ZBiotics Company, San Francisco, CA, United States of America.
PLoS One. 2024 Nov 7;19(11):e0312457. doi: 10.1371/journal.pone.0312457. eCollection 2024.
We have addressed critical challenges in probiotic design to develop a commercially viable bacterial strain capable of removing the intestinal toxin, acetaldehyde. In this study, we report the engineering of the hag locus, a σD-dependent flagellin expression site, as a stable location for robust enzyme production. We demonstrate constitutive gene expression in relevant conditions driven by the endogenous hag promoter, following a deletion of the gene encoding a post-translational regulator of σD, FlgM, and a point mutation to abrogate the binding of the translational inhibitor CsrA. Reporter constructs demonstrate activity at the hag locus after germination, with a steady increase in heterologous expression throughout outgrowth and vegetative growth. To evaluate the chassis as a spore-based probiotic solution, we identified the physiologically relevant ethanol metabolic pathway and the subsequent accumulation of gut-derived acetaldehyde following alcohol consumption. We integrated a Cupriavidus necator aldehyde dehydrogenase gene (acoD) into the hag locus under the control of the flagellin promoter and observed a rapid reduction in acetaldehyde levels in gut-simulated conditions post-germination. This work demonstrates a promising approach for the development of genetically engineered spore-based probiotics.
我们已经解决了益生菌设计中的关键挑战，开发出一种商业上可行的细菌菌株，能够去除肠道毒素乙醛。在这项研究中，我们报告了 hag 基因座的工程改造，该基因座是一个 σD 依赖性鞭毛表达位点，是稳定产生大量酶的位置。我们证明了在相关条件下，由内源性 hag 启动子驱动的组成型基因表达，这是在删除编码 σD 的翻译后调节因子 FlgM 的基因后实现的，并进行了点突变以消除翻译抑制剂 CsrA 的结合。报告基因构建体在发芽后在 hag 基因座上显示出活性，随着出芽和营养生长过程中的异源表达不断增加，活性稳定增加。为了评估底盘作为基于孢子的益生菌解决方案，我们确定了生理相关的乙醇代谢途径以及饮酒后肠道衍生的乙醛的后续积累。我们将 Cupriavidus necator 醛脱氢酶基因（acoD）整合到 hag 基因座中，由鞭毛启动子控制，并在发芽后模拟肠道条件下观察到乙醛水平迅速降低。这项工作展示了一种有前途的方法，用于开发基于基因工程改造的孢子益生菌。
