High GC Content Cas9-Mediated Genome-Editing and Biosynthetic Gene Cluster Activation in Saccharopolyspora erythraea

Abstract

The overexpression of bacterial secondary metabolite biosynthetic enzymes is the basis for industrial overproducing strains. Genome editing tools can be used to further improve gene expression and yield. Saccharopolyspora erythraea produces erythromycin, which has extensive clinical applications. In this study, the CRISPR-Cas9 system was used to edit genes in the S. erythraea genome. A temperature-sensitive plasmid containing the PermE promoter, to drive Cas9 expression, and the Pj23119 and PkasO promoters, to drive sgRNAs, was designed. Erythromycin esterase, encoded by S. erythraea SACE_1765, inactivates erythromycin by hydrolyzing the macrolactone ring. Sequencing and qRT-PCR confirmed that reporter genes were successfully inserted into the SACE_1765 gene. Deletion of SACE_1765 in a high-producing strain resulted in a 12.7% increase in erythromycin levels. Subsequent PermE- egfp knock-in at the SACE_0712 locus resulted in an 80.3% increase in erythromycin production compared with that of wild type. Further investigation showed that PermE promoter knock-in activated the erythromycin biosynthetic gene clusters at the SACE_0712 locus. Additionally, deletion of indA (SACE_1229) using dual sgRNA targeting without markers increased the editing efficiency to 65%. In summary, we have successfully applied Cas9-based genome editing to a bacterial strain, S. erythraea, with a high GC content. This system has potential application for both genome-editing and biosynthetic gene cluster activation in Actinobacteria.

RETRACTED: Optimizing sgRNA to Improve CRISPR/Cas9 Knockout Efficiency: Special Focus on Human and Animal Cell

“…In addition to verify that the GC content between 40% and 60% is favored for knockdown of a target gene, SOCS1 (suppressor of cytokine signaling 1), by sgRNA, this study also revealed that the positive traits in sgRNAs are the circumvention of both a C at position 3 and a G at position 16 (Bruegmann et al, 2019). Besides, the study of CRISPR/Cas9-mediated mutagenesis in Drosophila revealed that sgRNAs with four GC in the sequence of the six base pairs adjacent to the PAM sequence are severely favored (Liu et al, 2018). Further, Fu et al (2017) determined the efficacy of thousands of targets, applying this to the Escherichia coli type I-E Cascade (CRISPR-associated complex for antiviral defense) system.…”

Characterization of Cas proteins for CRISPR‐Cas editing in streptomycetes

“…Beyond genome editing, multiplexing with orthogonal catalytically inactive Cas proteins with different PAM specificities may allow transcriptional control of different gene subsets in actinomycetes using CRISPR interference (Rock et al, ; Tong et al, ; Zhao et al, ). Last but not least, we expect this expanded Cas toolbox to be relevant to the engineering of rare actinomycetes (Cohen & Townsend, ; Liu, Wei, & Ye, ; Wolf et al, ).…”

An Update on Molecular Tools for Genetic Engineering of Actinomycetes—The Source of Important Antibiotics and Other Valuable Compounds

“…These data revealed that the genomes encode several biosynthetic gene clusters (BGCs) for the production of diverse secondary metabolites. However, only a few of the BGCs are expressed at standard laboratory conditions and, therefore, the biosynthetic potential of many actinomycetes remains unexploited [ 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 ]. Numerous strategies were developed to induce the expression of the BGCs and access the corresponding products.…”