实验室适应性进化技术在光合蓝细菌底盘工程中的研究进展

Advances in using adaptive laboratory evolution technology for engineering of photosynthetic cyanobacteria

摘要:

蓝细菌是唯一可进行放氧光合作用的原核微生物，基于光合蓝细菌构建“自养型细胞工厂”具有广阔前景。但以蓝细菌作为底盘进行生物燃料及化学品的合成仍存在细胞耐受能力差、产量低等问题，导致实现工业化生产的经济可行性还比较低，亟需通过合成生物学等技术手段构建新的藻株。近年来，实验室适应性进化(adaptive laboratory evolution,ALE)已被用于底盘工程中，实现了优化生长速度、增加耐受性、加强底物利用和提高产品产量等目标。ALE在提高蓝细菌鲁棒性方面取得了一定进展，已获得了耐受高光、重金属离子、高盐和高浓度有机溶剂胁迫的进化藻株。但是，蓝细菌中的ALE策略效率相对较低，耐受各胁迫的分子机制并未阐释完全。本文综述了ALE相关技术策略及其在蓝细菌底盘工程中的应用，讨论了如何借鉴其他微生物中ALE手段，构建更大ALE突变文库、增加菌株的突变频率、缩短进化时间、探索多重胁迫耐受工程菌构建原则及研究策略等，高效解析进化菌株的突变体库，构建高产量、鲁棒性强的工程菌株等，以期未来促进蓝细菌底盘的改造及其工程菌的规模化应用。

关键词:光合蓝细菌;实验室适应性驯化;自养细胞工厂;合成生物学

Abstract:

Cyanobacteria are the only prokaryotes capable of oxygenic photosynthesis, which have potential to serve as “autotrophic cell factories”. However, the synthesis of biofuels and chemicals using cyanobacteria as chassis are suffered from poor stress tolerance and low yield, resulting in low economic feasibility for industrial production. Thus, it's urgent to construct new cyanobacterial chassis by means of synthetic biology. In recent years, adaptive laboratory evolution (ALE) has made great achievements in chassis engineering, including optimizing growth rate, increasing tolerance, enhancing substrate utilization and increasing product yield. ALE has also made some progress in improving the tolerance of cyanobacteria to high light intensity, heavy metal ions, high concentrations of salt and organic solvents. However, the engineering efficiency of ALE strategy in cyanobacteria is generally low, and the molecular mechanisms underpinning the tolerance to various stresses have not been fully elucidated. To this end, this review summarizes the ALE-associated technical strategies and their applications in cyanobacteria chassis engineering, following by discussing how to construct larger ALE mutation library, increase mutation frequency of strains and shorten evolution time. Moreover, exploration of the construction principles and strategies for constructing multi-stress tolerant cyanobacteria, and efficient analysis the mutant libraries of evolved strains as well as construction of strains with high yield and strong robustness are discussed, with the aim to facilitate the engineering of cyanobacteria chassis and the application of engineered cyanobacteria in the future.

Key words:photosynthetic cyanobacteria;adaptive laboratory evolution;autotrophic cell factories;synthetic biology