祖先序列重建结合计算筛选的黄酮合酶I挖掘与优化用于芹菜素合成

谢东奇; 张顺志; 邵茜; 朱国梁; 丁霈希; 温霖; 郑良振; 王晟

doi:10.12211/2096-8280.2026-059

您当前的位置：

首页 >

文章列表页 >

祖先序列重建结合计算筛选的黄酮合酶I挖掘与优化用于芹菜素合成

研究论文 | 更新时间：2026-10-15

- 祖先序列重建结合计算筛选的黄酮合酶I挖掘与优化用于芹菜素合成
- Discovery and Optimization of Flavone Synthase I for Apigenin Synthesis by Combining Ancestral Sequence Reconstruction with Computational Screening
- 合成生物学 2026年页码：1-23
- 作者机构：
  
  1.上海智峪生物科技有限公司，上海 201210
  2.中国新时代控股集团有限公司，北京 100034
- 作者简介：
  
  谢东奇（1994—），男，硕士，高级工程师，现任上海智峪生物科技有限公司研发经理。研究方向为合成生物学驱动的工业酶创制，涉及定向进化、蛋白制备及生物过程工程，聚焦高效生物催化剂开发与产业化工艺研究。 E-mail：xiedongqi@zelixir.com。
- 基金信息：
- DOI：10.12211/2096-8280.2026-059
  中图分类号： Q814
- 收稿：2026-07-01，
  
  修回：2026-10-10，
  
  网络首发：2026-10-15，
- 稿件说明：
移动端阅览
谢东奇，张顺志，邵茜，朱国梁，丁霈希，温霖，郑良振，王晟. 祖先序列重建结合计算筛选的黄酮合酶I挖掘与优化用于芹菜素合成［J］. 合成生物学， 2026， 7. DOI： 10.12211/2096-8280.2026-059

XIE Dongqi， ZHANG Shunzhi， SHAO Qian， ZHU Guoliang， DING Peixi， WEN Lin， ZHENG Liangzhen， WANG Sheng. Discovery and Optimization of Flavone Synthase I for Apigenin Synthesis by Combining Ancestral Sequence Reconstruction with Computational Screening［J］. Synthetic Biology Journal， 2026， 7. DOI： 10.12211/2096-8280.2026-059
谢东奇，张顺志，邵茜，朱国梁，丁霈希，温霖，郑良振，王晟. 祖先序列重建结合计算筛选的黄酮合酶I挖掘与优化用于芹菜素合成［J］. 合成生物学， 2026， 7. DOI： 10.12211/2096-8280.2026-059 DOI：

XIE Dongqi， ZHANG Shunzhi， SHAO Qian， ZHU Guoliang， DING Peixi， WEN Lin， ZHENG Liangzhen， WANG Sheng. Discovery and Optimization of Flavone Synthase I for Apigenin Synthesis by Combining Ancestral Sequence Reconstruction with Computational Screening［J］. Synthetic Biology Journal， 2026， 7. DOI： 10.12211/2096-8280.2026-059 DOI：

摘要

黄酮合酶I（FNS-I）催化柚皮素C2-C3位脱氢生成芹菜素，是酶法合成黄酮的关键步骤。为拓展有限的FNS-I序列空间并提高催化活性，本研究以欧芹PcFNS-I为起点，在伞形科内收集22条FNS-I候选现生序列，经祖先序列重建（ASR）获得AncFNS-I-48和AncFNS-I-50，并以CataPro排序结合物种代表性选取3条天然同源酶，连同PcFNS-I构成6个候选酶，以等量纯化蛋白测定相对活性；随后以AncFNS-I-48为亲本，利用PSSM、Pythia和CataPro筛选29个单点突变体，配套建立

380

终点法高通量筛选流程。在3次平行测定中，以AncFNS-I-48活性归一化为1.00，PcFNS-I、AncFNS-I-50、CcFNS-I、DcFNS-I和CmFNS-I的相对活性分别为0.63、0.63、0.26、0.06和0.05。50 mmol/L磷酸盐缓冲液中的pH响应表明，AncFNS-I-48在pH 6.0-8.5范围内保持较高产物生成能力。结合柚皮素在偏碱性条件下较好的溶解性及后续较高底物负荷与工艺评价的需要，后续条件优化统一在pH 8.0下进行；25–30 ℃更适合长时间反应，250 μmol/L FeSO₄时芹菜素生成量最高，为2.674 ± 0.101 mmol/L。采用

380

终点法，以SDS-PAGE目标条带净积分灰度校正的相对初始反应速率评价29个突变体；V159E和H261F分别为WT的3.06 ± 0.26和1.50 ± 0.04倍，均显著高于WT（p

0.01）。在采用HPLC检测的20 mL反应体系验证中，以WT归一化为1.00，I115V、V159E、M230D和N297D分别为2.15 ± 0.15、2.14 ± 0.11、2.02 ± 0.10和1.88 ± 0.13，均显著高于WT；M85L为0.35 ± 0.03，显著低于WT（p

0.01）。综合两类指标，本文优先推荐低酶投量下相对初始反应速率最高的V159E；I115V在20 mL制备级反应中的产物生成指标均值最高，但与V159E接近，且现有分析未检验二者间差异，因此将其作为侧重制备级产物生成的备选。本研究建立了“谱系序列收集-祖先重建-AI预测-计算突变-高通量筛选”的酶工程路线，为FNS-I及2-ODD家族酶的挖掘与改造提供方法参考。

Abstract

Flavone synthase I (FNS-I) catalyzes C2-C3 dehydrogenation of naringenin to form apigenin and is a key enzyme for enzymatic flavone synthesis. To expand the limited FNS-I sequence space and improve catalytic activity

22 extant Apiaceae FNS-I candidate sequences were collected

using parsley PcFNS-I as the reference. Ancestral sequence reconstruction (ASR) yielded AncFNS-I-48 and AncFNS-I-50

while CataPro ranking combined with lineage coverage identified three natural homologs; together with PcFNS-I

these formed six candidates that were evaluated with equal amounts of purified protein. AncFNS-I-48 was then used as the parent for PSSM-

Pythia-

and CataPro-guided prioritization of 29 single-site variants and

380

endpoint high-throughput screening. Across three independent biological replicates

with AncFNS-I-48 normalized to 1.00

the relative activities of PcFNS-I

AncFNS-I-50

CcFNS-I

DcFNS-I

and CmFNS-I were 0.63

0.63

0.26

0.06

and 0.05

respectively. Profiling in 50 mmol/L phosphate buffer showed that AncFNS-I-48 maintained comparatively high apigenin production from pH 6.0 to 8.5. Considering the improved apparent solubility of naringenin under mildly alkaline conditions and the needs of subsequent high-substrate-load and process evaluation

condition optimization was conducted at pH 8.0. Temperatures of 25–30 °C favored long-term product accumulation

and 250 μmol/L FeSO₄ gave the highest apigenin concentration (2.674 ± 0.101 mmol/L). At low enzyme input

380

endpoint measurements combined with SDS-PAGE target-band integrated-density correction were used to evaluate the relative initial reaction rates of 29 single-site variants. V159E and H261F reached 3.06 ± 0.26 and 1.50 ± 0.04 times the WT level

respectively

and were significantly higher than WT (p

0.01). In the HPLC-quantified 20 mL reaction validation system with higher enzyme input

I115V

V159E

M230D

and N297D reached 2.15 ± 0.15

2.14 ± 0.11

2.02 ± 0.10

and 1.88 ± 0.13 relative to WT

respectively

whereas M85L was significantly lower than WT (0.35 ± 0.03

0.01). Considering both metrics

V159E is the preferred variant because it showed the highest relative initial reaction rate at low enzyme input

whereas I115V remains an alternative for

preparative product formation

with a marginally higher mean in the 20 mL assay and no direct statistical comparison between the two variants. This study establishes an integrated enzyme-engineering route combining lineage-focused sequence collection

ancestral reconstruction

AI prediction

computational mutagenesis

and high-throughput screening

providing a methodological reference for the discovery and engineering of FNS-I and other 2-ODD enzymes.

关键词

Keywords

references

Salehi B , Venditti A , Sharifi-Rad M , et al . The therapeutic potential of apigenin [J ] . International Journal of Molecular Sciences , 2019 , 20 ( 6 ): 1305 .

Hostetler GL , Ralston RA , Schwartz SJ . Flavones: food sources, bioavailability, metabolism, and bioactivity [J ] . Advances in Nutrition , 2017 , 8 ( 3 ): 423 - 435 .

Williams CA , Grayer RJ . Anthocyanins and other flavonoids [J ] . Natural Product Reports , 2004 , 21 ( 4 ): 539 - 573 .

Winkel-Shirley B . Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology [J ] . Plant Physiology , 2001 , 126 ( 2 ): 485 - 493 .

He M , Min JW , Kong WL , et al . A review on the pharmacological effects of vitexin and isovitexin [J ] . Fitoterapia , 2016 , 115 : 74 - 85 .

Shukla S , Gupta S . Apigenin: a promising molecule for cancer prevention [J ] . Pharmaceutical Research , 2010 , 27 ( 6 ): 962 - 978 .

Madunić J , Madunić IV , Gajski G , et al . Apigenin: a dietary flavonoid with diverse anticancer properties [J ] . Cancer Letters , 2018 , 413 : 11 - 22 .

苑林宏 , 夏薇 , 赵秀娟 , 等 . 芹菜素通过抑制PKB/Akt激酶活性诱导人胃癌细胞凋亡 [J ] . 科学通报 , 2007 , 52 ( 13 ): 1525 - 1529 .

YUAN L H , XIA W , ZHAO X J , et al . Apigenin induces apoptosis of human gastric cancer cells by inhibiting PKB/Akt kinase activity [J ] . Science Bulletin , 2007 , 52 ( 13 ): 1525 - 1529 .

蔡婧 , 刘安文 , 赵向丽 , 等 . 芹菜素对肝癌细胞生长及基因表达的影响 [J ] . 世界华人消化杂志 , 2010 , 18 ( 6 ): 542 - 549 .

CAI J , LIU A W , ZHAO X L , et al . Effects of apigenin on hepatocellular carcinoma cell growth and gene expression [J ] . World Chinese Journal of Digestology , 2010 , 18 ( 6 ): 542 - 549 .

宋晓童 . 8-(6''-伞形酮)-芹菜素衍生物的抗糖尿病活性研究 [D ] . 天津 : 天津科技大学 , 2019 .

SONG X T . Antidiabetic activity of 8-(6''-umbelliferone)-apigenin derivatives [D ] . Tianjin : Tianjin University of Science and Technology , 2019 .

周云峰 , 罗飞 , 付欢欢 , 等 . 芹菜素对肺癌细胞氨肽酶N抑制作用机制的研究 [J ] . 解放军医药杂志 , 2015 , 27 ( 8 ): 21 - 24 .

ZHOU Y F , LUO F , FU H H , et al . Mechanism of apigenin-mediated inhibition of aminopeptidase N in lung cancer cells [J ] . Medical & Pharmaceutical Journal of Chinese People's Liberation Army , 2015 , 27 ( 8 ): 21 - 24 .

闻永举 , 申秀丽 . 柚皮苷半合成芹菜素的研究 [J ] . 江苏农业科学 , 2012 , 40 ( 5 ): 189 - 190 .

WEN Y J , SHEN X L . Semisynthesis of apigenin from naringin [J ] . Jiangsu Agricultural Sciences , 2012 , 40 ( 5 ): 189 - 190 .

陈平 , 韩俊杰 , 刘涛 , 等 . 水溶性芹菜素的合成 [J ] . 食品科学 , 2009 , 30 ( 18 ): 69 - 72 .

CHEN P , HAN J J , LIU T , et al . Synthesis of water-soluble apigenin [J ] . Food Science , 2009 , 30 ( 18 ): 69 - 72 .

陈新 , 李平 . 新的半合成芹菜素制备工艺 [P ] . 中国发明专利 , CN 200410000704 , 2004 .

CHEN X , LI P . A new semisynthetic process for preparing apigenin [P ] . Chinese invention patent , CN 200410000704 , 2004 .

肖金霞 , 靳莎 , 杨雪峰 . 天然产物芹菜素的化学全合成 [J ] . 生物技术世界 , 2014 ( 9 ): 10 - 11 .

XIAO J X , JIN S , YANG X F . Total chemical synthesis of the natural product apigenin [J ] . Biotechnology World , 2014 ( 9 ): 10 - 11 .

李凤霞 . 查尔酮异构酶基因过表达对新疆雪莲类黄酮生物合成的调控 [D ] . 北京 : 中国科学院植物研究所 , 2006 .

LI F X . Regulation of flavonoid biosynthesis in Saussurea involucrata by overexpression of chalcone isomerase [D ] . Beijing : Institute of Botany, Chinese Academy of Sciences , 2006 .

李春霞 . 黄酮类化合物芹菜素酶法合成体系的建立及条件优化 [D ] . 扬州 : 扬州大学 , 2017 .

LI C X . Establishment and optimization of an enzymatic system for apigenin synthesis [D ] . Yangzhou : Yangzhou University , 2017 .

Martens S , Mithofer A . Flavones and flavone synthases [J ] . Phytochemistry , 2005 , 66 ( 20 ): 2399 - 2407 .

Ayabe S , Akashi T . Cytochrome P450s in flavonoid metabolism [J ] . Phytochemistry Reviews , 2006 , 5 ( 2-3 ): 271 - 282 .

Gebhardt Y , Witte S , Forkmann G , et al . Molecular evolution of flavonoid dioxygenases in the family Apiaceae [J ] . Phytochemistry , 2005 , 66 ( 11 ): 1273 - 1284 .

Martens S , Forkmann G , Matern U , et al . Cloning of parsley flavone synthase I [J ] . Phytochemistry , 2001 , 58 ( 1 ): 43 - 46 .

Martens S , Forkmann G , Britsch L , et al . Divergent evolution of flavonoid 2-oxoglutarate-dependent dioxygenases in parsley [J ] . FEBS Letters , 2003 , 544 ( 1-3 ): 93 - 98 .

周志华 , 周金林 , 叶德晓 , 等 . 黄酮合酶及其应用 [P ] . 中国发明专利 , CN 110616204 B , 2019 .

ZHOU Z H , ZHOU J L , YE D X , et al . Flavone synthase and its application [P ] . Chinese invention patent , CN 110616204 B , 2019 .

Abramson J , Adler J , Dunger J , et al . Accurate structure prediction of biomolecular interactions with AlphaFold 3 [J ] . Nature , 2024 , 630 ( 8016 ): 493 - 500 .

Gumulya Y , Baek JM , Wun SJ , et al . Engineering highly functional thermostable proteins using ancestral sequence reconstruction [J ] . Nature Catalysis , 2018 , 1 ( 11 ): 878 - 888 .

Trudeau DL , Kaltenbach M , Tawfik DS . On the potential origins of the high stability of reconstructed ancestral proteins [J ] . Molecular Biology and Evolution , 2016 , 33 ( 10 ): 2633 - 2641 .

Wang Z , Xie D , Wu D , et al . Robust enzyme discovery and engineering with deep learning using CataPro [J ] . Nature Communications , 2025 , 16 ( 1 ): 2736 .

李贵祥 , 徐铮 , 李莎 , 等 . 基于同源建模和定点突变技术研究嗜热型L-阿拉伯糖异构酶与D-半乳糖的亲和作用 [J ] . 催化学报 , 2012 , 33 ( 10 ): 1701 - 1707 .

LI G X , XU Z , LI S , et al . Homology modeling and site-directed mutagenesis of thermophilic L-arabinose isomerase for interaction with D-galactose [J ] . Chinese Journal of Catalysis , 2012 , 33 ( 10 ): 1701 - 1707 .

郭发旭 , 冯全 , 张建华 , 等 . 人工智能驱动的酶改造与设计研究进展 [J ] . 生物技术通报 , 2025 , 41 ( 12 ): 50 - 65 .

GUO F X , FENG Q , ZHANG J H , et al . Progress in artificial-intelligence-driven enzyme engineering and design [J ] . Biotechnology Bulletin , 2025 , 41 ( 12 ): 50 - 65 .

Yang Z . PAML 4: phylogenetic analysis by maximum likelihood [J ] . Molecular Biology and Evolution , 2007 , 24 ( 8 ): 1586 - 1591 .

Gilchrist C L M , Mirdita M , Steinegger M . Multiple protein structure alignment at scale with FoldMason [J ] . Science , 2026 , 391 ( 6784 ): 485 - 488 .

Chai Discovery , Boitreaud J , Dent J , et al . Chai-1: decoding the molecular interactions of life [EB/OL ] . bioRxiv , 2024 : 2024 . 10 . 10 . 615955 . DOI: 10.1101/2024.10.10.615955 http://dx.doi.org/10.1101/2024.10.10.615955 .

Altschul SF , Madden TL , Schäffer AA , et al . Gapped BLAST and PSI-BLAST: a new generation of protein database search programs [J ] . Nucleic Acids Research , 1997 , 25 ( 17 ): 3389 - 3402 .

Sun J , Zhu T , Cui Y , et al . Structure-based self-supervised learning enables ultrafast protein stability prediction upon mutation [J ] . The Innovation , 2025 , 6 ( 1 ): 100750 .

浏览量

下载量

CSCD

文章被引用时，请邮件提醒。

提交

工具集

关联资源

生物传感器及其用于功能糖生物合成的研究进展

基于转录因子生物传感器的构建与应用进展

基于高通量液相色谱质谱技术的菌株筛选与关键分子定量分析研究进展

原位电离质谱技术在微生物菌株筛选中的应用进展

微液滴高通量筛选方法的研究与应用进展