Development of an Advanced Screening Platform

描述（由申请人提供）：这项工作旨在开发一种先进的筛选平台，该平台克服了用于发现新酶、蛋白质治疗剂和小分子药物的常规筛选方法的功能和通量限制。我们建议进一步开发百万孔GigaMatrix技术，在第一阶段中展示了筛选Diversa的大量不同环境DNA文库，以发现能够将头孢菌素C（CephC）转化为7-氨基头孢烷酸（7-ACA）的酰胺酶。7-ACA是许多广泛使用的广谱抗生素生产中的关键中间体。将CephC直接转化为7-ACA的有效生物催化方法将取代当前的工业方法，减少相关的化学废物流，并降低这些抗生素的成本。有证据表明，能够这种转换的酰胺酶存在，但很少见，因此，广泛的筛选高度多样化的基因库将需要他们的发现。第二阶段工作的第一年旨在完善百万井GigaMatrix平台，以实现如此大规模的筛选工作。这需要通过最大限度地减少导致下游瓶颈的假阳性来优化该技术的整体生产力，然后是孵育，平板操作，检测和命中恢复的规模化自动化。第二年将集中于候选酰胺酶的大规模筛选以及随后的酶表征和优化。这包括通过开发具有改进的荧光信号-背景性能的新底物来改进当前的筛选测定。从筛选工作中获得的候选酶将在实验室规模的应用条件下测试其直接将CephC转化为7-ACA的能力。Diversa的进化技术（基因位点饱和诱变TM和/或基因重组TM）将在必要时用于生产适用于大规模生产半合成头孢菌素抗生素的商业上可行的酰胺酶。预计CephC酰胺酶和百万孔GigaMatix平台都将成为这项工作成果的商业产品，每一个都代表着对新药物和治疗剂合成的重大贡献。

DESCRIPTION (provided by applicant): This effort is aimed at the development of an advanced screening platform that overcomes the functional and throughput limitations of conventional screening methodologies for the discovery of new enzymes, protein therapeutics, and small molecule drugs. We propose to further develop the million-well GigaMatrix technology demonstrated in Phase I toward screening Diversa's large collection of diverse environmental DNA libraries for the discovery of amidases capable of converting cephalosporin C (CephC) to 7- aminocephalosporanic acid (7-ACA). 7-ACA is a key intermediate in the manufacture of many widely used broad-spectrum antibiotics. An efficient biocatalytic method for direct conversion of CephC to 7-ACA would supplant current industrial methods, reduce the associated chemical waste stream, and lower the cost of these antibiotics. Evidence suggests that amidases capable of this conversion exist, yet are rare and therefore extensive screening of highly diverse gene libraries will be required for their discovery. The first year of the Phase II work is aimed at refining the million-well GigaMatrix platform to enable such massive screening efforts. This entails optimization of the overall productivity of the technology by minimizing false positives that lead to downstream bottlenecks, followed by scale-up automation of incubation, plate manipulation, detection and hit recovery. The second year will focus on large-scale screening for candidate amidases and subsequent enzyme characterization and optimization. This includes improving the current screening assay by developing a new substrate with improved fluorescent signal-to-background performance. Candidate enzymes obtained from the screening effort will be tested in lab-scale application conditions for their ability to directly convert CephC to 7-ACA. Diversa's evolution technologies (Gene Site Saturation Mutagenesis TM and/or GeneReassembly TM) will be employed, as necessary, to produce a commercially-viable amidase applicable to large-scale manufacture of semi-synthetic cephalosporin antibiotics. It is anticipated that both the CephC amidase and the million-well GigaMatix platform will be commercial products resulting from the outcome of this work, each representing a significant contribution to the synthesis of new pharmaceuticals and therapeutics.