Recent progress of enzymatic synthesis of flavonoid C-glycosides with C-glycosyltransferase
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摘要:
C-糖基化修饰是植物次级代谢产物修饰之一，能增加植物次级代谢产物稳定性、生物利用度、水溶性和生物活性。黄酮类碳苷化合物作为重要的植物次级代谢产物之一，在调控植物生长发育和抵御病虫害侵扰、抗紫外光辐射、抗菌等方面发挥重要作用。一般而言，这类化合物的生物合成通常是在C-糖基转移酶（C-glycosyltransferase, CGT）催化下，黄酮类化合物与UDP-Glucose等核苷二磷酸酯发生反应生成。本文综述了近年来CGT催化下生物合成黄酮类碳苷的文献报道，分别从原料黄酮类化合物、黄酮类酚苷和黄酮类化合物生源途径角度出发，论述其生物合成的3个策略。其中以黄酮类化合物为原料直接或者间接合成黄酮类碳苷是使用最普遍的合成策略；同时整理归纳最近报道的各种CGT的分类、植物来源和催化功能。
Abstract:C-Glycosylation is one of the modifications of plant secondary metabolites, which can increase the stability, bioavailability, water solubility and biological activities. Flavonoid C-glycosides, as one of the most important plant secondary metabolites, exhibit important biological activities in regulating plant growth and development, resisting pests and diseases, resisting ultraviolet radiation, and antibacterial etc. Generally, this kind of such compounds are biosynthesized by flavonoids reacting with UDP-Glucose and other nucleoside diphosphates catalyzed by C-glycosyltransferase (CGT). This paper reviews the literature reports on the biosynthesis of flavonoid C-glycosides catalyzed by CGT in recent years, and discusses three strategies for its biosynthesis from the perspectives of raw material flavonoids, flavonoid O-glycosides and flavonoid biosynthetic pathways. Among them, the direct or indirect synthesis of flavonoid C-glycosides using flavonoids as raw materials is the most common synthesis strategy, and the classification, plant origin and catalytic function of various CGTs reported recently were also summarized.
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Keywords:
- enzymatic synthesis /
- flavonoid C-glycoside /
- C-glycosyltransferase
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黄酮类碳苷化合物是一类以黄酮类化合物为苷元，通过Csp2-Csp3键连接到糖端基的一类化合物[1]。它们具有广泛多样的重要生物活性，包括抗氧化、抗炎、抗肿瘤、抗菌、抗病毒、神经保护、肝保护、降血糖、抗抑郁、心血管保护、镇痛和其他活性等[2−3]。作为天然产物，黄酮类碳苷化合物通常可以分为两类，单碳苷黄酮类化合物和双碳苷黄酮类化合物。其糖基连接位点通常是在黄酮类化合物A环6位或者和8位[4]。该类化合物在自然界中分布广泛，但提取分离步骤冗长且含量不高。化学合成黄酮类碳苷化合物面临许多困难和挑战，如β-立体选择性不高、合成步骤冗长和操作麻烦等缺点，因此生物合成黄酮类碳苷方法变得至关重要[5]。与化学合成黄酮类碳苷方法相比，生物合成方法具有条件温和、区域和立体选择性好、专一性强等特点。黄酮类碳苷化合物生物合成一般是通过CGT催化UDP-糖化合物与黄酮类化合物反应合成，因此CGT对黄酮类碳苷化合物的生物合成至关重要[6]。
CGT属于GT1大家族，催化底物与作为供体的核苷化合物（糖基供体UDP-sugar）作用生成碳苷。根据糖型，可以分为UDP-glucose (UDP-Glc), UDP-xylose (UDP-Xyl), UDP-galactose (UDP-Gal), UDP-arabinose (UDP-Ara), UDP-glucuronic acid (UDP-GlcA)和UDP-N-acetylglucosamine (UDP-GlcNAc)6种。自从2009年首个黄酮类CGT被报道以来，大约有44个黄酮类CGT被鉴定结构和功能表征[7]。这些CGT结构差异性大，只有不到30%的氨基酸序列相同或者相似。此外，与O-糖基转移酶（O-glycosyltransferase, OGT）相比，CGT在自然界数量要少很多，对其结构表征研究较少[8]。黄酮类CGT存在于各种属来源的植物中，如水稻[9]、玉米[10]、大豆[11]、荞麦[12]、葛根[13]、金橘[14]、芒果[15]、黄芩[16]、铁皮石斛[17]等。本文根据黄酮类碳苷的生物合成方法策略进行分类，总结最近生物合成碳苷的文献报道，探讨不同植物来源的CGT的分类及对黄酮类碳苷生物合成的影响，并讨论该领域存在的挑战和前景。
目前黄酮类碳苷化合物生物合成策略主要有3种：（1）以黄酮类化合物（包括中间体）为原料合成策略。在各种植物来源CGT或者人工克隆CGT催化作用下，与UDP-糖直接反应，生成产物；（2）以黄酮类酚苷（flavonoid O-glycoside）为原料的黄酮类碳苷合成策略。黄酮类酚苷在OGT作用下，释放出黄酮类化合物苷元，随后与UDP-糖在CGT催化下直接作用，生成产物；（3）多酶催化接力策略合成黄酮类碳苷（酶催化从头合成黄酮类碳苷）。该策略将黄酮类碳苷化合物生物合成途径中所有参与的酶催化接力，最终生成黄酮类碳苷目标产物。这些酶包括但不限于酪氨酸裂解酶（tyrosine amino lyase, TAL）、4-香豆酰辅酶A连接酶（4-coumaroyl-CoA ligase, 4CL）、查耳酮合成酶（chalcone synthase, CHS）、查耳酮异构酶（chalcone isomerase, CHI）、黄酮合成酶（flavone synthase, FNS）、黄酮醇合成酶（flavonol synthase, FLS）、黄烷酮3-羟化酶（flavanone 3-hydroxylase, F3H）、黄烷酮2-羟化酶（flavanone 2-hydroxylase, F2H）、脱水酶（dehydratase, DH）、黄酮6位C-糖基转移酶（flavone 6-C-glycosyltransferase, F6CGT）、黄酮8位C-糖基转移酶（flavone 8-C-glycosyltransferase, F8CGT）等。表1列出了近年来新发现报道的CGT，其中发现部分酶具有多种催化活性，除C-glycosylation外，还有如O-glycosylation, S-glycosylation, N-glycosylation等活性。Entries 1~21是以黄酮类化合物为原料，与UDP-糖在CGT催化下生成黄酮类碳苷化合物的例子；entries 22~23是以黄酮酚苷为原料合成黄酮类碳苷化合物的例子；entries 24~25为从头合成黄酮类碳苷化合物例子。总而言之，生物合成黄酮类碳苷核心就是依靠CGT催化黄酮类化合物（或中间体）与UDP-糖作用生成产物。
Table 1. Various C-glycosyltransferase(CGT) for flavonoid C-glycoside synthesisEntry CGT Source Function Substrates Ref. 1 UGT708D1 Glycine max Flavone 6-C-glycosyltransferase Chrysin, Luteolin [18] 2 GtUF6CGT1 Gentiana triflora Flavone 6-C-glycosyltransferase Chrysin, Luteolin [18] 3 TcCGT1 Trollius chinensis Flavone 8-C-glycosyltransferase Apigenin, Luteolin, et al. [19] 4 UGT708A60 Hordeum vulgare (barley) Benzene 3-C-glycosyltransferase Phloretin [20] 5 WjGT1(UGT84A57) Wasabi (Eutrema japonicum) Flavone 6-C-glycosyltransferase, Flavonol 6-C-glycosyltransferase Apigenin, Luteolin, Kaempferol, Quercetin [21] 6 PlCGT Pueraria lobata Isoflavone 8-C-glycosyltransferase Isoflavones, Phloretin [22] 7 GtUF6CGT1 Gentiana triflora Flavone 6-C-glycosyltransferase Apigenin, Luteolin [23] 8 UGT708D1 Glycine max 2-hydroxyflavanone C-glycosyltransferase 2-Hydroxynaringenin, Phloretin [11] 9 DcaCGT Dendrobium
catenatum (D. officinale)Di-C-glycosylation 2-Hydroxynaringenin, Phloretin, Apigenin [17] 10 ScCGT1 Stenoloma chusanum Flavone 6-C-glycosyltransferase Phloretin, 2-Hydroxynaringenin [24] 11 ZmCGT1 Zea mays L. 2-Hydroxyflavanone C-glycosyltransferase 2-Hydroxyflavanone [10] 12 UGT708N1, UGT708N2 Nelumbo
nucifera Gaertn.Flavone 6-C-glycosyltransferase, Flavone 8-C-glycosyltransferase 2-Hydroxyflavanone, 2-Hydroxynaringenin [25] 13 PlUGT43 P. lobata Isoflavone 8-C-glycosyltransferase Daidzein, Genistein [26] 14 FcCGT (UGT708G1) Fortunella crassifolia Flavone 6-C-glycosyltransferase, Flavone 8-C-glycosyltransferase 2-Hydroxyflavanones, Dihydrochalcone [14] 15 CuCGT (UGT708G2) Citrus unshiu Flavone 6-C-glycosyltransferase, Flavone 8-C-glycosyltransferase 2-Hydroxyflavanones, Dihydrochalcone [14] 16 FeCGTa (UGT708C1) Fagopyrum esculentum
M. (buckwheat)C-glucosylation 2-Hydroxyflavanones, Dihydrochalcone, Trihydroxyacetophenones [27] 17 FeCGTb (UGT708C2) Fagopyrum esculentum
M. (buckwheat)C-glucosylation 2-Hydroxyflavanones, Dihydrochalcone, [27] 18 AbCGT Aloe barbadensis Flavone 8-C-glycosyltransferase Phloretin, 2-Hydroxynaringenin [28] 19 GgCGT Glycyrrhiza glabra Di-C-glycosyltransferase Phloretin, Nothofagin [29] 20 OsCGT Oryza sativa
(rice)Benzene 3-C-glycosyltransferase Phloretin [30] 21 TcCGT Trollius chinensis Flavone 8-C-glycosyltransferase Luteolin, Apigenin, Naringenin [31] 22 Gt6CGT Gentiana triflora Flavone 6-C-glycosyltransferase 3’-O-methylluteolin [32] 23 MiCGTb Mangifera indica Acylphloroglucinol 3-C- glycosyltransferase 2-O-glucosides [15] 24 OsUGT708A2, OsUGT708A3, OsUGT708A4 Japonica rice C-glucosyltransferase, C-Arabinosylation (OsUGT708A2), Phloretin, 2OH-Nar [33] 25 OsUGT708A1, OsUGT708A39, OsUGT708A40 Indica rice C-arabinosylation (OsUGT708A40) Phloretin [33] 1. 以黄酮类化合物为原料的合成策略
该策略是生物合成黄酮类碳苷最常用方式。从作用模式上，可以分为直接方式和间接方式。直接方式是指黄酮类化合物在CGT催化下与UDP-糖直接生成黄酮类碳苷；间接方式是指黄酮类化合物中间体首先在CGT催化下与UDP-糖生成中间体碳苷，然后在脱水酶或者黄酮合成酶作用下生成黄酮类化合物碳苷（最终产物）[34]。目前该策略合成方法研究内容包括筛选CGT[16, 20]、优化反应条件参数如pH、温度、反应浓度等以提高产量[18, 35−36]、研究CGT晶体结构以及生物催化机制[12, 19, 29]、组合酶催化技术[30]、固定酶催化平台以及连续流合成（表 1, entry 20）等[30]。
1.1 CGT催化黄酮类化合物与UDP-糖直接合成
它通常是利用黄酮类化合物与UDP-糖在CGT作用下，生成C6或者C8黄酮类糖碳苷。以根皮素（phloretin）底物为例，在CGT催化下，黄酮类化合物底物与UDP-糖结合，一步生成黄酮类碳苷（图1）。大部分黄酮碳苷的生物合成途径都是经2-羟基黄酮烷与UDP-糖在CGT催化下生成黄酮烷碳苷，然后在脱水酶（DH）催化下脱去一分子水，最终生成黄酮碳苷[37]。各种植物来源的CGT发挥不同的催化功能，包括生成黄酮C6葡萄糖碳苷的CGTs：Glycine max来源的UGT708D1（表1, entry 1）、Gentiana triflora来源的GtUF6CGT1（entry 2）、Eutrema japonicum来源的WjGT1（entry 5）、Gentiana triflora来源的GtUF6CGT1（entry 7）、Zea mays L.来源的ZmCGT1（entry 11）和UGT708N1（entry 12）；生成黄酮和异黄酮C8葡萄糖碳苷的CGTs：Trollius chinensis来源的TcCGT1（entry 3）、Pueraria lobata来源的PlCGT（entry 6）、UGT708N2（entry 12）、Pueraria lobata来源的PlUGT43（entry 13）和Aloe barbadensis来源的AbCGT（entry 18）；催化Phloretin转化为Nothofagin的CGTs：Hordeum vulgare来源的UGT708A60（entry 4）和Pueraria lobata来源的PlCGT（entry 6）；生成二碳苷的CGTs：Dendrobium catenatum中分离得到的DcaCGT（entry 9）、金橘中鉴定了两种C-糖基转移酶FcCGT、CuCGT（entries 14~15）和Glycyrrhiza glabra来源的GgCGT（entry 19）等等。
Hirade等[11]报道了Glycine max来源的UGT708D1是一种有效的2-羟基黄酮烷的C-葡萄糖转移酶，催化2-hydroxynaringenin转化为C8或者C6-葡萄糖碳苷，脱水后生成vitexin和isovitexin。其结构中His20、Asp85和Arg292为保守氨基酸序列，若以Ala取代Asp85或者Arg292，则丧失C-葡萄糖转移酶功能；若以Ala取代His20，则催化生成碳苷活性变成催化生成O-苷活性（表1, entry 8）。
Ni等[24]报道了Stenoloma chusanum来源的ScCGT1能有效催化phloretin、2-hydroxynaringenin和2-hydroxyeriodictyol生成相应碳苷。突变实验表明，保守氨基酸残基His26和Asp14协调发挥作用，使得ScCGT1发挥CGT活性。突变型ScCGT1比野生型ScCGT1活性更高，应用突变型ScCGT1使得Nothofagin的产量达到38 mg/L，是野生型的2.3倍。组合CjFNS I/F2H和ScCGT1-P164T多酶催化，成功实现了将柚皮素（naringenin）转化成牡荆素（vitexin）和异牡荆素（isovitexin）（表1，entry 10）。
Nagatomo 等[27]报道了从荞麦子叶中分离得到的两种同工酶FeCGTa (UGT708C1)和FeCGTb (UGT708C2)，它们能催化2-羟基黄酮烷、二氢查耳酮、三羟基苯乙酮及其类似结构发生C-糖基化反应。重组的FeCGTa (UGT708C1)和FeCGTb (UGT708C2)要比分离纯化的FeCGTa (UGT708C1)和FeCGTb (UGT708C2)对糖基底物范围更宽。结构研究表明，UGT708C1中氨基酸Asp382, Gln383, Thr151和Thr150残基对糖基识别非常重要，而氨基酸Phe130, Tyr102和Phe198在结合和稳定黄酮底物中起到了重要的作用（表1, entries 16~17）。从金橘中鉴定了两种C-糖基转移酶FcCGT和CuCGT，FcCGT和CuCGT有98%的氨基酸序列相同，表明在柑橘属植物中这些氨基酸序列是高度保守的。它们能催化2-羟基黄酮烷、二氢查耳酮和它们的单糖单碳苷合成单糖二碳苷。FcCGT和CuCGT对糖底物范围较宽，能催化UDP-glucose、UDP-xylose、UDP-galactose生成相应碳苷，但对UDP-glucuronic acid无效。他们还揭示了从日本芥末中分离得到的WjGT1能催化异肥皂草苷（isosaponarin）的生物合成机制。首先WjGT1催化apigenin合成6-C-葡萄糖苷，然后再生成4'-O-葡萄糖苷产物[38]。Eutrema japonicum来源的WjGT1能有效地催化apigenin、luteolin、kaempferol和quercetin的C6葡萄糖苷化合成，采用分步给料的方式，isovitexin产量高达172 mg/L，转化率为99% [21]。
He等[19]报道了第1个黄酮C8位选择性CGT。Trollius chinensis来源的TcCGT1，能区域选择性催化生成黄酮C8位葡萄糖碳苷。对其I94E和G284K两个残基进行突变，成功将C-glycosylation活性转变为O-glycosylation活性。Glycyrrhiza glabra来源的GgCGT能显著催化Phloretin生成双碳苷，对其G389K突变能改变di-C-glycosylation活性到mono-C-glycosylation活性[29]。揭示了夏佛塔苷和异夏佛塔苷的两步碳苷化生物合成途径。对14种单子叶植物和双子叶植物来源的CGTa和CGTb进行了克隆和表征，揭示出CGTa第一步对2-羟基黄酮烷苷元进行C-葡萄糖苷化和CGTb第二步对前一步的产物进行C-阿拉伯糖苷化的生物合成途径[16]。Pueraria lobata来源的PlCGT能有效催化异黄酮C8葡萄糖碳苷化反应和phloretin转化为nothofagin。通过氨基酸位点突变研究表明，PlCGT窄的底物结合口袋和相关的氨基酸残基Asn16~Asp124对其催化功能具有关键作用[22]。
Xie等[28]报道了Aloe barbadensis分离得到的一种混杂C-糖基转移酶AbCGT，展现出强大的糖基转移能力。它能对一系列的苯酚类物质，包括苯二酚、苯三酚以及苯胺、苯硫酚等衍生物发生糖基化反应，生成C-苷、O-苷、N-苷和S-苷。具有类似功能的CGT还包括UGT708A6（催化生成C-苷和O-苷）、MiCGT（催化生成C-苷、O-苷和N-苷）、GgCGT（催化生成C-苷、O-苷、S-苷和N-苷）等。之所以出现多样的催化活性，是由底物结构和酶催化位点的特点所决定的。
1.2 糖基转移酶与蔗糖合成酶协同催化（组合酶催化）
由于UDP-糖价格比较昂贵，限制了CGT直接催化UDP-糖与黄酮类化合物发生反应生成碳苷的普适性。协同催化策略是利用蔗糖合成酶与CGT联合催化，构建催化循环生成产物。以phloretin为例，首先蔗糖在蔗糖合成酶的作用下，释放出葡萄糖分子。葡萄糖分子与UDP结合，生成UDP-Glucose。然后在CGT催化下生成产物，同时释放出UDP参与下一阶段催化循环（图2）。
Liu等[39]利用TcCGT1/SUSy组合催化，大规模地制备orientin和vitexin。筛选条件，得出TcCGT1酶活性最高的条件是pH为9.0，温度为37 ℃。通过筛选不同的蔗糖合成酶，得出orientin最高的产量为
2324.4 mg/L，vitexin产量达到5524.1 mg/L。Liu等[40]首先报道了利用组合酶催化合成天然产物Nothofagin。为了解决原料phloretin水溶性差的问题，采用周期性投放原料phloretin方式，组合OsCGT和GmSUSy两种酶，leloir糖基转移酶参与合成天然产物nothofagin，合成效率高达20 g/L。其后又报道了nothofagin的酶法连续流合成[30]。利用蔗糖合成酶SuSy和碳苷转移酶OsCGT共同固定在阴离子载体上，SuSy不断地从蔗糖分解出葡萄糖，葡萄糖和UDP结合生成UDP-glucose，随后在OsCGT作用下将phloretin转化成nothofagin，转化率不低于95%，合成效率可以高达52 mg/mL。该课题组报道了固定酶的方式，Z-CGT/Z-SuSy联合运用合成nothofagin。其中Z-CGT/Z-SuSy可以连续循环使用[41]。
Qiu等[31]报道了TcCGT和GmSUS催化接力的方法合成orientin。筛选了酶的比例、pH、反应温度等影响因素，最后以7.09 g/L得到orientin，以5.05 g/L得到vitexin。采用CEP/Gt6CGT酶接力方法，通过调控诱导策略，控制乙酸堆积含量，同时采用纤维二糖磷酸化酶催化纤维二糖磷酸化产生葡萄糖1-磷酸酯增加生成UDP-葡萄糖的方法，使得异荭草苷产量能达到
1371 mg/L，转化率高达97.4%，产率高达81.5% （表1, entry 21）。他们还报道了O-methylation/C-glycosylation策略合成3'-O-methylisoorientin。首先通过筛选5种O-甲基转移酶，成功合成了3'-O-methylluteolin。随后以3'-O-methylluteolin为原料，通过Gt6CGT/ GmSUS联用方法，成功合成了3'-O-methylisoorientin。合成产量高达（226±8）mg/L，转化率为98%（表1, entry 22）。2. 黄酮类酚苷为原料合成黄酮类碳苷策略
由于自然界中黄酮类酚苷化合物也是主要次级代谢产物之一[42]，因此利用酚苷为原料原位转化为黄酮类化合物，然后在CGT催化下生成碳苷或者在CGT催化下直接将酚苷转化为碳苷，也成为了合成策略之一。以phlorizin为例，在OGT作用下，转化为Phloretin；随后在CGT催化下与UDP-Glucose作用，生成nothofagin (图3)。
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Figure 3. Enzymatic synthesis strategy of flavonoid C-glycosides from phenolic glycosidesGutmann等[43]报道了一种“一锅法”合成二氢查耳酮碳苷方法。他们选用PcOGT/OsCGT互补酶接力催化方法，以二氢查耳酮2'-酚苷phlorizin为原料，O-葡萄糖转移酶PcOGT首先将phlorizin催化裂解成葡萄糖和苷元phloretin，随后葡萄糖与加入的催化量UDP结合，形成UDP-Glucose，最后在OsCGT催化下，与苷元phloretin生成碳苷nothofagin。将OsCGT的I121D突变体应用在本体系中，无须PcOGT参与，单酶催化即可将phlorizin转化成nothofagin。
Chen等[15]报道了Mangifera indica来源的新型C-糖基转移酶MiCGTb，它能高效地把phlorizin转化为nothofagin。MiCGTb展现出葡萄糖氧苷水解活性，无须OGT酶的参与，只需催化量的UDP参与反应，单酶作用就可以把酚苷转化为碳苷，反应收率高达95%以上（表1, entry 23）。
3. 从头合成黄酮类碳苷策略（酶接力催化）
该策略从各种黄酮类化合物生源途径入手，运用各种酶接力催化来合成黄酮类碳苷化合物[1, 8]。黄酮类化合物生源途径中的酶包括酪氨酸裂解酶、4-香豆酰辅酶A连接酶、查耳酮合成酶、查耳酮异构酶、黄酮合成酶、黄酮醇合成酶、黄烷酮3-羟化酶等（图4）。此外，在UDP-糖的生源途径中，包括glucokinase (glk-Z. mobilisi), phosphoglucomutase (pgm2-B. licheniformis DSM13), glucose 1-phosphate uridylyltransferase (galU-E. coli K-12), glucose facilitator diffusion protein (glf-Z. mobilisi)等[3, 18]。
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Figure 4. Analysis of the biosynthetic pathway of isovitexin, vitexin, kaempferol 6-C-glucoside and kaempferol 8-C-glucosideChong等[44]报道了4种黄酮类碳苷化合物的从头生物合成。从酪氨酸出发，经过5步转化，分别是SeTAL, R4CL, PeCHS, MsCHI, PcFNS转化生成apigenin，然后apigenin在TcCGT催化下生成vitexin（93.9 mg/L）；在WjGT1催化下生成isovitexin（30.2 mg/L）。从酪氨酸出发，经过6步转化，生成kaempferol。分别在TcCGT和WjGT1催化下得到kaempferol 8-C-glucoside（38.6 mg/L）和Kaempferol 6-C-glucoside（14.4 mg/L）。
Wang等[37]报道了“一锅法”酶接力催化生成黄酮类碳苷化合物策略。通过筛选黄酮合成酶（FNS I）、C-糖基转移酶（TcCGT）和蔗糖合成酶（GmSUS），成功实现了从黄酮烷Naringenin到黄酮碳苷Vitexin的酶法合成，合成效率高达935.6 mg/L，转化率为78.7%。黄酮烷naringenin 的生源合成途径是由苯丙氨酸（phenylalanine）经phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI)参与生成的。
4. 总结与展望
总而言之，CGT的氨基酸序列结构对CGT的功能至关重要，而这些功能影响着催化活性，包括转化率、催化速率、催化位点的选择性、底物的适用性和选择性。上述3种生物合成策略中，大多数文献报道都是采用以黄酮类化合物为原料的合成策略。该策略普适性最广；黄酮类酚苷为原料合成黄酮类碳苷策略，提供了另外一种原料底物选择性。黄酮酚苷相较于黄酮而言，有更好的底物稳定性和水溶性。但开发组合酶催化体系或者单一酶催化体系将酚苷转化为碳苷面临较大的挑战，该策略文献报道较少；酶接力催化策略需要多酶的联合使用，成本较为昂贵，且需要解决酶兼容性等问题。3种策略相比，第1种策略应用最多，也是第2种和第3种策略中最后一步CGT催化黄酮类化合物合成碳苷所必须采取的方式。目前CGT催化黄酮类碳苷生物合成存在的主要问题有：CGT催化效率不高以及选择性不高、CGT的成本较高以及CGT的底物范围很窄。努力的方向包括：（1）利用突变技术开发出普适性强、廉价易得的CGT用于生物合成黄酮类碳苷化合物；（2）提高CGT的专一性、选择性和催化效率；（3）降低UDP-糖的成本和提高黄酮类碳苷的产量。期待功能强大（位点选择性高、底物范围广泛、产量高和耐受性强）的新型CGT被发现和改造出来，更好地应用于黄酮类碳苷化合物的生物合成。
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Figure 3. Enzymatic synthesis strategy of flavonoid C-glycosides from phenolic glycosides
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Figure 4. Analysis of the biosynthetic pathway of isovitexin, vitexin, kaempferol 6-C-glucoside and kaempferol 8-C-glucoside
Table 1 Various C-glycosyltransferase(CGT) for flavonoid C-glycoside synthesis
Entry CGT Source Function Substrates Ref. 1 UGT708D1 Glycine max Flavone 6-C-glycosyltransferase Chrysin, Luteolin [18] 2 GtUF6CGT1 Gentiana triflora Flavone 6-C-glycosyltransferase Chrysin, Luteolin [18] 3 TcCGT1 Trollius chinensis Flavone 8-C-glycosyltransferase Apigenin, Luteolin, et al. [19] 4 UGT708A60 Hordeum vulgare (barley) Benzene 3-C-glycosyltransferase Phloretin [20] 5 WjGT1(UGT84A57) Wasabi (Eutrema japonicum) Flavone 6-C-glycosyltransferase, Flavonol 6-C-glycosyltransferase Apigenin, Luteolin, Kaempferol, Quercetin [21] 6 PlCGT Pueraria lobata Isoflavone 8-C-glycosyltransferase Isoflavones, Phloretin [22] 7 GtUF6CGT1 Gentiana triflora Flavone 6-C-glycosyltransferase Apigenin, Luteolin [23] 8 UGT708D1 Glycine max 2-hydroxyflavanone C-glycosyltransferase 2-Hydroxynaringenin, Phloretin [11] 9 DcaCGT Dendrobium
catenatum (D. officinale)Di-C-glycosylation 2-Hydroxynaringenin, Phloretin, Apigenin [17] 10 ScCGT1 Stenoloma chusanum Flavone 6-C-glycosyltransferase Phloretin, 2-Hydroxynaringenin [24] 11 ZmCGT1 Zea mays L. 2-Hydroxyflavanone C-glycosyltransferase 2-Hydroxyflavanone [10] 12 UGT708N1, UGT708N2 Nelumbo
nucifera Gaertn.Flavone 6-C-glycosyltransferase, Flavone 8-C-glycosyltransferase 2-Hydroxyflavanone, 2-Hydroxynaringenin [25] 13 PlUGT43 P. lobata Isoflavone 8-C-glycosyltransferase Daidzein, Genistein [26] 14 FcCGT (UGT708G1) Fortunella crassifolia Flavone 6-C-glycosyltransferase, Flavone 8-C-glycosyltransferase 2-Hydroxyflavanones, Dihydrochalcone [14] 15 CuCGT (UGT708G2) Citrus unshiu Flavone 6-C-glycosyltransferase, Flavone 8-C-glycosyltransferase 2-Hydroxyflavanones, Dihydrochalcone [14] 16 FeCGTa (UGT708C1) Fagopyrum esculentum
M. (buckwheat)C-glucosylation 2-Hydroxyflavanones, Dihydrochalcone, Trihydroxyacetophenones [27] 17 FeCGTb (UGT708C2) Fagopyrum esculentum
M. (buckwheat)C-glucosylation 2-Hydroxyflavanones, Dihydrochalcone, [27] 18 AbCGT Aloe barbadensis Flavone 8-C-glycosyltransferase Phloretin, 2-Hydroxynaringenin [28] 19 GgCGT Glycyrrhiza glabra Di-C-glycosyltransferase Phloretin, Nothofagin [29] 20 OsCGT Oryza sativa
(rice)Benzene 3-C-glycosyltransferase Phloretin [30] 21 TcCGT Trollius chinensis Flavone 8-C-glycosyltransferase Luteolin, Apigenin, Naringenin [31] 22 Gt6CGT Gentiana triflora Flavone 6-C-glycosyltransferase 3’-O-methylluteolin [32] 23 MiCGTb Mangifera indica Acylphloroglucinol 3-C- glycosyltransferase 2-O-glucosides [15] 24 OsUGT708A2, OsUGT708A3, OsUGT708A4 Japonica rice C-glucosyltransferase, C-Arabinosylation (OsUGT708A2), Phloretin, 2OH-Nar [33] 25 OsUGT708A1, OsUGT708A39, OsUGT708A40 Indica rice C-arabinosylation (OsUGT708A40) Phloretin [33] 
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