Colitis is a common disease of the colon that is very difficult to treat. Probiotic bacteria could be an effective treatment. The probiotic Escherichia coli Nissle 1917 (EcN) was engineered to synthesize the ketone body (R)-3-hydroxybutyrate (3HB) for sustainable production in the gut lumen of mice suffering from colitis. Components of heterologous 3HB synthesis routes were constructed, expressed, optimized, and inserted into the EcN genome, combined with deletions in competitive branch pathways. The genome-engineered EcN produced the highest 3HB level of 0.6 g/L under microaerobic conditions. The live therapeutic was found to colonize the mouse gastrointestinal tract over 14 days, elevating gut 3HB and short-chain-length fatty acid (SCFA) levels 8.7- and 3.1-fold compared to those of wild-type EcN, respectively. The sustainable presence of 3HB in mouse guts promoted the growth of probiotic bacteria, especially Akkermansia spp., to over 31% from the initial 2% of all the microbiome. As a result, the engineered EcN termed EcNL4 ameliorated colitis induced via dextran sulfate sodium (DSS) in mice. Compared to wild-type EcN or oral administration of 3HB, oral EcNL4 uptake demonstrated better effects on mouse weights, colon lengths, occult blood levels, gut tissue myeloperoxidase activity and proinflammatory cytokine concentrations. Thus, a promising live bacterium was developed to improve colonic microenvironments and further treat colitis. This proof-of-concept design can be employed to treat other diseases of the colon.

结肠炎是一种很难治疗的常见结肠疾病。益生菌可能是一种有效的治疗方法。益生菌大肠杆菌Nissle 1917 (EcN) 被设计用于合成酮体 (R)-3-羟基丁酸 (3HB)，用于在患有结肠炎的小鼠的肠腔中可持续生产。构建、表达、优化异源 3HB 合成途径的成分并将其插入 EcN 基因组，并结合竞争性分支途径中的缺失。基因组工程的 EcN 在微需氧条件下产生最高 0.6 g/L 的 3HB 水平。与野生型 EcN 相比，发现这种活的治疗剂在小鼠胃肠道中定植了 14 天，使肠道 3HB 和短链脂肪酸 (SCFA) 水平分别提高了 8.7 倍和 3.1 倍。3HB 在小鼠肠道中的持续存在促进了益生菌的生长，尤其是Akkermansiaspp.，从所有微生物组的最初 2% 增加到 31% 以上。结果，被称为 EcNL4 的工程化 EcN 改善了小鼠中通过硫酸葡聚糖钠 (DSS) 诱导的结肠炎。与野生型 EcN 或口服 3HB 相比，口服 EcNL4 对小鼠体重、结肠长度、潜血水平、肠组织髓过氧化物酶活性和促炎细胞因子浓度有更好的影响。因此，开发了一种有前途的活细菌来改善结肠微环境并进一步治疗结肠炎。这种概念验证设计可用于治疗结肠的其他疾病。