The Antitumor Agent Ansamitocin P-3 Binds to Cell Division Protein FtsZ in Actinosynnema pretiosum

Abstract

Ansamitocin P-3 (AP-3) is an important antitumor agent. The antitumor activity of AP-3 is a result of its affinity towards β-tubulin in eukaryotic cells. In this study, in order to improve AP-3 production, the reason for severe growth inhibition of the AP-3 producing strain Actinosynnema pretiosum WXR-24 under high concentrations of exogenous AP-3 was investigated. The cell division protein FtsZ, which is the analogue of β-tubulin in bacteria, was discovered to be the AP-3 target through structural comparison followed by a SPR biosensor assay. AP-3 was trapped into a less hydrophilic groove near the GTPase pocket on FtsZ by hydrogen bounding and hydrophobic interactions, as revealed by docking analysis. After overexpression of the APASM_5716 gene coding for FtsZ in WXR-30, the resistance to AP-3 was significantly improved. Moreover, AP-3 yield was increased from 250.66 mg/L to 327.37 mg/L. After increasing the concentration of supplemented yeast extract, the final yield of AP-3 reached 371.16 mg/L. In summary, we demonstrate that the cell division protein FtsZ is newly identified as the bacterial target of AP-3, and improving resistance is an effective strategy to enhance AP-3 production.

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

“…The results showed that the overexpression of ssgA _ 6663 did not improve AP-3 production, which was consistent with what was observed in S. griseus [ 26 , 28 ]. As expected, enhanced expression of ftsZ_5883 improved strain resistance against AP-3 [ 45 ], alleviated cell toxicity, and increased AP-3 production by 45% ( Figure 3 A). Moreover, overexpression of adpA_1075 increased AP-3 production by 85% without affecting dry cell weight (DCW) at the end of fermentation ( Figure 3 A,B).…”

“…Overexpression of ftsZ gene might also be used to alleviate AP-3 toxicity and improve resistance of A. pretiosum to AP-3. Since FtsZ was determined to be the intracellular binding target of AP-3 [ 45 ]. A high transcriptional level of ftsZ_5883 was observed in mutant with a denser mycelial interweaving pattern ( Figure 1 D).…”

“…FtsZ protein from A. pretiosum as the analogue of β-tubulin, was demonstrated to be the AP-3 binding target. Overexpression of APASM_5716 gene that encodes FtsZ resulted in AP-3 resistance and overproduction in A. pretiosum ATCC 31280 [ 45 ]. A two-component signal transduction system, PhoPR homolog was identified in the genome of the A. pretiosum X47 strain.…”

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

“…In this study, a minimum of 27% increase in AP-3 yield was obtained based on the objectiveoriented strategies. The AP-3 production obtained in mutant MD02 (365 mg/L) was higher than those recently reported, where improvement of AP-3 tolerance and enhancement of the efflux efficiency of AP-3 were mainly focused (Wu et al 2020;Wang et al 2021Wang et al , 2020b. We additionally constructed the strain with both the knockout of gene cluster T1PKS-15 and the knock-in of BDP promoter.…”

Recent Advances of Actinomycetes

“…The study by X. Wang et al [2] reveals that the antitumor agent named ansamitocin P-3 (AP-3) binds to cell division protein FtsZ in Actinosynnema pretiosum, a rare actinomycete. FtsZ, a β-tubulin analog present in bacteria, was discovered to be the AP-3 target through structural comparison and an Surface Plasmon Resonance (SPR) biosensor assay.…”