SBIR-STTR Award

Screening remains a major bottleneck in the discovery of new enzymes, protein therapeutics, and small molecule drugs. Many important high-value bioactives have yet to be discovered. This project is aimed at overcoming functional and throughput limitations of conventional screening methodologies by demonstrating the feasibility of a million-well screening platform for the discovery of rare bioactives from complex environmental gene libraries. Diversa's 100,000-well GigaMatrixTM platform has been successfully utilized to discover novel enzymes by expression screening. We now propose to make a significant advance in this approach to screening. By using million-well plates, we are aiming at screening throughputs of one billion clones per day using standard liquid-phase fluorescent assays. The necessity for this innovation derives from the requirement to screen complex, multi-organism (10 to the power of 7 to 10 to the power of 10) DNA libraries generated both from primary environmental samples and from laboratory evolution techniques. Furthermore, this development enables not only the discovery of rare novel enzymes but also antibody and protein therapeutic programs that require rapid screening of libraries with 10 to the power of 8 to 10 to the power of 9 antibody and protein variants. Commercialization of both bioactive molecules derived from screening and the GigaMatrix million-well technology itself is envisioned, each representing a significant contribution to the synthesis of new pharmaceuticals and therapeutics

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.

Thesaurus Terms:
amidase, drug screening /evaluation, genetic library, genetic screening, high throughput technology, technology /technique development cephalosporin, enzyme activity, expression cloning, molecular cloning biotechnology, site directed mutagenesis