• 特约评述 •
颜钊涛2,3, 周鹏飞4, 汪阳忠1, 张鑫1, 谢雯燕1, 田晨菲1,2, 王勇2
收稿日期:
2024-12-18
修回日期:
2025-02-24
出版日期:
2025-02-24
通讯作者:
田晨菲,王勇
作者简介:
基金资助:
Zhaotao YAN2,3, Pengfei ZHOU4, Yangzhong WANG1, Xin ZHANG1, Wenyan XIE1, Chenfei TIAN1,2, Yong WANG2
Received:
2024-12-18
Revised:
2025-02-24
Online:
2025-02-24
Contact:
Chenfei TIAN, Yong WANG
摘要:
植物细胞培养(Plant cell culture, PCC)作为一种极具发展潜力的生物合成平台,具有生长周期短、成本效益高、无病源危害、次生代谢产物丰富等优势,在医药、食品和保健等领域备受关注。然而,生产效率不足是限制PCC应用于商业化生产的最大阻碍,问题在于遗传转化困难、调控网络复杂、细胞结团及遗传稳定性差。合成生物学遵循自下而上的工程化建造理念,对天然植物细胞进行精准设计与改造,为开发高效、经济可行的植物细胞工厂提供了新的解决方案。本文首先回顾了PCC作为合成平台在生产重组蛋白和次生代谢产物中的研究现状。重点探讨了植物合成生物学对PCC的工业化发展中的推动作用,包括优质植物细胞系的构建、遗传转化体系的优化、表达系统的优化、生产效率与产能的提升以及赋予植物细胞合成异源产物的能力。最后,本文对PCC未来的发展前景进行了展望,强调了合成生物学理念和技术在突破当前技术瓶颈中的关键作用,为植物细胞大规模培养的进一步发展指明了方向。
中图分类号:
颜钊涛, 周鹏飞, 汪阳忠, 张鑫, 谢雯燕, 田晨菲, 王勇. 植物合成生物学:植物细胞大规模培养的新机遇[J]. 合成生物学, DOI: 10.12211/2096-8280.2024-095.
Zhaotao YAN, Pengfei ZHOU, Yangzhong WANG, Xin ZHANG, Wenyan XIE, Chenfei TIAN, Yong WANG. Plant synthetic biology: New opportunities for large-scale culture of Plant Cells[J]. Synthetic Biology Journal, DOI: 10.12211/2096-8280.2024-095.
产品 | 物种 | 应用 | 制造商 | 参考 |
---|---|---|---|---|
紫杉醇 | Taxus chinensis L. | 药物,抗癌 | Phyton Biotech | https://phytonbiotech.com/about-pcf/ |
迷迭香酸 | Melissa axillaris L. | 药物,抗氧化 | Aethera Biotech | https://www.aetherabiotech.it/en/ |
可可粉 | Theobroma cacao L. | 食品成分 | California Cultured | https://www.cacultured.com/ |
黄烷醇 | Theobroma cacao L | 医药、保健品成分 | AyanaBio | www.ayanabio.com |
海茴香细胞提取物 | Crithmum maritimum L. | 化妆品成分,抗氧化 | Ancelbio | http://ancelbio.cn/ |
火绒草细胞提取物 | Leontopodium alpinum L. | 化妆品成分,抗皱 | Ancelbio | http://ancelbio.cn/ |
GBL-Skin1 | Glycyrrhiza uralensis L. | 化妆品原料,乳化剂 | Green Bioactives | https://greenbioactives.com/ |
白藜芦醇 | Graptoveria amethorum L. | 化妆品、保健品成分 | Bioharvest Science | https://bioharvest.com/ |
Elelyso | Daucus carota L. | 戈谢病治疗性蛋白 | Protalix BioThera | http://protalix.comhttps://clinicaltrials.gov |
OPRX-100 | Daucus carota L. | 溃疡性结肠炎治疗性蛋白 | Protalix BioThera | http://protalix.comhttps://clinicaltrials.gov |
PRX-102 | Nicotiana tabacum L. | 法布里病治疗性蛋白 | Protalix BioThera | http://protalix.comhttps://clinicaltrials.gov |
新城疫病毒 疫苗 | Nicotiana tabacum L. | 新城疫病毒疫苗 | Dow AgroSciences | https://www.dow.com/ |
MOSS-FH | Physcomitrium patens L. | 溶血性尿毒症综合征治疗性蛋白 | Greenovation Biotech GmbH | http://www.greenovation.com/ developmental-pipeline.html |
表1 植物细胞大规模生产的商业化案例
Table 1 Commercial cases of mass production of plant cells
产品 | 物种 | 应用 | 制造商 | 参考 |
---|---|---|---|---|
紫杉醇 | Taxus chinensis L. | 药物,抗癌 | Phyton Biotech | https://phytonbiotech.com/about-pcf/ |
迷迭香酸 | Melissa axillaris L. | 药物,抗氧化 | Aethera Biotech | https://www.aetherabiotech.it/en/ |
可可粉 | Theobroma cacao L. | 食品成分 | California Cultured | https://www.cacultured.com/ |
黄烷醇 | Theobroma cacao L | 医药、保健品成分 | AyanaBio | www.ayanabio.com |
海茴香细胞提取物 | Crithmum maritimum L. | 化妆品成分,抗氧化 | Ancelbio | http://ancelbio.cn/ |
火绒草细胞提取物 | Leontopodium alpinum L. | 化妆品成分,抗皱 | Ancelbio | http://ancelbio.cn/ |
GBL-Skin1 | Glycyrrhiza uralensis L. | 化妆品原料,乳化剂 | Green Bioactives | https://greenbioactives.com/ |
白藜芦醇 | Graptoveria amethorum L. | 化妆品、保健品成分 | Bioharvest Science | https://bioharvest.com/ |
Elelyso | Daucus carota L. | 戈谢病治疗性蛋白 | Protalix BioThera | http://protalix.comhttps://clinicaltrials.gov |
OPRX-100 | Daucus carota L. | 溃疡性结肠炎治疗性蛋白 | Protalix BioThera | http://protalix.comhttps://clinicaltrials.gov |
PRX-102 | Nicotiana tabacum L. | 法布里病治疗性蛋白 | Protalix BioThera | http://protalix.comhttps://clinicaltrials.gov |
新城疫病毒 疫苗 | Nicotiana tabacum L. | 新城疫病毒疫苗 | Dow AgroSciences | https://www.dow.com/ |
MOSS-FH | Physcomitrium patens L. | 溶血性尿毒症综合征治疗性蛋白 | Greenovation Biotech GmbH | http://www.greenovation.com/ developmental-pipeline.html |
植物物种 | 拉丁名 | 蛋白名称 | 细胞材料 | 产量 | 参考文献 |
---|---|---|---|---|---|
水稻 | Oryza sativa L. | 人骨形态发生蛋白2(BMP2) | 水稻愈伤组织细胞 | 21.5 μg/mL培养液 | [ |
水稻 | Oryza sativa L. | 人类生长激素(hGH) | 水稻愈伤组织细胞 | 57 mg/L培养液 | [ |
水稻 | Oryza sativa L. | 合成牛胰蛋白酶原(Synthetic bovine trypsinogen) | 水稻愈伤组织细胞 | 15 mg/L培养液 | [ |
水稻 | Oryza sativa L. | 酸性葡萄糖苷酶(GAA) | 水稻愈伤组织细胞 | 37 mg/L培养液 | [ |
水稻 | Oryza sativa L. | 血管内皮生长因子(VEGFs) | 水稻愈伤组织细胞 | 19 mg/L培养液 | [ |
水稻 | Oryza sativa L. | 包膜糖蛋白(Envelope glycoprotein) | 水稻愈伤组织细胞 | 18.5 μg/g | [ |
水稻 | Oryza sativa L. | 贝伐单抗(Bevacizumab monoclonal antibody) | 水稻愈伤组织细胞 | 160.7–242.8 mg/kg | [ |
烟草 | Nicotiana tabacum L. | 人抗胰蛋白酶(Human α1-antitrypsin) | BY-2悬浮细胞 | 34.7 mg/L培养液 | [ |
烟草 | Nicotiana tabacum L. | 人生长激素(Human growth hormone) | BY-2悬浮细胞 | 5.2%总可溶蛋白 | [ |
表2 植物细胞异源合成重组蛋白
Table 2 Heterologous synthesis of recombinant proteins in plant cells
植物物种 | 拉丁名 | 蛋白名称 | 细胞材料 | 产量 | 参考文献 |
---|---|---|---|---|---|
水稻 | Oryza sativa L. | 人骨形态发生蛋白2(BMP2) | 水稻愈伤组织细胞 | 21.5 μg/mL培养液 | [ |
水稻 | Oryza sativa L. | 人类生长激素(hGH) | 水稻愈伤组织细胞 | 57 mg/L培养液 | [ |
水稻 | Oryza sativa L. | 合成牛胰蛋白酶原(Synthetic bovine trypsinogen) | 水稻愈伤组织细胞 | 15 mg/L培养液 | [ |
水稻 | Oryza sativa L. | 酸性葡萄糖苷酶(GAA) | 水稻愈伤组织细胞 | 37 mg/L培养液 | [ |
水稻 | Oryza sativa L. | 血管内皮生长因子(VEGFs) | 水稻愈伤组织细胞 | 19 mg/L培养液 | [ |
水稻 | Oryza sativa L. | 包膜糖蛋白(Envelope glycoprotein) | 水稻愈伤组织细胞 | 18.5 μg/g | [ |
水稻 | Oryza sativa L. | 贝伐单抗(Bevacizumab monoclonal antibody) | 水稻愈伤组织细胞 | 160.7–242.8 mg/kg | [ |
烟草 | Nicotiana tabacum L. | 人抗胰蛋白酶(Human α1-antitrypsin) | BY-2悬浮细胞 | 34.7 mg/L培养液 | [ |
烟草 | Nicotiana tabacum L. | 人生长激素(Human growth hormone) | BY-2悬浮细胞 | 5.2%总可溶蛋白 | [ |
物种 | 外植体部位 | 类型 | 激素 | 培养基及PH | 周期 | 参考文献 |
---|---|---|---|---|---|---|
水稻 | 胚乳 | 愈伤组织 | 1.0 mg/L 2,4-D; 1.0 mg/L 6BA | N6培养基 | 6个月 | [ |
葡萄 | 叶片 | 愈伤组织 | 0.05 mg/L NAA; 0.5 mg/L 2,4-D; 2.0 mg/L KT | B5培养基 | 21天 | [ |
胡萝卜 | 茎段 | 愈伤组织 | 0.5 mg/L 2,4-D | MS培养基 | 14天 | [ |
红豆杉 | 胚乳 | 愈伤组织 | 1.0 mg/L 2,4-D; 0.5 mg/L 6BA | B5培养基 | 15天 | [ |
人参 | 根部 | 愈伤组织 | 1.0 mg/L 2,4-D; 0.1 mg/L KT | MS培养基 | 90天 | [ |
地黄 | 根部 | 形成层干细胞 | 2.0 mg/L NAA; 2.0 mg/L 6BA | MS培养基 | 14天 | [ |
表 3 常见体外植物细胞系诱导条件
Table 3 Common induction conditions of plant cell lines in vitro
物种 | 外植体部位 | 类型 | 激素 | 培养基及PH | 周期 | 参考文献 |
---|---|---|---|---|---|---|
水稻 | 胚乳 | 愈伤组织 | 1.0 mg/L 2,4-D; 1.0 mg/L 6BA | N6培养基 | 6个月 | [ |
葡萄 | 叶片 | 愈伤组织 | 0.05 mg/L NAA; 0.5 mg/L 2,4-D; 2.0 mg/L KT | B5培养基 | 21天 | [ |
胡萝卜 | 茎段 | 愈伤组织 | 0.5 mg/L 2,4-D | MS培养基 | 14天 | [ |
红豆杉 | 胚乳 | 愈伤组织 | 1.0 mg/L 2,4-D; 0.5 mg/L 6BA | B5培养基 | 15天 | [ |
人参 | 根部 | 愈伤组织 | 1.0 mg/L 2,4-D; 0.1 mg/L KT | MS培养基 | 90天 | [ |
地黄 | 根部 | 形成层干细胞 | 2.0 mg/L NAA; 2.0 mg/L 6BA | MS培养基 | 14天 | [ |
图2 植物细胞产量提升策略从“黑箱”到“白箱”在“Black box”中,诱导子提升目标产物产量作用机制尚未完全明确;在“White box”中,通过不同基因线路可以分别实现精准的目标产物产量提升[89]
Fig.2 Strategies for increasing plant cell yield from "black box" to "white box"In the "Black box", the mechanism of the inducer to increase the yield of the target product has not been fully defined; In the "White box", the yield of target products can be increased precisely with different gene circuits[89]
植物 物种 | 拉丁名 | 改造策略 | 细胞材料 | 化合物 类别 | 作用效果 | 参考文献 |
---|---|---|---|---|---|---|
山葡萄 | Vitis amurensis Rupr. | 过表达VaCPK29 | 悬浮细胞 | 多酚类 | 白藜芦醇1.39 mg/L培养液 | [ |
柑橘 | CitrusreticulataL. | 过表达CsMADS6、PSY、PDS和CCD1 | 愈伤组织细胞 | 类胡萝卜素 | 类胡萝卜素23 µg/g DW | [ |
加州 藜芦 | Veratrum californicum var. | 过表达VnOSC1 | 愈伤组织细胞 | 生物碱 | 环巴胺6.14 mg/g DW | [ |
红豆杉 | Taxus baccata L. | 过表达NINV | 悬浮细胞 | 二萜类 | 紫杉醇94 μg/g FW | [ |
红豆杉 | Taxus baccata L. | 过表达BAPT、DBTNBT | 悬浮细胞 | 二萜类 | 紫杉醇310 mg/L培养液 | [ |
灌木状辣椒 | Capsicum frutescens L. | 过表达VpVAN | 悬浮细胞 | 芳香族化合物 | 香兰素 573.39±120.70 µg/g组织 | [ |
烟草 | Nicotiana tabacum L. | 过表达转录因子AmRos1和AmDel | BY-2悬浮细胞 | 黄酮类 | 花青素30 mg/g DW | [ |
甜菜 | Beta vulgaris L. | 过表达VpVAN | 毛状根 | 芳香族化合物 | 香兰素0.0430 ±0.003 mg/g DW | [ |
烟草 | Nicotiana tabacum L. | 过表达HCHL | 悬浮细胞 | 黄酮类 | 花青素75.4±6.1 µmol/g FW | [ |
竹 | Phyllostachys nigra L. | 过表达PpHCH | 悬浮细胞 | 酚类 | 4-羟基苯甲醇1.7 g/L培养液 | [ |
烟草 | Nicotiana tabacum L. | 过表达CqCYP76AD1、CqDODA、CqCDOPA5GT和CqAmaSy | BY-2悬浮细胞 | 苷类 | 苋菜苷13.67±4.13 µM;甜菜苷26.60±1.53 µM | [ |
烟草 | Nicotiana tabacum L. | 过表达CqCYP76AD1-1和CqDODA-1 | BY-2悬浮细胞 | 类黄酮 | 甜菜苷19.53±8.60 µM | [ |
烟草 | Nicotiana tabacum L. | 过表达VoGES | 悬浮细胞 | 单萜类 | 香叶醇16 µg/g DW | [ |
烟草 | Nicotiana tabacum L. | 过表达PgDDS | 悬浮细胞 | 三萜类 | 达玛烯二醇-II 573 µg/g DW | [ |
烟草 | Nicotiana tabacum L. | 过表达PgDDS 和 CYP716A47 | 悬浮细胞 | 三萜类 | 原人参二醇980.9 µg/g DW | [ |
烟草 | Nicotiana tabacum L. | CRISPRi抑制NtC4H | 悬浮细胞 | 苯丙 素类 | 绿原酸1799.69 ng/mL培养液; 乔松酮384.19 ng/mL培养液; 柚皮素597.53 ng/mL培养液 | [ |
水稻 | Oryza sativa L. | 修饰近靶顺式作用元件,激活PHYTOENE SYNTHASE 1启动子 | 愈伤组织细胞 | 类胡萝卜素 | 八氢番茄红素7.13 µg/g DW | [ |
水飞蓟 | Silybum marianum L. | 过表达STS | 悬浮细胞 | 多酚类 | 白藜芦醇50 ng/g FW | [ |
连翘 | Forsythia koreana L. | 过表达CYP81Q1;RNAi抑制UGT71A18和PLR | 悬浮细胞 | 木质素 | 芝麻素10.83±0.35 µg/g DW | [ |
表4 植物细胞合成次生代谢产物案例
Table 4 Cases of secondary metabolites synthesized by plant cells
植物 物种 | 拉丁名 | 改造策略 | 细胞材料 | 化合物 类别 | 作用效果 | 参考文献 |
---|---|---|---|---|---|---|
山葡萄 | Vitis amurensis Rupr. | 过表达VaCPK29 | 悬浮细胞 | 多酚类 | 白藜芦醇1.39 mg/L培养液 | [ |
柑橘 | CitrusreticulataL. | 过表达CsMADS6、PSY、PDS和CCD1 | 愈伤组织细胞 | 类胡萝卜素 | 类胡萝卜素23 µg/g DW | [ |
加州 藜芦 | Veratrum californicum var. | 过表达VnOSC1 | 愈伤组织细胞 | 生物碱 | 环巴胺6.14 mg/g DW | [ |
红豆杉 | Taxus baccata L. | 过表达NINV | 悬浮细胞 | 二萜类 | 紫杉醇94 μg/g FW | [ |
红豆杉 | Taxus baccata L. | 过表达BAPT、DBTNBT | 悬浮细胞 | 二萜类 | 紫杉醇310 mg/L培养液 | [ |
灌木状辣椒 | Capsicum frutescens L. | 过表达VpVAN | 悬浮细胞 | 芳香族化合物 | 香兰素 573.39±120.70 µg/g组织 | [ |
烟草 | Nicotiana tabacum L. | 过表达转录因子AmRos1和AmDel | BY-2悬浮细胞 | 黄酮类 | 花青素30 mg/g DW | [ |
甜菜 | Beta vulgaris L. | 过表达VpVAN | 毛状根 | 芳香族化合物 | 香兰素0.0430 ±0.003 mg/g DW | [ |
烟草 | Nicotiana tabacum L. | 过表达HCHL | 悬浮细胞 | 黄酮类 | 花青素75.4±6.1 µmol/g FW | [ |
竹 | Phyllostachys nigra L. | 过表达PpHCH | 悬浮细胞 | 酚类 | 4-羟基苯甲醇1.7 g/L培养液 | [ |
烟草 | Nicotiana tabacum L. | 过表达CqCYP76AD1、CqDODA、CqCDOPA5GT和CqAmaSy | BY-2悬浮细胞 | 苷类 | 苋菜苷13.67±4.13 µM;甜菜苷26.60±1.53 µM | [ |
烟草 | Nicotiana tabacum L. | 过表达CqCYP76AD1-1和CqDODA-1 | BY-2悬浮细胞 | 类黄酮 | 甜菜苷19.53±8.60 µM | [ |
烟草 | Nicotiana tabacum L. | 过表达VoGES | 悬浮细胞 | 单萜类 | 香叶醇16 µg/g DW | [ |
烟草 | Nicotiana tabacum L. | 过表达PgDDS | 悬浮细胞 | 三萜类 | 达玛烯二醇-II 573 µg/g DW | [ |
烟草 | Nicotiana tabacum L. | 过表达PgDDS 和 CYP716A47 | 悬浮细胞 | 三萜类 | 原人参二醇980.9 µg/g DW | [ |
烟草 | Nicotiana tabacum L. | CRISPRi抑制NtC4H | 悬浮细胞 | 苯丙 素类 | 绿原酸1799.69 ng/mL培养液; 乔松酮384.19 ng/mL培养液; 柚皮素597.53 ng/mL培养液 | [ |
水稻 | Oryza sativa L. | 修饰近靶顺式作用元件,激活PHYTOENE SYNTHASE 1启动子 | 愈伤组织细胞 | 类胡萝卜素 | 八氢番茄红素7.13 µg/g DW | [ |
水飞蓟 | Silybum marianum L. | 过表达STS | 悬浮细胞 | 多酚类 | 白藜芦醇50 ng/g FW | [ |
连翘 | Forsythia koreana L. | 过表达CYP81Q1;RNAi抑制UGT71A18和PLR | 悬浮细胞 | 木质素 | 芝麻素10.83±0.35 µg/g DW | [ |
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