干扰素基因激活蛋白（STING）是用于肿瘤治疗的重要和有前途的先天免疫靶点。然而，STING激动剂的不稳定性以及它们引起全身免疫激活的倾向是一大障碍。来自改良后的大肠杆菌Nissle 1917的STING激活剂，在抗肿瘤活性方面表现出较高的效果，并有效地减少了STING通路激活所引起的“离靶”系统效应。在这项研究中，我们使用合成生物学的方法来优化在体外催化CDA合成的双腺苷酸环化酶的翻译水平。我们开发了2个工程菌株，CIBT4523和CIBT4712，用于产生高水平的CDA，同时保持其浓度在不影响生长的范围内。尽管CIBT4712表现出与体外CDA水平相对应的更强的STING通路诱导作用，但在同种移植瘤模型中，其抗肿瘤活性低于CIBT4523，这可能与存活在肿瘤组织中的细菌稳定性有关。CIBT4523表现出完全的肿瘤消退、小鼠生存时间延长和拒绝再次挑战的肿瘤，为更有效的肿瘤治疗提供了新的可能性。我们展示了在工程细菌菌株中适当的CDA产生对于平衡抗肿瘤效能和自毒性至关重要。

The stimulator of interferon genes (STING) protein is an important and promising innate immune target for tumor therapy. However, the instability of the agonists of STING and their tendency to cause systemic immune activation is a hurdle. The STING activator, cyclic di-adenosine monophosphate (CDA), produced by the modified Escherichia coli Nissle 1917, shows high antitumor activity and effectively reduces the systemic effects of the "off-target" caused by the activation of the STING pathway. In this study, we used synthetic biological approaches to optimize the translation levels of the diadenylate cyclase that catalyzes CDA synthesis in vitro. We developed 2 engineered strains, CIBT4523 and CIBT4712, for producing high levels of CDA while keeping their concentrations within a range that did not compromise the growth. Although CIBT4712 exhibited stronger induction of the STING pathway corresponding to in vitro CDA levels, it had lower antitumor activity than CIBT4523 in an allograft tumor model, which might be related to the stability of the surviving bacteria in the tumor tissue. CIBT4523 exhibited complete tumor regression, prolonged survival of mice, and rejection of rechallenged tumors, thus, offering new possibilities for more effective tumor therapy. We showed that the appropriate production of CDA in engineered bacterial strains is essential for balancing antitumor efficacy and self-toxicity.