自动化高通量技术在天然产物生物合成中的应用

doi:10.12211/2096-8280.2023-035

合成生物学 ›› 2023, Vol. 4 ›› Issue (5): 932-946.DOI: 10.12211/2096-8280.2023-035

自动化高通量技术在天然产物生物合成中的应用

胡哲辉¹^,², 徐娟², 卞光凯¹

^1.中国科学院深圳先进技术研究院合成生物学研究所，广东深圳 518055
^2.华中农业大学园艺林学学院，果蔬园艺作物种质创新与利用全国重点实验室，湖北武汉 430071

收稿日期:2023-05-06 修回日期:2023-07-24 出版日期:2023-10-31 发布日期:2023-11-15
通讯作者: 卞光凯
作者简介:胡哲辉（1998—），男，博士研究生。研究方向为植物来源萜类天然产物生物合成。E-mail：zh.hu2@siat.ac.cn
卞光凯（1986—），男，研究员，博士生导师。研究方向为天然产物生物合成及高值天然产物高效合成；利用合成生物学手段改造丝状真菌生产高性能生物材料。E-mail：gk.bian@siat.ac.cn
基金资助:
国家重点研发计划“绿色生物制造”重点专项(2022YFC2106100);国家自然科学基金面上项目(32070063)

Application of automated high-throughput technology in natural product biosynthesis

Zhehui HU¹^,², Juan XU², Guangkai BIAN¹

^1.Insititute of Syntheitc Biology，Shenzhen Institute of Advanced Technology，Chinese Academy of Sciences，Shenzhen 518055，Guangdong，China
^2.National Key Laboeratory for Germplasm Innovation & Utilization of Horticultural Crops，College of Horticulture and Forestry，Huazhong Agricultural University，Wuhan 430071，Hubei，China

Received:2023-05-06 Revised:2023-07-24 Online:2023-10-31 Published:2023-11-15
Contact: Guangkai BIAN

摘要/Abstract

摘要：

天然产物与人们的日常生活息息相关，是药物、食品、营养添加剂、色素、化妆品等产品的重要来源。发掘新的天然产物并实现其高效合成对于提升人们的生活水平至关重要。然而，研究通量低和产物产量低已成为限制该领域快速发展的两大瓶颈。为了克服这些问题，自动化高通量技术的引入在天然产物生物合成领域具有重要意义。该技术可以将高度依赖人力资源的低通量和随机性手工操作的实验过程转变为基于先进设备的自动化、标准化和高效的研究过程。因此，将自动化高通量技术引入天然产物生物合成领域可以有效解决研究瓶颈，加速研发进程。本文综述了自动化高通量技术在天然产物生物合成领域的应用，阐述了其在天然产物挖掘、高效生物合成和快速检测等方面的优势。最后，讨论了该技术现存缺陷，包括仪器设备造价高昂、操作复杂，并且一些装置尚未得到推广。相信在合成生物、信息技术、自动化等多领域的共同协作下，自动化高通量技术具备的独特优势将为新型天然产物功能产品的开发提供可持续来源。

关键词: 合成生物学, 天然产物, 自动化技术, 高产底盘, 生物铸造

Abstract:

Natural products are usually secondary metabolites derived from animals, plants, or microorganisms. They are closely related to people's daily lives and important sources of drugs, food and nutrition additives, pigments, and cosmetics. The exploration of novel natural products and the efficient synthesis of value-added products are key to enriching and improving people's living standards. Researchers have exploited a large number of natural products through strategies such as direct product isolation, homologous activation, and heterologous expression. However, low throughput and low titer have become the two major bottlenecks of this field. With the rapid development of synthetic biology, automated high-throughput technology has been developed to transform the low throughput, stochastic manual experimental process that relies on human resources into an automated, standardized, and efficient research process, which is regarded as a leading technology in natural product biosynthesis. This review compares and highlights the advantages of automated high-throughput technology over the traditional techniques within the context of natural products biosynthesis, provides an overview of the components and principles of automated high-throughput workstations, and summarizes the existing and upcoming automated high-throughput facilities. In addition, this review highlights the applications of automated high-throughput technology, including the batch mining of natural product biosynthetic genes and gene clusters, overproduction, and efficient detection of value-added natural products. These examples effectively illustrate the benefits of this technology in the field of natural product biosynthesis. In conclusion, the use of automated high-throughput technology in synthetic biology has shown promising results, particularly in the high-throughput discovery and biosynthesis of natural products. Finally, we discuss the existing shortcomings of the technology. Despite the cost and operational challenges associated with the automated high-throughput technology, it is expected to significantly advance both basic and applied research in synthetic biology, while also provide a sustainable source for the development of new functional products based on newly discovered natural products.

Key words: synthetic biology, natural products, automation technology, chassis, biofoundry

中图分类号:

Q816

胡哲辉, 徐娟, 卞光凯. 自动化高通量技术在天然产物生物合成中的应用[J]. 合成生物学, 2023, 4(5): 932-946.

Zhehui HU, Juan XU, Guangkai BIAN. Application of automated high-throughput technology in natural product biosynthesis[J]. Synthetic Biology Journal, 2023, 4(5): 932-946.

图/表 5

表1 传统方法与自动化高通量方法在天然产物生物合成中的比较

Table 1 A comparison of automated high-throughput and traditional methods in natural product biosynthesis

直接分离

同源激活

异源表达

生物样本单一

局限于单个基因的异源表达

产物丰度低、筛选通量低

打造特定微生物高产底盘

基于海量数据库批量挖掘

高通量工作站自动化验证

不受限于特定的生物样本

充分释放酶的产物合成潜力

规模化、自动化

天然产物高效合成

突变体文库平板筛选

关键酶的定点诱变

人工改造代谢通路

突变体间微小差异分辨率低

试错过程需要消耗大量劳动力

无法完全释放酶的催化潜力

基于auto-HTP对突变体文库高效筛选

自动化改造底盘细胞

限速酶定向进化

微生物适应性进化

自动化实现海量试错

多维度改造底盘细胞

缩短菌株改造研发周期

天然产物的快速检测

气相色谱、质谱

液相色谱、质谱

核磁共振

基于紫外/可见光谱检测

耗时长、通量低

对目标产物的产量和纯度要求高

基于荧光光谱检测

基于生物传感器的荧光检测

基于先进光谱技术的检测

便捷、高效、省时

对目标产物的动态、实时、自动化检测

图1 不同通量筛选方法的比较［41-42］（a）传统平板筛选法，筛选通量为103～104；（b）基于自动化设备的微孔板筛选法，筛选通量为104～105；（c）基于流式细胞仪的荧光激活细胞分选，筛选通量为108～109；（d）基于微流控芯片和分选设备的液滴微流控分选，筛选通量为108～109

Fig. 1 A comparison of different screening methods[41-42](a) Traditional screening method based on agar plates, with a screening throughput of 103～104; (b) Microplate screening method based on automation equipment, with a screening throughput of 104～105; (c) Fluorescence activated cell sorting, with a screening throughput of 108～109; (d) Droplet-based microfluidic sorting, with a screening throughput of 108～109

图2 自动化高通量工作站（a）自动化高通量工作站平面示意图；（b）自动化高通量工作流程（以酵母为例）

Fig. 2 Automated high-throughput workstation(a) Schematic diagram of the automated high-throughput workstation; (b) A representative workflow of automated high-throughput process, with yeast engineering shown as an example

图3 基于生物铸造平台对新功能萜类基因（簇）的高效挖掘及抗炎新分子mangicol J的高效合成［51， 56］（a） 34个新嵌合萜类合酶（PTTC）的批量挖掘；（b） 39个萜类基因簇重建为208个突变株；（c）通过高通量筛选从185个萜类产物中筛选到高抗炎活性70号（mangicol J）化合物；（d） mangicol J生物合成途径解析；（e）构建通用米曲霉底盘实现mangicol J高效合成

Fig. 3 The application of biocasting in efficient mining of new functional terpenoid genes (gene clusters) and the biosynthesis of mangicol J, a new anti-inflammatory molecule[51, 56](a) The batch mining of 34 new chimeric terpenoid synthases (PTTC); (b) The reconstruction of 39 terpenoid gene clusters into 208 mutants; (c) High-throughput screening of 185 terpenoids leading to the isolation of compound 70 (mangicol J) with high anti-inflammatory activity; (d) The elucidation of the biosynthetic pathway of mangicol J; (e) The construction of a general Aspergillus oryzae chassis for efficient biosynthesis of mangicol J

图 4 高通量筛选检测天然产物的不同策略［42， 95］

Fig. 4 Different strategies for high throughput detection of natural products[42, 95]

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自动化高通量技术在天然产物生物合成中的应用

Application of automated high-throughput technology in natural product biosynthesis

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