Why Natural Products?

The world of natural products (NPs) is a treasure trove of molecular diversity, harboring immense potential across various applications, particularly in therapeutics. Recent advancements in omics, computational sciences, and biotechnology have breathed new life into NP research, enabling access to the vast and untapped chemical potential within Nature through new innovative strategies for natural product biosyntheses.

Planning For a Diverse Microbial Collection

While these strategies offer promise, they are not always foolproof. Often, metabolic conditions within the host organism are inadequate to support the production of desired NPs. Furthermore, traditional efforts to up-regulate and activate biosynthetic gene clusters (BGCs) have been largely focused on specific genomic or structural aspects. However, what do you do when faced with a large diverse microbial collection –for example, the 160K strong natural organism library located in the Agency for Science, Technology and Research (A*STAR), Singapore. Is there a higher throughput and more generalized approach to interrogate these precious but silent natural producers?

This led us to ponder: instead of targeting specific BGCs, could a multi-faceted approach be employed to prime native strains, typically low producers, into a permanent optimal state for NP production?

The proposed solution was to leverage the innate regulatory and metabolic requirements of these strains for biosynthesis, thereby sidestepping time-consuming traditional methods of precise engineering, refactoring, and assembly.

To achieve this goal across a diverse native strain collection, a generalized and highly successful editing method was required. In this respect, phiC31-derived integrase emerged as our trusty tool of choice. Our editing strategy involved the examination of five different regulators (“activators”) in 54 diverse and non-domesticated actinobacterial strains. Eventually, we ended up with 124 unique strain-activator combinations which we fermented in 3-5 media. (Side note: molecular engineering of such scale in these actinobacteria would be unthinkable ten years ago, all these are made possible with great leaps in analytics, automation, synthetic biology (for actinobacteria),  metabolic engineering, and data science.)

Double The Products, Half The Effort (Next Time)

Armed with this data, we posed the question: Did our efforts yield the desired results? Yes! In fact, going beyond our initial expectations – we not only saw a nearly 2-fold increase in production of new metabolites but also observed upregulation of metabolites already produced in the native strains. Serendipitously, the process also enabled us to discover a tetramic acid analog with novel gram-negative bioactivity!

Next, contemplating future endeavors, we pondered: What steps should be taken if we were to embark on this journey anew? By meticulously analyzing the impact of "activators" and media on strain enhancement, we identified an initial minimal set of conditions necessary for maximizing productivity. Through extensive experience studying both known and novel multi-regulators, and multi-media cultivation experiments, we can now provide recommendations for a cost-effective approach to expand the chemical space with minimal experimentation. It turned out to be a 3 by 3 combination for maximal effect – 3 “activators” x 3 media!

In conclusion, this journey of revisiting something old (media optimization) and something new (genome engineering) illuminates the incredible potential of natural products, and how harnessing the intrinsic capabilities of native strains can unlock a treasure trove of diverse chemical compounds. The critical insights gleaned from this research are poised to revolutionize the use of native strain collections, guiding future exploration into the vast and rich world of Nature's potent pharmacy.