Advanced microbiome therapeutics have emerged as a powerful approach for the treatment of numerous diseases. While the genetic instability of genetically engineered microorganisms is a well-known challenge in the scale-up of biomanufacturing processes, it has not yet been investigated for advanced microbiome therapeutics. Here, the evolution of engineered Escherichia coli Nissle 1917 strains producing Interleukin 2 and Aldafermin were investigated in two strain backgrounds with and without the three error-prone DNA polymerases polB, dinB, and umuDC, which contribute to the mutation rate of the host strain. Whole genome short-read sequencing revealed the genetic instability of the pMUT-based production plasmid after serial passaging for approximately 150 generations using an automated platform for high-throughput microbial evolution in five independent lineages for six distinct strains. While a reduction of the number of mutations of 12%–43% could be observed after the deletion of the error-prone DNA polymerases, the interruption of production-relevant genes could not be prevented, highlighting the need for additional strategies to improve the stability of advanced microbiome therapeutics.

中文翻译：

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放大模拟过程中工程化大肠杆菌 Nissle 1917 菌株的遗传异质性


先进的微生物组疗法已成为治疗多种疾病的有力方法。虽然基因工程微生物的遗传不稳定性是生物制造工艺放大中众所周知的挑战，但尚未针对先进的微生物组疗法进行研究。在这里，在两种菌株背景下研究了产生白细胞介素 2 和 Aldafermin 的工程化大肠杆菌 Nissle 1917 菌株的进化，其中有和没有三种容易出错的 DNA 聚合酶 polB、dinB 和 umuDC，它们有助于宿主菌株的突变率。全基因组短读长测序揭示了基于 pMUT 的生产质粒在连续传代后大约 150 代的遗传不稳定性，该平台使用自动化平台在 6 个不同菌株的 5 个独立谱系中进行高通量微生物进化。虽然在删除易出错的 DNA 聚合酶后可以观察到突变数量减少 12%-43%，但无法阻止生产相关基因的中断，这凸显了需要额外的策略来提高先进微生物组疗法的稳定性。