SBIR Phase I: Using enzymes secreted by soil amoebae to fight bacterial biofilms in agriculture

这个小企业创新研究（SBIR）第一阶段项目的更广泛影响/商业潜力是开发一种新的技术平台，用于治疗在田间和收获储存期间对植物健康构成持续威胁的细菌病原体。许多细菌通过在植物表面形成生物膜来杀死宿主。这一建议的核心是发现生物膜赋予细菌对抗菌剂的耐受性，无论这些抗菌剂是抗生素、消毒剂还是杀菌剂。分散这些生物膜对于清除植物表面的污染细菌是必要的。该提案通过从五大洲和几个岛屿国家分离的3000多种独特的微生物中开发安全和天然的生物膜降解酶来解决这些挑战。这个原理验证项目的重点是发现用于治疗细菌性马铃薯软腐病的酶。这种疾病对我们国家的社会和商业都有影响，因为马铃薯是美国消费最多的蔬菜，总市场价值超过35亿美元。拟议的项目推进了Dictyostelid菌株的使用，这些菌株能够分散Dickeya和Pectobacterium形成的生物膜，并以从相应细菌生物膜中释放的浮游细菌为食。为了能够破坏这些生物膜，它们必须能够破坏生物膜的完整性。该项目使用授权公司在全球农业市场上使用的专有技术。糖苷水解酶家族（GHF）的疑似成员已经通过生物信息学和粗分泌产物被鉴定出来。该项目有两个目标：(1)利用多核苷酸链反应扩增编码GHF的Dictyostelids DNA，并将其克隆到大肠杆菌兼容载体中，并使用专用亲和柱纯化框架内组氨酸标签序列；(2)亲和纯化酶将用于分散建立在96孔聚苯乙烯板上的Dickeya和Pectobacterium生物膜。表现最好的酶将进一步检查其在马铃薯表面分散生物膜和防止软腐的能力。该奖项反映了美国国家科学基金会的法定使命，并通过基金会的知识价值和更广泛的影响审查标准进行评估，认为值得支持。

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) Phase I project is development of a novel technology platform for the treatment of bacterial pathogens that pose a persistent threat to plant health both in the field and during storage of the harvest. Many bacteria kill their host by forming biofilms on the plant surface. Central to this proposal are findings that biofilms confer tolerance of bacteria to antimicrobial agents, whether these antimicrobial agents are antibiotics, disinfectants, or germicides. Dispersing these biofilms is necessary to remove contaminating bacteria from the plant surface. This proposal is addressing these challenges by developing safe and natural biofilm degrading enzymes from the collection of over 3,000 unique microorganisms called Dictyostelids isolated from five continents and several island nations. The focus of this proof-of-principle project is discovery of enzymes for treatment of bacterial potato soft rot. This disease is socially and commercially impacting our nation as potatoes are the most consumed vegetable in the U.S. with a total market value exceeding $3.5 B. The proposed project advances the use of Dictyostelid strains capable of dispersing biofilms formed by Dickeya and Pectobacterium and feeding on planktonic bacteria liberated from the corresponding bacterial biofilms. To be able to destroy those biofilms they must be able to break down biofilm integrity. This project uses proprietary technology licensed to the firm for a world-wide use in the agricultural markets. Suspected members of Glycoside Hydrolase Family of enzymes (GHF) have been already identified by bioinformatics and in crude secreted products. This project has two objectives: (1) Using polynucleotide chain reaction it will amplify Dictyostelids DNA encoding GHF and clone it into an Escherichia coli compatible vector with an in-frame Histidine-Tag sequence for purification using a specialized affinity column, and (2) Affinity purified enzymes will be used to disperse Dickeya and Pectobacterium biofilms established on 96-well polystyrene plates. The best performing enzymes will be further examined for their ability to disperse biofilms on a potato surface and prevent soft rot.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.