Logo Beilstein Institut

Cytochrome P450 monooxygenase-mediated tailoring of triterpenoids and steroids in plants

  1. 1 ORCID Logo and
  2. 1,2 ORCID Logo
1Institute of Botany, Leibniz University Hannover, Herrenhäuser Str. 2, 30419 Hannover, Germany
2Centre of Biomolecular Drug Research, Leibniz University Hannover, Schneiderberg 38, 30167 Hannover, Germany
  1. Corresponding author email
This article is part of the thematic issue "Enzymes in biosynthesis".
Associate Editor: J. S. Dickschat
Beilstein J. Org. Chem. 2022, 18, 1289–1310. https://doi.org/10.3762/bjoc.18.135
Received 29 Jun 2022, Accepted 02 Sep 2022, Published 21 Sep 2022

Abstract

The cytochrome P450 monooxygenase (CYP) superfamily comprises hemethiolate enzymes that perform remarkable regio- and stereospecific oxidative chemistry. As such, CYPs are key agents for the structural and functional tailoring of triterpenoids, one of the largest classes of plant natural products with widespread applications in pharmaceuticals, food, cosmetics, and agricultural industries. In this review, we provide a full overview of 149 functionally characterised CYPs involved in the biosynthesis of triterpenoids and steroids in primary as well as in specialised metabolism. We describe the phylogenetic distribution of triterpenoid- and steroid-modifying CYPs across the plant CYPome, present a structure-based summary of their reactions, and highlight recent examples of particular interest to the field. Our review therefore provides a comprehensive up-to-date picture of CYPs involved in the biosynthesis of triterpenoids and steroids in plants as a starting point for future research.

Keywords: biosynthesis; CYPs; cytochrome P450 monooxygenases; plants; steroid; sterol; triterpene; triterpenoid

Introduction

Triterpenoids are a large class of natural products derived from precursors containing 30 carbon atoms and composed of six isoprene units (C5). The structural variety of triterpenoids found in plants is particularly astonishing, and so are their biological activities. To date, more than 20,000 different plant triterpenoids have been identified, and many of these have found applications in agronomic [1], food [2], cosmetics [3] and pharmaceutical industries [4]. Plant triterpenoids include primary metabolites such as phytosterols or steroidal hormones such as brassinosteroids, but also specialised metabolites that convey diverse biological functions [5]. A key factor for the structural richness of triterpenoids and steroids from plants lies in their extensive oxidative tailoring, which in many cases is carried out by cytochrome P450 monooxygenases (CYPs). CYPs represent one of the largest superfamilies of enzymes in plants; in many species, around 1% of all genes encode CYPs [6]. CYPs are well-known for their capacity to catalyse highly regio- and stereospecific reactions on complex substrates. Besides simple hydroxylations, they can also introduce oxo, carboxy, or epoxy moieties or double bonds. Such decorations often also enable additional layers of diversification by glycosyltransferases or acyltransferases [7]. Hence, there is considerable interest in CYPs involved in triterpenoid and steroid metabolism in plants not only for improving our understanding of plant specialised metabolism, but also for synthetic biology and chemoenzymatic synthesis. In this review, we will provide an extensive overview of CYPs involved in tailoring of triterpenoids and steroids in plants. We will first introduce the nomenclature and mechanistic properties of these enzymes, before we describe the phylogenetic distribution of triterpenoid-modifying CYPs and summarise their reaction space. Lastly, we will highlight selected recent examples of multifunctional CYPs that catalyse particularly remarkable modifications of triterpenoids. We therefore hope to provide an up-to-date overview over these key enzymes in plant triterpenoid and steroid metabolism since the last comparable endeavour from Ghosh in 2017 [7]. In addition, readers might also be interested in other excellent reviews or resources providing a more general overview over plant CYPs or CYPs from other plant pathways [6,8-13].

Review

Nomenclature

Considering the enormous numbers of genes encoding cytochrome P450 monooxygenases in plants, a universal naming system is crucial to group related CYPs and to facilitate functional predictions. Hence, CYPs from all kingdoms are systematically named according to their amino acid identity by the cytochrome P450 nomenclature committee (David Nelson: dnelson@uthsc.edu). CYPs are grouped into clans, families, and subfamilies. The broadest hierarchy level is represented by clans, which comprise one or multiple families. An example CYP name is CYP51G1; here, “CYP51” designates the family, “G” refers to the subfamily within the CYP51 family, and “1” represents the isoform of CYPs in this subfamily [14]. Typically, all CYPs in the same subfamily share more than 55% amino acid sequence identity, and all CYPs in the same family more than 40%, although exceptions exist [15,16]. These thresholds also underline the remarkable sequence variety of CYPs, as even enzymes with only 60–70% amino acid identity can display almost identical biochemical activity.

Enzymatic mechanism

As monooxygenases, CYPs catalyse the transfer of a single oxygen atom from molecular oxygen to their substrates (Figure 1A). Decades of research on CYPs led to detailed insights into their mechanistic properties based on a variety of biochemical, biophysical and computational methods [17-21]. Key for the oxidative chemistry performed by CYPs is a heme prosthetic group that activates molecular oxygen using electrons from an electron donor such as NADPH. A central Fe(III) ion is coordinated by the heme porphyrine system as well as a cysteine thiolate ligand from the protein backbone (Figure 1B). The generally accepted catalytic cycle for hydroxylations is shown in Figure 1C.

[1860-5397-18-135-1]

Figure 1: Enzyme function of cytochrome P450 monooxygenases (CYPs). A) Typical net reaction of CYPs, resulting in hydroxylation of a substrate. As monooxygenases, CYPs catalyse transfer of only one oxygen atom from molecular oxygen to their substrate. B) Heme prosthetic group containing the reactive Fe ion. Also shown is the abbreviated form of the cofactor used in the catalytic cycle. C) Catalytic cycle of CYPs. The key intermediates F, G, and H, called compound 0, compound I, and compound II, respectively, are highlighted. For details see main text. Figure 1 was created with BioRender.com. This content is not subject to CC BY 4.0.

In the resting state A, the central ferric ion is coordinated by six ligands, four from the porphyrin ring system, one cysteine thiolate group and an aqua ligand (water), resulting in an octahedral complex [17]. The oxidative reaction is initiated by displacement of the axial water molecule by the substrate (step 1), which pushes the Fe(III) ion out of the porphyrin plane in intermediate B [17]. This geometrical distortion promotes electron transfer from a reductase partner protein (step 2). The most common electron donors in plants are cytochrome P450 reductases which employ NADPH for the electron transfer, but several other electron transfer systems are known [22]. The reduced ferrous intermediate C, bearing an overall negative charge, can then efficiently bind molecular oxygen (step 3), leading to dioxygen adduct D. Transfer of an additional electron from a reducing partner such as cytochrome P450 reductase (step 4) generates peroxo intermediate E, which upon protonation (step 5) gives hydroperoxo intermediate F, also called compound 0. This nucleophilic and basic intermediate is prone to dehydration (step 6), leading to the strongly electrophilic and oxidising key intermediate G, which is commonly known as compound I (cpd I). Although there has been a lot of debate regarding the exact structure and electronic properties of compound I (intermediate G), it is now generally accepted as a ferryl (Fe(IV)) oxo species with a radical cation in the porphyrin system [18,23]. In the case of hydroxylations, the oxygen from compound I (intermediate G) can then be transferred by an oxygen rebound mechanism (steps 7 and 8) via the ferryl hydroxy intermediate H, also known as compound II. This leads to the hydroxylated product coordinated to ferric ion (intermediate I). Lastly, displacement of the product by water regenerates the resting state A (step 9). In addition to simple hydroxylations, slight deviations from this general mechanistic cycle can also lead to different reaction outcomes, e.g., rearrangements, desaturations or epoxidations. Multiple oxidation rounds, leading to aldehydes/ketones or carboxylic acids, are also commonly observed. Together, this versatile oxidative chemistry makes CYPs key enzymes in specialised metabolism in general [8,11], and crucial agents for the structural diversity of triterpenes.

Phylogenetic distribution of triterpenoid- and steroid-modifying plant cytochrome P450 monooxygenases

To assess the phylogenetic distribution of triterpenoid-modifying CYPs in comparison to other CYPs from plants, we collected a total of 149 CYPs from plants reported in the literature which are involved in triterpenoid or steroid metabolism (Table 1), and generated a neighbour-joining tree together with 266 non-triterpenoid CYPs with a different substrate scope (Figure 2). Notably, our analysis highlights that triterpenoid CYPs do not seem to occur randomly in various CYP clans and families; instead, certain clans and families represent “hotspots” of triterpenoid/steroid-modifying CYPs.

Table 1: List of characterised plant cytochrome P450 monooxygenases (CYPs) modifying triterpenoids or steroids.

Name Clan Family Species Accession number Scaffold Substrate Reaction Product Ref.
CYP51G1 51 51 Sorghum bicolor XM_0214610212.1 steroid obtusifoliol C14α demethylation 4α-methyl-
5α-ergosta-8,
14,24(28)-
trien-3β-ol 		[24]
CYP51G1 51 51 Arabidopsis thaliana AB014459 steroid obtusifoliol C14α demethylation 4α-methyl-
5α-ergosta-8,
14,24(28)-
trien-3β-ol 		[25]
CYP51H10 51 51 Avena strigosa DQ680852 pentacyclic oleanane β-amyrin C12–C13β epoxidation / C16 β hydroxlation 12,13-β-
epoxy-16-β-
hydroxy-
amyrin 		[1]
CYP51H14 51 51 Brachy-
podium distachyon 		ON108677 pentacyclic triterpene 19-hydroxy-
isoarborinol 		C7 and C28 hydroxylation 7,19,28-tri-
hydroxyiso-
arborinol 		[26]
CYP51H15 51 51 Brachy-
podium distachyon 		ON108678 pentacyclic triterpene isoarborinol C19 hydroxylation 19-hydroxy-
isoarborinol 		[26]
CYP51H16 51 51 Brachy-
podium distachyon 		ON108679 pentacyclic triterpene 7,19,28-tri-
hydroxyiso-
arborinol 		C1 hydroxylation 1,7,19,28-
tetrahydroxy-
isoarborinol 		[26]
CYP51H35 51 51 Triticum aestivum ON108669 pentacyclic triterpene isoarborinol C19 hydroxylation 19-hydroxy-
isoarborinol 		[26]
CYP51H37 51 51 Triticum aestivum ON108670 pentacyclic triterpene 19-hydroxy-
isoarborinol 		C25 hydroxylation and C2 oxidation ellarinacin [26]
CYP71A16 71 71 Arabidopsis thaliana NM_123623.5 monocyclic triterpene aldehyde marneral /
marnerol 		C23 hydroxylation 23-hydroxy-
marneral /
23-hydroxy-
marnerol 		[27,28]
CYP71BQ5 71 71 Melia azedarach MK803264.1 tirucallane triterpenoid dihydroniloticin C21 hydroxylation melianol [29]
CYP71CD2 71 71 Melia azedarach MK803271 tirucallane triterpenoid tirucalla-7,24-
dien-3β-ol 		C23 hydroxylation and C24–C25 epoxidation dihydro-
niloticin 		[29]
CYP71D353 71 71 Lotus japonicus KF460438 pentacyclic lupane dihydrolupeol /
20-hydroxy-
lupeol 		C20 hydroxylation / C28 oxidation 20-hydroxy-
lupeol /
20-hydroxy-
betulinic acid 		[30]
CYP71D443 71 71 Ajuga reptans LC066937 steroid 3β-hydroxy-5β-
cholestan-6-one 		C22 hydroxylation 3β,22R-
dihydroxy-5β-
cholestan-6-
one 		[31]
CYP81AQ19 71 81 Momordica charantia LC456843 tetracyclic triterpenoid cucurbitadienol C23α hydroxylation cucurbita-
5,24-dien-
3,23α-diol 		[32]
CYP81Q58 71 81 Cucumis sativus KM655856 tetracyclic triterpenoid 19-hydroxy-
cucurbitadienol 		C25 hydroxylation / double bond shift 19,25-
dihydroxy-
cucurbita-
dienol 		[33]
CYP81Q59 71 81 Cucumis melo Melo3C022375 tetracyclic triterpenoid 11-carbonyl-
20β-hydroxy-
cucurbitadienol 		C2β hydroxylation 11-carbonyl-
2β,20β-
dihydroxy-
cucurbita
dienol 		[34]
CYP82J17 71 82 Trigonella foenum-
graecum 		MK636709 steroid 16S-hydroxy-
22-oxo-
cholesterol 		C27 hydroxy-
lation / spiro-
ketalisation 		diosgenin [35]
CYP93A220 / IaAO5 71 93 Ilex asprella MZ508433 pentacyclic oleanane β-amyrin C24 oxidation α-boswellic acid [36]
CYP93E1 71 93 Glycine max AB231332 pentacyclic oleanane β-amyrin /
sophoradiol 		C24 hydroxylation 24-hydroxy-
β-amyrin /
soyasapo-
genol B 		[37]
CYP93E2 71 93 Medicago truncatula DQ335790 pentacyclic oleanane β-amyrin C24 hydroxylation 24-hydroxy-
β-amyrin 		[38]
CYP93E3 71 93 Glycyrrhiza uralensis AB437320 pentacyclic oleanane β-amyrin C24 hydroxylation 24-hydroxy-
β-amyrin 		[39]
CYP93E4 71 93 Arachis hypogaea KF906535 pentacyclic oleanane β-amyrin C24 hydroxylation 24-hydroxy-
β-amyrin 		[40]
CYP93E5 71 93 Cicer arietinum KF906536 pentacyclic oleanane β-amyrin C24 hydroxylation 24-hydroxy-
β-amyrin 		[40]
CYP93E6 71 93 Glycyrrhiza glabra KF906537 pentacyclic oleanane β-amyrin C24 hydroxylation 24-hydroxy-
β-amyrin 		[40]
CYP93E7 71 93 Lens culinaris KF906538 pentacyclic oleanane β-amyrin C24 hydroxylation 24-hydroxy-
β-amyrin 		[40]
CYP93E8 71 93 Pisum sativum KF906539 pentacyclic oleanane β-amyrin C24 hydroxylation 24-hydroxy-
β-amyrin 		[40]
CYP93E9 71 93 Phaseolus vulgaris KF906540 pentacyclic oleanane β-amyrin C24 hydroxylation 24-hydroxy-
β-amyrin 		[40]
CYP705A1 71 705 Arabidopsis thaliana NM_001341032.1 tricyclic triterpenoid arabidiol C15–C16 cleavage 14-apo-
arabidiol 		[28]
CYP705A5 71 705 Arabidopsis thaliana NM_124173.3 tricyclic triterpenoid 7β-hydroxy-
thalianol 		C15–C16 desaturation desaturated
(C15–C16)
7β-hydroxy-
thalianol 		[41]
CYP712K1 71 712 Tripterygium wilfordii MN621243 pentacyclic triterpenoid friedelin C29 oxidation polpunonic
acid and
29-hydroxy-
friedelin 		[42]
CYP712K2 71 712 Tripterygium wilfordii MN621244 pentacyclic triterpenoid friedelin C29 oxidation polpunonic
acid and
29-hydroxy-
friedelin 		[42]
CYP712K3 71 712 Tripterygium wilfordii MN621245 pentacyclic triterpenoid friedelin C29 oxidation polpunonic
acid and
29-hydroxy-
friedelin 		[42]
CYP712K4 71 712 Maytenus ilicifolia MK829814 pentacyclic triterpenoid friedelin C29 oxidation polpunonic
acid or
maytenoic
acid 		[43]
CYP72A61 72 72 Medicago truncatula DQ335793 pentacyclic oleanane 24-hydroxy-
β-amyrin 		C22 hydroxylation soyasapo-
genol B 		[44]
CYP72A61v2 72 72 Medicago truncatula XM_003605422 pentacyclic oleanane 24-hydroxy-
β-amyrin 		C22 hydroxylation soyasapo-
genol B 		[44]
CYP72A62v2 72 72 Medicago truncatula AB558147 pentacyclic oleanane β-amyrin C29 oxidation 29-hydroxy-
β-amyrin /
epi-katonic
acid 		[45]
CYP72A63 72 72 Medicago truncatula AB558146 pentacyclic oleanane β-amyrin C30 oxidation 11-deoxy-
glycyrrhen-
tinic acid 		[46]
CYP72A64v2 72 72 Medicago truncatula MK534548 pentacyclic oleanane β-amyrin C29 oxidation 29-hydroxy-
β-amyrin /
epi-katonic
acid 		[45]
CYP72A65v2 72 72 Medicago truncatula XM_003628012.4 pentacyclic oleanane β-amyrin C21 hydroxylation 21-hydroxy-
β-amyrin 		[45]
CYP72A67 72 72 Medicago truncatula DQ335780 pentacyclic oleanane oleanolic acid /
hederagenin /
gypsogenic acid
/ gypsogenin 		C2β hydroxylation 2β-hydroxy-
oleanolic acid
/ bayogenin /
medicagenic
acid / 2β,3β-
dihydroxy-
olean-12-en-
23-oxo-28-
oic acid 		[47,48]
CYP72A68 72 72 Medicago truncatula DQ335782 pentacyclic oleanane oleanolic acid /
hederagenin /
gypsogenin 		C23 oxidation hederagenin
/ gypsogenin
/ gypsogenic
acid 		[48]
CYP72A68v2 72 72 Medicago truncatula XM_013608494.3 pentacyclic oleanane oleanolic acid /
hederagenin /
gypsogenin 		C23 oxidation hedera-
genin /
gypsogenin /
gypsogenic acid 		[44]
CYP72A69 72 72 Glycine max LC143440 pentacyclic oleanane soyasapogenol B C21 hydroxylation soyasapo-
genol A 		[49]
CYP72A141 72 72 Glycine max MK534532 pentacyclic oleanane β-amyrin C29 hydroxylation 29-hydroxy-
β-amyrin 		[45]
CYP72A154 72 72 Glycyrrhiza uralensis AB558153 pentacyclic oleanane β-amyrin /
11-oxo-β-amyrin 		C30 oxidation 30-hydroxy-
β-amyrin /
glycyrrhetinic acid 		[46]
CYP72A302 72 72 Phaseolus vulgaris MK534537 pentacyclic oleanane β-amyrin C29 hydroxylation 29-hydroxy-
β-amyrin 		[45]
CYP72A397 72 72 Kalopanax septemlobus KT150517 pentacyclic oleanane oleanolic acid C23 oxidation hederagenin [50]
CYP72A552 72 72 Barbarea vulgaris MH252571 pentacyclic oleanane oleanolic acid C23 oxidation hederagenin [51]
CYP72A557 72 72 Medicago truncatula MK534544 pentacyclic oleanane β-amyrin C21 hydroxylation 21-hydroxy-
β-amyrin 		[45]
CYP72A558 72 72 Medicago truncatula MK534545 pentacyclic oleanane β-amyrin C21 hydroxylation 21-hydroxy-
β-amyrin 		[45]
CYP72A559 72 72 Medicago truncatula MK534546 pentacyclic oleanane β-amyrin C21 hydroxylation 21-hydroxy-
β-amyrin 		[45]
CYP72A613 72 72 Trigonella foenum-
graecum 		MK636708 steroid 16S-hydroxy-
22-oxo-
cholesterol 		C27 hydroxylation / spiro-
ketalisation 		diosgenin [35]
CYP72A616 72 72 Paris polyphylla MK636705 steroid 16S-hydroxy-
22-oxo-
cholesterol 		C27 hydroxy-
lation / spiro-
ketalisation 		diosgenin [35]
CYP72A694 72 72 Vigna angularis MK534538 pentacyclic oleanane β-amyrin /
29-hydroxy-
β-amyrin 		C29 oxidation 29-hydroxy-
β-amyrin /
epi-katonic
acid 		[45]
CYP72A697 72 72 Lotus japonicus MK534539 pentacyclic oleanane β-amyrin C29 hydroxylation 29-hydroxy-
β-amyrin 		[45]
CYP72A699 72 72 Trifolium pratense MK534549 pentacyclic oleanane β-amyrin /
29-hydroxy-
β-amyrin 		C29 oxidation 29-hydroxy-
ß-amyrin /
epi-katonic
acid 		[45]
CYP714E19 72 714 Centella asiatica KT004520 pentacyclic oleanane / ursane oleanolic acid /
ursolic acid 		C23 oxidation hederagenin
/ 23-
hydroxy-
ursolic acid 		[52]
CYP714E88 / IaAO4 72 714 Ilex asprella MZ508437 pentacyclic oleanane / ursane ursolic acid /
oleanolic acid 		C23 oxidation 23-carboxyl-
ursolic acid /
gypsogenic
acid 		[36]
CYP734A7 72 734 Solanum lycoper-
sicum 		AB223041 steroid castasterone /
28-nor-
castasterone /
brassinolide 		C26 hydroxylation 26-hydroxy-
castaster-
one /
26-hydroxy-
norcastaster-
one / 26-hydroxy-
brassinolide 		[53]
CYP749A63 72 749 Crataegus pinnatifida MF596155 pentacyclic oleanane oleanolic acid C24 hydroxylation 4-epi-
hederagenin 		[54]
CYP85A1 85 85 Arabidopsis thaliana AB035868 steroid 6-deoxoteaster-
one / 3-dehydro-
6-deoxoteaster-
one / 6-deoxo-
typhasterol /
6-deoxo-
castasterone 		C6 oxidation teasterone /
3-dehydro-
teasterone /
typhasterol /
castasterone 		[55]
CYP85A1 85 85 Solanum lycoper-
sicum 		U54770 steroid 6-deoxoteaster-
one / 3-dehydro-
6-deoxoteaster-
one / 6-deoxo-
typhasterol /
6-deoxo-
castasterone 		C6 oxidation teasterone /
3-dehydro-
teasterone /
typhasterol /
castasterone 		[56]
CYP85A2 85 85 Arabidopsis thaliana AB087801 steroid castasterone /
6-deoxo-
castaster-
one / 6-deoxo-
typhasterol /
3-dehydro-6-
deoxo-
teaster-
one 		Baeyer-
Villiger oxidation / C6 oxidation 		brassinolide /
castaster-
one /
typhasterol /
3-dehydro-
teasterone 		[57,58]
CYP85A3 85 85 Solanum lycoper-
sicum 		AB190445 steroid 6-deoxocastas-
terone /
castasterone 		Baeyer-
Villiger oxidation / C6 oxidation 		castasterone
/ brassinolide 		[58]
CYP87D16 85 87 Maesa lanceolata KF318735 pentacyclic oleanane β-amyrin C16α hydroxylation 16α-hydroxy-
β-amyrin 		[59]
CYP87D18 85 87 Siraitia grosvenorii HQ128570 tetracyclic triterpenoid cucurbitadienol /
11α-hydroxy-
cucurbitadienol/
24,25-di-
hydroxy-
cucurbitadienol 		C11 oxidation 11α-hydroxy-
cucurbita-
dienol /
11-oxo-
cucurbita-
dienol /
mogrol 		[34]
CYP87D20 85 87 Cucumis sativus Csa1G044890 tetracyclic triterpenoid cucurbitadienol /
11-oxo-cucur-
bitadienol 		C11 oxidation / C20β hydroxylation 11-oxocucur-
bitadienol /
11-carbonyl-
20β-hydroxy-
cucurbita-
dienol 		[34]
CYP88D6 85 88 Glycyrrhiza uralensis AB433179 pentacyclic oleanane β-amyrin C11 oxidation 11-oxo-β-
amyrin 		[39]
CYP88L2 85 88 Cucumis sativus Csa3G903540 tetracyclic triterpenoid cucurbitadienol /
11-oxo-cucur-
bitadienol 		C19 hydroxylation 19-hydroxy-
cucurbita-
dienol 		[34]
CYP88L7 85 88 Momordica charantia LC456844 tetracyclic triterpenoid cucurbitadienol C19 hydroxylation, C5 and C19 ether bridge cucurbita-
5,24-dien-
3β,19-diol
and 5β,19-
epoxy-
cucurbita-
6,24-dien-
3β-ol 		[32]
CYP88L8 85 88 Momordica charantia LC456845 tetracyclic triterpenoid cucurbitadienol C7β hydroxylation cucurbita-
5,24-dien-
3β,7β-diol 		[32]
CYP90A1 85 90 Arabidopsis thaliana X87367 steroid 6-deoxocat-
hasterone /
6-deoxoteaster-
one / 22S-hydroxy-
campesterol /
22R,23R-di-
hydroxycam-
pesterol 		C3 oxidation 22S-hydroxy-
5α-campes-
tan-3-one /
3-dehydro-6-
deoxo-
teasterone/
22S-hydroxy-
campest-4-
en-3-one /
22R,23R-
dihydroxy-
campest-4-
en-3-one 		[60]
CYP90B1 85 90 Arabidopsis thaliana NM_114926.4 steroid campesterol /
24R-ergost-4-
en-3-one /
24R-5α-ergos-
tan-3-one /
campestanol /
6-oxocampes-
tanol 		C22 hydroxylation 22S-hydroxy-
campesterol /
22S-hydroxy-
24R-ergost-
4-en-3-one /
22S-hydroxy-
24R- 5α-
ergostan-3-
one / 6-
deoxocat-
hasterone /
cathasterone 		[61]
CYP90B2 85 90 Oryza sativa AB206579 steroid campesterol /
campestanol 		C22 hydroxylation 22S-hydroxy-
campesterol /
6-deoxo-
cathasterone 		[62]
CYP90B3 85 90 Solanum lycoper-
sicum 		NM_001279330.2 steroid campesterol /
24R-ergost-4-
en-3-one /
24R-5α-ergos-
tan-3-one /
campestanol 		C22 hydroxylation 22-hydroxy-
campesterol /
22S-hydroxy-
24R-ergost-
4-en-3-one /
22S-hydroxy-
24R-5α-
ergostan-3-
one / 6-deoxo-
cathasterone 		[63]
CYP90B27 85 90 Veratrum californicum KJ869252 steroid cholesterol /
26-hydroxy-
cholesterol /
7ß-hydroxy-
cholesterol 		C22 hydroxylation 22R-hydroxy-
cholesterol /
22,26-di-
hydroxy-
cholesterol /
7ß,22-di-
hydroxy-
cholesterol 		[64]
CYP90B27 85 90 Paris polyphylla KX904822 steroid cholesterol C22 hydroxylation 22R-hydroxy-
cholesterol 		[65]
CYP90B50 85 90 Trigonella foenum-
graecum 		MK636707 steroid cholesterol C22R, C16 dihydroxy-
lation 		16S,22R-
dihydroxy-
cholesterol 		[35]
CYP90B51 85 90 Trigonella foenum-
graecum 		MK636706 steroid cholesterol C22S hydroxylation / C22R hydroxylation 22S-hydroxy-
cholesterol /
22R-hydroxy-
cholesterol 		[66]
CYP90B52 85 90 Paris polyphylla MK636701 steroid cholesterol C22S hydroxylation 22S-hydroxy-
cholesterol 		[35]
CYP90B71 85 90 Dioscorea
zingi-
berensis 		MN829441 steroid cholesterol C22R hydroxylation 22R-hydroxy-
cholesterol 		[66]
CYP90C1 85 90 Arabidopsis thaliana NM_001342408.1 steroid 22S-hydroxy-
24R-5α-ergost-
an-3-one /
3-epi-6-
deoxocat-
hasterone 		C23 hydroxylation 3-dehydro-6-
deoxoteas-
terone /
6-deoxo-
typhasterol 		[67]
CYP90D1 85 90 Arabidopsis thaliana NM_112223 steroid 22S-hydroxy-
24R-5α-ergost-
an-3-one /
3-epi-6-
deoxocat-
hasterone 		C23 hydroxylation 3-dehydro-6-deoxoteas-
terone /
6-deoxo-
typhasterol 		[67]
CYP90D2 85 90 Oryza sativa NM_001409071 steroid 22S-hydroxy-
24R-5α-ergost-
an-3-one /
3-epi-6-
deoxocat-
hasterone 		C23 hydroxylation 3-dehydro-6-
deoxoteas-
terone /
6-deoxo-
typhasterol 		[68]
CYP90D3 85 90 Oryza sativa AAT44310 steroid 22S-hydroxy-
24R-5α-ergost-
an-3-one /
3-epi-6-
deoxocat-
hasterone 		C23 hydroxylation 3-dehydro-6-
deoxoteas-
terone /
6-deoxo-
typhasterol 		[68]
CYP90G1v1 85 90 Veratrum californicum KJ869258 steroid 22R-hydroxy-
cholesterol /
22,26-di-
hydroxycholes-
terol /
22-hydroxy-26-
aminocholes-
terol 		C22 hydroxylation 22-keto-
cholesterol /
22-keto-26-
hydroxy-
cholesterol /
verazine 		[64]
CYP90G1v2 85 90 Veratrum californicum KJ869261 steroid 22R-hydroxy-
cholesterol /
22,26-di-
hydroxycholes-
terol /
22-hydroxy-26-
aminocholes-
terol 		C22 hydroxylation 22-keto-
cholesterol /
22-keto-26-
hydroxy-
cholesterol /
verazine 		[64]
CYP90G1v3 85 90 Veratrum californicum KJ869260 steroid 22R-hydroxy-
cholesterol /
22,26-di-
hydroxycholes-
terol /
22-hydroxy-26-
aminocholes-
terol 		C22 hydroxylation 22-keto-
cholesterol /
22-keto-26-
hydroxy-
cholesterol /
verazine 		[64]
CYP90G4 85 90 Paris polyphylla MK636702 steroid 22R-hydroxy-
cholesterol 		C16 oxidation 16S,22R-
dihydroxy-
cholesterol 		[66]
CYP90G6 85 90 Dioscorea
zingi-
berensis 		MN829442 steroid 22R-hydroxy-
cholesterol 		C16 oxidation 16S,22R-
dihydroxy-
cholesterol 		[66]
CYP708A15 85 708 Iberis amara MW514550 tetracyclic triterpenoid 16β-hydroxy-
cucurbitadienol 		C22 hydroxylation 16β,22-
dihydroxy-
cucurbita-
dienol 		[69]
CYP708A15v2 85 708 Iberis amara MW514551 tetracyclic triterpenoid 16β-hydroxy-
cucurbitadienol 		C22 hydroxylation 16β,22-
dihydroxy-
cucurbita-
dienol 		[69]
CYP708A16 85 708 Iberis amara MW514556 tetracyclic triterpenoid cucurbitadienol C16 hydroxylation 16β-hydroxy-
cucurbita-
dienol 		[69]
CYP708A2 85 708 Arabidopsis thaliana NM_001344756.1 tricyclic triterpenoid thalianol C7β hydroxylation 7β-hydroxy-
thalianol 		[41]
CYP716A1 85 716 Arabidopsis thaliana NM_123002.2 pentacyclic ursane / oleanane / lupane α-amyrin /
β-amyrin /
lupeol 		C28 oxidation ursolic acid /
oleanolic
acid /
betulin 		[70]
CYP716A2 85 716 Arabidopsis thaliana LC106013.1 pentacyclic ursane / oleanane / lupane α-amyrin /
β-amyrin /
lupeol 		C16/C22α/
C28 hydroxylation 		uvaol /
C22α-
hydroxy-β-
amyrin /
erythrodiol /
betulin 		[70]
CYP716A12 85 716 Medicago truncatula FN995113 pentacyclic ursane / oleanane / lupane α-amyrin /
β-amyrin /
betulin 		C28 oxidation ursolic acid /
oleanolic
acid /
betulinic acid 		[71]
CYP716A14v2 85 716 Artemisia annua KF309251 pentacyclic ursane / oleanane α-amyrin /
β-amyrin 		C3 oxidation α-amyrone /
β-amyrone 		[72]
CYP716A15 85 716 Vitis vinifera AB619802 pentacyclic ursane / oleanane / lupane α-amyrin /
β-amyrin /
betulin 		C28 oxidation ursolic acid /
oleanolic
acid /
betulinic acid 		[73]
CYP716A17 85 716 Vitis vinifera AB619803 pentacyclic oleanane β-amyrin C28 oxidation oleanolic acid [73]
CYP716A44 85 716 Solanum
lycoper-
sicum 		XM_004239248.4 pentacyclic ursane / oleanane α-amyrin /
β-amyrin 		C28 oxidation ursolic acid /
oleanolic acid 		[74]
CYP716A46 85 716 Solanum
lycoper-
sicum 		XM_004243858 pentacyclic ursane / oleanane α-amyrin /
β-amyrin 		C28 oxidation ursolic acid /
oleanolic acid 		[74]
CYP716A51 85 716 Lotus japonicus AB706297 pentacyclic ursane / oleanane / lupane α-amyrin /
β-amyrin /
lupeol 		C28 oxidation ursolic acid /
oleanolic
acid /
betulinic acid 		[75]
CYP716A52v2 85 716 Panax ginseng JX036032 pentacyclic oleanane β-amyrin C28 oxidation oleanolic acid [76]
CYP716A75 85 716 Maesa lanceolata KF318733 pentacyclic oleanane β-amyrin C28 oxidation oleanolic acid [59]
CYP716A78 85 716 Cheno-
podium
quinoa 		KX343075 pentacyclic oleanane β-amyrin C28 oxidation oleanolic acid [77]
CYP716A79 85 716 Cheno-
podium
quinoa 		KX343076 pentacyclic oleanane β-amyrin C28 oxidation oleanolic acid [77]
CYP716A80 85 716 Barbarea vulgaris KP795926 pentacyclic ursane / oleanane / lupane α-amyrin /
β-amyrin /
lupeol 		C28 oxidation ursolic acid /
oleanolic
acid /
betulinic acid 		[78]
CYP716A81 85 716 Barbarea vulgaris KP795925 pentacyclic ursane / oleanane / lupane α-amyrin /
β-amyrin /
lupeol 		C28 oxidation ursolic acid /
oleanolic
acid /
betulinic acid 		[78]
CYP716A83 85 716 Centella asiatica KU878849 pentacyclic ursane / oleanane α-amyrin /
β-amyrin 		C28 oxidation ursolic acid /
oleanolic acid 		[79]
CYP716A86 85 716 Centella asiatica KU878848 pentacyclic oleanane β-amyrin C28 oxidation oleanolic acid [79]
CYP716A94 85 716 Kalopanax septemlobus KT150521 pentacyclic oleanane β-amyrin C28 oxidation oleanolic acid [50]
CYP716A110 85 716 Aquilegia coerulea KU878864 pentacyclic oleanane β-amyrin C28 oxidation oleanolic acid [79]
CYP716A111 85 716 Aquilegia coerulea KY047600 pentacyclic oleanane β-amyrin C16β hydroxylation 16β-hydroxy-
β-amyrin 		[79]
CYP716A113 85 716 Aquilegia coerulea KU878866 tetracyclic triterpenoid cycloartenol unknown regio-
selectivity 		hydroxy-
cyclo-
artenol,
performs non-
specific reaction of endogenous
yeast
compounds 		[79]
CYP716A140 85 716 Platycodon grandiflorus KU878853 pentacyclic oleanane / ursane β-amyrin /
16β-hydroxy-
β-amyrin /
12,13α-epoxy-
β-amyrin 		C28 oxidation oleanolic
acid /
16β-hydroxy-
oleanolic acid 		[79]
CYP716A140v2 85 716 Platycodon grandiflorus LC209199 pentacyclic oleanane β-amyrin C28 oxidation oleanolic acid [80]
CYP716A141 85 716 Platycodon grandiflorus KU878855 pentacyclic oleanane β-amyrin /
oleanolic acid 		C28 oxidation / C16β hydroxylation oleanolic
acid /
16β-hydroxy-
oleanolic acid 		[79,80]
CYP716A154 85 716 Catharan-
thus roseus 		JN565975 pentacyclic ursane / oleanane / lupane α-amyrin /
β-amyrin /
betulin 		C28 oxidation ursolic acid /
oleanolic
acid /
betulinic acid 		[81]
CYP716A155 85 716 Rosmarinus officinalis MK592859 pentacyclic lupane lupeol C28 oxidation betulinic acid [82]
CYP716A175 85 716 Malus domestica XM_008392874 pentacyclic ursane / oleanane / lupane α-amyrin /
β-amyrin /
lupeol /
germanicol 		C28 oxidation ursolic acid /
oleanolic
acid /
betulinic
acid /
morolic acid 		[83]
CYP716A179 85 716 Glycyrrhiza uralensis LC157867 pentacyclic ursane / oleanane / lupane α-amyrin /
β-amyrin /
betulin 		C28 oxidation / C22α hydroxylation ursolic acid /
C22α-
hydroxy-
amyrin /
oleanolic
aicd /
betulinic acid 		[84]
CYP716A180 85 716 Betula platyphylla KJ452328 pentacyclic lupane lupeol C28 oxidation betulinic acid [85]
CYP716A210 / IaAO1 85 716 Ilex asprella MK994507 pentacyclic ursane / oleanane α-amyrin /
β-amyrin 		C28 oxidation ursolic acid /
oleanolic acid 		[86]
CYP716A244 85 716 Eleuthero-
coccus
senticosus 		KX354739 pentacyclic oleanane β-amyrin C28 oxidation oleanolic acid [87]
CYP716A252 85 716 Ocimum basilicum JQ958967 pentacyclic ursane / oleanane α-amyrin /
β-amyrin 		C28 oxidation ursolic acid /
oleanolic acid 		[88]
CYP716A253 85 716 Ocimum basilicum JQ958968 pentacyclic ursane / oleanane α-amyrin /
β-amyrin 		C28 oxidation ursolic acid /
oleanolic acid 		[88]
CYP716A265 85 716 Lager-
stroemia
speciosa 		MG708187 pentacyclic ursane / oleanane / lupane α-amyrin /
β-amyrin /
lupeol 		C28 oxidation ursolic acid /
oleanolic
acid /
betulinic acid 		[89]
CYP716A266 85 716 Lager-
stroemia
speciosa 		MG708188 pentacyclic ursane / oleanane / lupane α-amyrin /
β-amyrin /
lupeol 		C28 oxidation ursolic acid /
oleanolic
acid /
betulinic acid 		[89]
CYP716C11 85 716 Centella asiatica KU878852 pentacyclic oleanane / ursane oleanolic acid /
ursolic acid /
6β-hydroxy-
oleanolic acid 		C2α hydroxylation maslinic
acid /
2α-hydroxy-
ursolic acid /
6β-hydroxy-
maslinic acid 		[79]
CYP716C49 85 716 Crataegus pinnatifida MF120282 pentacyclic oleanane / ursane / lupane oleanolic acid /
ursolic acid /
betulinic acid 		C2α hydroxylation maslinic
acid /
corosolic
acid /
alphitolic acid 		[54]
CYP716C55 85 716 Lager-
stroemia speciosa 		MG708191 pentacyclic ursane / oleanane ursolic acid /
oleanolic acid 		C2α hydroxylation corosolic
acid /
maslinic acid 		[89]
CYP716E26 85 716 Solanum lycoper-
sicum 		XM_004241773 pentacyclic ursane / oleanane α-amyrin /
β-amyrin 		C6β hydroxylation 6β-hydroxy-
α-amyrin /
daturadiol 		[74]
CYP716E41 85 716 Centella asiatica KU878851 pentacyclic oleanane / ursane oleanolic acid /
ursolic acid /
maslinic acid 		C6β hydroxylation 6β-hydroxy-
oleanolic
acid /
6β-hydroxy-
ursolic acid /
6β-hydroxy-
maslinic acid 		[79]
CYP716S1 85 716 Panax ginseng JX036031 tetracyclic triterpene protopanaxadiol C6 hydroxylation protopanaxa-
triol 		[76]
CYP716S5 85 716 Platycodon grandiflorus KU878856 pentacyclic oleanane β-amyrin /
oleanolic acid 		C12-C13α epoxidation C12-C13α-
epoxy-β-
amyrin /
C12-C13α-
epoxy-
oleanolic acid 		[79]
CYP716U1 85 716 Panax ginseng JN604536 tetracyclic triterpene dammarene-
diol-II 		C12 hydroxylation protopanaxa-
diol 		[76]
CYP716Y1 85 716 Bupleurum falcatum KC963423 pentacyclic ursane / oleanane α-amyrin /
β-amyrin 		C16α hydroxylation 16α-hydroxy-
α-amyrin /
16α-hydroxy-
β-amyrin 		[38]
IaAO2 85 716 Ilex asprella OL604227 pentacyclic ursane / oleanane α-amyrin /
β-amyrin 		C28 oxidation ursolic acid /
oleanolic acid 		[36]
CYP724A1 85 724 Arabidopsis thaliana NM_001343334.1 steroid possibly brassinosteroids C22 hydroxylation   [90]
CYP724B1 85 724 Oryza sativa AB158759 steroid campesterol /
campestanol 		C22 hydroxylation 22S-hydroxy-
campesterol /
6-deoxo-
cathasterone 		[62]
CYP724B2 85 724 Solanum
lycoper-
sicum 		XM_004243170 steroid campesterol /
24R-ergost-4-
en-3-one /
24R-5α-ergos-
tan-3-one /
campestanol 		C22 hydroxylation 22-hydroxy-
campesterol /
22S-hydroxy-
24R-ergost-
4-en-3-one /
22S-hydroxy-
24R-5α-
ergostan-
3-one /
6-deoxo-
cathasterone 		[63]
CYP94D108 86 94 Paris polyphylla MK636703 steroid 16S-hydroxy-
22-oxo-
cholesterol 		C27 hydroxy-
lation / spiro-
ketalisation 		diosgenin [35]
CYP94D109 86 94 Paris polyphylla MK636704 steroid 16S-hydroxy-
22-oxo-
cholesterol 		C27 hydroxy-
lation / spiro-
ketalisation 		diosgenin [35]
CYP94N1 86 94 Veratrum californicum KJ869255 steroid 22R-hydroxy-
cholesterol 		C26 hydroxylation 22,26-di-
hydroxy-
cholesterol and
22-hydroxy-
cholesterol-
26-al 		[64]
CYP710A1 710 710 Arabidopsis thaliana AB219423 steroid β-sitosterol C22 desaturation stigmasterol [91]
CYP710A2 710 710 Arabidopsis thaliana AB233425 steroid β-sitosterol /
24-epi-
campesterol 		C22 desaturation stigmasterol/
brassicaster-
ol 		[91]
CYP710A4 710 710 Arabidopsis thaliana NM_128444.2 steroid β-sitosterol C22 desaturation stigmasterol [91]
CYP710A11 710 710 Solanum
lycoper-
sicum 		NM_001247585.2 steroid β-sitosterol C22 desaturation stigmasterol [91]
[1860-5397-18-135-2]

Figure 2: Phylogenetic distribution of CYPs acting on triterpenoid and steroid scaffolds (red nodes) compared to other CYPs from higher plants [15]. Numbers and black bars mark CYP families containing CYPs known to act on triterpenoids and steroids. Amino acid sequences (149 triterpenoid CYPs, 266 non-triterpenoid CYPs) were aligned using MUSCLE [92], and a neighbour-joining consensus tree of 1,000 bootstrap replicates was generated using the Jukes–Cantor model. The final tree was visualised in Python using the ete3 package. Annotations in Figure 2 were created with BioRender.com. This content is not subject to CC BY 4.0. A high resolution version with tip labels is available as Supporting Information File 1.

Probably the most well-known example of a triterpenoid-biased CYP family are the CYP716s (part of the CYP85 clan) [79], but also other families of the CYP85 clan such as CYP87, CYP85, or CYP90 contain mostly triterpene-modifying CYPs to date. The small clans CYP51 and CYP710 are other important examples of groups with a high preference for triterpenoid substrates. The highly diverse CYP71 clan, in contrast, only contains a few triterpene-modifying CYPs, particularly in the families CYP93, CYP712 and CYP705. The CYP72 family (CYP72 clan) also contains several known representatives. In other clans, however, not a single triterpene-modifying CYP has been identified so far, for example CYP97, CYP74, or CYP711.

The discovery of biosynthetic genes in plants often involves the screening of large pools of gene candidates derived from sequencing studies [93-96]. Hence, efficient approaches are needed to select the most promising gene candidates, particularly for large gene families such as CYPs. Our summarised phylogenetic distribution of known triterpenoid-modifying CYPs therefore might facilitate the discovery of new CYPs in triterpenoid and steroid pathways in plants by highlighting CYP families with a known propensity to participate in these pathways.

Major reaction types of triterpenoid- and steroid-modifying CYPs

The basic polycyclic skeletons of triterpenoids and steroids are created by oxidosqualene cyclases (OSCs) from the universal substrate 2,3-oxidosqualene [5]. As different folding modes (chair–boat–chair vs chair–chair–chair) and different ring sizes can occur during this cyclisation cascade, resulting triterpene and sterol scaffolds have drastically different three-dimensional shapes. For this reason, CYPs are typically specific to a certain group of triterpenoid scaffolds. Hence, we summarised our list of 149 triterpenoid/steroid CYPs (Table 1) according to their target scaffold.

Figure 3 covers plant CYPs acting on steroid, cucurbitacin, or simple tetracyclic triterpenoid scaffolds. Important scaffolds here are campesterol (1), β-sitosterol (2), cholesterol (3), cucurbitadienol (4), and dammarenediol-II (5). Not surprisingly, CYPs involved in the biosynthesis of essential sterols in plants are highly conserved and play a crucial role in their growth and development. For example, members of the CYP710A subfamily were characterised as C22 desaturases in Arabidopsis and tomato [91,97]. Three CYPs, CYP710A1, CYP710A2 and CYP710A4 were identified in Arabidopsis and CYP710A11 was identified in tomato. All four CYPs could produce stigmasterol from β-sitosterol (2) in enzyme assays performed in vitro. However, Arabidopsis CYP710A2 showed substrate flexibility towards campesterol (1) epimers and could also produce brassicasterol from 24-epicampesterol in vitro. Enzymes of the CYP51G subfamily (CYP51 clan) function as sterol 14α-demethylases in green plants [24,25,98]. These enzymes catalyse oxidation of the C14α methyl group to trigger elimination of formic acid [24,25]. The sister subfamily CYP51H, on the other hand, is only found in monocots. AsCYP51H10 from Avena sativa (oat) is a multifunctional CYP that performs hydroxylation and epoxidation reactions of the β-amyrin (6) scaffold to produce 12,13β-epoxy-16β-hydroxy-β-amyrin [1,99]. Thus, CYP51H10 is an example of a neofunctionalised CYP recruited from primary sterol metabolism.

[1860-5397-18-135-3]

Figure 3: CYPs modifying steroid (A), cucurbitacin steroid (B) and tetracyclic triterpene (C) backbones. Substructures in grey indicate regions where major structural differences occur between different substrates of the same class. Representative substrate skeletons (not exact substrates) are shown in the dotted boxes. For exact substrate specificity see Table 1.

Two members of the CYP87D subfamily decorate the tetracyclic scaffold in plants from the Cucurbitaceae family (Figure 3B). CYP87D18 (CYP85 clan) was identified as a multifunctional C11 oxidase involved in the biosynthetic pathway of mogrosides. Mogrosides, isolated from ripe fruits of Siraitia grosvenorii (Cucurbitaceae) are glycosylated triterpenoid saponins with rare C24 and C25 hydroxylation [100]. Based on feeding assays in yeast it was found that CYP87D18 catalyses a two-step sequential C11 oxidation of cucurbitadienol (4) to 11-hydroxycucurbitadienol and 11-oxo-cucurbitadienol [101]. CYP87D18 also catalysed C11 hydroxylation of trans-24,25-dihydroxycucurbitadienol to form trihydroxylated mogrol in yeast [102].

CYPs acting on pentacyclic 6-6-6-6-6 triterpenes, which include the extremely important and widespread scaffolds β-amyrin (6), α-amyrin (7), and friedelin (8), are summarised in Figure 4. Of particular relevance in this area is the CYP716 family, which plays a central role in the diversification of triterpenoids in eudicots [79]. Members of the CYP716A subfamily were mostly identified as C28 oxidases that catalyse three-step oxidation of α-amyrin (7), β-amyrin (6) and lupeol (10) to ursolic acid, oleanolic acid, and betulinic acid, respectively [71,73]. Nonetheless, other CYP716 enzymes have evolved to perform a wider range of modifications of triterpenoids; several CYP716 enzymes were found to catalyse C3 oxidation of α-amyrin (7) and β-amyrin (6), C16α oxidation of β-amyrin (6), or C22α oxidation of α-amyrin (7) [40,72,79]. Some members even act on triterpenoid scaffolds other than the 6-6-6-6-6 pentacyclic triterpenes. For example, two CYP716 enzymes from Panax ginseng act on tetracyclic scaffolds; CYP716U1 hydroxylates dammarenediol-II (5) to protopanaxadiol, and CYP716S1 hydroxylates the C6 of protopanaxadiol to form protopanaxatriol [76,103]. CYP716A113v1 from Aquilegia coerulea hydroxylates cycloartenol with unknown regiospecificity when expressed in a yeast strain harbouring a tomato cycloartenol synthase gene [79].

[1860-5397-18-135-4]

Figure 4: CYPs modifying pentacyclic 6-6-6-6-6 triterpenes. Substructures in grey indicate regions where major structural differences occur between different substrates of the same class. Representative substrate skeletons (not exact substrates) are shown in the dotted boxes. For exact substrate specificity see Table 1.

CYP712 family members (clan 71) were first identified in the biosynthetic pathway of nor-triterpenoid celastrol, a potent anti-obesity metabolite [42,43]. In two independent studies, transcriptome mining and functional studies in Nicotiana benthamiana were used to identify the CYPs CYP712K1, CYP712K2, CYP712K3, and CYP712K4 capable of oxidising friedelin (8) into polpunonic acid via an aldehyde intermediate [42,43].

Members of the CYP93E subfamily are restricted to legumes and are involved in the biosynthesis of triterpenoid saponins. So far, nine CYP93E members were identified from different legume species [37,40]. All of these perform C24 hydroxylation of β-amyrin (6) to form 24-hydroxy-β-amyrin. CYP93E1 also catalyses the conversion of sophoradiol to soyasapogenol B [37,40,46]. Members of other CYP93 subfamilies (CYP93A, B, C and G) are ubiquitous in flowering plants and are mostly involved in flavonoid biosynthesis [25,26].

Lastly, CYPs acting on either pentacyclic 6-6-6-6-5 scaffolds, such as isoarborinol (9) or lupeol (10), or on unusual triterpene scaffolds such as arabidiol (11) or thalianol (12) are grouped in Figure 5. Enzymes from the CYP705 and CYP708 family catalyse Brassicaceae-specific reactions. The corresponding genes were found in operon-like gene clusters and catalyse the modification of monocyclic marnerol and tricyclic thalianol (12) in Arabidopsis [27,41]. Marneral synthase (MRN1) produces two oxidation products, one is marneral (aldehyde) and the other marnerol (alcohol). Arabidopsis CYP71A16 hydroxylates the allylic methyl side-chain of monocyclic marneral/marnerol to 23-hydroxymarneral/23-hydroxymarnerol. Modification of thalianol (12) involves CYPs from two clans. Genes encoding CYP708A2 (clan 85) and CYP705A5 (clan 71) are physically clustered with the thalianol synthase (THAS) gene, encoding the corresponding oxidosqualene cyclase. CYP708A2 oxidises the tricyclic thalianol (12) scaffold to 7β-hydroxythalianol, while CYP705A5 is a desaturase and introduces a double bond at C15 [41]. The related Arabidopsis CYP705A1 (also from clan 71) accepts a slightly different scaffold, arabidiol (11), triggering cleavage of the side chain at the same C15 instead of dehydrogenation. This shows that even closely related CYPs from the same subfamily can exhibit distinct differences in their substrate and reaction profiles.

[1860-5397-18-135-5]

Figure 5: CYPs modifying pentacyclic 6-6-6-6-5 triterpenes (A) and unusual triterpenes (B). Substructures in grey indicate regions where major structural differences occur between different substrates of the same class. Representative substrate skeletons (not exact substrates) are shown in the dotted boxes. For exact substrate specificity see Table 1.

Recent examples of triterpenoid and steroid cytochrome P450 monooxygenases

In this last section, we will illustrate selected examples that showcase the enzymatic versatility of CYPs in plant triterpenoid and steroid metabolism (Figure 6).

[1860-5397-18-135-6]

Figure 6: Recent examples of multifunctional CYPs in triterpenoid and steroid metabolism in plants that install complex oxidative modifications; in addition, their discovery also showcases modern approaches to elucidate plant specialised metabolism. A) CYPs from different plants producing diosgenin (13) (Tf: Trigonella foenum-graecum; Pp: Paris polyphylla; Dz: Dioscorea zingiberensis) [35,66]. B) Formation of the defence compound ellarinacin (15) in bread wheat [26]. Stereochemistry of ellarinacin (15) is shown as published. C) Biosynthesis of the key intermediate melianol (21) in the pathway to the limonoid limonin (18) [29]. The stereochemistry is shown as published.

Diosgenin (13) is a specialised plant natural product with a unique 5,6-spiroketal moiety that serves as an inexpensive raw material for the industrial synthesis of steroidal drugs. Diosgenin (13) biosynthesis from cholesterol (3) was explored in Paris polyphylla (Pp; monocot), Trigonella foenum-graecum (Tf; dicot) and Dioscorea zingiberensis (Dz; monocot) (Figure 6A) [35,66]. Multifunctional CYPs PpCYP90G4/TfCYP90B50 were independently recruited from the ancient CYP90B subfamily involved in brassinosteroid biosynthesis to catalyse the initial C22,16 dihydroxylation of cholesterol (3) [35]; in contrast, the related CYP DzCYP90B71 was found to catalyse only the first hydroxylation at C22 [66]. This step is followed by a rate-limiting cyclisation step through unstable furostanol intermediate 14 that involves CYP-catalysed oxidative ring closure, leading to a hemiketal bridge between C16 and C22. Following these initial hydroxylations, CYPs from multiple families catalyse end-of-chain hydroxylation at C27 which is followed by spontaneous spiroketalisation to form diosgenin (13). The CYP pairs PpCYP90G4-PpCYP94D108 in P. polyphylla and TfCYP90B50-TfCYP82J17 in T. foenum-graecum resulted in the highest diosgenin (13) production. Diosgenin (13) biosynthesis in distantly related plants is an example of catalytic plasticity embedded within the ancient CYP90Bs. Especially CYPs from large families often show high substrate promiscuity which facilitates duplication events resulting in neofunctionalisation [15].

Ellarinacin (15) is a defence-related arborinane-type triterpenoid that was recently discovered in bread wheat (Triticum aestivum) by genome mining (Figure 6B) [26]. The ellarinacin gene cluster encodes the three CYP enzymes TaCYP51H35, TaCYP51H37 and TaCYP51H13P, with the latter carrying a premature stop codon. TaCYP51H35 catalyses the C19-hydroxylation of isoarborinol (9) to form 19-hydroxyisoarborinol (16), which is oxidised to ketone 17 by a dehydrogenase (TaHID). TaCYP51H37 then carries out a remarkable double oxidation at the methyl group C25 as well as C2, leading to the highly unusual acetal-epoxide proposed for ellarinacin (15). This work therefore not only represents an important example how a CYP51H evolved by gene duplication and neofunctionalisation from a sterol biosynthetic gene, but also demonstrates the capacity of CYPs to catalyse unique enzymatic cascades.

Limonoids are highly oxidised, modified and truncated triterpenoids; one of the most well-known limonoids is the eponymous compound limonin (18), which contributes to the bitter taste of citrus products (Figure 6C) [104]. The first steps of limonoid biosynthesis were recently explored by functional characterisation in heterologous hosts [29,105-107]. There, CYP enzymes MaCYP71CD2 and MaCYP71BQ5 from Melia azedarach initiate the ring formation on the side chain of the triterpene precursor tirucalla-7,24-dien-3β-ol (19) in a sequential manner. MaCYP71CD2 is a bifunctional CYP that hydroxylates C23 and additionally introduces a C24–C25 epoxide on the side chain of tirucalla-7,24-dien-3β-ol (19), yielding dihydroniloticin (20). MaCYP71BQ5 then oxidises the methyl group C21 to a formyl group, leading to spontaneous hemiacetal ring formation in the product melianol (21). It is believed that these transformations are the starting point for formation of the characteristic furan ring of limonoids [29].

Taken together, these case studies not only represent impressive examples how CYPs create chemical complexity in plant triterpenoid and steroid metabolism, but also illustrate state-of-the-art approaches to discover and characterise new CYPs by genome mining, co-expression analyses, and efficient heterologous expression systems.

Conclusion

In this review, we provided a comprehensive overview over the phylogenetic distribution and diverse metabolic reactions catalysed by CYPs involved in the tailoring of triterpenoids and steroids from plants, covering 149 CYPs that have been functionally characterised to date (Table 1). Considering that up to 1% of all plant genes encode CYPs and that triterpenoids are one of the largest natural product classes in plants, we expect that this number will rise quickly in years to come. Several of our examples highlight the substrate promiscuity embedded within ancient CYP families, which enables rapid functional extension to acquire unique catalytic functions during duplication events [15,26,79]. The increasing availability of high-quality transcriptome and genome data even of non-model plants together with reliable and efficient expression systems in yeast and in Nicotiana benthamiana will facilitate future approaches to fully harness the diversity of triterpenoids and steroids found in plants. In combination with ground-breaking machine learning approaches for protein structure prediction such as AlphaFold2 [108], we anticipate that the catalytic repertoire of CYPs will be exploited much more for the biotechnological production of tailor-made triterpenoids and steroids in the near future. We hope that our review provides a good starting point for such further studies.

Supporting Information

Supporting Information File 1: High-quality version of the phylogenetic tree shown in Figure 2 with tip labels.
Format: PDF Size: 519.0 KB Download

Acknowledgements

We thank the entire Franke group for helpful discussions. The graphical abstract was created with BioRender.com. This content is not subject to CC BY 4.0.

Funding

We gratefully acknowledge financial support by the Emmy Noether programme of the Deutsche Forschungsgemeinschaft (FR 3720/3-1) and the SMART BIOTECS alliance between the Technische Universität Braunschweig and the Leibniz Universität Hannover, supported by the Ministry of Science and Culture (MWK) of Lower Saxony.

References

  1. Qi, X.; Bakht, S.; Qin, B.; Leggett, M.; Hemmings, A.; Mellon, F.; Eagles, J.; Werck-Reichhart, D.; Schaller, H.; Lesot, A.; Melton, R.; Osbourn, A. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 18848–18853. doi:10.1073/pnas.0607849103
    Return to citation in text: [1] [2] [3]
  2. Kitagawa, I. Pure Appl. Chem. 2002, 74, 1189–1198. doi:10.1351/pac200274071189
    Return to citation in text: [1]
  3. Zhang, M.; Sun, X.; Ren, R.; Su, L.; Xu, M.; Zheng, L.; Li, H. Phytochem. Lett. 2022, 49, 145–151. doi:10.1016/j.phytol.2022.03.020
    Return to citation in text: [1]
  4. Xiong, J.; Kashiwada, Y.; Chen, C.-H.; Qian, K.; Morris-Natschke, S. L.; Lee, K.-H.; Takaishi, Y. Bioorg. Med. Chem. 2010, 18, 6451–6469. doi:10.1016/j.bmc.2010.06.092
    Return to citation in text: [1]
  5. Thimmappa, R.; Geisler, K.; Louveau, T.; O'Maille, P.; Osbourn, A. Annu. Rev. Plant Biol. 2014, 65, 225–257. doi:10.1146/annurev-arplant-050312-120229
    Return to citation in text: [1] [2]
  6. Nelson, D.; Werck-Reichhart, D. Plant J. 2011, 66, 194–211. doi:10.1111/j.1365-313x.2011.04529.x
    Return to citation in text: [1] [2]
  7. Ghosh, S. Front. Plant Sci. 2017, 8, 1886. doi:10.3389/fpls.2017.01886
    Return to citation in text: [1] [2]
  8. Nguyen, T.-D.; Dang, T.-T. T. Front. Plant Sci. 2021, 12, 682181. doi:10.3389/fpls.2021.682181
    Return to citation in text: [1] [2]
  9. Bathe, U.; Tissier, A. Phytochemistry 2019, 161, 149–162. doi:10.1016/j.phytochem.2018.12.003
    Return to citation in text: [1]
  10. Zhang, Y.; Ma, L.; Su, P.; Huang, L.; Gao, W. Crit. Rev. Biotechnol. 2021, 1–21. doi:10.1080/07388551.2021.2003292
    Return to citation in text: [1]
  11. Mizutani, M.; Sato, F. Arch. Biochem. Biophys. 2011, 507, 194–203. doi:10.1016/j.abb.2010.09.026
    Return to citation in text: [1] [2]
  12. Miettinen, K.; Iñigo, S.; Kreft, L.; Pollier, J.; De Bo, C.; Botzki, A.; Coppens, F.; Bak, S.; Goossens, A. Nucleic Acids Res. 2018, 46, D586–D594. doi:10.1093/nar/gkx925
    Return to citation in text: [1]
  13. Shang, Y.; Huang, S. Plant J. 2019, 97, 101–111. doi:10.1111/tpj.14132
    Return to citation in text: [1]
  14. Bak, S.; Beisson, F.; Bishop, G.; Hamberger, B.; Höfer, R.; Paquette, S.; Werck-Reichhart, D. Arabidopsis Book 2011, 9, e0144. doi:10.1199/tab.0144
    Return to citation in text: [1]
  15. Hansen, C. C.; Nelson, D. R.; Møller, B. L.; Werck-Reichhart, D. Mol. Plant 2021, 14, 1244–1265. doi:10.1016/j.molp.2021.06.028
    Return to citation in text: [1] [2] [3] [4]
  16. Nelson, D. R. Cytochrome P450 Nomenclature, 2004. In Cytochrome P450 Protocols; Phillips, I. R.; Shephard, E. A., Eds.; Methods in Molecular Biology; Humana Press: Totowa, NJ, USA, 2006; pp 1–10. doi:10.1385/1-59259-998-2:1
    Return to citation in text: [1]
  17. Meunier, B.; de Visser, S. P.; Shaik, S. Chem. Rev. 2004, 104, 3947–3980. doi:10.1021/cr020443g
    Return to citation in text: [1] [2] [3]
  18. Poulos, T. L. Chem. Rev. 2014, 114, 3919–3962. doi:10.1021/cr400415k
    Return to citation in text: [1] [2]
  19. Guengerich, F. P. ACS Catal. 2018, 8, 10964–10976. doi:10.1021/acscatal.8b03401
    Return to citation in text: [1]
  20. Rittle, J.; Green, M. T. Science 2010, 330, 933–937. doi:10.1126/science.1193478
    Return to citation in text: [1]
  21. Dubey, K. D.; Shaik, S. Acc. Chem. Res. 2019, 52, 389–399. doi:10.1021/acs.accounts.8b00467
    Return to citation in text: [1]
  22. Chen, C.-C.; Min, J.; Zhang, L.; Yang, Y.; Yu, X.; Guo, R.-T. ChemBioChem 2021, 22, 1317–1328. doi:10.1002/cbic.202000705
    Return to citation in text: [1]
  23. Ortiz de Montellano, P. R. Substrate Oxidation by Cytochrome P450 Enzyme. In Cytochrome P450: Structure, Mechanism, and Biochemistry; Ortiz de Montellano, P. R., Ed.; Springer International Publishing: Cham, Switzerland, 2015; pp 111–176. doi:10.1007/978-3-319-12108-6_4
    Return to citation in text: [1]
  24. Bak, S.; Kahn, R. A.; Olsen, C. E.; Halkier, B. A. Plant J. 1997, 11, 191–201. doi:10.1046/j.1365-313x.1997.11020191.x
    Return to citation in text: [1] [2] [3]
  25. Kushiro, M.; Nakano, T.; Sato, K.; Yamagishi, K.; Asami, T.; Nakano, A.; Takatsuto, S.; Fujioka, S.; Ebizuka, Y.; Yoshida, S. Biochem. Biophys. Res. Commun. 2001, 285, 98–104. doi:10.1006/bbrc.2001.5122
    Return to citation in text: [1] [2] [3] [4]
  26. Polturak, G.; Dippe, M.; Stephenson, M. J.; Chandra Misra, R.; Owen, C.; Ramirez-Gonzalez, R. H.; Haidoulis, J. F.; Schoonbeek, H.-J.; Chartrain, L.; Borrill, P.; Nelson, D. R.; Brown, J. K. M.; Nicholson, P.; Uauy, C.; Osbourn, A. Proc. Natl. Acad. Sci. U. S. A. 2022, 119, e2123299119. doi:10.1073/pnas.2123299119
    Return to citation in text: [1] [2] [3] [4] [5] [6] [7] [8] [9]
  27. Field, B.; Fiston-Lavier, A.-S.; Kemen, A.; Geisler, K.; Quesneville, H.; Osbourn, A. E. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 16116–16121. doi:10.1073/pnas.1109273108
    Return to citation in text: [1] [2]
  28. Castillo, D. A.; Kolesnikova, M. D.; Matsuda, S. P. T. J. Am. Chem. Soc. 2013, 135, 5885–5894. doi:10.1021/ja401535g
    Return to citation in text: [1] [2]
  29. Hodgson, H.; De La Peña, R.; Stephenson, M. J.; Thimmappa, R.; Vincent, J. L.; Sattely, E. S.; Osbourn, A. Proc. Natl. Acad. Sci. U. S. A. 2019, 116, 17096–17104. doi:10.1073/pnas.1906083116
    Return to citation in text: [1] [2] [3] [4] [5]
  30. Krokida, A.; Delis, C.; Geisler, K.; Garagounis, C.; Tsikou, D.; Peña‐Rodríguez, L. M.; Katsarou, D.; Field, B.; Osbourn, A. E.; Papadopoulou, K. K. New Phytol. 2013, 200, 675–690. doi:10.1111/nph.12414
    Return to citation in text: [1]
  31. Tsukagoshi, Y.; Ohyama, K.; Seki, H.; Akashi, T.; Muranaka, T.; Suzuki, H.; Fujimoto, Y. Phytochemistry 2016, 127, 23–28. doi:10.1016/j.phytochem.2016.03.010
    Return to citation in text: [1]
  32. Takase, S.; Kera, K.; Nagashima, Y.; Mannen, K.; Hosouchi, T.; Shinpo, S.; Kawashima, M.; Kotake, Y.; Yamada, H.; Saga, Y.; Otaka, J.; Araya, H.; Kotera, M.; Suzuki, H.; Kushiro, T. J. Biol. Chem. 2019, 294, 18662–18673. doi:10.1074/jbc.ra119.009944
    Return to citation in text: [1] [2] [3]
  33. Shang, Y.; Ma, Y.; Zhou, Y.; Zhang, H.; Duan, L.; Chen, H.; Zeng, J.; Zhou, Q.; Wang, S.; Gu, W.; Liu, M.; Ren, J.; Gu, X.; Zhang, S.; Wang, Y.; Yasukawa, K.; Bouwmeester, H. J.; Qi, X.; Zhang, Z.; Lucas, W. J.; Huang, S. Science 2014, 346, 1084–1088. doi:10.1126/science.1259215
    Return to citation in text: [1]
  34. Zhou, Y.; Ma, Y.; Zeng, J.; Duan, L.; Xue, X.; Wang, H.; Lin, T.; Liu, Z.; Zeng, K.; Zhong, Y.; Zhang, S.; Hu, Q.; Liu, M.; Zhang, H.; Reed, J.; Moses, T.; Liu, X.; Huang, P.; Qing, Z.; Liu, X.; Tu, P.; Kuang, H.; Zhang, Z.; Osbourn, A.; Ro, D.-K.; Shang, Y.; Huang, S. Nat. Plants (London, U. K.) 2016, 2, 16183. doi:10.1038/nplants.2016.183
    Return to citation in text: [1] [2] [3] [4]
  35. Christ, B.; Xu, C.; Xu, M.; Li, F.-S.; Wada, N.; Mitchell, A. J.; Han, X.-L.; Wen, M.-L.; Fujita, M.; Weng, J.-K. Nat. Commun. 2019, 10, 3206. doi:10.1038/s41467-019-11286-7
    Return to citation in text: [1] [2] [3] [4] [5] [6] [7] [8] [9] [10]
  36. Li, L.; Lin, S.; Chen, Y.; Wang, Y.; Xiao, L.; Ye, X.; Sun, L.; Zhan, R.; Xu, H. Front. Plant Sci. 2022, 13, 831401. doi:10.3389/fpls.2022.831401
    Return to citation in text: [1] [2] [3]
  37. Shibuya, M.; Hoshino, M.; Katsube, Y.; Hayashi, H.; Kushiro, T.; Ebizuka, Y. FEBS J. 2006, 273, 948–959. doi:10.1111/j.1742-4658.2006.05120.x
    Return to citation in text: [1] [2] [3]
  38. Moses, T.; Pollier, J.; Almagro, L.; Buyst, D.; Van Montagu, M.; Pedreño, M. A.; Martins, J. C.; Thevelein, J. M.; Goossens, A. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 1634–1639. doi:10.1073/pnas.1323369111
    Return to citation in text: [1] [2]
  39. Seki, H.; Ohyama, K.; Sawai, S.; Mizutani, M.; Ohnishi, T.; Sudo, H.; Akashi, T.; Aoki, T.; Saito, K.; Muranaka, T. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 14204–14209. doi:10.1073/pnas.0803876105
    Return to citation in text: [1] [2]
  40. Moses, T.; Thevelein, J. M.; Goossens, A.; Pollier, J. Phytochemistry 2014, 108, 47–56. doi:10.1016/j.phytochem.2014.10.002
    Return to citation in text: [1] [2] [3] [4] [5] [6] [7] [8] [9]
  41. Field, B.; Osbourn, A. E. Science 2008, 320, 543–547. doi:10.1126/science.1154990
    Return to citation in text: [1] [2] [3] [4]
  42. Hansen, N. L.; Miettinen, K.; Zhao, Y.; Ignea, C.; Andreadelli, A.; Raadam, M. H.; Makris, A. M.; Møller, B. L.; Stærk, D.; Bak, S.; Kampranis, S. C. Microb. Cell Fact. 2020, 19, 15. doi:10.1186/s12934-020-1284-9
    Return to citation in text: [1] [2] [3] [4] [5]
  43. Bicalho, K. U.; Santoni, M. M.; Arendt, P.; Zanelli, C. F.; Furlan, M.; Goossens, A.; Pollier, J. Plant Cell Physiol. 2019, 60, 2510–2522. doi:10.1093/pcp/pcz144
    Return to citation in text: [1] [2] [3]
  44. Fukushima, E. O.; Seki, H.; Sawai, S.; Suzuki, M.; Ohyama, K.; Saito, K.; Muranaka, T. Plant Cell Physiol. 2013, 54, 740–749. doi:10.1093/pcp/pct015
    Return to citation in text: [1] [2] [3]
  45. Fanani, M. Z.; Fukushima, E. O.; Sawai, S.; Tang, J.; Ishimori, M.; Sudo, H.; Ohyama, K.; Seki, H.; Saito, K.; Muranaka, T. Front. Plant Sci. 2019, 10, 1520. doi:10.3389/fpls.2019.01520
    Return to citation in text: [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11]
  46. Seki, H.; Sawai, S.; Ohyama, K.; Mizutani, M.; Ohnishi, T.; Sudo, H.; Fukushima, E. O.; Akashi, T.; Aoki, T.; Saito, K.; Muranaka, T. Plant Cell 2011, 23, 4112–4123. doi:10.1105/tpc.110.082685
    Return to citation in text: [1] [2] [3]
  47. Biazzi, E.; Carelli, M.; Tava, A.; Abbruscato, P.; Losini, I.; Avato, P.; Scotti, C.; Calderini, O. Mol. Plant 2015, 8, 1493–1506. doi:10.1016/j.molp.2015.06.003
    Return to citation in text: [1]
  48. Tzin, V.; Snyder, J. H.; Yang, D. S.; Huhman, D. V.; Watson, B. S.; Allen, S. N.; Tang, Y.; Miettinen, K.; Arendt, P.; Pollier, J.; Goossens, A.; Sumner, L. W. Metabolomics 2019, 15, 85. doi:10.1007/s11306-019-1542-1
    Return to citation in text: [1] [2]
  49. Yano, R.; Takagi, K.; Takada, Y.; Mukaiyama, K.; Tsukamoto, C.; Sayama, T.; Kaga, A.; Anai, T.; Sawai, S.; Ohyama, K.; Saito, K.; Ishimoto, M. Plant J. 2017, 89, 527–539. doi:10.1111/tpj.13403
    Return to citation in text: [1]
  50. Han, J. Y.; Chun, J.-H.; Oh, S. A.; Park, S.-B.; Hwang, H.-S.; Lee, H.; Choi, Y. E. Plant Cell Physiol. 2018, 59, 319–330. doi:10.1093/pcp/pcx188
    Return to citation in text: [1] [2]
  51. Liu, Q.; Khakimov, B.; Cárdenas, P. D.; Cozzi, F.; Olsen, C. E.; Jensen, K. R.; Hauser, T. P.; Bak, S. New Phytol. 2019, 222, 1599–1609. doi:10.1111/nph.15689
    Return to citation in text: [1]
  52. Kim, O. T.; Um, Y.; Jin, M. L.; Kim, J. U.; Hegebarth, D.; Busta, L.; Racovita, R. C.; Jetter, R. Plant Cell Physiol. 2018, 59, 1200–1213. doi:10.1093/pcp/pcy055
    Return to citation in text: [1]
  53. Ohnishi, T.; Nomura, T.; Watanabe, B.; Ohta, D.; Yokota, T.; Miyagawa, H.; Sakata, K.; Mizutani, M. Phytochemistry 2006, 67, 1895–1906. doi:10.1016/j.phytochem.2006.05.042
    Return to citation in text: [1]
  54. Dai, Z.; Liu, Y.; Sun, Z.; Wang, D.; Qu, G.; Ma, X.; Fan, F.; Zhang, L.; Li, S.; Zhang, X. Metab. Eng. 2019, 51, 70–78. doi:10.1016/j.ymben.2018.10.001
    Return to citation in text: [1] [2]
  55. Shimada, Y.; Fujioka, S.; Miyauchi, N.; Kushiro, M.; Takatsuto, S.; Nomura, T.; Yokota, T.; Kamiya, Y.; Bishop, G. J.; Yoshida, S. Plant Physiol. 2001, 126, 770–779. doi:10.1104/pp.126.2.770
    Return to citation in text: [1]
  56. Bishop, G. J.; Nomura, T.; Yokota, T.; Harrison, K.; Noguchi, T.; Fujioka, S.; Takatsuto, S.; Jones, J. D. G.; Kamiya, Y. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 1761–1766. doi:10.1073/pnas.96.4.1761
    Return to citation in text: [1]
  57. Shimada, Y.; Goda, H.; Nakamura, A.; Takatsuto, S.; Fujioka, S.; Yoshida, S. Plant Physiol. 2003, 131, 287–297. doi:10.1104/pp.013029
    Return to citation in text: [1]
  58. Nomura, T.; Kushiro, T.; Yokota, T.; Kamiya, Y.; Bishop, G. J.; Yamaguchi, S. J. Biol. Chem. 2005, 280, 17873–17879. doi:10.1074/jbc.m414592200
    Return to citation in text: [1] [2]
  59. Moses, T.; Pollier, J.; Faizal, A.; Apers, S.; Pieters, L.; Thevelein, J. M.; Geelen, D.; Goossens, A. Mol. Plant 2015, 8, 122–135. doi:10.1016/j.molp.2014.11.004
    Return to citation in text: [1] [2]
  60. Ohnishi, T.; Godza, B.; Watanabe, B.; Fujioka, S.; Hategan, L.; Ide, K.; Shibata, K.; Yokota, T.; Szekeres, M.; Mizutani, M. J. Biol. Chem. 2012, 287, 31551–31560. doi:10.1074/jbc.m112.392720
    Return to citation in text: [1]
  61. Fujita, S.; Ohnishi, T.; Watanabe, B.; Yokota, T.; Takatsuto, S.; Fujioka, S.; Yoshida, S.; Sakata, K.; Mizutani, M. Plant J. 2006, 45, 765–774. doi:10.1111/j.1365-313x.2005.02639.x
    Return to citation in text: [1]
  62. Sakamoto, T.; Morinaka, Y.; Ohnishi, T.; Sunohara, H.; Fujioka, S.; Ueguchi-Tanaka, M.; Mizutani, M.; Sakata, K.; Takatsuto, S.; Yoshida, S.; Tanaka, H.; Kitano, H.; Matsuoka, M. Nat. Biotechnol. 2006, 24, 105–109. doi:10.1038/nbt1173
    Return to citation in text: [1] [2]
  63. Ohnishi, T.; Watanabe, B.; Sakata, K.; Mizutani, M. Biosci., Biotechnol., Biochem. 2006, 70, 2071–2080. doi:10.1271/bbb.60034
    Return to citation in text: [1] [2]
  64. Augustin, M. M.; Ruzicka, D. R.; Shukla, A. K.; Augustin, J. M.; Starks, C. M.; O'Neil‐Johnson, M.; McKain, M. R.; Evans, B. S.; Barrett, M. D.; Smithson, A.; Wong, G. K.-S.; Deyholos, M. K.; Edger, P. P.; Pires, J. C.; Leebens‐Mack, J. H.; Mann, D. A.; Kutchan, T. M. Plant J. 2015, 82, 991–1003. doi:10.1111/tpj.12871
    Return to citation in text: [1] [2] [3] [4] [5]
  65. Yin, Y.; Gao, L.; Zhang, X.; Gao, W. Phytochemistry 2018, 156, 116–123. doi:10.1016/j.phytochem.2018.09.005
    Return to citation in text: [1]
  66. Zhou, C.; Yang, Y.; Tian, J.; Wu, Y.; An, F.; Li, C.; Zhang, Y. Plant J. 2022, 109, 940–951. doi:10.1111/tpj.15604
    Return to citation in text: [1] [2] [3] [4] [5] [6] [7]
  67. Ohnishi, T.; Szatmari, A.-M.; Watanabe, B.; Fujita, S.; Bancos, S.; Koncz, C.; Lafos, M.; Shibata, K.; Yokota, T.; Sakata, K.; Szekeres, M.; Mizutani, M. Plant Cell 2006, 18, 3275–3288. doi:10.1105/tpc.106.045443
    Return to citation in text: [1] [2]
  68. Sakamoto, T.; Ohnishi, T.; Fujioka, S.; Watanabe, B.; Mizutani, M. Plant Physiol. Biochem. 2012, 58, 220–226. doi:10.1016/j.plaphy.2012.07.011
    Return to citation in text: [1] [2]
  69. Dong, L.; Almeida, A.; Pollier, J.; Khakimov, B.; Bassard, J.-E.; Miettinen, K.; Stærk, D.; Mehran, R.; Olsen, C. E.; Motawia, M. S.; Goossens, A.; Bak, S. Mol. Biol. Evol. 2021, 38, 4659–4673. doi:10.1093/molbev/msab213
    Return to citation in text: [1] [2] [3]
  70. Yasumoto, S.; Fukushima, E. O.; Seki, H.; Muranaka, T. FEBS Lett. 2016, 590, 533–540. doi:10.1002/1873-3468.12074
    Return to citation in text: [1] [2]
  71. Carelli, M.; Biazzi, E.; Panara, F.; Tava, A.; Scaramelli, L.; Porceddu, A.; Graham, N.; Odoardi, M.; Piano, E.; Arcioni, S.; May, S.; Scotti, C.; Calderini, O. Plant Cell 2011, 23, 3070–3081. doi:10.1105/tpc.111.087312
    Return to citation in text: [1] [2]
  72. Moses, T.; Pollier, J.; Shen, Q.; Soetaert, S.; Reed, J.; Erffelinck, M.-L.; Van Nieuwerburgh, F. C. W.; Vanden Bossche, R.; Osbourn, A.; Thevelein, J. M.; Deforce, D.; Tang, K.; Goossens, A. Plant Cell 2015, 27, 286–301. doi:10.1105/tpc.114.134486
    Return to citation in text: [1] [2]
  73. Fukushima, E. O.; Seki, H.; Ohyama, K.; Ono, E.; Umemoto, N.; Mizutani, M.; Saito, K.; Muranaka, T. Plant Cell Physiol. 2011, 52, 2050–2061. doi:10.1093/pcp/pcr146
    Return to citation in text: [1] [2] [3]
  74. Yasumoto, S.; Seki, H.; Shimizu, Y.; Fukushima, E. O.; Muranaka, T. Front. Plant Sci. 2017, 8, 21. doi:10.3389/fpls.2017.00021
    Return to citation in text: [1] [2] [3]
  75. Suzuki, H.; Fukushima, E. O.; Shimizu, Y.; Seki, H.; Fujisawa, Y.; Ishimoto, M.; Osakabe, K.; Osakabe, Y.; Muranaka, T. Plant Cell Physiol. 2019, 60, 2496–2509. doi:10.1093/pcp/pcz145
    Return to citation in text: [1]
  76. Han, J.-Y.; Hwang, H.-S.; Choi, S.-W.; Kim, H.-J.; Choi, Y.-E. Plant Cell Physiol. 2012, 53, 1535–1545. doi:10.1093/pcp/pcs106
    Return to citation in text: [1] [2] [3] [4]
  77. Fiallos-Jurado, J.; Pollier, J.; Moses, T.; Arendt, P.; Barriga-Medina, N.; Morillo, E.; Arahana, V.; de Lourdes Torres, M.; Goossens, A.; Leon-Reyes, A. Plant Sci. 2016, 250, 188–197. doi:10.1016/j.plantsci.2016.05.015
    Return to citation in text: [1] [2]
  78. Khakimov, B.; Kuzina, V.; Erthmann, P. Ø.; Fukushima, E. O.; Augustin, J. M.; Olsen, C. E.; Scholtalbers, J.; Volpin, H.; Andersen, S. B.; Hauser, T. P.; Muranaka, T.; Bak, S. Plant J. 2015, 84, 478–490. doi:10.1111/tpj.13012
    Return to citation in text: [1] [2]
  79. Miettinen, K.; Pollier, J.; Buyst, D.; Arendt, P.; Csuk, R.; Sommerwerk, S.; Moses, T.; Mertens, J.; Sonawane, P. D.; Pauwels, L.; Aharoni, A.; Martins, J.; Nelson, D. R.; Goossens, A. Nat. Commun. 2017, 8, 14153. doi:10.1038/ncomms14153
    Return to citation in text: [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15]
  80. Tamura, K.; Teranishi, Y.; Ueda, S.; Suzuki, H.; Kawano, N.; Yoshimatsu, K.; Saito, K.; Kawahara, N.; Muranaka, T.; Seki, H. Plant Cell Physiol. 2017, 58, 874–884. doi:10.1093/pcp/pcx043
    Return to citation in text: [1] [2]
  81. Huang, L.; Li, J.; Ye, H.; Li, C.; Wang, H.; Liu, B.; Zhang, Y. Planta 2012, 236, 1571–1581. doi:10.1007/s00425-012-1712-0
    Return to citation in text: [1]
  82. Huang, J.; Zha, W.; An, T.; Dong, H.; Huang, Y.; Wang, D.; Yu, R.; Duan, L.; Zhang, X.; Peters, R. J.; Dai, Z.; Zi, J. Appl. Microbiol. Biotechnol. 2019, 103, 7029–7039. doi:10.1007/s00253-019-10004-z
    Return to citation in text: [1]
  83. Andre, C. M.; Legay, S.; Deleruelle, A.; Nieuwenhuizen, N.; Punter, M.; Brendolise, C.; Cooney, J. M.; Lateur, M.; Hausman, J.-F.; Larondelle, Y.; Laing, W. A. New Phytol. 2016, 211, 1279–1294. doi:10.1111/nph.13996
    Return to citation in text: [1]
  84. Tamura, K.; Seki, H.; Suzuki, H.; Kojoma, M.; Saito, K.; Muranaka, T. Plant Cell Rep. 2017, 36, 437–445. doi:10.1007/s00299-016-2092-x
    Return to citation in text: [1]
  85. Zhou, C.; Li, J.; Li, C.; Zhang, Y. BMC Biotechnol. 2016, 16, 59. doi:10.1186/s12896-016-0290-9
    Return to citation in text: [1]
  86. Ji, X.; Lin, S.; Chen, Y.; Liu, J.; Yun, X.; Wang, T.; Qin, J.; Luo, C.; Wang, K.; Zhao, Z.; Zhan, R.; Xu, H. Front. Plant Sci. 2020, 11, 612. doi:10.3389/fpls.2020.00612
    Return to citation in text: [1]
  87. Jo, H.-J.; Han, J. Y.; Hwang, H.-S.; Choi, Y. E. Phytochemistry 2017, 135, 53–63. doi:10.1016/j.phytochem.2016.12.011
    Return to citation in text: [1]
  88. Misra, R. C.; Sharma, S.; Sandeep; Garg, A.; Chanotiya, C. S.; Ghosh, S. New Phytol. 2017, 214, 706–720. doi:10.1111/nph.14412
    Return to citation in text: [1] [2]
  89. Sandeep; Misra, R. C.; Chanotiya, C. S.; Mukhopadhyay, P.; Ghosh, S. New Phytol. 2019, 222, 408–424. doi:10.1111/nph.15606
    Return to citation in text: [1] [2] [3]
  90. Zhang, R.; Xia, X.; Lindsey, K.; da Rocha, P. S. C. F. J. Plant Physiol. 2012, 169, 421–428. doi:10.1016/j.jplph.2011.10.013
    Return to citation in text: [1]
  91. Morikawa, T.; Mizutani, M.; Aoki, N.; Watanabe, B.; Saga, H.; Saito, S.; Oikawa, A.; Suzuki, H.; Sakurai, N.; Shibata, D.; Wadano, A.; Sakata, K.; Ohta, D. Plant Cell 2006, 18, 1008–1022. doi:10.1105/tpc.105.036012
    Return to citation in text: [1] [2] [3] [4] [5]
  92. Edgar, R. C. Nucleic Acids Res. 2004, 32, 1792–1797. doi:10.1093/nar/gkh340
    Return to citation in text: [1]
  93. Jacobowitz, J. R.; Weng, J.-K. Annu. Rev. Plant Biol. 2020, 71, 631–658. doi:10.1146/annurev-arplant-081519-035634
    Return to citation in text: [1]
  94. Chuang, L.; Franke, J. Rapid Combinatorial Coexpression of Biosynthetic Genes by Transient Expression in the Plant Host Nicotiana Benthamiana. In Engineering Natural Product Biosynthesis: Methods and Protocols; Skellam, E., Ed.; Methods in Molecular Biology; Springer US: New York, NY, USA, 2022; pp 395–420. doi:10.1007/978-1-0716-2273-5_20
    Return to citation in text: [1]
  95. Dang, T.-T. T.; Franke, J.; Carqueijeiro, I. S. T.; Langley, C.; Courdavault, V.; O’Connor, S. E. Nat. Chem. Biol. 2018, 14, 760–763. doi:10.1038/s41589-018-0078-4
    Return to citation in text: [1]
  96. Nett, R. S.; Lau, W.; Sattely, E. S. Nature 2020, 584, 148–153. doi:10.1038/s41586-020-2546-8
    Return to citation in text: [1]
  97. Arnqvist, L.; Persson, M.; Jonsson, L.; Dutta, P. C.; Sitbon, F. Planta 2008, 227, 309–317. doi:10.1007/s00425-007-0618-8
    Return to citation in text: [1]
  98. Cabello-Hurtado, F.; Taton, M.; Forthoffer, N.; Kahn, R.; Bak, S.; Rahier, A.; Werck-Reichhart, D. Eur. J. Biochem. 1999, 262, 435–446. doi:10.1046/j.1432-1327.1999.00376.x
    Return to citation in text: [1]
  99. Geisler, K.; Hughes, R. K.; Sainsbury, F.; Lomonossoff, G. P.; Rejzek, M.; Fairhurst, S.; Olsen, C.-E.; Motawia, M. S.; Melton, R. E.; Hemmings, A. M.; Bak, S.; Osbourn, A. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, E3360–E3367. doi:10.1073/pnas.1309157110
    Return to citation in text: [1]
  100. Kasai, R.; Nie, R.-L.; Nashi, K.; Ohtani, K.; Zhou, J.; Tao, G.-D.; Tanaka, O. Agric. Biol. Chem. 1989, 53, 3347–3349. doi:10.1080/00021369.1989.10869815
    Return to citation in text: [1]
  101. Zhang, J.; Dai, L.; Yang, J.; Liu, C.; Men, Y.; Zeng, Y.; Cai, Y.; Zhu, Y.; Sun, Y. Plant Cell Physiol. 2016, 57, 1000–1007. doi:10.1093/pcp/pcw038
    Return to citation in text: [1]
  102. Itkin, M.; Davidovich-Rikanati, R.; Cohen, S.; Portnoy, V.; Doron-Faigenboim, A.; Oren, E.; Freilich, S.; Tzuri, G.; Baranes, N.; Shen, S.; Petreikov, M.; Sertchook, R.; Ben-Dor, S.; Gottlieb, H.; Hernandez, A.; Nelson, D. R.; Paris, H. S.; Tadmor, Y.; Burger, Y.; Lewinsohn, E.; Katzir, N.; Schaffer, A. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, E7619–E7628. doi:10.1073/pnas.1604828113
    Return to citation in text: [1]
  103. Han, J.-Y.; Kim, H.-J.; Kwon, Y.-S.; Choi, Y.-E. Plant Cell Physiol. 2011, 52, 2062–2073. doi:10.1093/pcp/pcr150
    Return to citation in text: [1]
  104. Li, L.-J.; Tan, W.-S.; Li, W.-J.; Zhu, Y.-B.; Cheng, Y.-S.; Ni, H. Int. J. Mol. Sci. 2019, 20, 6194. doi:10.3390/ijms20246194
    Return to citation in text: [1]
  105. Pandreka, A.; Chaya, P. S.; Kumar, A.; Aarthy, T.; Mulani, F. A.; Bhagyashree, D. D.; B, S. H.; Jennifer, C.; Ponnusamy, S.; Nagegowda, D.; Thulasiram, H. V. Phytochemistry 2021, 184, 112669. doi:10.1016/j.phytochem.2021.112669
    Return to citation in text: [1]
  106. Lian, X.; Zhang, X.; Wang, F.; Wang, X.; Xue, Z.; Qi, X. Physiol. Plant. 2020, 170, 528–536. doi:10.1111/ppl.13189
    Return to citation in text: [1]
  107. Chuang, L.; Liu, S.; Biedermann, D.; Franke, J. Front. Plant Sci. 2022, 13, 958138. doi:10.3389/fpls.2022.958138
    Return to citation in text: [1]
  108. Jumper, J.; Evans, R.; Pritzel, A.; Green, T.; Figurnov, M.; Ronneberger, O.; Tunyasuvunakool, K.; Bates, R.; Žídek, A.; Potapenko, A.; Bridgland, A.; Meyer, C.; Kohl, S. A. A.; Ballard, A. J.; Cowie, A.; Romera-Paredes, B.; Nikolov, S.; Jain, R.; Adler, J.; Back, T.; Petersen, S.; Reiman, D.; Clancy, E.; Zielinski, M.; Steinegger, M.; Pacholska, M.; Berghammer, T.; Bodenstein, S.; Silver, D.; Vinyals, O.; Senior, A. W.; Kavukcuoglu, K.; Kohli, P.; Hassabis, D. Nature 2021, 1–11. doi:10.1038/s41586-021-03819-2
    Return to citation in text: [1]

© 2022 Malhotra and Franke; licensee Beilstein-Institut.
This is an open access article licensed under the terms of the Beilstein-Institut Open Access License Agreement (https://www.beilstein-journals.org/bjoc/terms), which is identical to the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0). The reuse of material under this license requires that the author(s), source and license are credited. Third-party material in this article could be subject to other licenses (typically indicated in the credit line), and in this case, users are required to obtain permission from the license holder to reuse the material.

Other Beilstein-Institut Open Science Activities
Journal Logo
Institut Logo
Preprint Logo
Symposia Logo