Rational approach to improve ansamitocin P‐3 production by integrating pathway engineering and substrate feeding in <i>Actinosynnema pretiosum</i>

Du, Zhiqiang; Zhong, Jian‐Jiang

doi:10.1002/bit.26775

Cited by 12 publications

(13 citation statements)

References 27 publications

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“…As an important drug precursor, various strategies have been applied to improve AP-3 yield [12][13][14][15][16][17]. Previously in our lab, a high-yield AP-3 producing strain Actinosynnema pretiosum NXJ-24 was constructed by engineering the post-modification steps, which resulted in a 5-fold increase in AP-3 yield to 246 mg/L [14].…”

Section: Introductionmentioning

confidence: 99%

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

Wang

Kang

et al. 2020

Biomolecules

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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.

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Section: Introductionmentioning

confidence: 99%

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

Wang

Kang

et al. 2020

Biomolecules

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“…Using maytansinol as a payload, the deacylated product of AP-3, antibody–drug conjugates have been recently developed as new strategy for cancer treatment [ 29 ], e.g., the Food and Drug Administration (FDA)-approved T-DM1 (Kadcyla ® ) for Human Epidermal Growth Factor Receptor 2 (HER2)-positive metastatic breast cancer [ 30 ]. Aided by the in-depth biosynthetic studies [ 31 , 32 ], the yield improvement of AP-3 has been intensively conducted through random mutagenesis and screening, process engineering, and pathway engineering, which includes the optimization of post-PKS modifications, enhancement of precursor supplies, improved gene expression, and so on [ 33 , 34 , 35 , 36 , 37 , 38 , 39 ]. However, the yield of AP-3 is still low and insufficient for supporting the clinical trials of drug leads in pipeline and subsequent clinical treatment.…”

Section: Introductionmentioning

confidence: 99%

Subtilisin-Involved Morphology Engineering for Improved Antibiotic Production in Actinomycetes

Kang

Zhang

et al. 2020

Biomolecules

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In the submerged cultivation of filamentous microbes, including actinomycetes, complex morphology is one of the critical process features for the production of secondary metabolites. Ansamitocin P-3 (AP-3), an antitumor agent, is a secondary metabolite produced by Actinosynnema pretiosum ATCC 31280. An excessive mycelial fragmentation of A. pretiosum ATCC 31280 was observed during the early stage of fermentation. Through comparative transcriptomic analysis, a subtilisin-like serine peptidase encoded gene APASM_4178 was identified to be responsible for the mycelial fragmentation. Mutant WYT-5 with the APASM_4178 deletion showed increased biomass and improved AP-3 yield by 43.65%. We also found that the expression of APASM_4178 is specifically regulated by an AdpA-like protein APASM_1021. Moreover, the mycelial fragmentation was alternatively alleviated by the overexpression of subtilisin inhibitor encoded genes, which also led to a 46.50 ± 0.79% yield increase of AP-3. Furthermore, APASM_4178 was overexpressed in salinomycin-producing Streptomyces albus BK 3-25 and validamycin-producing S. hygroscopicus TL01, which resulted in not only dispersed mycelia in both strains, but also a 33.80% yield improvement of salinomycin to 24.07 g/L and a 14.94% yield improvement of validamycin to 21.46 g/L. In conclusion, our work elucidates the involvement of a novel subtilisin-like serine peptidase in morphological differentiation, and modulation of its expression could be an effective strategy for morphology engineering and antibiotic yield improvement in actinomycetes.

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“…1 The general pathway map of AP-3 biosynthesis. Ru5p ribose 5-phosphate; 6PGA 6-phosphogluconic acid; G6P glucose-6-phosphate; G1P glucose 1-phosphate; UDPG uridine diphosphate glucose; F6P fructose-6-phosphate; G3P glyceraldehyde 3-phosphate; 1,3-BPG 1,3-bisphosphoglycerate; MM-ACP methoxymalonyl-ACP; AHBA 3-amino-5-hydroxybenzoic acid; PND-3 N-demethylansamitocin P-3; ACGP-3 4″-O-carbamoylansamitocinoside P-3; AGP-3 ansamitocinoside P-3 contribute significantly to the increase of AP-3 biosynthesis (Du et al 2017;Du and Zhong 2018).…”

Section: Introductionmentioning

confidence: 99%

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

Guo

Sun

et al. 2022

Bioresour. Bioprocess.

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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

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Rational approach to improve ansamitocin P‐3 production by integrating pathway engineering and substrate feeding in Actinosynnema pretiosum

Cited by 12 publications

References 27 publications

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

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

Subtilisin-Involved Morphology Engineering for Improved Antibiotic Production in Actinomycetes

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

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