Methylation of Flavonoids: Chemical Structures, Bioactivities, Progress

Enzyme and Microbial Technology 86 (2016) 103–116

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Enzyme and Microbial Technology

j ournal homepage: www.elsevier.com/locate/emt

Review

Methylation of ﬂavonoids: Chemical structures, bioactivities, progress

and perspectives for biotechnological production

a b a a

Niranjan Koirala , Nguyen Huy Thuan , Gopal Prasad Ghimire , Duong Van Thang ,

a,∗

Jae Kyung Sohng

Department of BT-Convergent Pharmaceutical Engineering, Institute of Biomolecule Reconstruction, Sun Moon University, 100, Kalsan-ri, Tangjeonmyun,

Asansi, Chungnam 336-708, Republic of Korea

Center for Molecular Biology, Institute of Research and Development, Duy Tan University, K7/25 Quang Trung Street, Haichau District, Danang City, Viet Nam

a r a

t i b s

c l e i n f o t r a c t

Article history: Among the natural products, ﬂavonoids have been particularly attractive, highly studied and become one

Received 9 November 2015

of the most important promising agent to treat cancer, oxidant stress, pathogenic bacteria, inﬂamma-

Received in revised form 2 February 2016

tions, cardio-vascular dysfunctions, etc. Despite many promising roles of ﬂavonoids, expectations have

Accepted 9 February 2016

not been fulﬁlled when studies were extended to the in vivo condition, particularly in humans. Instabil-

Available online 11 February 2016

ity and very low oral bioavailability of dietary ﬂavonoids are the reasons behind this. Researches have

demonstrated that the methylation of these ﬂavonoids could increase their promise as pharmaceuti-

Keywords:

cal agents leading to novel applications. Methylation of the ﬂavonoids via theirs free hydroxyl groups

Flavonoids

Bioavailability or C atom dramatically increases their metabolic stability and enhances the membrane transport, lead-

ing to facilitated absorption and highly increased oral bioavailability. In this paper, we concentrated

Pharmaceutical agents

Methylation on analysis of ﬂavonoid methoxides including O- and C-methoxide derivatives in aspect of structure,

Synthetic biology bioactivities and description of almost all up-to-date O- and C-methyltransferases’ enzymatic character-

Metabolic engineering istics. Furthermore, modern biological approaches for synthesis and production of ﬂavonoid methoxides

using metabolic engineering and synthetic biology have been focused and updated up to 2015. This

review will give a handful information regarding the methylation of ﬂavonoids, methyltransferases and

biotechnological synthesis of the same.

Contents

1. Introduction ...... 104

2. Structure and bioactivity of methylated ﬂavonoids ...... 104

2.1. O-Methylated ﬂavonoids ...... 104

2.2. C-Methylated ﬂavonoids ...... 104

2.3. Several typical methylated ﬂavonoids and their bioactivities ...... 107

2.3.1. Isoﬂavonoids and their methylation ...... 107

2.3.2. Flavones and theirs methylation...... 107

2.3.3. Flavonols and theirs methylation ...... 107

2.3.4. Flavanones and theirs methylation ...... 107

3. Recent biotechnological progress for methylation of ﬂavonoid ...... 109

3.1. Methyltransferaseas a biological tool for synthesis of methylated ﬂavonoid ...... 109

3.2. Synthesis of methylated ﬂavonoids by in vitro enzymatic reaction ...... 109

∗

Corresponding author. Fax: +82 41 544 2919.

E-mail addresses: [email protected] (N. Koirala),

[email protected] (N.H. Thuan), [email protected]

(G.P. Ghimire), [email protected] (D.V. Thang), [email protected]

(J.K. Sohng).

http://dx.doi.org/10.1016/j.enzmictec.2016.02.003

104 N. Koirala et al. / Enzyme and Microbial Technology 86 (2016) 103–116

3.3. Approaches for modern biotechnological synthesis of methylated ﬂavonoids ...... 109

4. Conclusions and prospects ...... 112

Competing interest ...... 112

Acknowledgements ...... 112

References ...... 112

1. Introduction and biological activity, and since most of the glycosylated prod-

ucts showed only the increase in solubility and a lack of prominent

Polyphenols such as ﬂavonoids, stilbenes are widely presented biological activity (not in all cases though), methylation of these

in plant kingdom and involved in various defense mechanisms pharmaceutically signiﬁcant ﬂavonoids may give the compounds a

including auto-defending against herbivores, stress tolerance, competitive advantage.

water-lost resistance, etc. Among them ﬂavonoid have gained Methylation of free hydroxyl groups in ﬂavonoids dramatically

much interests due to their importantly medicinal and cos- increases their metabolic stability and enhances their membrane

metic properties [1]. Till date around 8000 naturally occurring transport, facilitating absorption and greater oral bioavailability

flavonoids have been identified and characterized which are abun- [14,15]. Supporting this, 7-hydroxyflavone, 7,4 -dihydroxyflavone,

dantly deposited in vegetables, stems, fruits, seeds, and other and 5,7-dihydroxyﬂavone (chrysin) were undetectable in tissue

organs [2]. Structurally, ﬂavonoids contain ﬁfteen carbon atoms levels after administration to rats, whereas the corresponding

in their basic nucleus: two six membraned rings linked with methylated derivatives reached high tissue levels [16]. Mono

a three carbon unit which may or may not be a part of the and dimethylated ﬂavones showed potent antiproliferative activ-

third ring. For convenience, the rings are labeled A, B, and C ity [17]; they inhibited carcinogenic-activating cytochrome P450

[3]. (CYP) transcription and activities [18], benzo[a]pyrene activating

As most plants contain ﬂavonoids, they are considered as enzymes and DNA binding in human bronchial epithelial BEAS-2B

traditional herbs to treat various types of diseases for long cells [19], and aromatase, an important target in hormone-sensitive

time such as increasing in immunological system via anti- cancers [20]. Similarly, 7-O-methyl genistein and 7-O-methyl

oxidant, anti-inﬂammatory, anti-allergenic, anti-cancer properties daidzein signiﬁcantly inhibited TNF-␣-induced invasion of HUVECs

[4–6]. Nowadays, several types of ﬂavonoids such as quercetin, at 20 ␮M, a concentration at which no cytotoxicity was observed

rutin, apigenin, etc. have been extensively used in single or [21]. Furthermore, there have been several reports on compounds

mixed form to make functional food, cosmetic or drug and like rhamnetin [22–24], sakuranetin [25,26] and genkwanin

widely commercialized whole the world such as Quercetin [27,28]. These compounds are the methylated metabolites of

B5 Plus Complex (viridian), rutin and vitamin C (Lamberts), quercetin, naringenin and apigenin, respectively, and quercetin

etc. is already in the clinical trial phase. These results and current

Naturally, ﬂavonoids are often in the type of glycosylated or research suggests that methylated forms have higher metabolic sta-

methylated form in plants due to those structures are more stability, oral bioavailabity and biological activity than unmethylated

ble, bioavailability as well as bioactivity. Glycosylation of ﬂavonoids forms. The emphasis is the effect of methylation modiﬁcation on

have been carried out by a biological tool, glycosyltransferase, in original compounds, including increase in metabolic stability and

which the enzyme catalyzes for the attachment of sugar molecule enhancement of pharmaceutical properties. In this review, we have

into aglycone resulting in glycosides [7,8]. In the similar manner, summarized the structure, bioactivities as well as strategies for

methylation of hydroxyl group in ﬂavonoids occurs in the presence biosynthesis of methylated ﬂavonoids using systematic metabolic

of methyltransferase that attaches methyl moieties to aglycone to engineering.

form methoxides. Methylation can occur via oxygen or carbon atom

to form O-methylated or C-methylated compounds, respectively.

2. Structure and bioactivity of methylated ﬂavonoids

Experimental data revealed that methylation of ﬂavonoids resulted

in dramatic change in pharmacological and biochemical proper-

2.1. O-Methylated ﬂavonoids

ties of methylated compound in compared with its parent [9,10].

Thereby, it is one of the most effective ways to modify of natural

O-methylated derivatives are formed via attachment of methyl

products for drug discovery.

group with oxygen of hydroxyl moiety in ﬂavonoid skeleton and

Depending on the reacted substrates, positions and donor

considered as product of post-modiﬁcation [29]. Due to numer-

groups, glycosylation and methylation may have different effects.

ous hydroxyl groups in ﬂavonoid core, methylation positions

Many promising applications of glycosylated ﬂavonoids were not

of ﬂavonoids are various. For example, structure of several O-

achieved when studies were extended to in vitro biological activ-

methylated ﬂavonoids are presented in Fig. 1A, extracted from

ity tests. For instance, when nonbenzoquinone geldanamycin was

Cuphea and Diplusodon [30], Friesodielsia discolor [31] or Piper mon-

glycosylated, the glycosylated products demonstrated weaker bio-

tealegreanum [32].

logical activity compared to the original aglycone [11]. Additionally,

in our recent preliminary studies, glycosylated genistein was sub-

jected to biological activity tests. Not much improvement was seen 2.2. C-Methylated ﬂavonoids

in its anti-cancer activity, though it has an added advantage of hav-

ing higher solubility than its parent compounds [12]. There are

Numerous C-methylated ﬂavonoids have been mined from

many unpublished results due to a lack of signiﬁcant biological

plant extract such as in Pisonia grandis roots [33], in some Myri-

activity related to the glycosylation of ﬂavonoids. This “-enhances

taceae [34] or Cleistocalyx operculatus [35] (Fig. 1B). Bioactivities

solubility” tag of glycosylated analogues is true as exempliﬁed by

of C-methylated ﬂavonoid have been checked as neuraminidase

studies focused on the use of sugar conjugation: glycosylated com-

inhibitors for novel inﬂuenza H1N1 [35], antioxidant and radical

pounds can greatly enhance drug solubility (up to >2-fold) and

scavenging effect [36,37].

enhance uptake in vitro[13]. As the motive for various modiﬁca-

It will be important to discuss occurrence and biological

tions of natural products like ﬂavonoids is to increase their stability

activities for other selected ﬂavonoids and their methyl con-

N. Koirala et al. / Enzyme and Microbial Technology 86 (2016) 103–116 105

Table 1

Occurrence and bioactivities of several typical methylated ﬂavonoids.

No. Names of compounds Bioactivities Origin References

1 Genkwanin Anti-bacterial, anti-plasmodial, radical Daphene genkwa Sieb. Et. Zucc; [62,27,63,64]

(7-O-methylapigenin) scavenging, chemo preventive and Rosmarinus ofﬁcinalis L.; Cistus

anti-inﬂammatory effect in relation with laurifolius L.

the miR-101/MKP-1/MAPK pathway

2 Sakuranetin (7-O- Inhibition of platelet aggregation, a Oryza sativa L., Boesenbergia [128–130]

methylnaringenin) cytotoxic compound to KB nasopharyngeal pandurata, Eriodictyon californicum,

carcinoma cells Piper aduncum

3 Rhamnetin Anti-melanogenesis,inhibits the formation Rhamnus petiolaris, Coriandrum [22,80,82]

(7-O-methylated of ␤-amyloid,enhancing the radio sativum, Syzygium aromaticum,

quercetin) therapeutic efﬁcacy by inhibiting Prunus ceracus

radiation-induced Notch-1 signaling in

lung cancer cell lines

4 Hesperetin Anti-estrogen, inhibition of breast cancer Natsudaidai, Citrofortunella mitis, [131,132]

cell proliferation, delaying tumorigenesis Citrus reticulate, Citrus limon,

Lamium album, Lamium biﬁdum

Cirillo.

5 Isorhamnetin Inhibition of over-accumulation of Tagetes lucida, Brassica rapa var. [133–135]

triglyceride in HepG2 cells, antioxidant rapa), Solidago virgaurea, Brassica

and cytotoxicity against HepG2 cells, juncea, Ginkgo biloba

inhibition of neutral endopeptidase,

inhibits xanthine oxidase

6 Isosakuranetin Cytotoxic and fungicide properties Citrus sinensis, Citrus x paradisi, [136,137]

Inhibition on CYP1B1, CYP1A2, CYP1A1 Monarda didyma

cytochromes’ activity

7 Dihydrokaempferide Effective antioxidant and radio protectant, Prunus domestica, Salix caprea, [138–140]

prevention of hypertension and signiﬁcant Brazilian green propolis

decrease in blood pressure, inhibitors of

fungi

8 Kaempferide Inhibition of TRP1 mRNA expression in Kaempferia galanga [141,142]

B16-4A5 cells and pro-coagulant activity in

human monocyte, cytotoxicity against

A549, HeLa and HT-29 cells

9 Syringetin (3 ,5 -O- Antiviral activity against HCV JFH-1 Lysimachia congestiﬂor [143–145]

dimethylmyricetin) J399EM infected in human cells, a

signiﬁcant induction of differentiation in

MC3T3-E1 mouse calvaria osteoblasts and

osteoblastic 1.19 cell line.

10 Laricitrin (3 -O- Antioxidant activity Red grape, Vaccinium uliginosum [146,147]

dimethylmyricetin)

11 Acacetin Anti-aromatase and anti-estrogen Robinia pseudoacacia, Turnera [148,149]

activities, used for the treatment of atrial diffusa

ﬁbrillation (AF)

12 Sinensetin Radical-scavenging activity Orthosiphon stamineus and in [150,151]

orange oil

13 2 ,4 -Dihydroxy-3 ,5 - Anti-M. tuberculosis H37Rv activity, Campomanesia adamantium, [152,153]

dimethyl-6 - inhibition of KDR tyrosine kinase, Cleistocalyx operculatus

methoxychalcone mitogen-activated protein kinase (MAPK)

and AKT activation of KDR signal

transduction, inhibition of vascular

endothelial HDMEC cells

14 Matteucinol Antitumor activity against HL60, KB, Melastomataceae, Ericaceae, [154,155]

BGC823 and Bel7402cells, cytotoxic Dryopteridaceae, Rhododendron

activity against theHL-60 and SMMC-7721 hainanense

cell lines.

15 Europinidin Used for treatment of diabetes and Plumbag, Ceratostigma [156]

metabolic syndrome, inhibition for

production of hepatitis C virus

16 Hirsutidin Treatment of a cancerous or precancerous Catharanthus roseus [157]

lesion of the skin

17 Petunidin Inhibition of human RNase H and HIV-2 Aronia sp., Amelanchier alnifolia, [158]

RNase H Vitis vinifera, Vitis rotundifolia

18 Rosinidin Inhibition of natural cyclooxygenase Catharanthus roseus, Primula rosea [159,160]

19 Diosmetin Inhibition of horse BchE and STK33 activity Caucasian vetch [161–163]

20 Tricin Antihistaminic activity in rat RBL2H3 cells Rice bran [164–166]

assessed as inhibition of DNP-BSA-induced

beta-hexosaminidase release preincubated

Antioxidant activity assessed as DPPH

radical scavenging activity

Potently inhibits cyclooxygenase enzymes

and interferes with intestinal

carcinogenesis in ApcMin mice

21 Tangeritin Appears to counteract the anticancer drug Citrus peels, citrus juices [167,168]

tamoxifen and to suppress the activity of

natural killer cells

Cholesterol lowering agent

Effects againstParkinson’s disease

106 N. Koirala et al. / Enzyme and Microbial Technology 86 (2016) 103–116

Table 1 (Continued)

No. Names of compounds Bioactivities Origin References

22 Wogonin Inhibitors of CDK9 that induce apoptosis in Scutellaria baicalensis [169–171]

cancer cells by transcriptional suppression

of Mcl-1

Shown pharmacological effects that

indicate wogonin may have anti-tumor

properties. Possess anticonvulsant effects

23 Zapotin Potential anti-carcinogenic effects against Casimiroa edulis [172,173]

isolated colon cancer cells

Potent anticancer activity of zapotin and

suggests a role for zapotin both as a

chemopreventive and a chemotherapeutic

agent against colon cancer

24 Poriol Dose response conﬁrmation for Pseudotsuga menziesii [174,175]

Mcl-1/Noxa interaction inhibitors

HTS to ﬁnd inhibitors of pathogenic

pemphigus antibodies

25 Lupeol Antiprotozoal, antimicrobial, Syzygium samarangense, Acacia [176,177]

antiinﬂammatory, antitumor and visco and Abronia villosa.

chemopreventive properties, antimicrobial

26 Betulin Effective against a variety of tumors. Starts Syzygium samarangense, bark of [178–180]

a process of apoptosis and can slow the birch trees, birch sap

growth of several types of tumor cells.

Decreased the lipid contents in serum and

tissues, and increased insulin sensitivity.

Antiviral activity against HIV1 3B in human

H9 cells assessed as inhibition of viral

replication

27 Demethoxymatteucinol Effective concentration against HIV-1 Desmos, Myrica serrata [154,181,182]

replication in H9 lymphocytic cells,

concentration that inhibits uninfected H9

cell growth, antitumor activity against

human HL60 cells, KB cells, BGC823 cells,

Bel7402 cells

28 2 ,6 -Dihydroxy-4 - Antifungal and antibacterial Myrica gale, Myrica serrata [37,181]

methoxy-3 ,5 - Inhibition of lipid peroxidationinduced by

dimethyldihydrochalcone tert-butyl hydroperoxide

(myrigalone B) Inhibition of peroxidation,scavenging

activity against the diphenylpicrylhydrazyl

(DPPH) radical, and inhibition of enzymatic

lipid peroxidation in linoleic acid

29 Myrigalone Antifungal and antibacterial Syzygium samarangense, Myrica [181,183]

H/2 ,4 -dihydroxy-6 - (Cladosporiumcucumerinum, Bacillus serrata

methoxy-3 - subtilis, and Escherichia coli)

methyldihydrochalcone Exhibited signiﬁcant and selective

inhibition against prolyl endopeptidase

30 Desmosﬂavanone II Inhibition of HIV-1 replication in H9 Desmos cochinchinensis Lour [182,184]

((2S)-7-hydroxy-5- lymphocytic cells, therapeutic index

methoxy-6-methyl-4- determined by ratio of IC50 to EC50 oxo-2-phenyl-2,3- dihydrochromene-8-

carbaldehyde

31 Desmosdumotin C Cytotoxicity activity against bone (HOS), Desmos dumosus, Mitrella kentii [185,186]

breast (MCF) and ovarian (IA9) cancer cell

lines

Gastroprotective effect and

anti-Helicobacter pylori activity

32 (2S)-5-hydroxy-7- Inhibition of inﬂuenza A virus Cleistocalyx operculatus [35,182]

methoxy-6,8- neuraminidase, effective concentration

dimethylﬂavanone against HIV-1 replication in H9

lymphocytic cells

33 Cariphenone B Antioxidant activity against tumor cell Hypericum carinatum [187,188]

lines

34 Rottlerin Inhibitor of protein kinase Cdelta Mallotu philippensis [189,190]

(PKCdelta)

35 Zidovudine Inhibition of reverse-transcriptase Synthesized [191–193]

inhibitor (NRTI), a component of

HIV-treated drug AZT, antiviral activity

against humanHIV 1-infected C8166 cells

and human HIV 13B-infected H9 cell

36 Pisonivanone Inhibition on growth of Mycobacterium Pisonia aculeate [194]

tuberculosis

jugates which may play a significant role in pharmaceutical ities is given in Table 1. Pharmaceutically significant flavonoids

industries in near future. A comprehensive overview of selected and their methylated derivatives are described to some details

methylated ﬂavonoids focusing on their occurrence and bioactiv- herein.

N. Koirala et al. / Enzyme and Microbial Technology 86 (2016) 103–116 107

2.3. Several typical methylated ﬂavonoids and their bioactivities alkaline phosphatase reporter assay), inhibition of important signal

molecules in production of TNF-␣ and IL-6 [61].

2.3.1. Isoﬂavonoids and their methylation

Isoﬂavonoids are a very distinctive subgroup of ﬂavonoids that

2.3.2.3. Apigenin. Apigenin-7-O-methylether also known as

are signiﬁcantly occurred in soybeans and other leguminous plants.

genkwanin is one of the major non-glycosylated ﬂavonoids found

They are found to play an important roles as precursors for the

in some herbs such as Genkwa Flos (Daphene genkwa Sieb. Et. Zucc),

development of phytoalexins during plant micro interactions [38].

rosemary (Rosmarinus ofﬁcinalis L.) [62] and Cistus laurifolius L. [63].

The metabolism of isoﬂavonoids initiates with the ﬁxed carbon

Genkwanin has been proven as a multi-fuctional pharmaceutical

gone through the phenylpropanoid pathway. Following multi-

agents such as anti-bacterial, antiplasmodial, radical scavenging,

ple enzymatic processes, phenolic compounds, isoﬂavonoids are

chemopreventive, anti-inﬂammatory effect [63,64], inhibitor for

generated [39]. Szkudelska and Nogowski reviewed the effect of

dehydrogenase type 1 in the 17␤-hydroxysteroid pathway [65].

genistein inducing hormonal and metabolic changes, by virtue of

which they can inﬂuence various disease pathways [40] or high

2.3.3. Flavonols and theirs methylation

pharmalogical bioactivities such as anti-cancer, reducing of cardio-

2.3.3.1. Myricetin. Myricetin (3,5,7-trihydroxy-2-(3,4,5-

vascular diseases [41,42] prevention of osteoporosis, attenuation

trihydroxyphenyl)-4-chromenone) is a natural ﬂavonol found

of post-menpopausal problems [43] and loss body mass and fat

in fruits, vegetables, tea, strawberries, red wine, and herbs [66]

tissue [44]. Also it has been studied that ingestion of dietary gen-

that involve in the inhibition of superoxide anions or restrict the

estein showed concentration changes of hormones, such as insulin,

bioactivities of xanthine oxidase [67], or suppressed the cancer for-

thyroid hormones, adrenocorticotropic hormone, cortisol and cor-

mation in rat [68]. Further studies mentioned myricetin as inhibitor

ticosterone, and lipid metabolic changes [45]. Similarly, it has been

of myeloperoxidase [69] or reduction of colon carcinogenesis [66].

shown that daidzein exhibits similar effects with those of genistein

Methylated myricetin derivative such as syringetin and

[46]. Furthermore, daidzein restricted bioactivities of enzymes in

laricetrin have been found throughout the plant kingdom [70] and

the biosynthesis of protein and DNA replication in osteoblasts that

play roles as antioxidants, anti-inﬂammatory, anti-artherosclerotic

inﬂuencing on the bone density [47–49]. It is because of these bio-

agents, etc. [71].

logical properties that isoﬂavonoids ﬁnd numerous applications

and makes them priceless in nutraceutical as well as cosmetic

applications and are important constituent of various dietary sup-

2.3.3.2. Kaempferol. Kaempferol (3,5,7-trihydroxy-2-(4-

plements, creams, ointments, moisturizing lotions and gels [50].

hydroxyphenyl)-4H-1-benzopyran-4-one) is a ﬂavonoid widely

7-O-methylation of the bioactive isoﬂavonoids can enhance

occurred in many edible plants and ingredient of traditional

their biological activities. In our recent study, although the 7-O-

medicine [72]. This compound involves in the apoptosis in human

methylation of genistein did not contribute to a better anti-cancer

lung cancer cell [73] or inhibition of cancer cell line under in vitro

and anti-angiogenic activity compared to genistein, the 7-O-

conditions [73,74].

methylation of daidzein provided more elevated chemosensitivity

7-O-Methyl dihydrokaempferol so-called 7-O-methyl aro-

than that of original compound. These results suggest that 7-O-

madendrin is found in plants that effecting on the development

methyl daidzein may have great potential as a chemotherapeutic

of liver carcinoma cells and adipocytes in vitro[75]. Considering all

agent for human cancers, and the regiospeciﬁc methylation strat-

these medicinal applications and the importance of methylation

egy could provide a novel starting point for pre-clinical and clinical

on biological activity of kaempferol, more experimental studies

applications of isoﬂavonoid compounds in cancer therapy [12,21].

are required for discovering the pharmacological effectiveness of

kaempferide in near future.

2.3.2. Flavones and theirs methylation

2.3.2.1. 7,8-Dihydroxyflavone. These flavonoids are significantly 2.3.3.3. Quercetin. Quercetin is a well-known flavonoid and its bio-

present in some parts of plant such as fruits and vegetables [51]. logical activities have been broadly well documented. Even though

It plays role as neurotrophin in mammalian [52], reducing angio- the use of quercetin in cancer prevention and as chemotherapy

genesis [53], a potent agonist against TrkB [54], antioxidant agent, adjuvant [1] as well as their relevance for the well-being of the

resistance against aging-induced morphological changes. In addi- cardiovascular system [76] for neuroprotection [77] and for the

tion, it can reduce the chances of neurodegenerative diseases improvement of cognitive functions has long been known [78], its

induced by ROS via increase the cellular glutathione level [55]. use as a therapeutic is limited because of its poor bioavailability and

Furthermore, 7,8-DHF was proven to be having a vasorelaxing solubility. The solution for this could be methylation of quercetin

and antihypertensive properties [56] suggesting its use in treat- which will be described in the paragraphs below taking rhamnetin

ment of cardiovascular diseases. 7,8-Dihydroxyﬂavone (ﬂavone) as an example.

and its methylation possess the cytoprotective effect against Rhamnetin, a naturally occurring 7-O-methylated quercetin

oxidative stress by scavenging intracellular ROS and 7-hydroxy-8- is abundantly occurred in Rhamnus petiolaris [79], Coriandrum

methoxyﬂavone has potent antioxidant activity without affecting sativum (Apiaceae) [80], and Prunus ceracus (Rosaceae) [81]. It pos-

endothelial cell viability even at long period of treatment [57]. sesses strong anti-inﬂammatory [82], anti-tumor, anti-cholesterol,

anti-bacterial and anti-melanogenesis [83] activities and inhibits

the formation of ␤-amyloid [23] or enhancing the radiotherapeutic

2.3.2.2. Luteolin. Luteolin (3 ,4 ,5,7-tetrahydroxyﬂavone) belongs

efﬁciency in lung cancer cell lines [24].

to a group of naturally occurring ﬂavonoids that are found widely

in the plant kingdom. Vegetables and fruits such as celery, parsley,

broccoli, onion leaves, carrot, peppers, cabbages, apple skins, and 2.3.4. Flavanones and theirs methylation

chrysanthemum ﬂowers are rich in luteolin [58–60]. Thereby these 2.3.4.1. Naringenin. 7-O-Methylnaringenin also known as saku-

ﬂavonoid are important ingredients of Chinese herbs for treatment ranetin is a chiral ﬂavanone [84] present in rice (Oryza sativa L.)

of hypertension, inﬂammatory diseases, etc. [51]. [85], ﬁnger root (Boesenbergia pandurata) [86], yerba santa (Eriod-

Double methylations of luteolin at 7- and 3 -OH position pro- ictyon californicum) [84], spiked pepper (Piper aduncum) [87] and

duces velutin. Velutin has been demonstrated as a potent inhibitory Populous davidiana [88]. Sakuranetin has been shown engage in

agent in nuclear factor (NF)-␬B activation (as assessed by secreted various plant defense roles due to its anti-bacterial, anti-fungal,

108 N. Koirala et al. / Enzyme and Microbial Technology 86 (2016) 103–116

Fig. 1. (A) Chemical structures of O-methylated ﬂavonoids and (B) C-methylated ﬂavonoids.

and anti-inﬂammatory activities [89] and cytotoxicity in carcinoma have been extensively studied such as reducing cardiac arrhyth-

cells [87]. mia and infarct size in rats [90] or anti-fungal [91], anti-bacteria

[92], anti-inﬂamatory [93], enhancing tyrosine activity of B16F10

2.3.4.2. Pinocembrin. Like naringenin, pinocembrin is a type of ﬂa- melanoma cells [94]. Thereby they have become more important

vanone widely spread in vascular plants, and their bioactivities in science and clinical uses. Considering all these medicinal appli-

N. Koirala et al. / Enzyme and Microbial Technology 86 (2016) 103–116 109

cations and the importance of pinocembrin, more experimental SAM-binding domain and metal-dependent catalytic site have been

studies are required for discovering the pharmacological effective- identiﬁed in LiOMT (Leptospira interrogans O-methyltransferase)

ness of methylated pinocembrin in near future. [108]. In addition, structure of several enzymes have been analyzed

as wheat ﬂavone O-methyltransferase [109], caffeic acid/5-

hydroxyferulic acid 3/5-O-methyltransferase [110]. However, no

3. Recent biotechnological progress for methylation of detail information on the characterization of C-methyltransferase

ﬂavonoid is has been presented till now. Most of amino acid sequences

are annotated as putative C-methyltransferases being awaited for

3.1. Methyltransferaseas a biological tool for synthesis of studying as presented in Table 2.

methylated ﬂavonoid

3.2. Synthesis of methylated ﬂavonoids by in vitro enzymatic

S-Adenosyl-l-methionine (AdoMet) dependent O- reaction

methyltransferases (OMT) operates by transferring methyl

moietyto a speciﬁc hydroxyl group of an acceptor compound Numerous putative O-methyltransferase have been mined from

resulted in the formation of its methyl ether derivative and bacteria (Bacillus, Streptomyces, Listeria, etc.), fungi (Phanerochaete

S-adenosyl- -homocysteine [95]. Generally, OMTs acting on the chrysosporium) and plant for characterization of their substrate

natural products are classiﬁed into three classes [96]. In particular, as ﬂavonoids. For example, luteolin, luteolin7-O-glucoside, eri-

class I and class II function in methylation of phenolic hydroxyl odictyol, dihydroquercetin, etc. were used as substrate for an

residues, while class III OMTs works on carboxyl groups to obtain O-methyltransferase puriﬁed from soybean suspension cell culture

methyl esters. Furthermore, those classes are subdivided by using [111]. In other ways, those were cloned and heterologous expressed

their biochemical properties such as structure, molecular size and in Escherichia coli, or Bacillus, for example, several regiospe-

cation requirement, etc. For example, cation dependent OMTs sub- ciﬁc ﬂavonoid 3 /5 -O-methyltransferases from tomato [112], or

group of class I (so-called caffeoyl coenzyme A OMTs or CCoAOMTs) Chrysosplenium americanum OMT1 and OMT2 were characterized

includes a group of low molecular mass (23–27 kDa) enzymes and as 3 -O-methylation and 3/5-O-methylation of ﬂavonoid, respec-

class II (also known as caffeic acid OMT or COMT) comprises OMTs tively [113]. This method offers some merits like valuable tools to

with a higher subunit molecular mass (38–45 kDa) and no cation study catalytic mechanism, activity as well as kinetic of enzyme.

dependency [97,98]. However, methyltransferases can simply be Furthermore, product is formed regio-selectively and the forma-

catagorized as O-methyltransferase (OMT), N-methyltransferase tion of by-products are extremely less with lower waste emission

(NMT) and C-methyltransferase (CMT) based on their target and energy requirements. However, it requires highly puriﬁed sub-

attachments such as oxygen, nitrogen and carbon, respectively strates, hard to scale up the reaction, thus mostly used in the

[99]. Up to now, OMTs are found ubiquitously in nature. Most laboratory.

plant-originated OMTs are characterized to use phenolic com-

pounds like stilbene and ﬂavonoids as their substrate [57,100,101]. 3.3. Approaches for modern biotechnological synthesis of

Five highly conserved regions are proposed as a signature for plant methylated ﬂavonoids

O-methyltransferases, two of which (regions I and IV) are believed

to be involved in S-adenosyl-l-methionine and metal binding, Although much information on biosynthetic pathways of plant

respectively [95]. However, just a few microorganisms-originated methylated ﬂavonoids have been obtained so far it is difﬁcult to

OMTs and their working mechanisms have been biochemically carry out mutagenesis or optimization for production of secondary

characterized. Biochemical and molecular characterization of metabolites using plants as direct hosts due to their huge size

several OMTs have been outlined as in Table 2. genome and complex regulatory network. This resulted in the wide

To investigate the structure-activity relationship, several spreading of direct extraction from plant and chemical synthesis of

ﬂavonoid methyltransferases have been overexpressed, crys- desired compounds before biotechnology still less developed. How-

tallized and analyzed the molecular structure, for instance, ever, directed extraction method is restricted by type of plant, low

Bacillus cereus BcOMT2 – a ﬂavonoid Mg – dependent O- concentration of methylated ﬂavonoids as well as their mixture

methyltransferase [102,103]. In another study, modeling of wheat in natural resource such that this method requires many tedious

(Triticuma estivum L.) O-methyltransferase (TaOMT2), a tricetin experiment skills and facilities [114–116]. Chemical synthesis also

O-methyltransferase. Stepwise tricetin methylation was proven often occurs following severe and complex reaction conditions and

via involvement of deprotonation of its hydroxyl groups by a produces many un-wanted by-products leading to impenetrability

His262-Asp263 pair. Futhermore Val309, a conserved amino acid in puriﬁcation, low yield of product, for example chemical synthesis

in a number of graminaceous ﬂavone OMTs, was determined the of silybin glycoside at C-23 [117].

enzyme TaOMT2 fortricetin as the preferred substrate [104]. In the Biosynthetic methods based on the reconstruction of recom-

similar manner, amino acid of rice-based ﬂavonoid OMT (ROMT9) binant plasmids harboring genes from various sources and uses

was aligned with the Medicago truncatula O-methyltransferase genetically engineered E. coli as host leading to generation of

(COMT) and resulted several speciﬁc amino acid residues regard- numerous natural and un-natural products. Such methods provide

ing enzymatic activities such as Asn-275, His-328 [105]. Recently, several advantages such as high yield of product owing to substrate-

Podospora anserina PaMTH1, a putative O-methyltransferase is speciﬁc enzyme, easily-occurred reaction, less type of secondary

objective for studying its structure and function. The authors metabolites produced by E. coli limiting formation of by-products.

showed that PaMTH1 is responsible for transferring of the methyl In addition, genetically engineered E. coli is straightforwardly con-

group from SAM to one hydroxyl group of the myricetin in a cation- trolled due to its simple genetical machine. Furthermore, it is easy

dependent manner [106]. to scale up the production of desired compound by using fermenter

Studying on crystal structure of ChOMT (chalcone SAM- [1,8,118].

dependent O-methyltransferase) and IOMT (isoﬂavone SAM- Most of methylated derivatives of ﬂavonoids can be produced by

dependent O-methyltransferase) provided a structural basis for biotransformation of ﬂavonoids. For example, two types of Strep-

understanding the substrate speciﬁcity of the diverse family of tomyces were used for methylation of genistein [119]. Kim et al.

plant OMTs and facilitates the engineering of novel activities in reported the biotransformation of apigenin and quercetin using

this extensive class of natural product biosynthetic enzymes [107]. regioselective 7-O-methyltransferase POMT7 having more than

110 N. Koirala et al. / Enzyme and Microbial Technology 86 (2016) 103–116

Table 2

Origin, accession numbers and biochemical properties of ﬂavonoid methyltransferases.

No. Origins Accession Protein size in Enzymatic activities References

number length (aa)

1 Hordeum vulgare subsp. CAA54616 390 Flavonoid 7-O-methyltransferase, [195,196]

vulgare kinetic parameters of KSAM = 10.9 ␮M,

Kapigenin = 4.6 ␮M, Vmax = 0.45 kat/g

2 Hordeum vulgare subsp. ABQ58825 356 Flavone-speciﬁc O-methyltransferase. Prefer [197]

vulgare substrate ﬂavone tricetin at 3 ,5 -O position

into tricin

3 Triticuma estivum ABB03907 356 Flavonoid O-methyltransferase (prefer 3 , 4 , 5 [198]

position of ﬂavone in to mono, di,tri methyl ether)

4 Glycine max C6TAY1 358 Flavonoid 4 -O-methyltransferase [29]

5 Vitis vinifera NP 001290011 235 3 ,5 -O-Methyltransferase showing strong [179,180]

preference foranthocyanins and glycosylated ﬂavonols

6 Catharanthus roseus AAR02419 359 Flavonoid 4 -O-methyltransferase [201]

7 Catharanthus roseus AAM97497 348 Sequential methylations at the 3 - and [202]

5 -positions of the B-ring in myricetin

(ﬂavonol) and

dihydromyricetin(dihydroﬂavonol)

Preferred substrates ﬂavonol glycosides and

anthocyanins

8 Theobroma cacao EOY28137 380 Flavonoid O-methyltransferase [203]

9 Zea mays NP 001106047 364 Catechol and ﬂavonoid O-methyltransferase [204]

10 Mesembryanthemum AAN61072 237 Flavonoids and caffeoyl-CoA [205] crystallinum

11 Glycyrrhiza echinata Q84KK6 367 Isoﬂavone 4 -O-methyltransferase [206,207]

12 Lechevalieriaaero Q8KZ94 283 Rebeccamycin sugar 4 -O-methyltransferase [208,209]

colonigenes

13 Synthetic construct AFQ94040 368 Monolignol 4-O-methyltransferase [210]

14 Triticuma estivum Q84N28 360 Caffeic acid O-methyltransferase [211]

15 Oryza sativa Japonica ABB90678 368 O-Methyltransferase [212]

Group

16 Medicago sativa 1FP1 D 372 Chalcone O-methyltransferase. [107]

17 Streptomyces peucetius Q06528 356 Carminomycin 4-O-methyltransferase DnrK [193,194]

18 Streptomyces avermitilis BAB69281 359 7-O-Methyltransferase [215]

19 Streptomyces peucetius AGU42206 223 Flavonoids O-methyltransferase [57]

ATCC 27952

20 Marinithermus AEB11491 395 C-Methyltransferase [216]

hydrothermalis DSM

14884

21 Meiothermus silvanus ADH64788 391 C-Methyltransferase [217]

DSM 9946

22 Haliscomenobacterhydrosis AEE53273 433 C-Methyltransferase [218]

DSM 1100

23 Marinithermus AEB11490 409 C-Methyltransferase [216]

hydrothermalis DSM

14884

24 Thermomonospora ACY98305 405 C-Methyltransferase [219]

curvata DSM 43183

25 Calditerrivibrio ADR19070 408 C-Methyltransferase [219]

nitroreducens DSM

19672

90% conversion [23]. Through the combination of optimized POMT7 to this research, a series of ﬂavonoid biosynthetic gene has been

expression and cofactor production, the production of rhamnetin cloned into recombinant plasmids to convert p-coumaric acid into

(7-O-methyl-quercetin) was increased upto 111 mg/L via using naringenin and dihydrokaempferol. Simultanously, biosynthetic

genetically engineered E. coli as host [120]. The same group genes for enhanced production of malonyl-CoA (a precursor of chal-

had characterized another regiospeciﬁc 7-O-methyltyransferase cone synthase) was also reconstructed to increase the heterologous

SaOMT2 showing the conversion of quercetin as 119% compared to production of targeted ﬂavonoids. Later on, 7-O-MT (Streptomyces

naringenin [121]. Biochemically, biosynthesis of those compounds avermitilis MA4680) and ﬂavanone-3-hydroxylase has been used

have been illustrated in Fig. 2. to produce sakuranetin as intermediate and ﬁnally 7-O-methyl-

Our group has recently optimized the conditions for pro- aromadendrin. Also, the authors can generate dihydrokaempferol

duction of methylated derivatives of pharmaceutically important using naringenin as substrate before converting this compound

ﬂavonoids using engineered E. coli. Through the combination of into ﬁnal 7-O-methyl-aromadendrin. The yield of 7-OMA depended

optimized conditions and cofactor production, the maximum yield on fed concentration of p-coumaric acid and achieved 30 mg/L

of 7-O-methyl genistein was 164 ␮M (46.74 mg/L) and 7-O-methyl (99.2 ␮M) after 24 h [122]. Rational design was used for enhanced

daidzein was 382 ␮M (102.75 mg/L) when 200 ␮M of genistein and intracellular tyrosine, a precursor for synthesis of chalcone, was

400 ␮M of daidzein was supplemented in the fed batch culture strategy for production of ﬂavonoid by Kim et al. Consequently,

[21]. To add more data to the current research on methylated com- they used the similar biosynthetic genes of ﬂavonoid such as

pounds, recently, system metabolic engineering strategy has been tyrosine ammonialyase (TAL), 4-coumaroyl CoA ligase (4CL), chal-

applied to produce 7-O-methyl-aromadendrin in E. coli. Regarding cone synthase (CHS) to generate naringenin as an intermediate.

N. Koirala et al. / Enzyme and Microbial Technology 86 (2016) 103–116 111

OH OH

HOOC H OOC

HOOC Cinnamic acid p-coumaric acid Caffeic acid

4CL 4CL 4CL OH OCH O- S CoA OH 3 ACS HO O C O C O CoAS HO O

CH3 CH3 SOMT2

O OH ACC Acid-CoA complex OH Acetate Acetyl-CoA OH O OH O

Kaempferol Kaempferide

O O

CoA

HO S CHS FLS OH 3x Malonyl-CoA OH OH OCH3 HO O HO OH H3CO O HO O SaOMT2 OH OH

OH O

S OH O OH O

OH O M H Dihydrokaempferol

T F3 7-O-methyl 2 Naringenin chalcone (Aromadendrin) Ponciretin aromadendrin CHI OH F3'H OH OH HO O HO O H CO O OH 3 NOMT

OH OCH3 SaOM T2 OH O OH O

OH O OH

Naringe nin Dihydroquercetin

7-O-methy l-naringein

(Taxi folin) H3CO O (Sakuran etin) NA

IFS DP T2 OH FNS M H aO OH O CRP FLS +S T9 M Rhamnazin NA RO HO O

OH DP OH OH

+ OH HO O OH OH

OH O HO O H3CO O

POMT7 OH O Genistei n OH OH

Apigenin OH O SaOMT2 OH O

Quercetin 7-O-meth yl-quercetin

SaOMT2 SaOMT2 POMT7 (Rhamneti n) OCH3 OH ROMT9 OH H3CO O H3CO O HO O OH OH O OH

OH O OH O

7- O-methyl-genistein

7-O-methyl-apigenin 3'-O-methyl-quercet in

(genkw anin) (Iso-rhamnetin)

Fig. 2. Metabolic engineering for biological synthesis of various methylated ﬂavonoid. CHS: chalcone synthase, CHI: chalcone isomerase, IFS: isoﬂavone synthase, 4CL:

4-coumarate-CoA ligase, RS: stilbene synthase (STS), FLS: flavonol synthase, F3’H: flavonoid-3 -hydroxylase, F3H: flavanone-3-hydroxylase, ACS: acetyl-CoA synthase, ACC:

acetyl-CoA carboxylase, CRP: cytochrome P450, FNS: ﬂavone synthase, POMT7: apigenin-7-O-methyltransferase; SaOMT2: Streptomyces avermitilis O-methyltransferase;

NOMT: naringenin O-methyltransferase; SOMT2: soybean O-methyltransferase; ROMT9: rice O-methyltransferase.

However, in order to increase the supply the coenzyme A (CoA), CoA, the yields of naringenin and pinocembrin reached about

one gene (icdA, isocitrate dehydrogenase) was deleted. Finally, 60 mg/L [124]. Later on, introduction of Photorabdus luminescens –

O-methyltransferase (OMT) was applied to produce sakuranetin originated acetyl – CoA carboxylase (ACC) and biotin ligase in com-

(7-O-methyl-naringenin) and ponciretin (4 -O-methyl naringenin) bination with redesigned acetate assimilation pathways in E. coli

with maximal yield of 42.5 mg/L and 40.1 mg/L, respectively [123]. led to much improvement of those ﬂavonoids during 36 h of cul-

Miyahisa et al. constructed recombinants harboring PAL (pheny- ture. And, yields of pinocembrin, naringenin and eriodictyol were

lalanine ammonia-lyase), ScCCL (cinnamate/coumarate:CoA lig- achieved as 429 mg/L, 119 mg/L, and 52 mg/L, respectively [125].

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from different sources and heterologously expressed in E. coli. This brin from glucose using system metabolic engineering in E. coli.

resulted in successful production of (2S)-naringenin from tyro- In particular, they designed recombinant vectors harboring genes

sine and (2S)-pinocembrin from phenylalanine. Furthermore by encoding for converting of glucose, synthesis of phenylalanine,

introduction of acetyl-CoA carboxylase (ACC) to enhance malonyl- phenylpropanoid, chalcone, etc. and a recombinant vector support-

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