Biotechnological Exploration of Transformed Root Culture for Value-Added Products

Hairy roots are useful tools for studying the biosynthesis of different plant-derived valuable compounds.Hairy roots could be preferred hosts when the desired compounds mainly accumulate in roots.Hairy roots are being considered as an alternative system to microbial hosts, including Escherichia coli and Saccharomyces cerevisiae, for producing plant-derived natural secondary metabolites because they are more similar to the native host plant.Hairy roots have emerged as valuable tools for the rapid characterization of plant gene function and enzyme activity in vivo because hairy roots naturally maintain many cofactors and precusor substrates, and the encoded plant-derived protein is more likely to be properly folded in hairy roots compared with in microbes. Medicinal plants produce valuable secondary metabolites with anticancer, analgesic, anticholinergic or other activities, but low metabolite levels and limited available tissue restrict metabolite yields. Transformed root cultures, also called hairy roots, provide a feasible approach for producing valuable secondary metabolites. Various strategies have been used to enhance secondary metabolite production in hairy roots, including increasing substrate availability, regulating key biosynthetic genes, multigene engineering, combining genetic engineering and elicitation, using transcription factors (TFs), and introducing new genes. In this review, we focus on recent developments in hairy roots from medicinal plants, techniques to boost production of desired secondary metabolites, and the development of new technologies to study these metabolites. We also discuss recent trends, emerging applications, and future perspectives. Medicinal plants produce valuable secondary metabolites with anticancer, analgesic, anticholinergic or other activities, but low metabolite levels and limited available tissue restrict metabolite yields. Transformed root cultures, also called hairy roots, provide a feasible approach for producing valuable secondary metabolites. Various strategies have been used to enhance secondary metabolite production in hairy roots, including increasing substrate availability, regulating key biosynthetic genes, multigene engineering, combining genetic engineering and elicitation, using transcription factors (TFs), and introducing new genes. In this review, we focus on recent developments in hairy roots from medicinal plants, techniques to boost production of desired secondary metabolites, and the development of new technologies to study these metabolites. We also discuss recent trends, emerging applications, and future perspectives. Medicinal plants produce numerous functionally diverse secondary metabolites (see Glossary), including terpenoids, phenylpropanoids, and alkaloids. Many of these compounds are of great pharmaceutical importance. For instance, some alkaloids have anticancer (i.e., camptothecin, taxol, and vinblastine), analgesic (i.e., morphine and codeine), or anticholinergic properties (i.e., atropine and scopolamine) [1.Carqueijeiro I. et al.Beyond the semi-synthetic artemisinin: metabolic engineering of plant-derived anti-cancer drugs.Curr. Opin. Biotechnol. 2020; 65: 17-24Crossref PubMed Scopus (23) Google Scholar, 2.Li F.S. Weng J.K. Demystifying traditional herbal medicine with modern approaches.Nat. Plants. 2017; 3: 17109Crossref PubMed Scopus (135) Google Scholar, 3.Srinivasan P. Smolke C.D. Engineering a microbial biosynthesis platform for de novo production of tropane alkaloids.Nat. Commun. 2019; 10: 3634Crossref PubMed Scopus (37) Google Scholar]. Ginsenosides have antiaging, antioxidative, adaptogenic, and anticancer properties [4.Nag S.A. et al.Ginsenosides as anticancer agents: in vitro and in vivo activities, structure–activity relationships, and molecular mechanisms of action.Front. 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Terpenoids (otherwise known as isoprenoids), a class of natural compounds with ~50 000 different structures, are produced in plants via the cytosolic mevalonate (MVA) and plastidial methylerythritol phosphate (MEP) pathways [7.Ashour M. et al.Biochemistry of terpenoids: monoterpenes, sesquiterpenes and diterpenes.Annual Plant Rev. 2010; 40: 258-303Google Scholar,8.Liao P. et al.The potential of the mevalonate pathway for enhanced isoprenoid production.Biotechnol. Adv. 2016; 34: 697-713Crossref PubMed Scopus (107) Google Scholar]. Sesquiterpenoids (artemisinin) and triterpenoids (ginsenosides) are mainly derived from the MVA pathway [6.Ma Y.N. et al.Jasmonate promotes artemisinin biosynthesis by activating the TCP14-ORA complex in Artemisia annua.Sci. Adv. 2018; 4eaas9357Crossref PubMed Scopus (42) Google Scholar,9.Kim Y.J. et al.Biosynthesis and biotechnological production of ginsenosides.Biotechnol. 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Nutr. 2019; 59: 953-964Crossref PubMed Scopus (101) Google Scholar,12.Dudareva N. et al.Biosynthesis, function and metabolic engineering of plant volatile organic compounds.New Phytol. 2013; 198: 16-32Crossref PubMed Scopus (684) Google Scholar]. Many secondary metabolites are present at low levels in planta, including plant-derived anticancer compounds [1.Carqueijeiro I. et al.Beyond the semi-synthetic artemisinin: metabolic engineering of plant-derived anti-cancer drugs.Curr. Opin. Biotechnol. 2020; 65: 17-24Crossref PubMed Scopus (23) Google Scholar]. Furthermore, many of these metabolites are synthesized only in specific tissues [9.Kim Y.J. et al.Biosynthesis and biotechnological production of ginsenosides.Biotechnol. Adv. 2015; 33: 717-735Crossref PubMed Scopus (193) Google Scholar,17.Sun M.H. et al.The biosynthesis of phenolic acids is positively regulated by the JA responsive transcription ERF115 in Salvia miltiorrhiza.J. Exp. 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Vinblastine and vincristine are produced exclusively in the aerial parts of plants, and catharanthine accumulates in all organs of Catharanthus roseus [20.Schweizer F. et al.An engineered combinatorial module of transcription factors boosts production of monoterpenoid indole alkaloids in Catharanthus roseus.Metab. Eng. 2018; 48: 150-162Crossref PubMed Scopus (32) Google Scholar]. Glandular secretory trichomes of Artemisia annua leaves are ‘biofactories’ for artemisinin biosynthesis and accumulation [6.Ma Y.N. et al.Jasmonate promotes artemisinin biosynthesis by activating the TCP14-ORA complex in Artemisia annua.Sci. Adv. 2018; 4eaas9357Crossref PubMed Scopus (42) Google Scholar]. The natural sources of these compounds often grow slowly or produce these metabolites in very small quantities over an extended growth periods (several years) before their roots can be harvested [21.Atanasov A.G. et al.Discovery and resupply of pharmacologically active plant-derived natural products: a review.Biotechnol. Adv. 2015; 33: 1582-1614Crossref PubMed Scopus (1232) Google Scholar]. In addition, medicinal plants grow in various ecological environments and often have bacterial or pesticide residues, leading to degradation of their quality [22.Normile D. Asian medicine: the new face of traditional Chinese medicine.Science. 2003; 299: 188-190Crossref PubMed Scopus (504) Google Scholar,23.Zhang J.H. et al.Quality of herbal medicines: challenges and solutions.Complement. Ther. Med. 2012; 20: 100-106Crossref PubMed Scopus (180) Google Scholar]. Therefore, it is important to explore other methods for producing these valuable and beneficial compounds. The production of genetically transformed root cultures (so-called ‘hairy roots’) represents a good alternative approach to produce target compounds in medicinal plants. Hairy roots grow faster than the adventitious roots, or even conventional plant cultures [24.Paek K.Y. et al.Large scale culture of ginseng adventitious roots for production of ginsenosides.Adv. Biochem. Eng. Biotechnol. 2009; 113: 151-176PubMed Google Scholar,25.Yu K.W. et al.Ginsenoside production by hairy root cultures of Panax ginseng: influence of temperature and light quality.Biochem. Eng. J. 2005; 23: 53-56Crossref Scopus (133) Google Scholar] and accumulate higher levels of certain valuable compounds compared with adventitious roots and native-grown plant roots [18.Kai G.Y. et al.Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures.Metab. 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For instance, novel cadaverine and natural triterpene saponins compounds have been found in Brugmansia candida and Medicago truncatula hairy roots, respectively, perhaps resulting from transformation or stress, but they were not identified in the leaves or roots of intact plants [28.Carrizo C.N. et al.Occurrence of cadaverine in hairy roots of Brugmansia candida.Phytochemistry. 2001; 57: 759-763Crossref PubMed Scopus (19) Google Scholar,29.Pollier J. et al.Metabolite profiling of triterpene saponins of Medicago truncatula hairy roots by liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry.J. Nat. Prod. 2011; 74: 1462-1476Crossref PubMed Scopus (71) Google Scholar]. Hairy root cultures are also excellent model systems for identifying novel genes and TFs or rapidly characterizing gene function. Moreover, hairy roots can be genetically modified, thereby allowing modulation of metabolite production through genetic engineering or genome editing. Finally, hairy root cultures can be artificially designed to produce unnatural compounds by blocking the biotransformation of an original precursor through RNAi or genome-editing of biosynthetic genes, combined with the feeding of exogenous substrates. For example, several unnatural fluorinated alkaloids, such as fluoro-ajmalicine, fluoro-serpentine, fluoro-catharanthine, and fluoro-tabersonine, were produced in C. roseus hairy roots when the tryptamine biosynthesis was suppressed by RNA silencing of tryptophan decarboxylase and feeding with the unnatural starting substrate 5-fluorotryptamine [30.Runguphan W. et al.Silencing of tryptamine biosynthesis for production of nonnatural alkaloids in plant culture.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 13673-13678Crossref PubMed Scopus (83) Google Scholar]. Hairy root cultures also offer benefits compared with microbes. For instance, they provide an alternative platform that is more similar to that in the native host plant compared with microbes, including Escherichia coli and Saccharomyces cerevisiae, and the encoded plant-derived protein is more likely to be properly folded in hairy roots than in microbes. Other applications of transformed root cultures include the production of high-value proteins, therapeutic vaccines, and antimicrobial peptides [31.Cardon F. et al.Brassica rapa hairy root based expression system leads to the production of highly homogenous and reproducible profiles of recombinant human alpha-L-iduronidase.Plant Biotechnol. J. 2019; 17: 505-516Crossref PubMed Scopus (13) Google Scholar, 32.Chahardoli M. et al.Recombinant production of bovine Lactoferrin-derived antimicrobial peptide in tobacco hairy roots expression system.Plant Physiol. 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Early research on hairy roots, including culture methods, conditions, the use of bioreactors, elicitation, and metabolic engineering to produce valuable secondary metabolites, has been reviewed elsewhere [37.Banerjee S. et al.Biotransformation studies using hairy root cultures - a review.Biotechnol. Adv. 2012; 30: 461-468Crossref PubMed Scopus (84) Google Scholar, 38.Georgiev M.I. et al.Genetically transformed roots: from plant disease to biotechnological resource.Trends Biotechnol. 2012; 30: 528-537Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 39.Hidalgo D. et al.Tailoring tobacco hairy root metabolism for the production of stilbenes.Sci. Rep. 2017; 7: 17976Crossref PubMed Scopus (9) Google Scholar, 40.Thakore D. et al.Mass production of ajmalicine by bioreactor cultivation of hairy roots of Catharanthus roseus.Biochem. Eng. 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Here, we review new strategies, including multigene engineering, the combined use of elicitors and genetic engineering, the use of newly identified key genes or TFs for the metabolic engineering of valuable secondary metabolites, the use of newly emerging Clustered and regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology, and proteomics and metabolomics to explore hairy roots from medicinal plants. We also discuss other applications and functions of hairy roots and address future prospects for using hairy roots as ‘green factories’ (i.e., plant-based production systems).Table 1Selected Secondary Metabolites Produced in Hairy Root Cultures from Various Plant SpeciesaAbbreviations: 1/2 B5, half-strength B5 solid medium; 1/2MS, half-strength Murashige and Skoog medium; B5, Gamborg B5 medium; MS, Murashige and Skoog medium; WPM, Woody-plant medium.Metabolite or functionPlants speciesStrainExplantCulture medium (liquid)RefsCamptothecinOphiorrhiza pumilaC58C1StemB5[44.Cui L.J. et al.Co-overexpression of geraniol-10-hydroxylase and strictosidine synthase improves anti-cancer drug camptothecin accumulation in Ophiorrhiza pumila.Sci. Rep. 2015; 5: 8227Crossref PubMed Scopus (63) Google Scholar]TanshinonesSalvia miltiorrhizaC58C1Leaf1/2MS[18.Kai G.Y. et al.Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures.Metab. Eng. 2011; 13: 319-327Crossref PubMed Scopus (187) Google Scholar]Phenolic acidsS. miltiorrhizaC58C1Leaf1/2MS[17.Sun M.H. et al.The biosynthesis of phenolic acids is positively regulated by the JA responsive transcription ERF115 in Salvia miltiorrhiza.J. Exp. Bot. 2019; 70: 243-254Crossref PubMed Scopus (67) Google Scholar]LariciresinolIsatis indigoticaC58C1Leaf1/2MS[59.Ma R.F. et al.AP2/ERF transcription factor, Ii049, positively regulates lignan biosynthesis in Isatis indigotica through activating salicylic acid signaling and lignan/lignin pathway genes.Front. Plant Sci. 2017; 8: 1361Crossref PubMed Scopus (36) Google Scholar]HyoscyamineAnisodus acutangulusC58C1Leaf1/2MS[40.Thakore D. et al.Mass production of ajmalicine by bioreactor cultivation of hairy roots of Catharanthus roseus.Biochem. Eng. J. 2017; 119: 84-91Crossref Scopus (23) Google Scholar]ScopolamineAtropa belladonnaC58C1LeafMS[46.Qiu F. et al.Functional genomics analysis reveals two novel genes required for littorine biosynthesis.New Phytol. 2020; 225: 1906-1914Crossref PubMed Scopus (27) Google Scholar]Withanoloide AWithania somniferaR1000; ATCC15834Leaf; cotyledonMS[34.Shasmita et al.Exploring plant tissue culture in Withania somnifera (L.) Dunal: in vitro propagation and secondary metabolite production.Crit. Rev. Biotechnol. 2018; 38: 836-850Crossref PubMed Scopus (12) Google Scholar]FlavonesScutellaria baicalensisA4LeafB5[19.Zhao Q. et al.A specialized flavone biosynthetic pathway has evolved in the medicinal plant, Scutellaria baicalensis.Sci. Adv. 2016; 2e1501780Crossref PubMed Scopus (94) Google Scholar]TaxolTaxus mediaLBA9402; C58C1Leaf; stemB5[92.Exposito O. et al.Metabolic responses of Taxus media transformed cell cultures to the addition of methyl jasmonate.Biotechnol. Prog. 2010; 26: 1145-1153PubMed Google Scholar]RheinPolygonum multiflorumR1601LeafMS[93.Huang B. et al.Optimal inductive and cultural conditions of Polygonum multiflorum transgenic hairy roots mediated with Agrobacterium rhizogenes R1601 and an analysis of their anthraquinone constituents.Pharmacogn. Mag. 2014; 10: 77-82Crossref PubMed Scopus (11) Google Scholar]ScutellarinErigeron breviscapusC58C1LeafB5[94.Chen R. et al.Integrated transcript and metabolite profiles reveal that EbCHI plays an important role in scutellarin accumulation in Erigeron breviscapus hairy roots.Front. Plant Sci. 2018; 9: 789Crossref PubMed Scopus (5) Google Scholar]SalidrosideRhodiola crenulataC58C1Leaf1/2MS[95.Lan X. et al.Engineering salidroside biosynthetic pathway in hairy root cultures of Rhodiola crenulata based on metabolic characterization of tyrosine decarboxylase.PLoS ONE. 2013; 8e75459Crossref PubMed Scopus (41) Google Scholar]GinsenosidePanax ginsengA4Root1/2MS[96.Zhang R. et al.Enhancement of ginsenoside Rg1 in Panax ginseng hairy root by overexpressing the α-L-rhamnosidase gene from Bifidobacterium breve.Biotechnol. Lett. 2015; 37: 2091-2096Crossref PubMed Scopus (20) Google Scholar]ParthenolideTanacetum partheniumATCC15834LeafMS[97.Pourianezhad F. et al.Effects of combined elicitors on parthenolide production and expression of parthenolide synthase (TpPTS) in Tanacetum parthenium hairy root culture.Plant Biotechnol. Rep. 2019; 13: 211-218Crossref Scopus (7) Google Scholar]Chicoric acidEchinacea purpureaR15834LeafWPM[98.Salmanzadeh M. et al.Heterologous expression of an acid phosphatase gene and phosphate limitation leads to substantial production of chicoric acid in Echinacea purpurea transgenic hairy roots.Planta. 2019; 251: 31Crossref PubMed Scopus (6) Google Scholar]Farnesiferol BFerula pseudalliaceaATCC15834Leaf1/2MS[99.Khazaei A. et al.Hairy root induction and farnesiferol B production of endemic medicinal plant Ferula pseudalliacea.3 Biotech. 2019; 9: 407Crossref PubMed Scopus (6) Google Scholar]RishitinSolanum tuberosumATCC15834TuberMS[100.Komaraiah P. et al.Enhanced production of antimicrobial sesquiterpenes and lipoxygenase metabolites in elicitor-treated hairy root cultures of Solanum tuberosum.Biotechnol. Lett. 2003; 25: 593-597Crossref PubMed Scopus (38) Google Scholar]Flavonoids/IsoflavonoidsGlycine maxARqual1Cotyledon, hypocotylsB5[101.Han X. et al.GmMYB58 and GmMYB205 are seed-specific activators for isoflavonoid biosynthesis in Glycine max.Plant Cell Rep. 2017; 36: 1889-1902Crossref PubMed Scopus (14) Google Scholar]NicotineNicotiana tabacumATCC15834LeafB5[102.Zhao B. et al.Enhanced production of the alkaloid nicotine in hairy root cultures of Nicotiana tabacum L.Plant Cell Tissue Organ Cult. 2013; 113: 121-129Crossref Scopus (20) Google Scholar]GossypolGossypium hirsutumA4LeafB5[103.Verma P.C. et al.Efficient production of gossypol from hairy root cultures of cotton (Gossypium hirsutum L.).Curr. Pharm. Biotechnol. 2009; 10: 691-700Crossref PubMed Scopus (9) Google Scholar]Resistance to pathogen infectionVitis viniferaA4StemMS[104.Meteier E. et al.Overexpression of the VvSWEET4 transporter in Grapevine hairy roots increases sugar transport and contents and enhances resistance to Pythium irregulare, a soilborne pathogen.Front. Plant Sci. 2019; 10: 884Crossref PubMed Scopus (11) Google Scholar]a Abbreviations: 1/2 B5, half-strength B5 solid medium; 1/2MS, half-strength Murashige and Skoog medium; B5, Gamborg B5 medium; MS, Murashige and Skoog medium; WPM, Woody-plant medium. Open table in a new tab Secondary metabolites (or specialized metabolites) are derived from general precursors. Therefore, the availability of a precursor and/or substrate is an important factor affecting target compound production. The engineering of secondary metabolites in hairy roots has been achieved by the genetic manipulation of key genes for the biosynthesis of substrates and/or precursors, intermediate products, and end products (Table 2). For example, the expression of genes encoding geraniol 10-hydroxylase (G10H), secologanin synthase (SLS), phenyllactate UDP-glycosyltransferase, and littorine synthase for alkaloid production has been manipulated in the hairy roots of various medicinal plants, including O. pumila and A. belladonna [44.Cui L.J. et al.Co-overexpression of geraniol-10-hydroxylase and strictosidine synthase improves anti-cancer drug camptothecin accumulation in Ophiorrhiza pumila.Sci. Rep. 2015; 5: 8227Crossref PubMed Scopus (63) Google Scholar, 45.Shi M. et al.Targeted metabolic engineering of committed steps improves anti-cancer drug camptothecin production in Ophiorrhiza pumila hairy roots.Ind. Crop. Prod. 2020; 148: 112277Crossref Scopus (19) Google Scholar, 46.Qiu F. et al.Functional genomics analysis reveals two novel genes required for littorine biosynthesis.New Phytol. 2020; 225: 1906-1914Crossref PubMed Scopus (27) Google Scholar]. Overexpression of the valerendiene synthase gene VDS in Valeriana officinalis hairy roots resulted in 1.5–4-fold higher levels of the sesquiterpenoid valerenic acid compared with the control [47.Ricigliano V. et al.Regulation of sesquiterpenoid metabolism in recombinant and elicited Valeriana officinalis hairy roots.Phytochemistry. 2016; 125: 43-53Crossref PubMed Scopus (20) Google Scholar]. Co-introduction of the key gene DXS (encoding 1-deoxy-D-xylulose-5-phosphate synthase, a key enzyme in the MEP pathway), and GGPPS (encoding geranylgeranyl diphosphate synthase, an enzyme in the middle of the pathway, which provides general precursor geranylgeranyl diphosphate for diterpenoid production) in transgenic S. miltiorrhiza hairy roots yielded tanshinone levels as high as 12.93 mg/g DW, compared with 0.61 mg/g DW in the controls [48.Shi M. et al.Enhanced diterpene tanshinone accumulation and bioactivity of transgenic Salvia miltiorrhiza hairy roots by pathway engineering.J. Agric. Food Chem. 2016; 64: 2523-2530Crossref PubMed Scopus (74) Google Scholar]. This finding suggests that the MEP pathway is more important than the MVA pathway for tanshinone biosynthesis and that crosstalk exists between these pathways. A similar strategy was been used to enhance phenylpropanoid production [49.Xiao Y. et al.The c4h, tat, hppr and hppd genes prompted engineering of rosmarinic acid biosynthetic pathway in Salvia miltiorrhiza hairy root cultures.PLoS ONE. 2011; 6e29713Crossref PubMed Scopus (99) Google Scholar].Table 2Examples of Metabolicall