Research Project
6735878
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.
