PFI:AIR - TT: Using A. niger as a host to express recombinant endolysin

这个PFI:AIR技术翻译项目侧重于一个重要的农业和人类健康相关问题。50多年来，在动物饲料中添加低剂量的传统（即“医学上重要的”）抗生素以提高食用动物的生产效率一直是农业行业的普遍做法。虽然这些“抗生素生长促进剂”提供了巨大的经济效益，但这种做法也导致抗生素耐药菌株，这些菌株有可能感染人类。因此，美国食品和药物管理局已采取措施制止这种做法。虽然已经提出了许多替代医学上重要的抗生素的方法，但大多数都没有在商业上取得成功，因为它们不够有效。作为医学上重要的抗生素的替代品，抗菌蛋白的使用具有很大的前景，但其商业化生产的一个重大限制是成本。因此，经济地生产抗菌蛋白的能力将对农业工业产生重大利益，因为它将使农民保持高水平的生产力，同时不会产生对人类构成危险的耐抗生素菌株。本提案探讨了用抗菌蛋白（AMP）替代医学上重要的抗生素饲料添加剂的潜力。本文描述的工作旨在生成概念验证数据，利用重组丝状真菌以商业上可行的滴度表达模型amp。该项目采用了一种与商业相关的真菌物种（黑曲霉）来表达具有抗菌活性的模型内溶素蛋白。为了增加两种不同的内溶素酶的表达和细胞外分泌，将使用几种方法，探索基因组位置，转录启动子和分泌信号序列的影响。采用多种方法将增加成功的可能性，也将允许确定导致高产重组真菌菌株的最相关因素。这种理解将允许有效地产生额外的生产菌株，用于不同的蛋白质或肽。此外，参与该项目的人员，包括一名博士后科学家和几名本科生，将在跨学科的环境中接受培训，这将提高他们的研究能力。人员还将通过开展与此项目相关的客户发现活动，参与创业教育体验。这将主要涉及与整个价值链上的潜在利益相关者联系，以测试与未来商业化相关的假设。

This PFI:AIR Technology Translation project focuses on an important agricultural and human-health related problem. For over 50 years, it is has been common practice in the agricultural industry to add low doses of traditional (i.e., "medically important") antibiotics to animal feed to improve production efficiency of food animals. While these "antibiotic growth promoters" provide tremendous economic benefit, this practice also leads to antibiotic-resistant bacterial strains which have the potential to infect humans. As a result, the US Food and Drug Administration has implemented measures to stop this practice. While numerous alternatives to medically important antibiotics have been proposed, most have not been commercially successful as they are not adequately effective. As an alternative to medically important antibiotics, the use of antimicrobial proteins has great promise, but a significant limitation to their commercial production has been cost. Thus the ability to economically manufacture antimicrobial proteins would represent a significant benefit to the agricultural industry as it would allow farmers to maintain high levels of productivity while not generating antibiotic-resistant strains that pose a danger to humans. This proposal explores the potential of replacing medically-important antibiotic feed-additives with antimicrobial protein (AMP). Work described here seeks to generate proof-of-concept data, expressing model AMPs, at commercially viable titers, using recombinant filamentous fungi.This project employs a commercially-relevant fungal species (Aspergillus niger), to express model endolysin proteins with demonstrated bactericidal activity. To increase both expression and extracellular secretion of two different endolysin enzymes several approaches will be used, exploring the impact of genomic location, transcriptional promoter, and secretion signal sequence. Employing several approaches will increase the likelihood for success and will also allow identification of the most relevant factors leading to a high-producing recombinant fungal strain. This understanding will allow efficient generation of additional production strains for different proteins or peptides. In addition, personnel involved in this project, both a postdoctoral scientist and several undergraduate students, will be trained in a cross-disciplinary environment that will increase their research capacity. Personnel will also participate in entrepreneurial education experiences, through conducting customer discovery activities related to this project. This will primarily involve contacting potential stakeholders, along the entire value chain, to test hypotheses related to future commercialization.