Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, USA.
Molecular Education, Technology and Research Innovation Center (METRIC), North Carolina State University, Raleigh, NC, USA.
Microb Cell Fact. 2023 Jun 7;22(1):109. doi: 10.1186/s12934-023-02117-y.
The probiotic yeast Saccharomyces boulardii (Sb) is a promising chassis to deliver therapeutic proteins to the gut due to Sb's innate therapeutic properties, resistance to phage and antibiotics, and high protein secretion capacity. To maintain therapeutic efficacy in the context of challenges such as washout, low rates of diffusion, weak target binding, and/or high rates of proteolysis, it is desirable to engineer Sb strains with enhanced levels of protein secretion. In this work, we explored genetic modifications in both cis- (i.e. to the expression cassette of the secreted protein) and trans- (i.e. to the Sb genome) that enhance Sb's ability to secrete proteins, taking a Clostridioides difficile Toxin A neutralizing peptide (NPA) as our model therapeutic. First, by modulating the copy number of the NPA expression cassette, we found NPA concentrations in the supernatant could be varied by sixfold (76-458 mg/L) in microbioreactor fermentations. In the context of high NPA copy number, we found a previously-developed collection of native and synthetic secretion signals could further tune NPA secretion between 121 and 463 mg/L. Then, guided by prior knowledge of S. cerevisiae's secretion mechanisms, we generated a library of homozygous single gene deletion strains, the most productive of which achieved 2297 mg/L secretory production of NPA. We then expanded on this library by performing combinatorial gene deletions, supplemented by proteomics experiments. We ultimately constructed a quadruple protease-deficient Sb strain that produces 5045 mg/L secretory NPA, an improvement of > tenfold over wild-type Sb. Overall, this work systematically explores a broad collection of engineering strategies to improve protein secretion in Sb and highlights the ability of proteomics to highlight under-explored mediators of this process. In doing so, we created a set of probiotic strains that are capable of delivering a wide range of protein titers and therefore furthers the ability of Sb to deliver therapeutics to the gut and other settings to which it is adapted.
产朊假丝酵母（Sb）是一种有前途的底盘，可以将治疗性蛋白递送到肠道，因为 Sb 具有先天的治疗特性、对噬菌体和抗生素的抗性以及高蛋白分泌能力。为了在冲洗、扩散率低、靶标结合弱和/或蛋白水解率高的情况下保持治疗效果，需要对 Sb 菌株进行工程改造，以提高蛋白分泌水平。在这项工作中，我们探索了顺式（即分泌蛋白表达盒内）和反式（即 Sb 基因组内）的遗传修饰，以增强 Sb 分泌蛋白的能力，以艰难梭菌毒素 A 中和肽（NPA）作为我们的模型治疗药物。首先，通过调节 NPA 表达盒的拷贝数，我们发现微生物反应器发酵中上清液中的 NPA 浓度可以变化 6 倍（76-458mg/L）。在高 NPA 拷贝数的情况下，我们发现先前开发的一组天然和合成分泌信号可以进一步将 NPA 分泌量调节在 121-463mg/L 之间。然后，根据对 S. cerevisiae 分泌机制的先验知识，我们生成了一个纯合单基因缺失菌株文库，其中最具生产力的菌株可分泌 2297mg/L 的 NPA。然后，我们通过进行组合基因缺失并补充蛋白质组学实验来扩展该文库。最终，我们构建了一个四重蛋白酶缺陷的 Sb 菌株，可分泌 5045mg/L 的分泌型 NPA，比野生型 Sb 提高了 10 倍以上。总的来说，这项工作系统地探索了广泛的工程策略来提高 Sb 中的蛋白分泌，并强调了蛋白质组学在突出该过程中未充分探索的介质的能力。通过这样做，我们创建了一组益生菌菌株，它们能够递送广泛的蛋白滴度，从而进一步提高 Sb 将治疗药物递送到肠道和其他适应环境的能力。
