化学诱导大肠杆菌Nissle 1917染色体进化及其应用

Establishment and application of chemically inducible chromosomal evolution in Escherichia coli Nissle 1917

摘要:

大肠杆菌Nissle 1917 (EcN)因生物相容性高、易基因改造等特点，常常被用于肿瘤细菌疗法。目前，大多数研究利用质粒载体构建EcN工程菌，存在基因表达不稳定、需要利用抗生素选择性压力来保持高拷贝数等问题。本研究以EcN中光敏剂5-氨基乙酰丙酸(5-aminolevulinic acid, 5-ALA)的合成为例，利用噬菌体整合技术将5-ALA合成的关键基因hemA^M、hemL单拷贝整合EcN基因组，然后利用化学诱导染色体进化(chemically inducible chromosomal evolution, CIChE)的方法提高hemA^M、hemL拷贝数，进而促进5-ALA的稳定合成。通过体外细胞实验初步验证了EcN工程菌能将稳定合成的5-ALA递送至肿瘤细胞并抑制其生长，实验结果为EcN工程菌应用于肿瘤光动力治疗提供理论基础。

Abstract:

The probiotic strain Escherichia coli Nissle 1917 (EcN) with high biocompatibility and susceptibility to genetic modification is often applied in bacterial therapies for cancer. However, most studies have used plasmids as vectors to construct engineering strains from EcN. Plasmid-based expression systems suffer from genetic instability, and they need antibiotic selective pressure to maintain high copy number. This study aimed to employ EcN for synthesizing the photosensitizer 5-aminolevulinic acid (5-ALA). Firstly, the key genes of 5-ALA synthesis, hemA^M and hemL, were integrated into the EcN genome by the phage integration technique. Then, chemically inducible chromosomal evolution (CIChE) was adopted to increase the copy number of hemA^M and hemL and thus improved the stable synthesis of 5-ALA. The in vitro cell experiments verified that the constructed engineering strain can deliver stably synthesized 5-ALA to tumor cells and inhibit their growth. This study provided a basis for applying the engineering strains of EcN in the photodynamic therapy for tumors.