CRISPR-Cas9 in vivo editing enables rapid engineering of a complex antibiotic assembly line Wei Li Thong, Yingxin Zhang, Ying Zhuo, Katherine J. Robins, Joanna K. Fyans, Abigail J. Herbert, Brian J.C. Law and Jason Micklefield University of Manchester, UK The increasing prevalence of multidrug-resistant bacteria has poses a global health threat that has driven the need to diversify existing natural products to produce variants with improved pharmacological properties. Bioengineering of versatile biosynthetic pathways such as nonribosomal peptide synthetases (NRPS) to produce new antibiotic analogues in vivo is an attractive prospect. The modular nature of NRPS suggests a “mix and match” approach may be feasible, whereby exchanging or engineering adenylation (A) domains may lead to new NRP products with different amino acid combinations. We envisaged that with CRISPR-Cas9 technology, subdomain exchanges within NRPS could be performed at their native loci on the chromosome, thereby avoiding many of the problems associated with module/domain exchanges performed through in trans complementation. Here, we selected the complex lipopeptide antibiotic enduracidin as a model for CRISPR-Cas9-mediated bioengineering. Enduracidin is a lipopeptide antibiotic that is produced by an enormous 2.0 MDa NRPS that assembles 17 amino acid precursors (Figure 1). We demonstrated efficient in vivo replacement of A subdomains at their native loci in the enduracidin biosynthetic gene cluster (BGC), producing ten new enduracidin analogues in good yield. 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 from within the enduracidin BGC, a range of subdomains from NRPS enzymes of diverse bacterial origins were also successfully utilised. Whole-genome sequencing was performed on several of our edited strains, and we saw that the CRISPR-Cas9 editing did not result in any major off-target effects, demonstrating the precision of our method. In summary, we show that complex antibiotic assembly lines can be engineered in native producer strains using CRISPR-Cas9 editing to generate novel product analogues without impacting production efficiency.
Figure 1 ( Left ) Structure of wild type and engineered enduracidins, amino acids in blue were targeted for modification and percentages of yield for each analogue are presented in brackets. ( Right ) Organization of the enduracidin NRPS. References 1. Thong et al Nature Comm. 2021 , 12 , 6872 (https://doi.org/10.1038/s41467-021-27139-1)
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