Application of cell-free synthesis strategy in biomaterial research

doi:10.12211/2096-8280.2021-069

Abstract

Abstract:

As various issues such as global warming, environmental pollution and the exhaustion of fossil resources become increasingly severe, biomaterials have attracted increasing attention in the fields of biotechnology and biomedical engineering due to their many advantages such as renewable, degradable nature, and good biocompatibility. Based on the “bottom-up” design concept, cell-free expression system as a useful supplement to the cell synthesis system, greatly accelerates the development of biomaterials and expands their application range, becoming a frontier of the materials synthetic biology. This review first introduces the difference between cell-free expression system and conventional cell expression system, and emphasizes the unique advantages of cell-free expression system. Next, this review overviews the application of cell-free strategies in sustainable production, functionalization and innovative design of biomaterials, as well as promotion of new types by accelerating the Design-Build-Test (DBT) cycle. All of these examples fully demonstrate the advantages of cell-free systems in the design and synthesis of novel biomaterials. Although the use of cell-free expression systems for biomaterial synthesis encounters challenges such as cost, weak post-translational modification, and urgent need for cross-field cooperation, we still believe that in the near future, with the development of synthetic biology and the deepening of interdisciplinary research, cell free strategy will provide a faster, cheaper and more friendly way to produce new biomaterials, thereby protecting the earth environment on which we depend. In addition, the continuous development of smart materials that are combined with cell-free strategies, such as virus detection biosensors, will protect the health of human beings around the world to a great extent, and even save thousands of lives. All in all, the cell-free expression system will definitely provide a powerful boost to the research and industrialization of new biomaterials, and make a non-negligible contribution to the joint solution of global environmental problems and the protection of human health.

Key words: biomaterials, cell-free expression system, material intelligence and functionalization, DBT cycle, synthetic biology

摘要：

生物材料凭借其可再生、可降解及生物相容性好等诸多优点，在生物技术及生物医学应用领域备受关注。基于“自下而上”设计理念的无细胞表达系统，作为细胞合成体系的有益补充，大力加速了生物材料的开发生产及其应用范围，并成为了材料合成生物学发展的前沿。本文介绍了无细胞表达系统的独特优势，并列举了其用于生物材料可持续生产的策略、与材料结合后的创新设计、赋予材料功能化和智能化方法，以及通过加速设计-构建-测试（DBT）循环来促进新型生物材料开发的各项应用，而这些例证都充分表明无细胞策略在生物材料设计与合成中的创新优势。尽管无细胞表达系统用于生物材料合成仍然存在诸如成本、翻译后修饰较弱、亟需交叉领域合作等方面的挑战，但相信随着合成生物学的发展及多学科交叉研究的深入，无细胞表达系统将为新型生物材料研发及产业化提供强大的助力，并为共同解决全球环境问题、保护人类生命健康等方面作出不可忽视的贡献。

关键词: 生物材料, 无细胞表达系统, 材料智能化和功能化, DBT循环, 合成生物学

CLC Number:

Botao JI, Zhigang QIAN, Xiaoxia XIA. Application of cell-free synthesis strategy in biomaterial research[J]. Synthetic Biology Journal, 2022, 3(4): 658-675.

吉博涛, 钱志刚, 夏小霞. 无细胞合成策略在生物材料研究中的应用[J]. 合成生物学, 2022, 3(4): 658-675.

Figures/Tables 7

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特征	细胞表达系统	无细胞表达系统
转录和翻译	-具有细胞膜，细胞内表征和产物释放可能具有挑战性 -内源性调控难以预测和修改 +能够使用定向进化技术	+开放的环境易于实时操作和控制 +直接添加底物或收获产物 +易于使用来自多种生物体的酶途径
翻译后修饰	+简单	-困难
自我复制	+简单	-困难
DNA模板	-质粒（或者整合到基因组的DNA）	+质粒或 PCR 产物
引入非天然氨基酸	-困难	+简单
膜蛋白和复合蛋白的合成	-由于细胞内环境有限而难以合成	+通过添加表面活性剂或调整系统环境易于合成
对毒性的耐受力	-低，难以合成毒性蛋白 -若中间产物有毒性，则可行性受到限制	+高，可以合成毒性蛋白 +无限制（如某些途径酶使用体积分数为12%异丁醇）
设计-构建-测试（DBT）循环	-慢，1～2周	+快，1～2天
资源利用率	-较低，由于复杂的细胞代谢而将资源转向细胞维护和副产品	+较高，所有资源都可以直接用于生产产品
与材料整合的能力	-困难	+简单
生物制造	-适中的产率和产量 -纯化前需再次裂解细胞 -不易放大生产，工业规模发酵条件是不同的 -菌种污染可能会是灾难性的 +多年实践经验；完善的方法	+较高的产率和产量 +无须细胞裂解的简单的纯化过程 +易于放大生产，具有线性放大的可能 -较为年轻，最近成立
成本	+低到适中	-中高，主要为酶和辅因子成本
稳定性	-通过发酵条件影响细胞环境	+由工程师直接控制反应条件

特征	细胞表达系统	无细胞表达系统
转录和翻译	-具有细胞膜，细胞内表征和产物释放可能具有挑战性 -内源性调控难以预测和修改 +能够使用定向进化技术	+开放的环境易于实时操作和控制 +直接添加底物或收获产物 +易于使用来自多种生物体的酶途径
翻译后修饰	+简单	-困难
自我复制	+简单	-困难
DNA模板	-质粒（或者整合到基因组的DNA）	+质粒或 PCR 产物
引入非天然氨基酸	-困难	+简单
膜蛋白和复合蛋白的合成	-由于细胞内环境有限而难以合成	+通过添加表面活性剂或调整系统环境易于合成
对毒性的耐受力	-低，难以合成毒性蛋白 -若中间产物有毒性，则可行性受到限制	+高，可以合成毒性蛋白 +无限制（如某些途径酶使用体积分数为12%异丁醇）
设计-构建-测试（DBT）循环	-慢，1～2周	+快，1～2天
资源利用率	-较低，由于复杂的细胞代谢而将资源转向细胞维护和副产品	+较高，所有资源都可以直接用于生产产品
与材料整合的能力	-困难	+简单
生物制造	-适中的产率和产量 -纯化前需再次裂解细胞 -不易放大生产，工业规模发酵条件是不同的 -菌种污染可能会是灾难性的 +多年实践经验；完善的方法	+较高的产率和产量 +无须细胞裂解的简单的纯化过程 +易于放大生产，具有线性放大的可能 -较为年轻，最近成立
成本	+低到适中	-中高，主要为酶和辅因子成本
稳定性	-通过发酵条件影响细胞环境	+由工程师直接控制反应条件

组成生物材料的特殊蛋白质	使CFE系统提高生产力和/或生物活性的方法
对生产宿主有毒的蛋白质	额外的tRNA补充^[68] 选择不同的提取物（例如昆虫）^[69] 连续交换无细胞表达系统^[70] 纯化标签以提高溶解度^[71]
具有氨基酸组成偏好的蛋白质	稀有tRNA的添加^[72] 选择不同的提取物（例如小麦胚芽）^[73] 用同义密码子替换初始密码子^[74-75]
需要分子伴侣或特定化学环境合成的蛋白质	伴侣蛋白，蛋白质二硫键异构酶（PDI），还原和氧化的谷胱甘肽（glutathione）调整^[76] 前导肽和微粒体囊泡^[77] 对于膜蛋白，加入洗涤剂/表面活性剂^[78-79]
含有非天然氨基酸的蛋白质	通过基因组工程消除提取物中的负面效应物，如释放因子1（RF1）的竞争限制^[80] 去除同源tRNA^[81]
需要糖基化修饰的蛋白质	内切糖苷酶介导的N-糖基化蛋白的制备^[82-83] 原核寡糖基转移酶（OST）介导的无细胞糖蛋白生物合成^{[34, 84]} 糖基转移酶（GT）介导的蛋白质糖基化和聚糖加工^[85-86]
需要磷酸化的蛋白质	基因组重新编码的大肠杆菌菌株^[87]

组成生物材料的特殊蛋白质	使CFE系统提高生产力和/或生物活性的方法
对生产宿主有毒的蛋白质	额外的tRNA补充^[68] 选择不同的提取物（例如昆虫）^[69] 连续交换无细胞表达系统^[70] 纯化标签以提高溶解度^[71]
具有氨基酸组成偏好的蛋白质	稀有tRNA的添加^[72] 选择不同的提取物（例如小麦胚芽）^[73] 用同义密码子替换初始密码子^[74-75]
需要分子伴侣或特定化学环境合成的蛋白质	伴侣蛋白，蛋白质二硫键异构酶（PDI），还原和氧化的谷胱甘肽（glutathione）调整^[76] 前导肽和微粒体囊泡^[77] 对于膜蛋白，加入洗涤剂/表面活性剂^[78-79]
含有非天然氨基酸的蛋白质	通过基因组工程消除提取物中的负面效应物，如释放因子1（RF1）的竞争限制^[80] 去除同源tRNA^[81]
需要糖基化修饰的蛋白质	内切糖苷酶介导的N-糖基化蛋白的制备^[82-83] 原核寡糖基转移酶（OST）介导的无细胞糖蛋白生物合成^{[34, 84]} 糖基转移酶（GT）介导的蛋白质糖基化和聚糖加工^[85-86]
需要磷酸化的蛋白质	基因组重新编码的大肠杆菌菌株^[87]

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