基于无细胞体系的生物合成代谢模块设计、构建与快速途径原型

doi:10.12211/2096-8280.2022-024

摘要/Abstract

摘要：

随着代谢工程及合成生物学技术的发展，化学品高效生物合成与绿色制造成为可能。高效生物合成体系的设计与构建是绿色生物制造的核心，其理论体系建立及关键技术突破，将为实现绿色生物制造领域高效生产及资源与环境可持续发展提供有力支撑。本文借助代谢途径模块设计的案例，探讨化合物生物合成过程中潜在通用模块设计原则、设计工具，以及基于无细胞蛋白合成体系的代谢模块快速构建及测试的方法，将突破生物合成途径多基因、多模块“设计-构建-测试”（Design-Build-Test cycle，DBT cycle）高效循环迭代的技术瓶颈。结合机器学习方法的应用，将使“设计-构建-测试”向“设计-构建-测试-学习”（Design-Build-Test-Learn cycle，DBTL cycle）进一步延伸，对高效合成模块的“精准-鲁棒性”设计与构建、推动合成生物学科学与技术发展具有重要意义。

关键词: 生物合成模块, 无细胞蛋白合成, 无细胞快速原型, 机器学习

Abstract:

Bio-based chemical production has drawn attention regarding the realization of a sustainable society. With the development of metabolic engineering and synthetic biology, it is possible to make more efficient biosynthesis and scale-up commercial production of useful metabolites by metabolic pathway modification. Usually,some microorganisms are utilized as platforms by increasing the expression of desired genes and/or decreasing, that of undesired genes. Precise control of natural metabolism is, however, still challenging due to the complicated regulatory architecture at the levels of transcription, translation, and post-translation. Hence, various strategies of design and construction of biosynthesis modules have been proposed to optimize and expedite the design-build-test cycles of developing biosynthetic system for renewable chemical synthesis in vitro to avoid laborious and expensive ways for the optimization of metabolism pathway,strain and biocatalysts for each new product. In this review, we discuss the strategies of modules design and their rapid prototyping based on cell-free protein synthesis for assembling biosynthetic pathway in vitro. Biosynthetic modules could be sets of enzymes that catalyze a cascade reaction for a specific purpose or chemical, containing the conversion of starting materials to intermediary metabolites, biosynthesis of target products from the intermediates, cofactor balance and phosphorylation. Enzymes from distinct sources can be combined to construct desired reaction cascades with fewer biological constraints in one vessel, enabling easier pathway design with high modularity. Multiple modules could then been designed by different groups for different purpose with the help of metabolic pathway database,computational design tool and some general module design rules. Cell-free protein synthesis was here utilized to build and rapidly prototype the functionality of biosynthesis pathway and module. The further application of machine learning methods might also contribute to better “precision-robustness” design and construction of these modules. This process is also called cell-free pathway engineering which has been proved to be a powerful and flexible enabling technology, providing simpler and faster engineering solutions with an unprecedented freedom of design in an open environment than cell system.

Key words: biosynthesis module, cell-free protein synthesis, rapid prototyping, machine learning

中图分类号:

Q816

唐士茗, 胡纪元, 郑穗平, 韩双艳, 林影. 基于无细胞体系的生物合成代谢模块设计、构建与快速途径原型[J]. 合成生物学, 2022, 3(6): 1250-1261.

Shiming TANG, Jiyuan HU, Suiping ZHENG, Shuangyan HAN, Ying LIN. Designing, building and rapid prototyping of biosynthesis module based on cell-free system[J]. Synthetic Biology Journal, 2022, 3(6): 1250-1261.

图/表 5

表1 细胞生物合成与无细胞生物合成的比较

Tab. 1 Comparison of cellular and cell-free biosynthesis

衡量标准	细胞生物合成	无细胞生物合成
途径设计	胞内代谢网络复杂	自由灵活
生产效率	碳通量被用于细胞生长及产生副产物	碳通量最大程度流向目标化合物
物质传输	具有选择性屏障	直接添加底物，产物易提取
产物分离纯化	副产物较多	较容易
毒性影响	毒性物质影响细胞生长	没有生存限制
成本	较低	酶的制备和辅因子成本
成熟性	多年实践经验	初具规模

图1 淀粉生物基无细胞生物合成途径模块化设计GDH—葡萄糖脱氢酶；DHAD—二羟酸脱水酶；KDGA—2-酮-3-脱氧葡萄糖酸醛缩酶；ALDH—甘油醛脱氢酶；L-LDH—L-乳酸脱氢酶；PDC—丙酮酸脱羧酶；ADH—乙醇脱氢酶；ALS—乙酰乳酸合成酶； KARI—酮醇酸还原异构酶；KDC—2-酮酸脱羧酶

Fig. 1 Modular design of cell-free biosynthetic pathway from starch-base materialsGDH—glucose dehydrogenase; DHAD—dihydroxy acid dehydratase; KDGA—2-keto-3-desoxy gluconate aldolase; ALDH—glyceraldehyde dehydrogenase; L-LDH—L-lactate dehydrogenase; PDC—pyruvate decarboxylase; ADH—alcohol dehydrogenase; ALS—acetolactate synthase; KARI—ketolacid reductoisomerase; KDC—2-ketoacid decarboxylase

图2 酵母萜类化合物合成模块设计

Fig. 2 Modular design of terpenoids biosynthetic pathway in yeast

图3 基于无细胞生物合成体系的快速途径原型原理图

Fig. 3 Overview of cell-free biosynthesis pathway prototyping

图4 酵母萜类合成模块设计及代谢途径快速原型

Fig. 4 Design and rapid prototyping of isoprenoid synthesis modular in yeast

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