Design and in situ biomanufacturing of targeted peptide inhibitors via engineered probiotic yeast.

许多生物学过程，如癌细胞增殖、炎症和感染，都发生在人类肠道中。在治疗这些疾病和感染时，生物疗法特别有用，也就是使用从活体中提取的物质进行治疗。生物治疗中使用的生物分子与肠道内的特定分子或有机体紧密结合，以调节它们的行为。不幸的是，生物疗法的生产和分销极其复杂和昂贵，限制了公众的可获得性。一种可能的替代方法是使用未使用的饮食材料和工程微生物直接在患者的肠道内制造生物药物。由于人类的肠道比用于生产传统生物制剂的工业生物反应器要复杂得多，因此如何完成这一壮举仍有很多需要学习。该项目将研究使用工程益生菌酵母直接在大肠中生产氨基酸(肽)短链的策略。这些多肽将经过优化，与艰难梭菌产生的毒素结合，艰难梭菌是一种医院获得性胃肠道感染，通常被称为C.diff。一种与面包师酵母相关的酵母菌株，布拉酵母，将被改造成在人类肠道的严酷条件下生产多肽。这种方法的有效性将在模型器官中进行检验。这种使用工程微生物将药物分子直接输送到肠道的过程有望大幅降低生物疗法的成本，从而改善人类健康。研究人员还将从事与研究相关的教育和外联活动，包括为高中教师举办一个关于微生物组工程的周末研讨会。旨在扩大代表不足群体参与STEM的活动将包括指导和积极招聘工作。原位生物制造，即治疗分子由工程微生物直接在人体肠道中合成，是一种有吸引力的替代生物疗法的复杂制造和口服给药的方法。然而，人类的肠道不同于任何工业生物反应器，在原位生物制造成为现实之前，许多悬而未决的问题仍然存在。研究人员认为，在布氏酵母菌中制造针对艰难梭状芽胞杆菌感染的多肽类药物将成为开发设计策略的理想平台和模型系统，从而能够直接在肠道中高效地生产生物分子。将追求两个互补的目标来测试工程菌株和多肽。首先，将设计与艰难梭菌毒素A及其表层蛋白(SLpA)特异结合的多肽。这些多肽将通过结合固相库筛选和计算多肽设计/优化方法来设计。数以千计的多肽的效率将使用体外肠道模型进行测试。其次，在肠道模型中，布氏链球菌的生理状态将通过确定哪些基因在肠道中表达，哪些蛋白质在肠道中分泌来表征。这些知识将被应用于开发能够有效地分泌和展示第一个目标中设计的多肽的布氏链球菌工程菌株。工程菌株在3D有机物中对抗艰难梭菌致病的能力也将被确定。该项目将增进对就地生物制造的了解，并揭示确保多肽效果的最佳实践。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Many biological processes such as cancer cell proliferation, inflammation, and infection occur in the human gut. Biologic therapies, or treatments using substances made from living organisms, are particularly useful in treating these diseases and infections. The biological molecules used in biologic therapy tightly bind to specific molecules or organisms within the gut to modulate their behavior. Unfortunately, production and distribution of biologic therapies is incredibly complex and expensive, limiting availability to the public. A possible alternative approach is to make the biologic drugs directly in the patient's gut using unused dietary material and engineered microbes. Since the human gut is much more complex than an industrial bioreactor used to produce conventional biologics, there is still much to learn about how to accomplish this feat. This project will investigate strategies for biomanufacturing short chains of amino acids (peptides) directly in the large intestine using engineered probiotic yeast. The peptides will be optimized to bind with the toxins produced by Clostridioides difficile, the hospital-acquired gastrointestinal infection commonly referred to as C. diff. A yeast strain related to baker's yeast, Saccharomyces boulardii, will be engineered to produce the peptides under the harsh conditions of the human gut. The effectiveness of this approach will be examined in model organs, or 'miniguts.' This process of delivering drug molecules directly to the gut using engineered microbes promises to substantially reduce the cost of biologic therapeutics and, subsequently, improve human health. The investigators will also engage in research-related education and outreach, including developing a weekend symposium for high school teachers on microbiome engineering. Activities toward broadening the participation of underrepresented groups in STEM will include mentoring and active recruitment efforts. In situ biomanufacturing, wherein therapeutic molecules are synthesized directly in the human gut by engineered microbes, is an attractive alternative to the complex manufacturing and oral delivery of biologic therapies. However, the human gut is unlike any industrial bioreactor, and many outstanding questions remain before in situ biomanufacturing becomes reality. The investigators posit that manufacturing peptide-based drugs in the probiotic yeast Saccharomyces boulardii to target Clostridioides difficile infection will serve as an ideal platform and model system by which to develop design strategies enabling efficient production of biologic molecules directly in the gut. Two complementary objectives will be pursued toward testing the engineering strains and peptides. First, peptides that specifically bind to C. difficile toxin A and to its surface layer protein (SlpA) will be designed. The peptides will be designed by combining solid phase library screening and computational peptide design/optimization approaches. The efficiency of thousands of peptides will be tested using in vitro gut models. Second, the physiological state of S. boulardii in gut models will be characterized by determining which genes are expressed and which proteins are secreted while residing in the gut. This knowledge will be applied to develop engineered S. boulardii strains that can effectively secrete and display the peptides designed in the first objective. The ability of the engineered strains to counteract C. difficile pathogenesis in 3D organoids will also be determined. The project will advance understanding of in situ biomanufacturing and reveal best practices for ensuring peptide efficacy.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.