Development of ClusterBuild to Synthesize Biosynthetic Gene Clusters

About This Grant

Project Summary/Abstract Development of ClusterBuild to Synthesize Biosynthetic Gene Clusters Opportunity Number: PA-24-245 PIs: Christopher L. Warren and Mary S. Ozers The ability to synthesize large biosynthetic gene clusters (BGCs) is essential for unlocking the therapeutic potential of natural products, including next-generation antibiotics. Many promising BGCs are derived from unculturable microbes, making direct access to their DNA challenging. Existing methods for assembling large constructs, such as PCR assembly, Golden Gate cloning, and solid-phase synthesis, are prohibitively expensive, labor-intensive, and slow when scaled beyond 10 kb, presenting a critical bottleneck in natural product discovery. These limitations hinder broader participation in synthetic biology and delay translation from genomic data to biomedical applications. This project proposes ClusterBuild, a novel platform that enables cost-effective, high-fidelity synthesis of large DNA constructs (up to 100 kb) by combining DNA microarrays with peptide nucleic acid (PNA)-based error correction and isothermal assembly. PNAs can be used to correct DNA synthesis errors due to their high binding affinity and specificity for complementary DNA sequences, enabling them to selectively hybridize to mismatched regions and facilitate targeted removal of erroneous DNA strands. ClusterBuild leverages high-density photolithographic microarrays to generate massive oligo libraries, while complementary PNA arrays are used to selectively purify error-free sequences. Aim 1 will demonstrate that PNA microarrays can purify high-fidelity oligonucleotides generated from a commercial DNA microarray using sequence-specific melting temperature (Tm) elution. Aim 2 will verify that purified DNA oligos can be efficiently assembled into a 12 kb biosynthetic gene cluster using barcoded, overlapping oligonucleotides and multi-stage isothermal assembly. Aim 3 will confirm functional expression of the assembled cluster in E. coli to produce a class II lanthipeptide, a promising compound with low micromolar activity against the pathogen Klebsiella pneumoniae. In Phase II, the full end-to-end workflow will be implemented using a digital micromirror device (DMD)-based microarray synthesizer to scale synthesis up to 100 megabases. The ClusterBuild technology will offer three key advantages over commercially available options by achieving more than 100-fold cost savings, a 50-fold increase in construct length, and fewer errors of one per 10 kb length. This proposal supports the "Build" step in the Design-Build-Test-Learn cycle, unlocking broader participation in synthetic biology and natural product drug discovery.