Samra Basharat,Ziyang Huang,Mengyue Gong,Xueqin Lv,Aqsa Ahmed,Iftikhar Hussain,Jianghua Li,Guocheng Du,Long Liu

1 Science Center for Future Foods,Jiangnan University,Wuxi 214122,China

2 Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China

3 School of Food Science and Technology,Jiangnan University,Wuxi 214122,China

ABSTRACT Stevia rebaudiana Bertoni is commonly called stevia and mostly found in the north east regions of South America.It is an herbaceous and shrubby plant belonging to the Asteraceae family.Stevia is considered as a natural sweetener and a commercially important plant worldwide.The leaves of S.rebaudiana contain steviol glycosides(SGs)which are highly potent and non-caloric sweeteners.The sweetening property of S.rebaudiana is contributed to the presence of these high potency,calorie free steviol glycosides.SGs are considerably suitable for replacing sucrose and other artificial sweetening agents which are used in different industries and pharmaceuticals.SGs amount in the plant mostly varies from 8% to 10%,and the enhancement of SGs is always in demand.These glycosides have the potential to become healthier alternatives to other table sugars for having desirable taste and zero calories.SGs are almost 300 times sweeter than sucrose.Being used as alternative sugar intensifier the commercial value of this plant in biopharmaceutical,food and beverages industries and in international market is increasing day by day.SGs have made stevia an important part of the medicinal world as well as the food and beverage industry,but the limited production of plant material is not fulfilling the higher global market demand.Therefore,researchers are working worldwide to increase the production of important SGs through the intercession of different biotechnological approaches in S.rebaudiana.This review aims to describe the emerging biotechnological strategies and approaches to understand,stimulate and enhance biosynthesis of secondary metabolites in stevia.Conventional and biotechnological methods for the production of steviol glycosides have been briefly reviewed and discussed.

Keywords:Steviol glycosides Biosynthesis Secondary metabolites Stevia rebaudiana

1.Introduction

Human health is considered at risk in the present era,possibly due to the intake of different harmful sweeteners present in wide variety of food products [1].Due to the increasing risks of chronic diseases and public awareness of adverse health impacts of synthetic sweeteners,the demand of alternative low or zero calorie sweeteners have been increased.These sweeteners are mainly extracted from two plant species namely,Stevia rebaudianaandSiraitia grosvenorii,which have been highly profiled to identify the sweeteners with high sweetening properties.However,sweetening ability does not essentially make product feasible for commercial use.Therefore,a study of new and improved sweetener is required that can meet the needs of consumer demands [2].

Stevia(S.rebaudiana)is considered as an exotic plant due to its commercial and medicinal value [3].It is commonly known stevia that belongs to the familyAsteraceaeand mostly considered as shrubby and herbaceous plant [4].It is an indigenous plant and found to be perennial throughout the year in the highland,north east regions of South America[4,5].S.rebaudianais the only specie out of 230 others,besidesStevia phlebophylla,that is reported to possess the unique ability of accumulating low-calorie sweetening agents called steviol glycosides(SGs)and is considered as the most common and commercially important plant for food and health sector[6-8].SGs that are also known as steviosides are the natural sweeteners that are present in the leaves of plantS.rebaudiana(bertoni).SGs are mostly found inS.rebaudiana,Stevia phlebophylla,andRubus sauvissimus,which are the only plant species known to produce diterpene glycosides.Dry leaves of stevia are mostly 10–15 times sweeter than sucrose with zero glycemic index.Hence,these glycosides are proved to be a highly potent and nontoxic for human health [9,10].Stevia leaves and their extracts are now used as an alternative to other synthetic sugars and their demand is increasing day by day in the food market[10].

Some major glycosides that are present in stevia are rebaudiosides,dulcosides,steviosides,steviomonoside and steviolbioside.SGs along with some other secondary metabolites such as flavonoids,phenols and alkaloids increases the plant potency and make these plants suitable for different medicines and food additives[10,11].Among the different glycosides produced in stevia leaves,steviosides are the most abundant one followed by rebaudioside A.These two major glycosides have an ability to become a healthier replacement for table sugars as they have zero calories and a desirable taste profile [2,4,12].Being considered as low-sugar and low carbohydrate food alternative,stevia plant and its glycosides have garnered great attention both commercially and scientifically with rise in demand in the recent years [13].Also being introduced to European countries in 1899,SGs have been considered as a best alternative sugar due to their intense sweetness[14,15].Similarly,these glycosides are reported as unique and potent sweetener in many other countries worldwide including Japan,China,Australia,New Zealand,Korea [16,17].

Structurally SGs are tetracyclic diterpenes that have the same kaurenoid precursor with gibberellic acid and are mainly synthesized in stevia leaf tissue [6,7,12,18].The lower-concentration SGs include steviolbioside,rebaudioside B,C,D,E,F,I and dulcoside A,while stevioside and rebaudioside A are considered as two major glycosides of SGs [4,17].Due to intense sweetness,SGs are proved to be an attractive sugar alternative in the food industry and in health sector.Some clinical examinations performed on SGs have shown that these sweeteners can exert antidiabetic,hypotensive cardiotonic,inflammatory,anticarcinogenic and antibacterial actions [19].

Stevia leaves extracts have been recognized as safe by FDA(Food and drug authority) to use as dietary ingredients,but it is important to note that only purified stevia leaf extracts have been approved for use as dietary supplements.In 2007,a safe level of intake and specifications for SGs have been established by FAO(Food and agriculture organization) and WHO (world health organization)expert committee that included a minimum purity of 95%of seven different named steviol glycosides.In 2008,FDA issued new GRAS (Generally Recognized as Safe) notices that include alternative biotechnological methods for production of SGs.In December 2008,in response to Generally Recognized as Safe(GRAS) notifications submitted to the US Food and Drug Administration(FDA),in which FDA stated that SGs especially rebaudioside A is a GRAS for use as a general purpose and healthy sweetener for food products and beverages.In 2010,the European Food Safety Authority (EFSA) also assessed the safety of SGs and established an ADI for their safe use.High-Purity rebaudioside M (minimum purity 95%) has been approved GRAS status by FDA in 2014 [20].

In several countries of Asia,including China,Japan,and Korea,stevia derived-sweeteners are permitted as a food additive.The Food Standards Australia New Zealand (FSANZ) has completed its evaluation of an application for use of SGs in foods in 2008.FDA stated that the use of high-purity SGs (≥95%) is safe for human consumption.Recently the Food Safety and Standards Authority of India (FSSAI) has allowed the use of natural sweetener stevia(steviol glycoside)in selected products,including dairy-based beverages,soft drinks,and desserts [20].

New researches and ideas are emerging with the time in order to enhance new biotechnological strategies and approaches to improve and stimulate the biosynthesis of these glycosides in stevia.In recent years,different biotechnological techniques have been developed to enhance the biosynthesis of SGs in stevia plant.Among these techniques,transciptome has garnered more attention and has been proved as a promising and authentic method to evaluate the genetic diversity of stevia plant molecular structure and characteristics [21,22].Therefore,in new researches,most transcriptome based analyses are used to identify the expression of those genes that are involved in the biosynthesis of SGs.Similarly,there are some other biotechnological methods that are used to enhance and stimulate production of SGs such as elicitation,adventitious or hairy root cultures,callus and cell cultures,metabolomics and transcriptomics elucidation,micropropagation and induction of polyploidy.This review covers probably all the available information on biotechnological approaches and researches conducted worldwide for the enhancement and stimulation of steviosides biosynthesis inS.rebaudiana.

2.Stevia rebaudiana

Stevia is a genus belongs to the familyAsteraceae,that contains 200 species of herbs and shrubs.This plant can grow up to one meter tall [23,24].It has an extensive root system with delicate stems and small elliptic leaves [25].This genus of stevia is unique due to its flower morphology [26].It contains approximately 200–230 species of perennial herbaceous and shrubby plants [6].These species can survive naturally in multiple places like mountain regions,dry valleys and river borders [4].S.rebaudianais the only specie that produces highly potent and valuable sweetening agents with desirable taste.Stevia is probably known as short day plant because it has short and critical day lengthi.e.12–13 h approximately [17,27].Moreover,the critical day length sometimes may vary from 8 hours to 14 hours that depends on their photoperiod sensitivity [28,29].S.rebaudianaleaves contains several glycosides compounds in which 33 glycosylated diterpenes have been determined,whereas a small amount of these glycosides can also be found in roots,stems and leaves [30].SGs consist of a diterpenoid steviol backbone with one to three glucoses at the steviolC13-hydroxyl andC19-carboxylic acid positions (Fig.1).The maximum sweetness level is obtained by the total number of glucose residues present,whereas the substitution of a β-1,2-glucose with an α-1,2-rhamnose,greatly reduces sweetness [31].

All glycosides of stevia share the same nucleus(i.e.steviol)and they can only differentiate by the amount and layout of glucose molecules that are adhered to the nucleus [32].Leaves of stevia also contain some essential amino acids,phosphorus,minerals,potassium,calcium and sodium and also some other compounds like phytochemicals,alkaloids,hydroxycynamic acids,diterpenes and triterpenes[12].According to some recent researches,various secondary metabolites of stevia have different therapeutic propertiese.g.they are anti-hypertensive,anti-microbial,anticarcinogenic and anti 147 hyperglycemic [33](see Table 1).

The UGTs (UDP-glycosyl transferases) of stevia suggests that they are highly region specific and have the ability to recognize a particular substructure of acceptor molecule [34,35].Sweet glycosides of stevia can be analyzed by liquid chromatography with UV,MS and ELS detector[36].A variety of SGs are produced by glucosyl tranferases are located in cytosol of plant that play important roles in the latter part of pathway.According to the toxicological studies,the compounds or secondary metabolites produced by stevia neither have mutagenic carcinogenic or tetratogenic effects nor have any allergic reactions when used as sweetening agents [37].

Fig.1.Figure shows SGs with diterpenoid steviol backbone having one to three glucoses at the steviol C13-hydroxyl and C19-carboxylic acid positions.

Table 1 Major Biochemical constituents and Glycosides present in S.rebaudiana

Recently in 2020,For the first time,scientists have completed the sequencing of the stevia plant genome.Lead scientists from Pure Circle Stevia Institute and Key Gene have revealed this major breakthrough in research by showing the annotated,high-quality genome sequences of three stevia cultivars.Researchers have identified several million potentially new markers in the assembled genomes.CEO of KeyGene Arjen van Tunen said that,having a single high-quality reference genome is generally considered a major step forward for newly domesticated crops,such as stevia In present,the whole genome sequence and annotation of this species.A total of 146,838,888 paired-end reads consisting of 22.2G bases were obtained by sequencing one leaf from a commercially grown seedling.The reads can be assembled by a denovo method followed by alignment to related species.GenMark-ES was used for the purpose of annotation.A complete genome sequence for this species will assist with discovering markers for crop yields,disease and drought resistance,and determine the biochemical pathways for the relevant metabolites [38].

3.Biological Significance of S.rebaudiana

Due to the presence of important secondary metabolites and remarkable physiological activity,stevia is considered as an important medicinal and biologically valuable plant [39].The important bioactive compounds present in stevia includes sterol,flavonoids,labdanes,triterpenoids,chlorophylls,organic acids,inorganic salts,mono-disaccaridesetc.[40].The medicinal value of the plant lies in these chemical substances for having ability to produce specific physiological action on the human body [41].According to Savitaet al.[42],stevia leaf extracts have high percentage of antinutritional factors when dissolved in water,tannins and oxalic acid with an amount range of 0.010 and 2295.0 mg per 100 g(dry mass basis),respectively.Similarly,saponins are considered as amphipathic glycosides and are found in different seeds,plants and cereals.These glycosides have abilities to raise the testosterone levels,stimulate the muscle growth and also show immunological,antibacterial and antidiabetic properties [43].Oxalic acid,which is another important compound,that may obstruct the bioavailability of iron,calcium and other nutrients in case of green leafy vegetables.Furthermore,some compounds like tannins have been reported to have some medicinal and pharmacological propertiese.g.the spasmolytic activity in smooth muscle cells or the free radical scavenging and anti-oxidant properties [23,44,45].

The leaves of stevia contain large amount of SGs which are used in different formulation of various herbal medicines for human expenditures[46].Dry leaf powder,suspension or intact fresh leaves contain folic acid,vitamin C and flavonoids like catechin and quercetin [47].

4.Steviol Glycosides

Glycosides are the compounds mostly found in plants and are named“steviol glycosides”according to the type of sugar they contain.Steviol is the common name for the extracts and metabolites extracted from the leaves ofS.rebaudianaBertoni.It is a natural,sweet-taste and calorie-free compound as well as a renewable raw food ingredient in the world market that may be used as a sugar substitute and alternative to artificial sweeteners[48].These glycosides contain a carbohydrate molecule (sugar) bound to a non-sugar component(aglycone)and can be converted by hydrolytic cleavage,into a sugar and a non-sugar component (aglycone).The sweet glycosides of stevia contain glucosides(glucose),pentosides (pentose),fructosides (fructose)etc.[43].

About 9%(around 20 species)of stevia have been known to contain Diterpenes.Most commonly two types of diterpenes are found in steviae.g.ent-kaurane-type diterpenes like SGs andentlabdanes type diterpenes which contain non-glycosylated sterebins A–N and found in leaves and flowers ofS.rebaudiana.Many triterpenes have also been found inS.rebaudianaleaves after methanolic extraction along with some sterols like phytosterols,non-sweet glycosides and lupeol esters.Essential and volatile oils ofS.rebaudianacontain large numbers of compounds,with the range from 30 to more than 300,which are about 0.4%–3.5% w/w of dried leaves [30,49].According to one study,inflorescence contains three times more essential oils as compared to vegetative leaves.But the composition of these oils is according to their ontogenetic stage and genotype[50].As inflorescence essential oil composed of large concentration of compounds like trans-β-farnesene and nerolidol which can provide specific flavors to dried herbs and inflorescence along with nonoxygenated sesquiterpenes,and monoterpenes such as β-pinene and limonene.The essential oil extracted from roots ofS.rebaudianaare known to contain more than 60 components among which the main components are αlongipinene,α-isocomene,and modheph-2-ene.Various phenolic compounds like flavonol glycosides,and a large variety of coumarins and cinnamic acid derivatives are part of leaf extracts ofS.rebaudiana,and have significant impact on organoleptic properties of stevia based products.Another compound extracted from stevia,which affect their taste because of its bitterness is steviamine,which is a water soluble indolizidine alkaloid and first ever extracted alkaloid not only fromS.rebaudianabut also from species in the familyAsteraceae[30].

Steviol glycosides(SGs)are well-known nontoxic,high potency,nonnutritive secondary metabolites with important commercial uses in biopharmaceutical,food and beverages industries [51].SGs preparations are slightly yellowish or white,crystalline,odorless powders and are freely soluble in water and ethanol.SGs compounds are highly stable,can be easily extracted with an aqueous solvent,and cannot interact with other food components.Unlike other sugars,stevia sugars can withstand up-to 200EC of temperature which can caramelize at about 150–160 depending upon conditions.The sweetness of stevioside aqueous solution cannot be change even heated at 95EC for 2 h and it is superior to sugar in taste,refreshment and mildness [52].

SGs are composed of different metabolites mixturee.g.stevioside and rebaudioside constitute 95% of total composition of metabolites.Biosynthesis of stevioside and rebaudioside can be performed by using steviol as a common metabolite.These steviosides are proved to be safe,non-carcinogenic,non-genotoxic and cannot associated with other reproductive or developmental abnormalities [53]

Steviosides sweeteners are considered as healthy practice and safe food for diabetic patients as these sweeteners do not contain any allergen and reduce the level of sugar in the diet without creating any allergic reaction in the body[19].Moreover,the extracts of these glycosides show positive anti-hyperglycemic effects and have the ability to enhance insulin secretion by directly acting on β-cells without altering the channel activity of cAMP and K+ATP level in the islets.Thus,these glycosides act as potent antihyperglycemic agents [54].Enhanced insulin production alternatively suppresses the glycogenesis in the patient body [55].

Similarly,the biochemical analysis of stevia leaf powder shown that leave glycosides of stevia are superior source of carbohydrates,proteins and dietary fibers.Hence,these glycosides were considered as the vital substances for human nutrition and health[4,28,56].According to different research studies these glycosides are used as anticancer,antiproliferative and cancer chemo protective agents and are proved as non-hazardous for human health[57,58].Beside of being a natural sweetener,a wide variety of biological activities of SGs and other bioactive compounds isolated from different parts of stevia are reported in literature [59].Stevioside has now also been identified as glucogonostatic,antihyperglycemica and insulinotropic because of its ability to lower the blood pressure [51].Medicinal properties of steviol glycosides are shown in Fig.2.

4.1.Physiochemical features of steviol glycosides

Fig.2.Medicinal properties of steviol glycosides.

Almost all secondary metabolites,such as steviol,steviobioside,rebaudioside A,B,C,D,E and F accumulate in the leaves [51,60].Among these glycosides,stevioside and rebaudioside A are present in higher concentrations.Rebaudioside A is 150–320 times sweeter while stevioside is 110–270 times sweeter than sucrose [21].The increased content of stevioside gives a metallic aftertaste,while increased content of rebaudioside A reduces the metallic aftertaste and improves the taste.These glycosides are dissolved and extracted in aqueous solution and show increased stability under high pH from 2 to 10 and high thermostability [51,61].These glycosides show stability even at 200°C and produce synergistic effect upon addition to other sweeteners or flavors and hence,enhance the taste,sweetness and flavor of sweeteners [62].

4.2.Biological features of steviol glycosides

To determine the safety aspects of SGs,differentin vitroandin vivostudies have been carried out.In 2006,a research was conducted to determine the cytotoxic and genotoxic behavior of stevioside.In this study,E.colistrains with different concentrations of stevioside were incubated,followed by agarose electrophoresis and bacterial transformation analysis for survival analysis,and after that,a drop in the survival rate of microbe was observed[46].This experiment suggested that cytotoxicity of stevioside enable them to cause lesions in DNA and it can destroy the lesion recovery pathways.Also,it has been suggested that stevioside conversion to steviol or other genotoxic compound or metabolite can be responsible for DNA damage capability [46].Similarly,in other studies the leaf extract of stevia was examined for its antimicrobial activity.For this purpose,leaf extract was isolated by using different solvents and each extract was then examined against some microbial species.As a result,different inhibitory activities by different extracts were observed against various microbes to a large extent.This experiment confirmed that stevia leaf extracts has antibacterial as well as antifungal ability[23,63].It was also determined that stevia might be a source of new non antimicrobial agent [23].Against plant pathogen antifungal activity of stevia was estimated to be higher than standard fungicide.This extraordinary antimicrobial activity has presented it as valuable potent non-antibiotic pharmaceutical and efficient food preservatives.Further experiments were performed to examine stevioside alone i.e.without other secondary metabolites,which showed that stevioside has the ability to activate cytotoxic cells of the host and reduce the amount of inflammatory mediators.This result suggested that stevioside is safe as an antifungal,antibacterial and anti-tumorous agent and sometimes it may play synergistic role with the innate immunity of the host [50,64].It was also reported by Nikiforovet al.that rebaudioside A is clinically insignificant and described the analysis of glycosides successfully by the capillary electrophoresis [65].Steviobioside and rebaudioside A structure was elucidated by mass spectrometry while the isolation of these glycosides was done by semi preparative high performance liquid chromatography (HPLC).Similarly,during the injection of sample the effect of organic solvent was used to study electrophoretic solution [66,67].

In 2001,another analytical method was introduced to simplify sample handling,preparation and chromatographic analysis[68,69].Later in 2004,to determine the amount of steviol on large scale,a reverse phase HPLC was introduced.Similarly,in 2009,another research was conducted,and it was found that due to the presence of organic and inorganic compounds,raw material used for the isolation and evaluation of steviol glycoside lost its quality.Therefore,to enhance the quality of product,a ceramic membrane purification process was introduced.Hence the sweetener obtained was found to have purity greater than 90% [30,70].In the mid 2009 some other methods such as desorption electrospray ionization(DESI)mass spectrometry and infrared reflectance spectroscopy (NIRS) were also evaluated by the researchers to determine the level of SGs [26,71].DESI was considered more authentic and was found to be rapid,qualitative and semiquantitative method because it did not require any sample preparation for SGs estimation in stevia leaves [72].According to the studies conducted in 2010,it was stated that Ultra-HPLC methods with electron spray mass spectrometry (UHPLC-MS) can be used for the routine evaluations of SGs 21.Recently,it was reported that ultrasonically assisted extraction method can be used to increase the yield of rebaudioside A by a factor of 1.5.Since 2000,various modified techniques have been explored for glycoside extraction and evaluations [73].

4.3.Biosynthesis of steviol glycosides

Fig.3.Diagrammatic representation of genes involved in SGs biosynthesis pathway.

As the steviosides are high molecular weight compounds,the oral uptake of stevioside by human body is extremely low and are unable to degrade into steviol by digestive enzymes [74,75].The process SGs biosynthesis originally begins in the chloroplast(Fig.3),and there is an immense correlation between the biosynthesis of SGs and development of chloroplast membrane system.Therefore,it was considered that the synthesis of SGs take place certainly in specialized cells and structures.Moreover,only those plants that have well-developed leaves are able to synthesize SGs in desired quantities [76].To mediate and manipulate the specific genes that are responsible for the increased production of different secondary metabolites or biochemical constituents,it is necessary to understand the genetic and metabolic aspects of biosynthetic pathways.

Mostly,there are eight glycosides which are synthesizes byS.rebaudianaand these are the derivatives of tetracyclic diterpenes steviol.Biosynthetic pathway of SGs mostly consists of sixteen enzyme-catalyzed systems in which different steps are involved.These steps are similar to the steps that are involved in Gibberellic acid (GA) methylerythritol-4-phosphate (MEP) pathway,which is used for the synthesis of isoprenoid and kaurenoic acid[14,77].Initially gibberellic acid is synthesized by the cyclization of geranylgeranyl diphosphate (GGDP) to kaurene through kaurene synthase (KS),copalyl diphosphate synthase (CPS) and 2 terpene cyclase.By using an enzyme kaurenoic acid-13 hydroxylase(KAH),the kaurenoic acid undergoes hydroxylation and produces steviol [78].

Stevioside is converted to rebaudioside A by UGT76G1 gene and hence improved the organoleptic properties of SGs [79,80].Two important genes namely ketosynthase (KS) and Carbamoylphosphate synthase (CPS) were isolated fromS.rebaudiana,to observe that the recombinant versions of these genes are responsible for SGs synthesis.KS and CPS genes are mostly considered to express more in mature leaves and this process separates GA and steviol biosynthesis[8,34].UGTD192 is mainly involved in the biosynthesis of SGs in leaves,and it can convert the mono-glycoside steviolmonoside to the bi-glycoside steviolbioside.Furthemore,it can convert the bi-glycoside rubusoside to the tri-glycoside stevioside and ri-glycoside stevioside to the tetra-glycoside rebaudioside E.Similarly,conversion of tetra-glycoside rebaudioside A to the penta-glycoside rebaudioside E is also occur by UGT91D2 [81](see Table 2).

UGT91D2 and UGT76G1 are capable of catalyzing glucosylation of the two glucose moieties.These glucose moieties are directly attached to theC13-andC19-positions through the formation of 1,2-β-d-and 1,3-β-d glucoside linkages,respectively.Similarly,UGT76G1 has ability to catalyze 1,3-β-d-glucosylations on both mono-and 1,2-disaccharides attached to the steviolC13 andC19-positions,UGT91D2 cannot catalyse 1,2-β-d-glucosylation if a 1,3-disaccharide is already present on theC13-orC19-positionand it can give possibly high yield of 1,3-glucosylated sideproducts.UGT91D2 cannot glucosylate glucoside structures that harbor a glucose residue bound in a 1,3-glucosidic linkage.The UGT91D2 polypeptides have at least 90%identity to the amino acid sequence that has been set forth in SEQ ID.The UGT91D2 polypeptide can include at least one amino acid substitution at residues.It can transfer a second part of sugar to the C-2′of a glucose in a steviol.The method includes the linkage of SGs with a UGT91D2 polypeptide and a UDP-sugar under suitable conditions for the transfer of the second sugar moiety to the steviol [82].

Table 2 Genes involved in the biosynthesis of SGs

A suitable UGT91D2 polypeptide act as a uridine 5′-diphospho glucosyl steviol-13-O-glucoside transferase that can transfer a glucose moiety to the C-2′of the 13-O-glucose of the acceptor molecule,steviol-13-O-glucoside.Normally,a suitable UGT91D2 polypeptide also functions as a uridine 5′-diphospho glucosyl:rubusoside transferase that transfer a glucose moiety to the C-2′of the 13-O-glucose of the acceptor molecule,rubusoside [31,82].

Functional UGT91D2 polypeptides can transfer sugar moieties from donors other than uridine diphosphate glucose.For example,a functional UGT91D polypeptide can act as a uridine 5′-diphospho D-xylosyl:steviol-13-O-glucoside transferase,transferring a xylose moiety to the C-2′of the 13-O-glucose of the acceptor molecule,steviol-13-O-glucoside.As another example,a functional UGT91D2 polypeptide can act as a uridine 5′-diphospho L-rhamnosyl:steviol-13-O-glucoside transferase,transferring a rhamnose moiety to the C-2′of the 13-O-glucose of the acceptor molecule,steviol-13-O-glucoside [31,83].

At present,SGs pathway is considered as an area of immense interest for researchers,because it shares four intermediate steps with Gibberellic acid pathway which plays significant role in plant growth.Similarly,SGs synthesis can also utilize the combined metabolic flux of cytosolic mevalonic acid (MVA) and plastidal methyl erythritol 4-phosphate (MEP) pathways [53].These two important metabolites are mainly synthesized in the mesophyll cells of stevia leaves,and mostly untraceable in the roots.The most important precursori.e.Geranylgeranyl pyrophosphate (C-20) is produced by the enzyme called geranyl pyrophosphate synthase(GGPPS) after condensing four isoprene units [4,57].

The initial phase of biosynthesis occurs in plastid where the localized MEP Pathway is involved and the production of isopentenyl pyrophosphate occurs by the consecutive work of several enzymese.g.deoxyxylulose phosphate synthase DXS,4-diphospho cytidyl-2-C-methyl-d-erythritol synthase CMS,deoxyxylulose phosphate reductase DXR,kinase,4-diphosphocytidyl-2-C-methy l-d-erythritol,CMK,4-diphosphocytidyl-2-Cmethyl-d-erythritol 2,4-cyclodiphosphate synthase MCS,1-hydroxy-2–methyl-2(E)-b utenyl-4-diphosphate synthase (HDS) and 1-hydroxyl-2-methyl-2(E)-butenyl-4-diphosphate reductase (HDR)UGT74G1(UDP-glyco syltransferase 74G1,glycosyltransferase 85C2,UGT76G1(UDP glycosyltransferase 76G1),unknown UGT and KAO (ent-kaurenoic acid oxidase).All these enzymes consecutively work to catalyze the production of isopentenyl pyrophosphate and hence initial phase of the synthesis is completed [14,52,56].

The second phase of biosynthesis is the condensation of four isoprene units of isopentenyl pyrophosphate,that is further condense by the geranylgeranyl diphosphate synthase (GGDPS).As a result,geranylgeranyl diphosphate is produced.It is a common precursor for all diterpenoids synthesis,and this compound is further converted intoent-kaurenoic acid.This is done by the action of two specific enzymes called copalyl diphosphate synthase (CDPS)and kaurene oxidase (KO) [8].Gibberellin pathways and steviol glycoside diverge at a point where kaurene is present and two different endoplasmic reticulum-membranes are located.Cytochrome P450 monooxygenases (CYPs) action started for the conversion ofent-kaurenoic acid oxidase (KAO) into steviol by the enzyme called kaurenoic acid oxidase KA,kaurenoic acid hydroxylase KAH and gibberellic acid [51,84].

In the final phase of steviol biosynthesis,glycosylation of steviol occurs in the cytosol by several UDP glycosyl transferases (UGTs)like UGT74G1,UGT76G1 and UGT85C2.These UGTs are vacuolated there and forms different steviol glycosides.As most of the enzymes involved in the biosynthesis of SGs have been identified,the current researches should focus on genetic and metabolic engineering of these biosynthetic pathways to increase the production of steviol glycosides [60].According to recent researches,some new techniques have been found to increase the SGs production that involves the silencing of genes.In this process three major UGT genes are silenced through a specific system called agrobacterium mediated gene transformation system ofS.rebaudianaand increase the SGs production [58,85].

5.Conventional Approaches to Enhance Steviol Glycosides Biosynthesis

5.1.Enhancement of SGs biosynthesis by physical factors

As determined by Ceunenet al.(2012),the quality and quantity of light play an important role in the accumulation of SGs concentration.To increase the stevioside level,which was assumed to be of greater amount during short-day conditions,a relatively interruptive treatment of far-red LED light was carried out,and the growing plants was provided in long night condition both in field and phytotron.The increased concentrations of steviol glycosides were independent of cultivar.Growth stages of stevia seedlings were obtained to be phytochrome mediated and ceased for some time in the vegetative period.During this vegetative period,steviol glycosides were collected and stored for further use [17].

The concentration of these newly formed glycosides can be monitored by different physical treatments given to the plant.These treatments include daylight condition,abiotic stress [86],spectra of light [30]and change induced by gamma rays [87].Scientists found almost two-fold higher steviol accumulation in young stevia seedling(under short-day condition)that are treated with red LED light.Moreover,a general phenomenon of decreased content of SGs material was also not found in the plants treated with far-red LED light as observed in control plants.The midnight interruption by red LED light provides an easy and reasonable method to increase vegetative leaf biomass production with an increased steviol glycoside yield during short photoperiods[79].

5.2.Enhancement of SGs biosynthesis by chemical factors

In an experiment conducted by Modiet al.in 2018,in vivogrown plants of stevia were treated with different concentrations of gibberellic acid (15,30,and 60 μmol·L-1) which increased the concentration of stevioside more than two-folds.Different types of plant growth regulators could be usedin vivoto improve the steviol glycoside concentrations[10].Plants were treated with methyl jasmonate and elicitor with four different concentrations(50,100,150,and 200 μmol·L-1) in another study.The concentrations of stevioside increased from 8.14%(control)to 10.33%(200 μmol·L-1).Nevertheless,the gene working for the stevioside biosynthesis acted differentially during these treatments [10].[88].Conventional and biotechnological approaches used to enhance biosynthesis of SGs can also be seen in Fig.4.

6.Biotechnological Approaches to Enhance SGs Biosynthesis

Biotechnological techniques have provided large number of opportunities in the engineering and enhancement of SGs biosynthesis pathways,which include almost all the tools that can obtain metabolomics,proteomics and transcriptomics of the plant [85].These biotechnological approaches have an ability to extend and improve the overall structural and functional characteristics of the plante.g.agronomy,genome mapping,morphology and physiology [2].Different biotechnological approachese.g.micropropagation have the ability for the in-vitro propagation and maximum manufacturing of plant biomass and some medicinally or biologically active compounds

6.1.Enhancement through induction of polyploidy

Fig.4.Figure represents conventional and biotechnological approaches to enhance SGs.

One of the methods to enhance biosynthesis of SGs is the induction of polyploidy.In this method different stevia mutants were made using colchicines with the concentration of 0.25%,0.50%,0.75%,1.0%,1.50%,and 2.5%.DNA concentrations of these mutants were experimented to determine the mutation in ploidy.Mutation and manipulation of ploidy were tried on stevia (2n=22) to produce higher concentration of steviol glycosides.Ploidy level was identified by flow cytometry analysis and steviol glycoside content in the leaves was determined by HPLC [89].Some polyploids showed two times increment in the percentage of stevioside as well as rebaudioside-A,compared to control.Hence,the effectiveness of colchicines as a polyploidizing agent to create new variants with higher steviol glycosides(stevioside and rebaudioside-A)content contributed to crop improvement in stevia,which was confirmed by induction of polyploidy in stevia [89].

6.2.Biotransformation for taste improvement

To overcome the modification of stevioside in an intermolecular transglycosylation reaction,several methods and experiments were applied on stevia.Catalization of different enzymes were performed,during which other carbohydrates are attached at positions C13and C19[58,90].The taste of stevioside and rebaudioside A is slightly bitter,and has an unpleasant aftertaste that limits their role in pharmaceuticals and food products.Different transglycosylating enzymes can be used for these purposes suchaspullalanase,cyclodextringlucanotransferases,βgalactosidase105,isomaltase104 dextrin dextranase and(CGTases)with maltose,pullulan,lactose,partly hydrolyzed starch and cyclodextrins used as donors,respectively 84.Enzymes like CGTases manufactured byBacillus.maceransBIO-4 andBacillus stearothermophilusB-5076 served as an effective biocatalyst in the enzymatic transglycosylation of stevia glycosides and starch can be used as a donor[91].Gtase from alkalophilicBacillus.firmuswas observed as an effective biocatalyst in transglycosylation that can remove the aftertaste bitterness of stevioside and improved its sweetness index [58].Similarly,microwave-assisted reaction has also been approved as fast and easy approach.In this experiment,efficient 1,4-intermolecular transglycosylation with β-CGTase was accomplished under microwave conditions in the presence of cyclodextrin as glucose donor that resulted in the optimum yields of two α-glycosylated biotransformed products in 65% and 25%yield,respectively.The quality and taste of glycosides was determined to be enhanced by transglycosylation using other enzyme systems.These enzymes includetrans-a-glucosylation of stevioside with maltose and biozyme L (crude α-amylase preparation produced byAspergillus spp.) [26,92].Trans-3-2,6-fructofurarylation of stevioside with sucrose and p-fructofuranosidase fromArthrobactorspecies.K-1affordedatrans-2,6-fructofuranosylated 19-O-glucosyl moiety in high yield from stevioside102.Treatment of stevioside with sucrose and glucosyltransferase fromStreptococcusmutants afforded a better yield than that by the Biozyme L-maltose system.At the same time,dynamics of stevioside production has been investigated with culture growth in liquid suspensions [58].

6.3.Micropropagation

Micropropagation technique can surely increase the production of secondary metabolites.In dry plant material this technique can increase SGs level from 2%-3% while in field grown material it increases the level of SGs up to 10% [10,93].In contamination free environment,the strong attribute of a plant cell allowedin vitromicro propagation which gave best outcomes as compared to propagation through stem cuttings and seed germination [14,94].In medicinal plants,selection of suitable PGR,its concentration in a media,culture growth conditions,and type of explants considerably influenced the establishment and development of micropropagation.Successful micropropagation ofS.rebaudianapromoted with MS media were supplemented with specific cytokinins such as Kinetin (Kn) and Benzyl adenine (BA) [95].

The fingerprinting of the micropropagated stevia plants and inter simple sequence repeats (ISSR) confirmed the genetic stability of plant,similar to the mother plant[96].At MS media supplemented with kinetin(0.5 mg·L-1),and IAA(2 mg·L-1)best shooting per explant was observed,while MS media supplied with IBA(2 mg·L-1) was suitable media for roots formation.Similarly,in another experiment,leaf derived callus was used by placing callus on MS media augmented with a combination of 0.5 mg·L-1and1.5 mg·L-1BA[94].For the large scale production and multiplication ofS.rebaudiana,optimized efficient micropropagation can also be performed through which half or more than half of the million plants can be produced from even single node [94].

6.4.Elicitation of secondary metabolites

Although there are several methods that were used to enhance the production of secondary metabolites,elicitation was considered as the most widely used method because elicitation has the ability to stimulate the plant defense system and these methods can frequently reduce the effects of stresses.It resulted in the increased production of metabolites in the plant tissue [47,84].

Sometimes due to the low yield production of the potent compound other technologies are considered as less potent or incapacitate for this purpose.To enhance the production of important bioactive compounds at commercial scale various strategies has been introduced which have the capacity to enhance the yield of these bioactive compoundsin vitrocultures [97].In this strategy different elicitors including jasmonate,melatonin,inositol triphosphate,ethylene,cyclic nucleotides and abscisic acid were used.These elicitors act as signaling molecules to produce stress conditions in the plant cell,and initiate the production of ROS which enhance the accumulation of bioactive compounds or secondary metabolites directly or indirectly.Literature indicates that both biotic and abiotic elicitors [47]have fruitful effects on steviol glycosides biosynthesis [98,99].

It has also been observed that the use of AgNPs and salicyclic acid (SA) as a chemical elicitor can significantly enhance the stevioside contentin vitroand can enhance the level of stevia cultures[100].Similarly,supplementation of media with SA also increased the stevioside contentin vitroregenerated plants and the elicitors such as Spermidine(SPD)methyl jasmonate(Me-J)have the ability to enhance the stevioside production in hydroponic culture.Also other elicitors like glutamic acid,glucose,gibberellic acid,proline and 2-acetooxybenzoic acid could significantly increase the steviosides level in callus cultures of plant as compared with controls[100].Likewise,to enhance the bioactive compounds and sweet glycosides accumulation in differentin vitrocultures ofS.rebaudiana,some biotic elicitors can be used,such asPseudomonas putida,Bacillus polymixa,Azobacter chroococcumandarbuscular mycorrizalfungus indicated the positive impact of these biofertilizers on the production and improvement of secondary metabolites.Some biofertilizers likeVesicular Arbuscular Mycorrhiza(VAM),andAzospirillum(AZO) and Phosphorus Solubilizing Bacteria (PSB) has also assessed the effects on the stevioside production and enhancement.From different researches it has been concluded that the inoculants extensively enhanced the stevioside content and biomass formation [101,102].

6.5.Vegetative propagation

Conventional propagation ofS.rebaudianainclude seeds or stem cuttings,which is mostly called as vegetative propagation.[8,98].Generally,unsuitable cross-pollination and seed propagation will cause plants to be more heterogeneous [88].Stem cuttings was most likely to be used for propagation and it can easily develop root system[99,103].The seeds of stevia are mostly smaller in size and the germination percentage is very low.Due to smaller size,these seeds have less growth and less germination potential.Similarly,due to poor seed germination,crop cultivation cannot be done on large scale that can stop programs of breeding Therefore,modern techniques of propagation such asin vitroregeneration are needed to enhance the production of this important plant species[103,104].

Sometimes it is difficult to collect the vegetative portion of plant for propagation,due to the limited availability of healthy mother plants and high labor input.Beside propagation issues,stevia productivity was also reduced due to some fungal diseases such as powdery mildew (ErysiphecichoracearumDC),root rot(Sclerotiumrolfsii),Septoria leaf spot (Septoriasteviae),damping-off(Rhizoctoniasolani,stem rot (Sclerotiumdephinii,Sclerotinia sclerotoirum) and leaf spot (Alternariaalternata).The stem cuttings were changed by environment for which it shows variable survival rate[14,105].

6.6.Cell and adventitious root culture

Secondary metabolites derived from the plants were the bonafied source of different food additives,food flavors and some other important industrial and pharmaceutical materials.Among different technologies that were derived for the enhancement and increase production of glycosides,plant cell culture technology was also an important and globally accepted technology,which was recognized as a potential alternative for the production and enhancement of important secondary metabolites and their derivatives[14].The quality and quantity of stevia plants and secondary metabolite profiles were improved by plant cell culture technology,which plays a significant role in improvement of these metabolites.For the production of secondary metabolites and disease free germplasm,advances in the field of plant cell culture played a vital role such as in the establishment of callus and cell culture,growth medium optimization forin vitroplant production,elicitation,strain selection,micropropagation etc.[98].Large scale production of stevia was required to exploit its industrial application,but plant tissue culture was the only method for massive and rapid production ofS.rebaudiana,due to the limited availability of low seed germination frequency and healthy stem cuttings [6].Large quantities of sweet glycosides could be isolated fromin vitroraised plant tissues in contrast to the field-grownS.rebaudiana[93,106].Under controlled growth and aseptic conditions,cell cultures and callus could be oppressed for the sustainable production of SGs.Development of callus culture was dependent upon type and PGRs (plant growth regulators) concentration,vitrogrowth conditions and the type of explants inS.rebaudiana.For the production of desirable bioactive compounds callus tissues were suitable because these tissues can maintain the power of cell division for a longer period,genetic engineering,germplasm conservation,gene manipulation are main examples [107,108].

A significant number of studies reported the establishment of callus and cell culture protocols inS.rebaudiana[109].A study conducted on the evaluation of PGRs,which effected and explanted on callus induction,growth and production showed that Murrashige and Skoog[110]media containing 2,4-Dichlorophenoxyacetic acid(2,4-D) and α-naphthaleneacetic acid can increase the rate of proliferation and induction of callus [14,106].A sufficient amount of steviosides can be produced by same hormonal treatment in the callus tissues.In leaf explant ofS.rebaudiana,higher doses of NAA added to MS media promoted callus growth and induction[111].For the activation of particular metabolic products and the production of valuable secondary metabolites,plant cell cultures can be formed by changing the culture conditions and application of elicitors such as pectin,methyl jasmonate,chitosan,nanoparticles,and melatonin pathways [94].In some studies,stevioside was enhanced by augmentation of an elicitor in MS media in the leaf explant derived callus culture ofS.rebaudiana[111].Similarly,for the enhanced production of rebaudioside A(3.40 mg/g DW)and stevioside (32.34 mg·g-1DW) different elicitors ofS.rebaudianalike salicylic acid (SA) and silver nanoparticles (AgNPs) have been reported 78.Moreover,different concentrations of sodium carbonate (Na2CO3) and sodium chloride (NaCl) were observed for the growth ofS.rebaudianacallus and suspension cultures the effects.Also,the stress conditions and growth parameters created by sodium compounds on plant can significantly enhance the accumulation and production of steviol glycosides (SGs) in callus and suspension culture[14].In secondary metabolites production technology,adventitious roots (AR) cultures have revolutionized the role of plant cell culture.These cultures were easily maintainedin vitro,faster in growth,and possesses unique genetic stability[112].Using this culture technology,a wide range of natural bioactive metabolites have been synthesized in larger quantities including those that are not found in the naturally growing plants[98,98].

6.7.Enhancement through metabolomics proteomics and transcriptomics

(Metabolic Pathways and Genetic engineering)

Current developments and advances in the tissue culture technology of plants have opened new avenues,new molecular tools for the higher production of valued compounds and other potent nutraceutical and pharmaceutical substances.Also,these developments indicated that to improve the production of secondary metabolites,the most important tools were transcription factors,which were proved to be the most efficient in metabolic engineering [113,114].Transcription factors are those regulatory proteins that are supposed to control multiple pathway steps and modulate the expression of specific gene groups by specific DNA bindingprotein interactions.These regulatory proteins can interact with specific transcription machinery and other transcription factors,and can mediate either an increase or a decrease in the accumulation of messenger RNA.Furthermore,these TFs can become a cause of activator or repressor of gene expression [115].

Similarly,some other systems like fermentation,molecular biology or enzymology of plant tissue were also proved as viable platform for the increase production of various secondary metabolites.In addition,some researchers suggested that the transgenic plants have the ability to maintain the level of protein production without additional intervention whereas the genome manipulation resulted in considerably large amount of desired substances produced by the plants infected with an engineered virus[116].Moreover,among the transformed tissues that were expressed in stevia,66.7% of the stevia plants can survive at higher concentration of bialaphos herbicide [117].

Similarly,genetic engineering techniques also included amplified fragment length polymorphism AFLP,polymorphic DNA(RAPD),simple sequence repeat (SSR),inter simple sequence repeat ISSR,high performance fragment length polymorphism(HPTLC) and next generation sequencing (NGS) technology [34].These methods were widely used because of their significant characteristics e.g.being fast,efficient,cost effective,these glycosides are proved helpful and effective in improving the sweetener profile in stevia by up and down regulation of desired genes [8].

In current studies,metabolic engineering is considered a stepping stone to enhance the biotechnical production of SGs in the heterologous host likeS.cerevisiae,CynobacteriaandE.coli.MVA pathway was mostly used to initiate the process,andS.cerevisiaewas successfully constructed to enhance the flux to improve the heterologous production terpenoids[118].At the same time,E.colihas also been successfully rewired to express the two entkaurene genes ofS.rebaudianawhich encode the two enzymes namelyentkaurene synthase KS andent-copalyl diphosphate synthase CPPS1[119].New researches and emerging technologies are now focusing on DNA based molecular markers[1,120].Moreover,a number of new molecular marker technologies were used in current researches to develop functional molecular markers for the genetic improvement and diversity characterization of plant.Some other molecular approaches were also implemented to increase the dry content and weight of stevia leaves hence increased yield of sweetening compounds [121].

The production of steviosides through microbial cell factories has attracted a lot of attention,as it is considered as a promising alternative to conventional methods.Recent advances in the field of metabolic engineering have enabled the construction of microbial cell factories for the production of wide range of valuable compounds.A large number of efforts were made to produceentkaurene,ent-kaurenoic acid,steviol,and steviol glycosides in microorganisms by metabolic engineering.However,the amount and titer of steviol is relatively low in metabolically engineered microorganisms,therefore a biosynthetic pathway of steviol in heterologous microorganisms needs to be metabolically optimized[31 122].

The two main advantages of using microbial factories are as follows:

· These microbial factories have ability to control the levels of expression of the different pathway genes.

· They have inherent opportunity to modify the substrate specificity of the enzymes involved by mutational studies.

Screening of the mutant library should be performed to identify mutations with positive impact on the accumulation of SGs especially Reb D and Reb M.Screening made it possible to identify variants,such as UGT76G1Thr146Gly and UGT76G1His155Leu,which prevents the accumulation of unwanted side-products and gave increased specific accumulation of the desired sweeteners [122].

Transcriptomics has also emerged as one of the promising methods to stimulate and induce SGs biosynthesis in stevia leaves.In the biosynthesis route of SGs,transcriptomic profiling of genes was involved which were useful for metabolic engineering of plant and hence improved the compound content and biomass yield[123].Similarly,RNA-Seq was also a well know universal technology that was used to understand the plant’s genomics and assist in further developments of various field such as biochemistry,agronomy,genome mapping and evolutionary studies ofS.rebaudiana.This technology is a part of NGS technology and is robust and cost effective [4].

Although all the above methods are now widely used to enhance the production of SGs,above studies showed that the methods discussed have some negative sides,e.g.stem cutting and seed germination were mostly considered as laborious and non-suitable approaches for SGs and healthy biomass production because of the loose viability of stevia seeds during storage [76].

Plant cell culture was time consuming and expensive and gave low yield production.Therefore,this technique may not have been considered viable for the large scale production of SGs or natural low calorie sweetener [33,93].

Similarly,metabolic engineering was also considered challenging for the production of complex natural products due to the complexity of plant cells.Great deals of efforts to identify the genes responsible for biosynthesis,protein engineering,metabolic engineering and downstream processing are required to develop a sustainable manufacturing process of SGs.The “omics”technology is emerging as one of the promising method to enhance and induce SGs biosynthesis inS.rebaudianaleaves.

7.Future Prospects

Trancriptomic,metabolomic and proteomic studies can be performed in stevia for future prospects to understand the chemical processes e.g.gene regulation and conversion into functional products such as protein and steviosides.In order to improve the total glycoside content and high yield of stevia,breeding programs should be established and plant breeding efforts should be focused on leaf yield and concentration of SGs.Due to the low concentration of stevioside content in stem,high leaf:stem ratios are desirable in cultivated stevia and sufficient genetic variability exists to make genetic gains in leaf yield.Therefore,it has been indicated in the cultivar descriptions that there is an immense need to develop a cultivar that is enriched in stevioside content that can be produced by using a low-cost method,that should be based on transplants produced from seeds.As among all the glycosides,rebaudiosde A is of particular interest because of its desirable taste and <95% purity,therefore,rebaudioside A with improved genotypes need to be developed with respect to other glycosides.

Further new research and development need to be carried out to improve the potential of stevia as a crop by developing improved varieties with high quality steviaviaplant breeding methods and biotechnological approaches.

The major problem of large-scale cultivation is the lack of quality planting material.Stevia is a self-incompatible plant,and seedgrown plants vary in their growth,quality and quantity of diterpene glycosides and desirable ratio of rebaudioside-A and stevioside,which restrict its cultivation from seed.Generally,plants with desirable characteristics are propagated by stem cuttings and tissue culture practices,which limits the large-scale production of planting material.Future research should be emphasis on the development of new seed varieties with high adaptability to different climate conditions,viable seed production,better germination,better leaf:stem ratio for successful cropping and higher diterpene glycoside production.

Environmental concerns are another reason for developing alternative ways of producing SGs.Therefore,modern techniques of propagation such asin vitroregeneration or tissue culture are needed to enhance the production of this important specie.clinical evaluations require serious efforts for improved production of sweet SGs through modern technology.Besides improved production of SGs,major concerns regarding commercialization of these glycosides include the issue pertaining to their safety for human use should also be developed.Additional efforts are required to resolve the issues regarding the permitted and safe use ofSteviasweeteners.Similarly,to ensure their acceptance,preference and choice by the general public,qualities related issues such as the intensity,persistence of sweetness and the absence of other residual flavors should be resolved.

8.Conclusions

Both conventional and biotechnological approaches are currently applied to increase the level of SGs in the plant.Biotechnological methods such as callus,cell culture adventitious root culture,micropropagation and cell culture engineering can be employed to enhance biomass and production of sweet glycosides inS.rebaudiana.Among these biotechnological approaches,RNAbased study or transcriptomics has emerged as one of the promising methods to stimulate and induce SGs biosynthesis in stevia leaves.The transcriptomic profiling of genes involved in the biosynthesis route of SGs enables target genes to be identified,and is useful in metabolic engineering of the plant to improve the compounds content,biomass and yield.Understanding of the biosynthetic genes,their transcription and the regulatory steps in the metabolic pathways are important for the genetic engineering ofS.rebaudianato produce new varieties with enhanced agronomic and metabolomics traits.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (21676119,31671845,32021005)and the Key Research and Development Program of China(2018YFA0900300,2018YFA0900504).