The biosynthetic potency of CPT and their molecular regulatory processes to sustain the CPT productivity by fungi are unraveled by exploring the rate-limiting enzymes, epigenetic regulators and transcriptional factors of CPT biosynthesis.
Abstract
Camptothecin (CPT) is one of the common chemotherapies for tumors, for its superior affinity for inhibiting the Topoisomerase I activity via stabilizing the enzyme–DNA ternary complex, thus precluding the relaxation of DNA in the frequently replicated cells. CPT was initially derived from the bark of chinese Happy tree plants “Camptotheca acuminata”. However, availability of the CPT and their derivatives is the main challenge that halts the further implementation of this compound. The fungal biosynthetic potency of CPT raises the prospective for the production of CPT, due to their short lifespan and feasibility of bulk biomass bioprocessing, nevertheless, weakening of the CPT productivity with the fungal storage consecutive subculturing are the challenge. So, this review was to unravel the biosynthetic potency of CPT and their molecular regulatory processes to sustain the CPT productivity by fungi by exploring the rate-limiting enzymes, epigenetic regulators and transcriptional factors of CPT biosynthesis. As well as, to explore the expression of CPT biosynthetic gene clusters regarding to chromatin remodeling, microbial-microbial crosstalk, in relation to deciphering the CPT biosynthesis gene cluster by fungi.
Taxol (paclitaxel) is a frontline anticancer drug widely applied for the treatment of breast, ovarian and lung cancers. Currently, its supply mainly relies on the semi-synthesis using baccatin III from Taxus plants. Heterologous biosynthesis of baccatin III in microorganisms offers a promising solution to alleviate global Taxol supply shortage, but remains challenging due to pathway complexity. Here, we report a novel taxusin-mediated biosynthetic pathway for baccatin III production via the identification of C13 deacetylase, elucidation of the exact sequence underlying C1 hydroxylation, and stepwise enzymatic functional validation. Through protein engineering of the promiscuous C1 and C5 hydroxylases, coupled with the distribution of pathway modules in Saccharomyces cerevisiae and Escherichia coli, we achieved the de novo biosynthesis of baccatin III. Collectively, our findings remodel the current biosynthetic framework governing the formation of Taxol precursors and highlight the great potential of microbial cell factories for the production of complex plant-derived therapeutic compounds. Highlights • Discovery of C13 deacetylase reveals a novel biosynthetic route to baccatin III via taxusin • Stepwise verification of the complete biosynthetic route to baccatin III through taxusin and baccatin VI • Single-site mutation reversed the product selectivity of T1OH and converted T5OH into a specific taxoid C5 hydroxylase • Complete biosynthesis of baccatin III in engineered microbes
As a core family of hormone regulating plant growth and development, the biosynthesis and inactivation mechanisms of cytokinins (CKs) have been well studied in plants. However, these processes remain largely uncharacterized in other organisms, particularly fungal phytopathogens. Here, we identify a distinct pathway of CKs from Fusarium and unravel the chemical and enzymatic logic of biosynthesis and inactivation, which mainly includes: (1) the IPT-LOG fusion enzyme FexA catalyzes N6-dimethylallylation of AMP and cleavage of the C1″-N9 bond to form iP (1); (2) the CYP450 FexB not only hydroxylates 1 to generate the canonical plant-type CKs trans-zeatin (tZ, 2) and cis-zeatin (cZ, 3), but also sequentially catalyzes C4'-N6 cyclization to produce a pyrrole derivative (6); (3) the BBE-like oxidase FexC independently mediates oxidative cleavage of compounds 1-3 and unexpectedly converts 1 to C1'-keto-iP (16); (4) FexC and the NmrA-like SDR FexD cooperate to catalyze double bond isomerization, yielding two novel CKs (17 and 19), where FexD functions as a rare NADPH-dependent 1,4-reductase. Biological activity assays demonstrate that the noncanonical fungus-specific compounds 6, 17, and 19 represent new inactive CK forms for plants, whereas 16 exhibits unexpectedly high activity. Importantly, unlike plants, Fusarium employ double bond isomerization and pyrrole formation as novel strategies for CK inactivation. Our findings uncover unusual functions of fungal CK enzymes and reveal the molecular basis underlying CK biosynthesis and inactivation in plant pathogenic fungi, which provide new insights into the discovery and application of CK derivatives.
Jin-Mei Zhang, Guan-Yin Yuan, Qing-Dong Xu et al.· Journal of the American Chem...· 0 citations
The global rise of drug-resistant Mycobacterium tuberculosis (Mtb) underscores an urgent need for antitubercular agents with novel targets and mechanisms of action. Among these, the de novo purine biosynthesis pathway is essential for Mtb growth and survival, making its constituent enzymes attractive targets for therapeutic intervention. Within this pathway, adenylosuccinate (ADS) synthetase (ADSS) Rv0357c catalyzes the first committed step in biosynthesis of adenosine monophosphate (AMP) by converting inosine monophosphate (IMP) to ADS through a GTP-dependent reaction with l-aspartate. Despite its importance, Mtb ADSS remains poorly characterized at the biochemical level. In this study, we report the expression, purification, and enzymatic characterization of recombinant Mtb ADSS. To overcome the challenge of the enzyme being predominantly expressed as inclusion bodies in Escherichia coli, we established both protein refolding and chaperone-assisted expression strategies to obtain soluble, catalytically active protein. Using complementary spectrophotometric, colorimetric, and fluorescence-based assays, we determined steady-state kinetic parameters and confirmed robust ADSS activity consistent with Michaelis-Menten behaviour. Furthermore, we developed scalable, nonradioactive assays compatible with high-throughput screening (HTS), enabling the quantitative monitoring of ADSS activity via GTP hydrolysis and phosphate release. As a proof of concept, the MESG assay successfully detected inhibition of Mtb ADSS by the previously reported ADSS inhibitor Aurodox, demonstrating its utility for inhibitor characterization and screening. Collectively, these results provide the first comprehensive biochemical framework for studying Mtb ADSS and establish a foundation for structure-guided inhibitor discovery targeting purine biosynthesis as a novel antitubercular strategy.
Vigyasa Singh, Ran Zhang, Ke Chen et al.· Biochimica et Biophysica Act...· 0 citations
This study engineered an experimental callus system for inducible production of CPT, which enabled multi‐omics and deep learning analyses to identify candidate genes in CPT biosynthesis and provides a valuable foundation for the complete elucidation of the CPT biosynthetic pathway.
Shenqiu Wang, Xing Wu, Maria Moreno et al.· The Plant Genome· 0 citations
Thiopeptides are ribosomally synthesized and post-translationally modified peptides (RiPPs) that form complex bioactive scaffolds through extensive enzymatic tailoring. The polyglycosylated thiopeptides persiathiacins, exhibit potent activity against multidrug-resistant Mycobacterium tuberculosis (Mtb) and methicillin-resistant Staphylococcus aureus (MRSA). The persiathiacin biosynthetic gene cluster encodes six cytochrome P450 (CYP) enzymes, but the logic of their oxidative modifications was unknown. Here, we establish a protoplast-based genetic system for Actinokineospora and systematically assign functions to all P450s. We demonstrate that PerX hydroxylates the central thiazole, PerV installs the third indole–core crosslink required for macrocyclization, and PerT, not PerU, catalyses indole N-hydroxylation. Combined gene inactivation and metabolite profiling reveal a hierarchical enzymatic sequence leading to the mature scaffold prior to sugar installation. Notably, the intermediate accumulating in the ΩperX mutant exhibits enhanced anti-M. tuberculosis potency compared to persiathiacin A (IC50 = 0.07 vs 1.5 µg mL−1). These results define the enzymatic logic and temporal organization of persiathiacin biosynthesis, providing a conceptual framework for rational diversification of complex thiopeptide natural products.
F. A. Sumang, Maxwell T. Stevens, W. Britton et al.· bioRxiv· 0 citations
Monoterpenoids are an important class of plant volatile natural products with broad applications in the food, fragrance, pharmaceutical, and agricultural industries. However, their conventional production largely relies on plant extraction, which is often constrained by low efficiency, high cost, and limited sustainability. Structurally, monoterpenoids can be classified into acyclic, monocyclic, and bicyclic types, and their structural diversity is closely associated with differences in biosynthetic routes and regulatory mechanisms. Their biosynthesis depends on precursor supply from the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways, followed by terpene synthase (TPS)-mediated scaffold formation and subsequent modification reactions. In addition, monoterpenoid accumulation is regulated by multiple factors, including environmental cues, phytohormone signaling, transcriptional regulation, and epigenetic or post-transcriptional control. Meanwhile, substantial progress has been made in the heterologous production of monoterpenoids in microbial platforms such as Escherichia coli and Saccharomyces cerevisiae through metabolic engineering and synthetic biology. This review summarizes recent advances in monoterpenoid biosynthesis, multilevel regulation, and heterologous production, with particular emphasis on major bottlenecks and optimization strategies for sustainable and efficient biomanufacturing.
Jun-Chi Zhang, Jiale Cui, Shang Li et al.· Natural Products and Biopros...· 0 citations
