A genetic engineering strategy for the improved production of the anti-helminthic

描述（由申请人提供）：蠕虫感染是全球范围内人类和动物种群中严重健康问题的原因。据估计，20 - 30亿人，主要来自世界农村和贫困地区，患有由寄生线虫引起的疾病；然而，由于商业市场不佳，药物开发和发现工作缺乏对抗人类感染的承诺。相比之下，在有价值的家畜群中，有强有力的动物卫生发现工作，旨在根除感染。因此，动物健康项目经常依赖于为人类治疗提供候选药物。由于在动物身上进行了广泛试验，降低了源自动物健康的人类药物的成本和开发风险。虽然人类候选药物仍然需要超过严格的安全标准，但同样的挑战是尽量减少货物成本，使受感染人群能够获得有效的低成本治疗。因此，paraherquamide代表了一类新的抗寄生虫抗生素，具有广谱抗线虫活性，是最近推出的动物保健抗寄生虫剂2- desoxopparaherquamide （Derquantel(tm)）的前体。目前，对苯二甲酰胺是通过真菌菌株P. simplicissimum发酵生产的。为了最大限度地提高产量，制药商已经开始了菌株改进计划，随机进化出高产突变体；然而，该程序未能达到预期的输出。在这里，冲积提出了一个现代化的，基因组驱动的菌株改进策略，旨在最大限度地提高发酵对甲酰胺的产量。具体来说，冲积的方法是由真菌基因组的DNA序列指导的，其中已经确定了负责对甲酰胺生产的关键调控和/或生物合成基因。合理的基因工程策略，如基因缺失或基因扩增，将用于关键基因的努力

DESCRIPTION (provided by applicant): Helminth infections are responsible for severe health problems worldwide, in both human and animal populations. It is estimated that 2-3 billion people, mainly from rural and impoverished regions of the world suffer from disease due to parasitic nematodes; however, drug development and discovery efforts lack commitment to combat human infection due to the poor commercial market. In contrast, there are robust animal health discovery efforts aimed at eradicating infection in valuable domesticated livestock herds. Thus, animal health programs are often relied on to provide drug candidates for human therapeutics. Extensively tested in animals, the cost and development risk of human drugs originating from animal health are reduced. While human drug candidates still need to surpass stringent safety standards, an equal challenge is minimizing cost of goods to enable the infected population access to potent, low cost therapeutics. Accordingly, paraherquamide represents a new class of anti-helminth antibiotics that possesses broad-spectrum anti-nematodal activity and is a precursor to a recently launched animal health anti-parasitic agent 2- desoxoparaherquamide (Derquantel(tm)). Currently paraherquamide is manufactured through fermentation of the fungal strain P. simplicissimum. To maximize yields, the drug manufacturer has embarked on strain improvement programs to randomly evolve high producing mutants; however, this program has failed to achieve the desired output. Here, Alluvium proposes a modernized, genomics-driven strain improvement strategy aimed at maximizing paraherquamide production in fermentation. Specifically, Alluvium's approach is guided by the DNA sequence of the fungal genome in which key regulatory and/or biosynthetic genes responsible for paraherquamide production have been identified. Rational genetic engineering strategies, such as gene deletion or gene amplification, will be applied to key genes in efforts to rationally generate a high producing mutant phenotype. In this Phase I proposal, work is focused on rationally manipulating three genes that are predicted to regulate, both positively and negatively paraherquamide biosynthesis. In addition, Alluvium proposes to replicate the entire paraherquamide biosynthetic gene cluster within the fungal chromosome in order to boost production of anti-helminthic natural product. Following success in these initial studies, Phase II work will focus on engineering further efficiencies in paraherquamide production, including developing biocatalytic methods capable of converting paraherquamide to 2-desoxoparaherquamide as well as biotransformation tools that can be employed to generate synthetically challenging structural analogs. In sum, the strain engineering technology under development at Alluvium will result in an optimized paraherquamide manufacturing process that will lower the cost of goods required to access this commercially important anti- helminthic therapeutic, thereby enabling the potential development of a much-needed human therapeutic. PUBLIC HEALTH RELEVANCE: Helminths, especially parasitic nematodes, cause severe health problems in both humans and domesticated animals. Approximately 2-3 billion people in rural and impoverished regions are infected; however, affordable and effective treatment options are scarce. The proposed work seeks to improve upon the production of the promising anti-helminthic animal health chemotherapeutic agent, paraherquamide, so it can be generated in a more cost-effective and efficient manner, thereby stimulating further development activities for potential human treatment.