Synthetic Biology Journal ›› 2022, Vol. 3 ›› Issue (4): 658-675.DOI: 10.12211/2096-8280.2021-069
• Invited Review • Previous Articles Next Articles
Botao JI, Zhigang QIAN, Xiaoxia XIA
Received:
2021-06-30
Revised:
2021-07-22
Online:
2022-09-08
Published:
2022-08-31
Contact:
Xiaoxia XIA
吉博涛, 钱志刚, 夏小霞
通讯作者:
夏小霞
作者简介:
基金资助:
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.
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URL: https://synbioj.cip.com.cn/EN/10.12211/2096-8280.2021-069
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 |
Tab. 1 The advantages and disadvantages of cell-free expression systems
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[ 选择不同的提取物(例如昆虫)[ 连续交换无细胞表达系统[ 纯化标签以提高溶解度[ |
具有氨基酸组成偏好的蛋白质 | 稀有tRNA的添加[ 选择不同的提取物(例如小麦胚芽)[ 用同义密码子替换初始密码子[ |
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[ 前导肽和微粒体囊泡[ 对于膜蛋白,加入洗涤剂/表面活性剂[ |
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[ 去除同源tRNA[ |
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[ 原核寡糖基转移酶(OST)介导的无细胞糖蛋白生物合成[ 糖基转移酶(GT)介导的蛋白质糖基化和聚糖加工[ |
需要磷酸化的蛋白质 | 基因组重新编码的大肠杆菌菌株[ |
Tab. 2 Corresponding methods for special protein synthesis using CFE system
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[ 选择不同的提取物(例如昆虫)[ 连续交换无细胞表达系统[ 纯化标签以提高溶解度[ |
具有氨基酸组成偏好的蛋白质 | 稀有tRNA的添加[ 选择不同的提取物(例如小麦胚芽)[ 用同义密码子替换初始密码子[ |
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[ 前导肽和微粒体囊泡[ 对于膜蛋白,加入洗涤剂/表面活性剂[ |
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[ 去除同源tRNA[ |
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[ 原核寡糖基转移酶(OST)介导的无细胞糖蛋白生物合成[ 糖基转移酶(GT)介导的蛋白质糖基化和聚糖加工[ |
需要磷酸化的蛋白质 | 基因组重新编码的大肠杆菌菌株[ |
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