1–25	[26]
	Tremacron mairei	Tubercularia sp. TF5	185.4	[27]
	T. yunnanensis	Taxomyces sp.	2.3
	T. chinensis var. mairei	Ozonium sp. BT2	4–18	[28]
	T. cuspidata	Botrytis sp. HD181–23	206.34	[29]
	T. chinensis var. mairei	Botrytis sp. XT2	161.24
	T. chinensis var. mairei	Ectostroma sp. XT5	276.75
	T. chinensis var. mairei	Papulaspora sp. XT17	10.25
	T. chinensis	Alternaria alternata TPF6	84.5	[30]
	T. chinensis var. mairei	Fusarium mairei Y1117	2.7	[31]
	T. chinensis var. mairei	Ozonium sp. EFY-21	21	[32]
	Aegle marmelos	Bartalinia robillardoides AMB9	187.6	[33]
	Cardiospermum helicacabum	Pestalotiopsis pauciseta CHP-11	113.3	[34]
	T. chinensis	Fusarium mairei UH23	286.4	[35]
	Citrus medica	Phyllosticta citricarpa No.598	265	[36]
	Podocarpus sp	Aspergillus fumigatus EPTP-1	557.8	[37]
	T. baccata	Botryodiplodia theobromae BT115	280.5	[38]
	T. cuspidata	Fusarium arthrosporioides F-40	131	[39]
	Cupressus sp	Phyllosticta spinarum No.625	235	[40]
	Wrightia tinctoria	Phyllosticta tabernaemontanae	461	[41]
	Thysanophrys celebica	Fusarium solani	1.6	[42]
	T. cuspidata	Aspergillus niger var. taxi HD86–9	273.6	[43]
	T. chinensis	F. solaniI Tax- 3	163.35	[44]
	T. chinensis	Metarhizium anisopliae H- 27	846.1	[45]
	T. media	Cladosporium cladosporioides MD2	800	[46]
	T. media	Aspergillus candidus MD3	112	[47]
	T. chinensis	Mucor rouxianus DA10	ND	[48]
	Hibiscus rosa-sinensis	Phyllosticta dioscoreae No.605	298	[49]
	Terminalia arjuna	Chaetomella raphigera TAC15	79.6–211.1	[50]
	Terminalia arjuna	Pestalotiopsis terminaliae	211	[51]
	Taxus cuspidata	Nodulisporium sylviforme	450	[52]
	Morinda citrifolia	Lasiodiplodia theobromae	245	[53]
	Rhizosphere	Pestalotiopsis malicola	186	[54]
	Ginkgo biloba	Phoma betae	795	[55]
	T. baccata	Stemphylium sedicola SBU-16	6.9	[56]
	T. baccata	F. redolens	66	[57]
	Corylus avellana and T. baccata	Penicillium aurantiogriseum NRRL 62431	70	[58]
	T. wallichiana	P. medicaginis	1125	[59]
	T. chinensis var. mairei	Aspergillus aculeatinus Tax-6	334.92	[60]
	Podocarpus gracilior	Aspergillus terreus EFB108	20	[61]
	Rhizosphere	Aspergillus flavipes	185–850	[62]
	Rhizosphere	Penicillium chrysogenum	85	[62]
	Terminalia arjuna	Alternaria brassicicola	140.8	[63]
	Taxus sp.	Aspergillus fumigatus TPF-06	1590	[64]
	Catheranthus roseus	Cladosporium cladosporioides	700	[65]
	Tarenna asiatica	Aspergillus oryzae	95.04	[66]
	T. baccata	Epicoccum nigrum TXB502	61.35	[67]
	Sargassum polycystum	Bacillus flexus DMTMMB08*	ND	[68]
		Bacillus licheniformis; DMTMMB10*
	Acanthaphora specifera	Oceanobacillus picturae DMTMMB24*
	Ginkgo biloba	Penicillium polonicum AUMC14487	90.53	[69]
	Mangifera indica	Colletotrichum sp. MIP-5	ND	[70]
	T. wallichiana	Annulohypoxylon sp. MUS1	282.05	[71]
	Persea americana	Neopestalotiopsis clavispora KY624416	100.6	[72]
	Moringa, Hibiscus	Penicillium sp. No.5	54.42–184.3	[73]
		Aspergillus niger No.10	43.95
		Fusarium sp. No.8	26.8
	Corylus avellana	Stemphylium vesicarium CA18	1400	[74]
	Corylus avellana	Melanconium hedericola CA12	1000
	Calotropis procera, Catharanthus roseus	Penicillium singorense	13	[75]
	Millingtonia hortensis	Cochliobolus hawaiiensis	282	[76]
	T. wallichiana	Aspergillus sp. GBPI TWR F5	5450	[77]
baccatin III	T. chinensis	Didmyostilbe sp. DF110	ND	[78]
	T. wallichiana	Diaporthe phaseolorum	219	[79]
	T. wallichiana	Trichoderma sp. IRB54a	187.56	[80]
10-deacetyl baccatin III	Corylus avellana	Melanconium hedericola CA12	22,100	[74]
		Aspergillus microcysticus CA3	20,400
		Arthrinium arundinis CA2	16,400

Table 1.

Production of paclitaxel and some of its precursors by endophytic microorganisms; due to the multiplicity of different isolates from the same species, the name of the strain is also mentioned, as well as the amount of production in the strains noticed without subsequent manipulations and optimizations.

^*

Reveals bacterial producers.

Some strains that are only capable of producing precursors, such as baccatin III and 10-DAB, are listed separately.

In addition to paclitaxel production, some endophytes can increase paclitaxel production in plants. Endophytic Pseudodidymocyrtis lobariellae fermentation broth can effectively increase paclitaxel accumulation in T. chinensis by regulating phytohormone metabolism and signal transduction and further regulating the expression of several key genes involved in paclitaxel biosynthesis [86]. The fermentation broth of Kocuria sp., Micromonospora sp., and Sphingomonas sp. also significantly increased the accumulation of taxanes in the stem cells of T. yunnanensis [87].

3.2 Vinca alkaloids

Vinblastine and vincristine are vinca alkaloids from Catharanthus roseus plant [88]. These compounds were the first herbal anticancer agents that were introduced to the clinical market. In the 1960s, vinblastine was used to treat breast cancer, testicular cancer, and Hodgkin’s disease. Three years later, its oxidized derivative, vincristine, was introduced, which was widely used in the treatment of leukemia. These compounds inhibit the division spindle by irreversibly binding to microtubules and finally induce apoptosis. Vinblastine production from endophytic Alternaria was first described in 1998, followed by Lingqi et al. discovered an endophytic Fusarium oxysporum from C. roseus that successfully produced vincristine [89, 90]. These discoveries sparked a global hunt for new alternative sources of vinblastine and vincristine. Vincristine is most valuable as an anticancer agent. Endophytic F. oxysporum successfully biotransformed vinblastine to vincristine [91].

Palem et al. isolated an endophytic Thalaromyces radicus from C. roseus that could produce vinblastine and vincristine [92]. Ayob et al. isolated an endophyte Nigrospora sphaerica from C. roseus that can produce vinblastine. This fungus produced vinblastine with 10-fold better cytotoxicity to a breast cancer cell line compared to vinblastine extracted from C. roseus [93]. Endophytic fungal and bacterial species were found to have the ability to synthesize Vindoline —the precursor of vinca alkaloids— and have a high potential to be used as a biological elicitor in the production of vincristine [94, 95]. Also, a species of Streptomyces spp. was isolated from the rhizosphere soil of C. roseus, which can produce vinblastine and vincristine, (Table 2) [104].

Host plant	Endophyte	Vinca alkaloids	Strain	Ref
Catharanthus roseus	Alternaria sp.	Vinblastine	Fungal	[89]
	F. oxysporum	Vincristine	Fungal	[90]
	Mycelia sterilia 97CY-3	Vincristine	Fungal	[96]
	Fusarium solani	Vinblastine, Vincristine	Fungal	[97]
	F. oxysporum	Vinblastine, Vincristine	Fungal	[98]
	Talaromyces radicus	Vinblastine, Vincristine	Fungal	[92]
	Eutypella sp.	Vincristine	Fungal	[99]
	Nigrospora sphaerica	Vinblastine	Fungal	[93]
	Unidentified	Vinblastine, Vincristine	Fungal	[100]
	Microbacterium sp.	Vindoline	Bacterial	[94]
	Chaetomim globosum	Vinblastine	Fungal	[101]
	Curvularia verruculosa	Vinblastine	Fungal	[102]
	Botryosphaeria laricina	Vinblastine, Vincristine	Fungal	[103]
C. roseus; Rhizospheric Soil	Streptomyces spp.	Vinblastine, Vincristine	Bacterial	[104]
C. roseus	F. oxysporum	Vinblastine, Vincristine	Fungal	[105]
	Alternaria sesami	Vindoline	Fungal	[95]
	Nigrospora zimmermanii	Vincristine	Fungal	[106]

Table 2.

Microbial production of vinca alkaloids by endophytic microorganisms; endophytic producers of vinca alkaloids have so far only been isolated from C. roseus or its rhizosphere soil.

Some of these isolates can biotransform vinblastine into vincristine. Also, some isolates only can synthesize vindoline as a valuable precursor of anticancer drugs.

3.3 Camptothecin

Camptothecin (CPT) is a pentacyclic quinoline alkaloid isolated from the wood of Camptotheca acuminata and the root of Nothapodytes foetida. Several reports show the therapeutic potential of CPT and its derivatives for the treatment of colon, cervical, uterine, lung, and ovarian cancer. Most of the two promising anticancer activities [107] are related to its main derivatives, 9-methoxycamptothecin and 10-hydroxycamptothecin, because CPT is not directly used as an anticancer drug due to its low solubility, short half-life, and toxicity [108, 109, 110]. These cytotoxic agents act by selectively inhibiting topoisomerase 1. and thereby disrupting the DNA replication process.

In 2005, the CPT-producing endophytic fungus Entrophospora infrequens was isolated from N. foetida [109]. Endophyte Neurospora crassa and Nodulisporium sp. isolated from N. foetida produces CPT in culture medium [111, 112]. There are also examples of endophytes that can produce hydroxylated CPT derivatives, for example, Mycelia sterilia XK001 can produce 10-hydroxycamptothecin, which is the clinically active derivative of CPT [107]. Most CPT-producing endophytes are fungi; however, there are also reports of bacterial producers (Table 3) [116, 121, 123, 126]. A CPT-producing endophytic fungus from the marine sponge Cliona sp. It has been isolated that unlike other endophytes isolated from soil and plant environments, and it has been isolated and identified from the marine environment and aquatic organisms [131].

Secondary metabolite	Host plant	Endophyte	Strain	Ref
CPT	Nothapodytes foetida	Entrophospora infrequens	Fungal	[109]
	Neanotis foetida	Neurospora sp.	Fungal	[111]
	Camptotheca acuminata	F. solani	Fungal	[108]
	N. foetida	Nodulisporium sp.	Fungal	[112]
	N. nimmoniana	Botryosphaeria parva	Fungal	[113]
	Apodytes dimidiata	F. solani	Fungal	[114]
	C. acuminata	Trichoderma atroviride	Fungal	[115]
	Miquelia dentata Bedd.	Bacillus subtilis, Bacillus sp., Bacillus cereus, Lysinibacillus sp.	Bacterial	[116]
	M. dentata Bedd.	Fomitopsis sp., Alternaria alternata, Phomposis sp.	Fungal	[117]
	C. acuminata	Fusarium nematophilum, Alternaria; Alternata, Phomopsis vaccinii	Fungal	[118]
	N. foetida	Fusarium oxysporum	Fungal	[119]
	Catharanthus roseus	F. solani	Fungal	[120]
	C. acuminata	Paenibacillus polymyxa	Fungal	[121]
	N. nimmoniana	Colletotrichm fructicola, Corynespora cassiicola	Fungal	[122]
	N. nimmoniana	F. solani	Fungal	[110]
	Pyrenacantha volubilis	Bacillus sp., B. subtilis, Bacillus amyloliquefaciens	Fungal	[123]
	Piper betel L.	Aspergillus niger	Fungal	[124]
	Chonemorpha fragrans	F. solani	Fungal	[125]
	Ephedra foliata	Kytococcus schroeter	bacterial	[126]
	Ophiorrhiza mungos	Meyerozyma sp., Talaromyces sp.	Fungal	[127]
	N. nimmoniana	Alternaria alstroemeriae, Alternaria burnsii	Fungal	[128]
	Cipadessa baccifera	Phyllosticta elongata	Fungal	[129]
	Ficus elastica	Aspergillus terreus, Aspergillus flavus	Fungal	[130]
	sponge Cliona sp.	Penicillium chrysogenum	Fungal	[131]
	Cestrum parqui	Aspergillus terreus	Fungal	[132]
	No. nimmoniana	Diaporthe sp. F18	Fungal	[133]
10-hydroxyCPT	No. nimmoniana	Mycelia sterilia_ XK001	Fungal	[107]
Podophyllotoxin	Podophyllum hexandrum	Alternaria sp., Penicillium sp.	Fungal	[134]
	Diphylleia sinensis	Penicillium sp.	Fungal
	Dysosma veitchii	Monilia sp., Penicillium sp.	Fungal
	D. sinensis	Penicillium implicatum	Fungal	[135]
	D. veitchii	Penicillium implicatum	Fungal	[136]
	Juniperus vulgaris	Alternaria sp.	Fungal	[137]
	P. peltatum	Phialocephala fortinii	Fungal	[138]
	P. hexandrum	Trametes hirsuta	Fungal	[139]
	P. hexandrum	Alternaria neesex	Fungal	[140]
	Juniperus recurva	Fusarium oxysporum	Fungal	[141]
	P. hexandrum	F. solani	Fungal	[142]
	Solanum hexandrum	Mucor fragilis	Fungal	[143]
	Podophyllum emodi	Alternaria tenuissima	Fungal	[144]
	D. sinensis	Penicillium sp.	Fungal	[145]
	P. hexandrum	Chaetomium globosum, Pseudallescheria sp.	Fungal	[146]
	Dysosma versipellis	Fusarium sp.	Fungal	[147]
	P. hexandrum	Penicillium sp.	Fungal	[148]
	Dysosma difformis	Penicillium sp., Trametes sp., Purpureocillium sp., Aspergillus sp., Ganoderma sp.	Fungal	[149]
	D. difformis	Fusarium proliferatum	Fungal	[150]
Deoxypodophyllotoxin	J. communis	Aspergillus fumigates	Fungal	[151]
Huperzine A	Huperzia serrata	Acremonium sp.	Fungal	[152]
	Phlegmariurus cryptomerianus	Blastomyces sp.	Fungal	[153]
		Botrytis sp.	Fungal
	Lycopodium serratum	Penicillium chrysogenum	Fungal	[154]
	H. serrata	Shiraia sp.	Fungal	[155]
		Cladosporium cladosporioides	Fungal	[156]
		Aspergillus flavus	Fungal	[157]
		Shiraia bambusicola	Fungal	[158]
		Colletotrichum gloeosporioides	Fungal	[159]
		Trichoderma sp.	Fungal	[160]
		Paecilomyces tenuis	Fungal	[161]
		Penicillium sp.	Fungal	[162]
	Phlegmariurus phlegmaria	Ceriporia lacerate	Fungal	[163]
	H. serrata	Colletotrichum sp., Ascomycota sp., Sarcosomataceae sp., Dothideomycetes sp.	Fungal	[164]
		Penicillium sp.	Fungal	[165]
		Alternaria brassicae	Fungal	[166]
		Penicillium polonicum, Colletotrichum gloeosporioides	Fungal	[167]
		Mucor racemosus, M. fragilis, Fusarium verticillioides, F. oxysporum, Trichoderma harzianum	Fungal	[168]
	Phlegmariurus taxifolius	Fusarium sp.	Fungal	[169]
	H. serrata	Fusarium sp.	Fungal	[170]

Table 3.

Production of plant-derived secondary metabolites by endophytic microorganisms.

CPT and active derivative 10-hydroxyCPT, podophyllotoxin, deoxypodophyllotoxin act as anticancer, and Huperzine A approved for treatment of Alzheimer’s disease.

3.4 Podophyllotoxin

Podophyllotoxin is an aryltetralin lignin that uses in the synthesis of anticancer drugs. It is originally isolated from the resins of the Podophyllum emodi, which is traditionally used to treat genital warts [16]. Podophyllotoxin is a strong inhibitor of microtubules, while its derivatives inhibit topoisomerase 2. These derivatives are used to treat bronchial and testicular cancers. Podophyllotoxin production from endophytic fungi isolated from Podophyllum [syn. Sinopodophyllum] hexandrum, Diphylleia sinensis, and Dysosma veitchii were reported for the first time [134]. After that, two strains of the endophytic fungus Phialocephala fortinii from the rhizome of P. peltatum, which could produce podophyllotoxin under axenic culture conditions, were isolated and identified [138]. The fungus Trametes isolated from P. hexandrum is another endophyte capable of producing podophyllotoxin and podophyllotoxin glycosides [139]. In addition, F. oxysporum and Aspergillus endophytes isolated from Juniperus recurva and Juniperus communis produced podophyllotoxin and deoxypadophyllotoxin, respectively [141, 151]. Podophyllotoxin production has also been reported from Mucor fragilis, and Alternaria tenuissima isolated from P. emodi was found to produce podophyllotoxin [143, 144]. Podophyllotoxin-producing endophytic fungi Penicillium sp., Trametes sp., Purpureocillium sp., Aspergillus sp. Ganoderma sp., and Fusarium spp. were isolated from plants of Dysosma spp. [149, 150]. Most podophyllotoxin-producing fungi belong to Penicillium sp., Alternaria sp., and Fusarium spp. genera, respectively, While there is no report of podophyllotoxin production among endophyte bacteria (Table 3).

Fungal production of podophyllotoxin is promising for mass production, and it is possible to provide affordable resources for commercial production by optimizing the cultivation methods and genetic changes of the producing microorganisms and reducing the pressure of harvesting from plant resources and giving the chance to producing plants for save from extinction.

3.5 Huperzine A

The lycopod Huperzia serrata is the main source of a natural lycopodium alkaloid called Huperzine A (HupA), which has attracted worldwide attention for its potential in the treatment of Alzheimer’s disease. This compound is an acetylcholinesterase inhibitor that increases the availability of acetylcholine in central cholinergic synapses by highly selective and reversible inhibition of this enzyme and blocking its activity. The bulk of HupA is obtained from the Huperziaceae family. The H. serrata has a narrow geographical distribution, slow growth rate, and very low HupA content, which limits its natural harvest and HupA extraction. In the first report, the endophyte Acremonium sp. isolated from H. serrata has been capable of production of HupA [152]. Similarly, the endophyte Shiraia sp. Slf14 and Cladosporium cladosporioides isolated from H. serrata leaves also produced HupA [155, 156, 158]. In general, 32 endophytic fungi belonging to 15 genera were recorded to produce Hup A. These fungal endophytes were isolated from members of Huperziaceae family, including H. serrata, Phlegmariurus phlegmaria, and Phlegmariurus taxifolius (Table 3) [171].

Xia and colleagues isolated endophytic fungi Mucor racemosus, M. fragilis, Fusarium verticillioides, F. oxysporum, and Trichoderma harzianum from the H. serrata, which can inhibit acetylcholinesterase enzyme [168]. The endophyte Ceriporia lacerate successfully transformed HupA into five different compounds that showed potential acetylcholinesterase inhibitory activity [172]. Biotransformation, using fungal endophytes, is also a valuable approach to producing HupA derivatives. Microbial production of HupA has positive economic and environmental effects. This will be a practical strategy to meet the global market demand through microbial fermentation and genetic manipulation of the source fungi.

4. Industrial aspects

The role of plant compounds in the production of many clinically effective anticancer drugs is undeniable, but the production of herbal drugs is not always as expected. Because their production from plant resources faces serious challenges, many of these compounds are produced at a certain stage of plant growth or under certain environmental conditions, stress, or availability of nutrients. Also, the growth of plants is slow, and to collect and extract some products, they must reach acceptable growth. On the other hand, production in plant cell culture also faces technical challenges. Also, due to the extent and variety of bioactive in plants, the purification processes of the desired effective substances will be complicated and therefore expensive. Due to the limitations identified with the productivity and vulnerability of plant species as sources of new metabolites, microorganisms act as an available and inexhaustible resource of new pharmaceuticals [173].

Over many years, seasonal and climatic factors have caused failure in traditional methods of extracting bioactive from natural resources. The environmental issues that researchers face during the extraction of bioactive from plants make it necessary to adopt new approaches to obtain these compounds [174]. In the future, with the increase in population, the demand for pharmaceutical and agricultural products will increase day by day, and the future of endophytic fungi for the isolation of various beneficial compounds is bright. There is a great need to discover bioactive compounds from natural resources that can be used to treat various diseases. Recently, more attention has been paid to the production of bioactive from endophytic fungi because they are excellent for exploiting the biosynthetic pathway for the synthesis of bioactive. The main challenge is the low yield of desired active compounds obtained from endophytes. However, to meet the demand of pharmaceutical companies to increase the commercial production of drugs, genetic engineering technologies, drug design techniques, and microbial fermentation technology can be solutions to increase the rate of endophyte production [2]. In addition, the use of cell co-cultures of host plants and endophytes has improved the production rate. Some secondary metabolites may be produced by combined endophyte and host activity. Some endophytic bacteria produce secondary metabolites in medicinal plants. For example, Bacillus altitudinis, Burkholderia sp., and Flavobacterium sp. act as effective stimulators that increase ginsenoside concentrations by converting the major ginsenoside Rb1 to the minor ginsenoside Rg3 in the valuable medicinal plant ginseng [175, 176, 177]. Such biotransformations using endophytic bacteria have significant potential to intensify the accumulation of rare active substances in medicinal plants. The endophytic Pseudomonas fluorescens can increase the production of sesquiterpenoids in Macrocephala Atractylodes [178]. The endophyte Bacillus subtilis in the plant Chuanxiong Ligusticum enhances ligustrazine accumulation [179].

The interaction of endophytes with plant tissues asymptomatically increases the production of secondary metabolites. A double synthesis of podophyllotoxin was obtained from the interaction of endophytic fungi Phialocephala fortinii and rhizomes of P. peltatum [138]. Endophytic fungi Stemphylium amaranthi and Gliomastix masseei can be used as fungal stimulants to improve indole alkaloid production from C. roseus [180].

5. Conclusion

Throughout history, humans have used plants and plant-derived products to treat various ailments. Plant secondary metabolites or bioactive are known to be synthesized by plants. Microbes living inside host plant tissues are also known for their ability to synthesize substances similar to those synthesized by the host plant. Secondary metabolites, such as alkaloids, flavonoids, terpenoids, steroids, etc. synthesized by microbes, are known for their vital role as antioxidants and anticancer. The discovery of the ability to produce plant secondary metabolites in endophytes has raised many hopes for the production of these compounds on an industrial scale. Microorganisms reduce environmental concerns about the production of biological substances in plants because endophytic microbes have a high reproduction ability, the possibility of their genetic manipulation is easier, and the fermentation conditions for them are simpler, cheaper, and more diverse.