Two strategies to improve the supply of PKS extender units for ansamitocin P-3 biosynthesis by CRISPR–Cas9

Abstract

Ansamitocin P-3 (AP-3) produced by Actinosynnema pretiosum is a potent antitumor agent. However, lack of efficient genome editing tools greatly hinders the AP-3 overproduction in A. pretiosum. To solve this problem, a tailor-made pCRISPR–Cas9apre system was developed from pCRISPR–Cas9 for increasing the accessibility of A. pretiosum to genetic engineering, by optimizing cas9 for the host codon preference and replacing pSG5 with pIJ101 replicon. Using pCRISPR–Cas9apre, five large-size gene clusters for putative competition pathway were individually deleted with homology-directed repair (HDR) and their effects on AP-3 yield were investigated. Especially, inactivation of T1PKS-15 increased AP-3 production by 27%, which was most likely due to the improved intracellular triacylglycerol (TAG) pool for essential precursor supply of AP-3 biosynthesis. To enhance a “glycolate” extender unit, two combined bidirectional promoters (BDPs) ermEp-kasOp and j23119p-kasOp were knocked into asm12-asm13 spacer in the center region of gene cluster, respectively, by pCRISPR–Cas9apre. It is shown that in the two engineered strains BDP-ek and BDP-jk, the gene transcription levels of asm13-17 were significantly upregulated to improve the methoxymalonyl-acyl carrier protein (MM-ACP) biosynthetic pathway and part of the post-PKS pathway. The AP-3 yields of BDP-ek and BDP-jk were finally increased by 30% and 50% compared to the parent strain L40. Both CRISPR–Cas9-mediated engineering strategies employed in this study contributed to the availability of AP-3 PKS extender units and paved the way for further metabolic engineering of ansamitocin overproduction.

Keywords

Actinosynnema pretiosum / CRISPR–Cas9 / Bidirectional promoters / Ansamitocin P-3 (AP-3) / Extender unit

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Funding

Shanghai Super Postdoctoral Program 2020

National Natural Science Fund for Young Scholars(32001035)

National Key R&D Program of China(2021YFC2102805)