Gene editing enables rapid engineering of complex antibiotic assembly lines

Abstract

Re-engineering biosynthetic assembly lines, including nonribosomal peptide synthetases (NRPS) and related megasynthase enzymes, is a powerful route to new antibiotics and other bioactive natural products that are too complex for chemical synthesis. However, engineering megasynthases is very challenging using current methods. Here, we describe 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. Gene editing was used to exchange subdomains within the NRPS, altering substrate selectivity, leading to ten new lipopeptide variants in good yields. In contrast, attempts to engineer the same NRPS using a conventional homologous recombination-mediated gene knockout and complementation approach resulted in only traces of new enduracidin variants. In addition to exchanging subdomains within the enduracidin NRPS, subdomains from a range of NRPS enzymes of diverse bacterial origins were also successfully utilized.

Type S Non‐Ribosomal Peptide Synthetases for the Rapid Generation of Tailormade Peptide Libraries**

“…For this reason, the entire natural product community is interested in finding new and efficient ways to unlock the hidden treasures that Nature has in store for us [17d,g,42] . Recent examples are the CRISPR‐Cas9‐based NRPS engineering [13] or DNA‐templated NRPSs [17e] . For easy and fast repurposing of biosynthetic modular assembly line pathways, we recently introduced the concept of type S NRPSs [17c] .…”

“…As a result, and due to the lack of adequate solutions, the pharmaceutical industry withdrew from traditional natural product research. With the rapid emergence of antimicrobial resistance (AMR) [2,7] and recent technological advances addressing the challenges, such as advances in cultivation, [8] DNA sequencing, [9,10] bioinformatics, [11] and synthetic biology [12] interest in natural product research has been reignited [6,13] …”

“…But as rational reprogramming efforts have been met with limited success, progress in the synthetic biology of NPs is of great importance. Therefore, several labs develop tools to enable the reproducible, rapid, and simple genetic manipulation of biosynthetic gene clusters (BGCs) for the biosynthesis of NRP derivatives and even new peptides [13,17] …”

“…With the rapid emergence of antimicrobial resistance (AMR) [2,7] and recent technological advances addressing the challenges, such as advances in cultivation, [8] DNA sequencing, [9,10] bioinformatics, [11] and synthetic biology [12] interest in natural product research has been reignited. [6,13] Despite the progress made, the complexity of NP structures make it difficult to generate synthetic NP derivatives, for example to explore structure-activity relationships and develop hits to leads. Thus, many clinical derivatives have been created by means of semi-synthesis, [2c] i. e. azithromycin [14] and cephalosporin.…”

Knowledge-guided data mining on the standardized architecture of NRPS: subtypes, novel motifs, and sequence entanglements

“…This subdomain starts from the middle of A3 and A4, and ends from the middle of A and A6 (the ending is exactly the G-motif, VPIGAPI in the PheA). Recently, another research team Thong et al also used this A subdomain swapping strategy by CRISPR-Cas9 and successfully reengineered A domain substrate specificity even subdomains from a range of NRPS enzymes of diverse bacterial origins 63 . By structural analysis, we suspected that the conserved glycine in the loop may provide crucial structure flexibility as a hinge during the large conformation changes when the NRPS exerting its multistep synthetase function.…”