Abstract
One major bottleneck in natural product drug development is derivatization, which is pivotal for fine tuning lead compounds. A promising solution is modifying the biosynthetic machineries of middle molecules such as macrolides. Although intense studies have established various methodologies for protein engineering of type I modular polyketide synthase(s) (PKSs), the accurate targeting of desired regions in the PKS gene is still challenging due to the high sequence similarity between its modules. Here, we report an innovative technique that adapts in vitro Cas9 reaction and Gibson assembly to edit a target region of the type I modular PKS gene. Proof-of-concept experiments using rapamycin PKS as a template show that heterologous expression of edited biosynthetic gene clusters produced almost all the desired derivatives. Our results are consistent with the promiscuity of modular PKS and thus, our technique will provide a platform to generate rationally designed natural product derivatives for future drug development.
Highlights
Introduction of edited BACs intoS. avermitilis SUKA
Based on the heterologous expression system we established that is applicable to biosynthetic clusters with over 200 kb cloned into the BAC vector[14], we developed the idea of in vitro module editing, which applies a combination of both CRISPR-Cas[915] and Gibson assembly[16] to the BAC clone in vitro (Fig. 1a)
If we can accurately edit the modular polyketide synthase(s) (PKSs) of rapamycin, whose modules show higher homology to each other than those of other systems such as the erythromycin PKS (86.1% homology between 6 KS domains), this technique will be applicable for most natural products
Summary
Introduction of edited BACs intoS. avermitilis SUKA. All clones were introduced into S. lividans TK24 carrying the SAP1 vector (containing the synthetic sequence of attBφK38-1, attBR4, attBφBT1, attBφC31 and attBTG1 of S. avermitilis MA4680). All clones were introduced into S. lividans TK24 carrying the SAP1 vector (containing the synthetic sequence of attBφK38-1, attBR4, attBφBT1, attBφC31 and attBTG1 of S. avermitilis MA4680). All clones were integrated into the SAP1 vector because the bacteriophage attachment sites (attB) were located in the SAP1 vector but not in the chromosome. The transfer of the SAP1 vector containing the edited gene cluster for rapamycin was conducted as described in ref. The objective transconjugants were confirmed by the antibiotic-resistance phenotype and the size of the linear plasmid by contour-clamped homogeneous electric field (CHEF)[43] electrophoresis using SAP1::pKU503rap as the control linear plasmid. Cloning of rapH and expression in SUKA33, 34. PKU565tsr-2cistron[23] was digested with HindIII and NheI, and a small fragment was cloned into the same site of pKU592AT-aac(3)IV to give the expression vector pKU592AT-aac(3) IV-2cistron
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