薛飛燕，楊明峰，馬蘭青

微生物法合成紅景天苷

薛飛燕，楊明峰，馬蘭青

北京農(nóng)學(xué)院 生物與資源環(huán)境學(xué)院 農(nóng)業(yè)部華北都市農(nóng)業(yè)重點(diǎn)實(shí)驗(yàn)室，北京 102206

紅景天苷是紅景天屬植物的主要有效成分之一，具有耐缺氧、抗輻射、抗疲勞、抗腫瘤、降血糖、提高免疫力等多重功效。隨著其需求量的日益增加和植物資源的不斷減少，微生物法合成紅景天苷因具有周期短和易調(diào)控等優(yōu)勢(shì)而倍受關(guān)注。目前微生物法合成紅景天苷尚處于基礎(chǔ)研發(fā)階段，為了方便相關(guān)領(lǐng)域研究者系統(tǒng)了解其研究現(xiàn)狀和探討其未來發(fā)展方向，文中對(duì)紅景天苷生物合成途徑、糖基轉(zhuǎn)移酶、野生菌/天然酶資源和工程菌/重組酶體系進(jìn)行了綜述。

紅景天苷，生物合成途徑，野生菌/天然酶，工程菌/重組酶

紅景天苷 (Salidroside) 亦稱紅景天甙，其化學(xué)結(jié)構(gòu)式為酪醇8--β-D-葡萄糖苷 (C14H20O7)，是以酪醇 (4-羥基苯乙醇，Tyrosol，C8H10O2) 為苷元的醇羥基與尿苷二磷酸葡萄糖 (Uridine diphosphate glucose, UDP-glucose, C15H24N2O17P2) 半縮醛羥基脫水后形成的糖苷 (圖1)[1-2]。作為紅景天屬藥用植物的主要活性成分，紅景天苷被證實(shí)具有耐缺氧、抗輻射、抗疲勞、抗腫瘤、降血糖、提高免疫力和記憶力等重要生理功效[1,3]。隨著人們對(duì)紅景天苷藥理作用認(rèn)識(shí)的不斷深入，其需求量與日俱增。

最初人們依靠野生紅景天屬植物提取紅景天苷，深受資源有限和含量低的約束[4]；后來利用組織培養(yǎng)和細(xì)胞懸浮培養(yǎng)等技術(shù)克服了野生資源的不足[5-6]，可仍然存在生產(chǎn)周期長(zhǎng)、產(chǎn)量低等問題；

近些年，相關(guān)研究者通過不斷嘗試和比較各種方法，普遍認(rèn)為微生物法合成紅景天苷具有潛在工業(yè)化應(yīng)用價(jià)值[7-8]。

1 紅景天苷生物合成途徑

圖2 UDP-葡萄糖的生物合成途徑(參照陳圣等[9])

微生物法合成植物天然產(chǎn)物在實(shí)現(xiàn)產(chǎn)業(yè)化的進(jìn)程中面臨的挑戰(zhàn)之一就是理清其生物合成途徑[7]。如圖1所示，紅景天苷生物合成途徑的最后一步反應(yīng)已經(jīng)被證實(shí)為尿苷二磷酸葡萄糖基轉(zhuǎn)移酶 (Uridine diphosphate glucosyltransferase, UGT) 催化底物酪醇與尿苷二磷酸葡萄糖合成紅景天苷[6-7]。反應(yīng)中涉及的尿苷二磷酸葡萄糖 (UDP-glucose) 其生物合成途徑研究較成熟，屬于初級(jí)代謝范疇，微生物合成UDP-glucose通常選擇如圖2的代謝途徑，通過弱化競(jìng)爭(zhēng)途徑或過表達(dá)關(guān)鍵酶 (如PGM和GalU等) 保障其供應(yīng)量[9-11]。因此，關(guān)于紅景天苷生物合成途徑的研究，人們一方面致力于途徑中催化反應(yīng)的關(guān)鍵酶UGT相關(guān)的研究：如本課題組率先獲得催化活性較高的植物UGT73B6[12]、UGT72B14[2]；隨后通過調(diào)研植物UGT超家族晶體結(jié)構(gòu)，介紹了UGTs的整體結(jié)構(gòu)特點(diǎn) (如保守的PSPG結(jié)構(gòu)單元) 以及蛋白與底物相互作用的細(xì)節(jié) (如口袋結(jié)構(gòu)結(jié)合糖基供體)[13]；Fan等根據(jù)植物UGTs的PGSG結(jié)構(gòu)特征進(jìn)一步篩選獲得高活性微生物UGT[14]。另一方面，研究者還重點(diǎn)關(guān)注了底物酪醇 (Tyrosol) 生成途徑，研究表明其來源于莽草酸途徑 (Shikimate pathway) 所生成的阿羅酸 (Arogenate)，阿羅酸合成酪醇的生物途徑因體系差異而有所不同[15-18]：根據(jù)植物芳香族天然產(chǎn)物代謝特點(diǎn)和規(guī)律，研究者最初認(rèn)為酪醇來源于苯丙氨酸解氨 (PAL) 途徑 (圖3中支路①) 和酪氨酸脫羧 (TyrDC) 途徑 (圖3中支路③)[18]，如Keski-Saaris等和Hu等證明了PAL的活性對(duì)紅景天苷積累有重要影響[19-20]，Landtag等和Lan等研究均表明TyrDC的活性對(duì)紅景天苷的積累很重要[21-22]，本課題組前期工作確認(rèn)高山紅景天植物中其苷元酪醇的主要合成途徑為酪氨酸脫羧途徑，其中TyrDC為關(guān)鍵酶和限速酶[23]；最近，Michael等發(fā)現(xiàn)紅景天植物中含有一種依賴于磷酸吡哆醛的羥基苯乙醛合酶 (HPAAS) 可直接催化酪氨酸獲得4-羥基苯乙醛然后還原生成酪醇 (圖3中支路④)[24]；另外，釀酒酵母以酪氨酸為底物經(jīng)過轉(zhuǎn)氨脫羧反應(yīng)合成酪醇機(jī)制很早就有報(bào)道，符合圖3中支路⑤TAT途徑[25]，Satoh等則在大腸桿菌中同時(shí)構(gòu)建了圖3中支路③和⑤實(shí)現(xiàn)了酪醇的有效合成[26]；按照?qǐng)D3中支路②TAL途徑合成酪醇的案例暫未見報(bào)道，但利用TAL催化活性實(shí)現(xiàn)由酪氨酸 (Tyrosine) 合成4-香豆酸/對(duì)羥基肉桂酸/對(duì)羥基苯丙烯酸 (-coumaric acid) 已有研究，如Kim等在闡述類苯乙醇合成現(xiàn)狀時(shí)介紹了TAL催化合成4-香豆酸進(jìn)而生產(chǎn)咖啡酸苯乙酯的途徑[8]；Rodriguez 等研究表明在釀酒酵母中超量表達(dá)約氏黃桿菌的TAL能夠增加4-香豆酸合成量[27]；Vannelli等和Vargas-Tah等研究均表明利用粘紅酵母雙功能酶PAL/TAL可以實(shí)現(xiàn)對(duì)羥基肉桂酸的有效合成[28-29]。為此，本課題組在發(fā)現(xiàn)粘紅酵母代謝產(chǎn)物中有酪醇組分后已經(jīng)開展了其酪醇合成途徑包括圖3中支路①和②的驗(yàn)證工作，以期更進(jìn)一步闡述酪醇的生物合成途徑，為建立新的微生物法合成紅景天苷體系提供基礎(chǔ)。

圖3 酪醇可能的生物合成途徑

2 微生物法合成紅景天苷

2.1 野生菌/天然酶合成紅景天苷

利用野生菌或天然酶合成紅景天苷的文獻(xiàn)報(bào)道中涉及的微生物以真菌為主 (表1)。最初的研究思路是以紅景天植物浸出物為培養(yǎng)基成分發(fā)酵獲得微生物菌體后提取酶液進(jìn)行體外催化酪醇合成紅景天苷：如賈艷萍等利用犁頭霉sp.的粗提酶液催化15 g/L的酪醇合成約1.5 g/L的紅景天苷[30]；Zhang等利用黑曲霉提純酶催化1.5%的酪醇合成紅景天苷可達(dá)10%[31]；王夢(mèng)亮等從紅景天植物根系土壤中篩選獲得微生物菌株米曲霉能夠利用5 g/L的酪醇合成0.7 g/L的紅景天苷[32]。后來研究者發(fā)現(xiàn)可以利用雙菌株共培養(yǎng) (Coculture) 的協(xié)同效應(yīng)或微生物細(xì)胞融合 (Cell fusion) 的方式來提高紅景天苷的含量：宋偉舟等利用雙菌株協(xié)同液體發(fā)酵紅景天使其紅景天苷含量提高86.29%[33]；馮敏等利用細(xì)胞融合雙親菌株固體發(fā)酵大花紅景天粉末，使其紅景天苷含量提高140%[34]。近期有利用植物內(nèi)生真菌合成紅景天苷的報(bào)道：如曲霉和鐮刀霉[35]，深色有隔內(nèi)生真菌培養(yǎng)7 d后可將紅景天苷產(chǎn)量提高到2.339 g/L[36]。

2.2 工程菌/重組酶合成紅景天苷

2.2.1 糖基化酪醇合成紅景天苷

隨著基因工程技術(shù)的發(fā)展、紅景天苷生物合成途徑的不斷明晰和代謝途徑中限速酶的逐步明確，利用微生物工程菌或重組酶合成紅景天苷的研究取得了突破性進(jìn)展，特別是以利用模式微生物 (如大腸桿菌和釀酒酵母) 為宿主的研究發(fā)展迅速 (表2)。2011年Yu等報(bào)道了3個(gè)高山紅景天UGTs在大腸桿菌中實(shí)現(xiàn)了重組表達(dá)，重組酶體外酶促反應(yīng)均獲得了紅景天苷產(chǎn)物[2]。2016年，Xue等利用密碼子優(yōu)化 (Codon optimization) 的辦法實(shí)現(xiàn)了UGT72B14在大腸桿菌的高效表達(dá)，并利用分批-補(bǔ)料 (Fed-batch) 的策略得到微生物紅景天苷產(chǎn)量為6.7 mg/L[37]。2017年，F(xiàn)an等通過基因挖掘進(jìn)一步獲得高活性的地衣芽胞桿菌UGT1并構(gòu)建了重組大腸桿菌，工程菌全細(xì)胞催化24 h后可將紅景天苷的產(chǎn)量提高到1.04 g/L[14]。

表1 野生菌/天然酶合成紅景天苷

PDB: Potato dextrose broth.

2.2.2 從頭合成紅景天苷

為了進(jìn)一步增強(qiáng)微生物合成紅景天苷的應(yīng)用可行性，人們充分利用基因工程、代謝工程和發(fā)酵工程技術(shù)發(fā)展了紅景天苷以葡萄糖為底物的從頭合成 () 技術(shù)。2014年，Bai等通過引入釀酒酵母ARO10和高山紅景天UGT73B6等關(guān)鍵酶，采用菌體生長(zhǎng)和產(chǎn)物合成在不同培養(yǎng)基分段發(fā)酵重組大腸桿菌的策略，以葡萄糖為底物可得紅景天苷產(chǎn)量為56.9 mg/L[38]。2017年，Chung等通過引入香芹AAS和擬南芥UGT85A1，以葡萄糖為底物同樣采用分段培養(yǎng)重組大腸桿菌可使紅景天苷的產(chǎn)量提高到288 mg/L[39]。2018年，Liu等通過引入釀酒酵母ARO10、畢赤酵母KDC4和擬南芥UGT85A1等關(guān)鍵基因構(gòu)建兩株大腸桿菌，采用兩株菌的共培養(yǎng)方式和分批-補(bǔ)料的調(diào)控策略，以葡萄糖和木糖為底物發(fā)酵129 h可以將紅景天苷的產(chǎn)量進(jìn)一步提高到6.03 g/L[40]。

表2 工程菌/重組酶合成紅景天苷

:; UGT: Uridine diphosphate dependent glycosyltransferase;c:; AAS: Aromatic aldehyde synthase;:;:;:;:; ARO10: Pyruvate decarboxylase; KDC: Decarboxylase;:; 4HPAAS: 4-hydroxyphenylacetaldehyde synthase; T8GT: Tyrosol:UDP-glucose 8-O-glucosyltransferase; “+”: Substrate was fed during fermentation, but the feeding titer was not reported.

隨著大腸桿菌合成紅景天苷的技術(shù)不斷成熟，人們又開始致力于釀酒酵母合成紅景天苷體系的研發(fā)。Torrens-Spence等通過引入紅景天4HPAAS和T8GT，采用密碼子優(yōu)化策略在釀酒酵母中構(gòu)建了紅景天苷合成途徑，獲得產(chǎn)量為1.5 mg/L[24]。Jiang等利用基因整合技術(shù)在釀酒酵母中引入香芹AAS和擬南芥UGT85A1，通過分批-補(bǔ)料的發(fā)酵調(diào)控策略發(fā)酵168 h同樣可實(shí)現(xiàn)紅景天苷的產(chǎn)量為732.5 mg/L[41]。

3 展望

紅景天苷作為紅景天的有效成分，其抗缺氧、抗疲勞、抗衰老、防輻射、增強(qiáng)心血管系統(tǒng)功能及對(duì)腫瘤的抑制等功效越來越受到關(guān)注。國(guó)內(nèi)外相關(guān)知識(shí)產(chǎn)權(quán)也由初期集中在植物紅景天苷提取方法及綜合應(yīng)用[42-43]，逐漸延伸至紅景天苷相關(guān)產(chǎn)品制備方法及針對(duì)性應(yīng)用效果[44-46]，并進(jìn)一步擴(kuò)展至生物酶催化法[47-49]及微生物發(fā)酵法[50-51]合成紅景天苷的技術(shù)研發(fā)。我國(guó)植物紅景天苷提取及制劑開發(fā)技術(shù)已經(jīng)邁向產(chǎn)業(yè)發(fā)展階段 (如公安部昆明警犬基地公布了利用紅景天苷制備工作犬用抗高原反應(yīng)藥物組合物的方法與應(yīng)用，四川康美保寧制藥有限公司公布了一種乙醇快速提取紅景天的方法，西安惠博生物科技有限公司公布了固定化酶催化制備紅景天苷的方法等)[52-54]，而微生物法合成紅景天苷目前還處于基礎(chǔ)研發(fā)階段，雖然國(guó)內(nèi)在“高活性酶和潛力宿主菌的篩選”、“關(guān)鍵基因和代謝途徑改造”、“培養(yǎng)方式和調(diào)控模式設(shè)計(jì)”等方面的研究處于領(lǐng)先地位[36,40-41]，但需要繼續(xù)攻克高效合成及高效利用等瓶頸技術(shù)。

一方面要充分發(fā)掘利用更多生物資源如植物基因資源構(gòu)建類似于圖3中支路②或④的高效途徑；或者同時(shí)啟動(dòng)多條途徑如圖3中支路①和②提高工作效率；或者借鑒UDP-葡萄糖原位再生體系增強(qiáng)糖苷合成思路提高紅景天苷合成效率[55]。

另一方面要充分利用現(xiàn)代生物學(xué)技術(shù)如Liu等利用基因組分析和合成生物學(xué)手段構(gòu)建了酵母高效合成燈盞花素[56]，同理可以充分利用基因編輯技術(shù)、合成生物學(xué)和生物信息學(xué)手段增強(qiáng)紅景天苷微生物細(xì)胞工廠運(yùn)行效率；或者利用代謝組學(xué)和微生物發(fā)酵聯(lián)產(chǎn)技術(shù)實(shí)現(xiàn)紅景天苷和其他活性成分 (如絡(luò)緦及其衍生物[6]、香豆素[16]、羥基酪醇[39]或淫羊藿次苷D2[24,38,51]等) 聯(lián)產(chǎn)以提高紅景天苷生產(chǎn)和應(yīng)用效率。

綜上，微生物法合成紅景天苷已有良好基礎(chǔ)，相信通過相關(guān)領(lǐng)域研究者深度挖掘豐富的生物資源和充分利用現(xiàn)代生物學(xué)技術(shù)，能夠早日實(shí)現(xiàn)產(chǎn)業(yè)化。

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Microbial synthesis of salidroside

Feiyan Xue, Mingfeng Yang, and Lanqing Ma

Key Laboratory of Urban Agriculture (North China), Ministry of Agriculture,College of Bioscience and Resource Environment, Beijing University of Agriculture, Beijing 102206, China

Salidroside, as one of the main active ingredients ofplant, has the effects of anti-hypoxia, anti-radiation, anti-fatigue, anti-tumor, hypoglycemia and improving immunity. With the increasing demand for salidroside and the decreasing of plant resources, microbial production of salidroside has attracted much attention due to its advantages of short period and easy controlling. At present, microbial production of salidroside is still at the basic research stage. In order to make it easier for researchers to understand the advances of microbial synthesis of salidroside, the biosynthesis pathways, uridine diphosphate glucosyltransferases, wild strain/natural enzymes and engineered strain/recombinant enzymes were reviewed.

salidroside, biosynthesis pathways, wild strains or natural enzymes, engineered strains or recombinant enzymes

November 1, 2018;

February 27, 2019

National Natural Science Foundation of China (Nos. 21606020, 31370674).

Lanqing Ma. Tel/Fax: +86-10-80797305; E-mail: lanqingma@bua.edu.cn

國(guó)家自然科學(xué)基金(Nos. 21606020, 31370674) 資助

薛飛燕, 楊明峰, 馬蘭青. 微生物法合成紅景天苷. 生物工程學(xué)報(bào), 2019, 35(7): 1184–1192.

Xue FY, Yang MF, Ma LQ. Microbial synthesis of salidroside. Chin J Biotech, 2019, 35(7): 1184–1192.

(本文責(zé)編 陳宏宇)