New methods to develop novel antibiotics are desperately needed. Now, researchers have developed a way to manipulate key assembly line enzymes in bacteria, using CRISPR-Cas9 gene editing, which could pave the way to production of a new generation of complex antibiotics. Re-engineering biosynthetic assembly lines, including nonribosomal peptide synthetases (NRPS) and related megasynthase enzymes, may offer a powerful new route to develop future medicines to combat antimicrobial resistance.

This work is published in Nature Communications in the paper, “Gene editing enables rapid engineering of complex antibiotic assembly lines.

“The emergence of antibiotic-resistant pathogens is one of the biggest threats we face today,” noted Jason Micklefield, PhD, professor of chemical biology at the Manchester Institute of Biotechnology in the U.K. He continued, “The gene-editing approach we developed is a very efficient and rapid way to engineer complex assembly line enzymes that can produce new antibiotic structures with potentially improved properties.”

The U.K. government suggests that antimicrobial resistance (AMR) infections are estimated to cause 700,000 deaths each year globally and are predicted to rise to 10 million, costing the global economy $100 trillion, by 2050. AMR also threatens many of the UN’s Sustainable Development Goals (SDGs), with an extra 28 million people that could be forced into extreme poverty by 2050 unless AMR is contained.

The new paper describes how CRISPR-Cas9 gene editing can be used to create new NRPS enzymes that deliver clinically important antibiotics. More specifically, how CRISPR-Cas9 gene editing can be exploited to rapidly engineer one of the most complex megasynthase assembly lines in nature—the 2.0 MDa NRPS enzymes that deliver the lipopeptide antibiotic enduracidin.

In this work, gene editing was used to exchange subdomains within the NRPS, altering substrate selectivity, leading to ten new lipopeptide variants in good yields. NRPS enzymes are prolific producers of natural antibiotics such as penicillin. However, up until now, manipulating these complex enzymes to produce new and more effective antibiotics has been a major challenge.

Microorganisms in our environment, such as soil-dwelling bacteria, have evolved NRPS that assemble amino acids into peptides which often have very potent antibiotic activity. Many of the most therapeutically important antibiotics, used in the clinic today (penicillin, vancomycin, and daptomycin), are derived from these NRPS enzymes.

Unfortunately, pathogens continue to emerge that carry resistance to existing antibiotic drugs. One solution could be to create new antibiotics with improved properties that can evade the resistance mechanisms of the pathogens. However, the nonribosomal peptide antibiotics are very complex structures that are difficult and expensive to produce by normal chemical methods. To address this, the Manchester team used gene editing to engineer the NRPS enzymes, swapping domains that recognize different amino acid building, leading to new assembly lines that can deliver new peptide products.

The researchers say that the gene-editing process could be used to produce improved antibiotics and possibly lead to the development of new treatments helping in the fight against drug-resistant pathogens and illnesses in the future.

“We are now able to use gene editing to introduce targeted changes to complex NRPS enzymes,” added Micklefield, “introducing alternative amino acids precursors into peptide structures. We are optimistic that our new approach could lead to new ways of making improved antibiotics which are urgently needed to combat emerging drug-resistant pathogens.”

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