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. Graphical Abstract

Mechanistic insight for improving butenyl-spinosyn production through combined ARTP/UV mutagenesis and ribosome engineering in Saccharopolyspora pogona

“…Increased transcription levels of propanoate metabolism pathway may favor the accumulation of propionyl-CoA and MM-CoAs, thereby promoting the butenyl-spinosyn biosynthesis. ( Guo et al, 2022 ).…”

Global Regulator AdpA_1075 Regulates Morphological Differentiation and Ansamitocin Production in Actinosynnema pretiosum subsp. auranticum

“…The previously constructed mutant MD15 with tandem deletion of two gene clusters (cluster T1PKS-15 and T1PKS/NRPS-5) [ 47 ] showed excellent fermentation stability ( Figure 1 A). To better understand the improvement of fermentation performance, the mycelia from culture broth were collected at 16, 24 and 72 h, and aerial hyphae were examined on YMG medium.…”

“…auranticum ATCC 31565 by atmospheric and room temperature plasma (ARTP) mutagenesis [ 12 ]. Strain L40 and its derivatives in this study were cultivated as described previously [ 47 ]. YMG agar plates (for solid culture) and TSBY broth (for liquid culture) were employed for strain culture.…”

“…CRISPR-Cas9 mediated gene inactivation. Mutants with gene ssgA_6663, adpA_1075, or asm28 disruption were performed by pCRISPR-Cas9apre with a unified construction process [ 47 ]. As an example, the construction of mutant with gene ssgA_6663 deletion was described briefly.…”

“…E. coli ET12567 (pUZ8002) was employed to introduce the resulting plasmid into L40. According to protocol described elsewhere [ 47 ], the conjugants were induced and screened for the correct constructs by colony PCR and Sanger sequencing ( Supplementary Figure S1 ).…”