Practical Insights and Emerging Trends for Strategic Cloning of Large Biosynthetic Gene Clusters from Bacteria

Cloning large biosynthetic gene clusters (BGCs) is fundamental to unlocking microbial natural product potential for drug discovery and biotechnology. These clusters encode diverse bioactive compounds, but their size, high GC content, and complex architecture pose significant technical challenges. This review scrutinises recent advances in BGC cloning strategies, categorising them into three major groups: (1) direct release-and-capture methods, (2) genome-integrated preconditioning systems, and (3) CRISPR-assisted hybrid platforms. This review compares the strengths, limitations, and reported efficiencies of BGC cloning strategies, highlighting trade-offs in precision, scalability, and workflow complexity. Emerging trends, such as AI-driven genome mining, modular synthetic biology toolkits, and high-throughput automation, are reshaping the cloning landscape, enabling predictive design and streamlined assembly of clusters exceeding 100 kb. By integrating comparative analysis with future perspectives, this review provides outlines on how next-generation strategies will accelerate heterologous expression, natural product discovery, and sustainable biomanufacturing.