Site-Directed Mutagenesis of Large Biosynthetic Gene Clusters via Oligonucleotide Recombineering and CRISPR/Cas9 Targeting

Abstract

Genetic engineering of natural product biosynthetic gene clusters represents an attractive approach to access new and complex bioactive small molecules. However, due to the large number and size of some genes involved in specialized metabolism, notably those encoding modular polyketide synthase and nonribosomal peptide synthetase megaproteins, it remains difficult to introduce precise genetic mutations to probe domain activity or alter chemical product formation. Here, we report the development and validation of a robust method combining oligonucleotide recombineering and CRISPR/Cas9 targeting for rapid site-directed mutagenesis of cloned pathways, which can be directly transferred to a heterologous host for expression. We rapidly generated 12 point mutations and identified several important determinants of successful mutagenesis, including the protospacer/PAM sequence and presence of regions of local homology. Our approach may be broadly applicable for researchers interested in probing natural product biosynthesis or performing pathway engineering.

Keywords: CRISPR-Cas9; biosynthetic gene cluster; heterologous expression; natural product; recombineering; site-directed mutagenesis.

Figure 1.

Oligonucleotide recombineering and CRISPR/Cas9 targeting for site-directed mutagenesis of the violacein BGC. (A) Sequences of vioX and vio. Base changes in vio (red bold) restore the vioB start codon (starred) and eliminate the PAM (highlighted turquoise) associated with the protospacer (highlighted yellow) in vioX. (B) Workflow for site-directed mutagenesis. pJZ002::vioX, which contains the spacer highlighted in A, and vio_F, a 70 nt oligo that restores the vio sequence, were cointroduced to E. coli HME68/pCB-P_blavioX by electroporation following induction of recombination and plated on different antibiotics for selection. Bla, ampicillin resistance gene; neo, kanamycin resistance gene; chl^R, chloramphenicol resistant. (C) Plate images. E. coli HME68/pCB-P_blavioX was transformed with vio_F and pJZ002 (empty vector control) or pJZ002::vioX and plated on kanamycin (kan^R) following 1000-fold dilution (10^–3) or kanamycin and ampicillin (kan^R + amp^R). Purple clones indicate production of the pigmented bis-indole violacein and thus harbor successfully mutated constructs. Faint purple clones on kanamycin only plates are pointed out with arrows. Bottom right panel shows highly efficient mutagenesis when CRISPR/Cas9 targeting is combined with oligo recombineering.

Figure 2.

Site-directed mutagenesis of ttc (A) and ttm (B). (A) Position of mutations in ttcA–C (~25.5 kb), with corresponding enzyme module, DNA and oligo sequences, and number of correct versus sequenced clones specified in chart. Protospacer and PAM sequences used for CRISPR/Cas9 targeting are highlighted in yellow and turquoise, respectively, in wild-type DNA sequence, which shows only mutated nucleotides. Full sequence of 70 nt oligo with mutations in red bold is displayed below. (B) Position of mutations in ttmA−B (~19.1 kb), with details specified in chart. *Sequencing data for experiments in which at least 1 correct clone was recovered is provided in Figure S4. **Sequencing data for chimeric T₄ modules generated through T_4a and T_4b targeting are provided in Figure S5.