Abstract Camptothecin derivatives are first-line anticancer drugs used worldwide for the treatment of diverse malignant tumors. However, the biosynthetic pathway of camptothecin has remained elusive for five decades. Here, we fully map its entire biosynthetic route. We discovered five key missing enzymes (OpCAR, OpSDR11, OpCS, OpGH1, and OpSTR) via the combination of MALDI mass spectrometry imaging, single-cell RNA sequencing and co-expression analysis. Meanwhile, we demonstrated a free flavin mononucleotide triggered the non-enzymatic 6-5-6 to 6-6-5 fused-ring skeleton rearrangement, filling the last gap in camptothecin biosynthesis. Finally, we validated this identified pathway and achieved the de novo biosynthesis of camptothecin in Saccharomyces cerevisiae . These discoveries uncover the long-standing mystery underlying camptothecin and pave the way for manufacturing camptothecin and its derivatives through synthetic biology approaches.

ABSTRACT 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

Trichoderma reesei decomposes renewable cellulose into soluble sugars by secreting cellulase and hemicellulase. Heterotrimeric G protein signaling regulates cellulase production. Here, we assessed the effects of G protein subunit deletion on cellulase production in T. reesei. The results revealed that G proteins bidirectionally regulate cellulase production, activation, and repression; GNA1, GNA3, TGB1, and GBG1 downregulate cellulase synthesis, whereas GNA2 upregulates it. To understand the role of the ACY1-cAMP-PKA pathway and G protein signaling in cellulase expression, we overexpressed/knocked out acy1 in G protein subunit-knockout strains. GNA1 orchestrates the G protein signaling pathway, with its core function being the transmission of glucose repression signals. GNA3 serves as a complement to GNA1. GNA2 is the only Gα subunit that transduces cellulose-induced signals. The ACY1-cAMP-PKA pathway can act as an activator, a repressor, or an inactive regulator in G protein signal transduction. Our study highlights avenues for constructing cellulase-hyperproducing strains.

•China's National Botanical Gardens (NGBs) target to protect 70% of threatened species by 2035.•NBGs implement ex situ conservation, scientific research, resource utilization, public education, and horticultural display.•NBGs and China's National Parks create a robust two-pronged approach that combines ex situ and in situ conservation.•NBGs contribute to global biodiversity targets by sharing conservation technologies and germplasm resources.

Flavonoids are natural products with high biological activity and potential applications. Prenylation increases the lipophilicity of flavonoids, endowing them with specific functions, selectivity, and pharmacological properties. However, traditional methods of plant extraction and chemical synthesis are insufficient to meet the demand for prenylflavonoids. Heterologous biosynthesis of prenylflavonoids in microorganisms provides an alternative approach. Compared with plant prenyltransferases, microbial prenyltransferases showed broad substrate specificity, which is more conducive to the biosynthesis of diverse prenylflavonoids. In this study, we cloned 31 dimethylallyltryptophan synthase prenyltransferases from five fungal species and tested candidate substrates. The products of Ad03 and Ao01 were identified, resulting in two unnatural prenylflavonoids and four natural prenylflavonoids. We constructed the isopentenol utilization pathway in Escherichia coli to develop the efficient dimethylallyl diphosphate synthesis pathway for 6-prenylsilybin (6-PS) synthesis. By optimizing the whole cell catalysis and two-phase reaction system, the 6-PS production titer reached 176 mg/L and the yield of silybin was 88%. Our study provides an efficient method for prenylflavonoids production.

The sesquiterpene lactone parthenolide is a promising anticancer drug. Its biosynthesis via a microbial cell factory has been considered as a sustainable alternative to plant extraction. Herein, systematic metabolic engineering approaches, as well as the introduction of a novel noncanonical tricarboxylic acid (TCA) cycle, were employed to enhance the production of the key precursor germacrene A. By identifying two new dehydrogenases and controlling the expression of parthenolide synthase, we further achieved the elimination of byproducts and enhanced parthenolide production. A two-stage fermentation approach and in situ product extraction using macroreticular resin were further applied to relieve the nocuous effect of costunolide and parthenolide on the growth of yeast cell factories, ultimately achieving a titer of 549.7 mg/L for parthenolide and 972.7 mg/L for costunolide in a 10 L fermenter, which represents the highest reported titer obtained by microbial fermentation. The strategies should also contribute to the microbial cell factory-construction for other natural products exhibiting toxicity.

Ergot alkaloids (EAs) are a class of indole derivatives used as prescription drugs for the treatment of neurological diseases. Due to the limited production of EAs by Claviceps and the enantioselective difficulties encountered in chemical synthesis, a sustainable supply of EAs remains challenging. Recently, numerous attempts have been made to produce EAs using heterologous hosts. However, these efforts have only resulted in the production of the precursor, lysergic acid (LA), with low efficiency. Here, we report the de novo high-efficient biosynthesis of LA and a series of LA-derived EAs in Aspergillus oryzae cell factories. Based on genome sequencing of the EA-producing strain, C. purpurea 22.07, an EA biosynthetic gene cluster was annotated and characterized. After introducing and optimizing the agroclavine (AG) biosynthetic pathway in A. oryzae, we constructed an efficient chassis strain for AG production. We then confirmed the function of the annotated CloA ' to catalyze the successive oxidation of AG into LA and isolysergic acid (ILA) in this AG-producing chassis and realize their de novo production with titers of 52.68 +/- 1.49 and 6.32 +/- 2.08 mg L-1, respectively. The subsequent introduction of the downstream non-ribosomal peptide synthetase genes LpsB ' and LpsC ' enabled the complete biosynthesis of ergometrine and a series of its analogs, achieving a total titer of more than 160 mg L-1. The unexpected biosynthesis of isolysergyl-glycine and lysergyl-glycine revealed a novel function of LpsC ', which utilizes glycine as a substrate. Our work successfully realized the complete biosynthesis of a series of EAs in an industrially feasible fungus, which will open new avenues for manufacturing EAs in a green and sustainable manner.

The downstream biosynthetic route of camptothecin has remained unclear for more than half a century. Here, we discovered a P450 enzyme involved in camptothecin biosynthesis from plant by chemoproteomics, which lays the foundation for synthesis of camptothecin and highlights the effectiveness of chemoproteomic profiling for discovering unknown enzymes.

Taxol is a widely-applied anticancer drug that inhibits microtubule dynamics in actively replicating cells. Although a minimum 19-step biosynthetic pathway has been proposed and 16 enzymes likely involved have been characterized, stepwise biosynthetic reactions from the well-characterized di-oxygenated taxoids to Taxol tetracyclic core skeleton are yet to be elucidated. Here, we uncover the biosynthetic pathways for a few tri-oxygenated taxoids via confirming the critical reaction order of the second and third hydroxylation steps, unearth a taxoid 9 α -hydroxylase catalyzing the fourth hydroxylation, and identify CYP725A55 catalyzing the oxetane ester formation via a cascade oxidation-concerted acyl rearrangement mechanism. After identifying a acetyltransferase catalyzing the formation of C7-OAc, the pathway producing the highly-oxygenated 1 β -dehydroxybaccatin VI with the Taxol tetracyclic core skeleton is elucidated and its complete biosynthesis from taxa-4(20),11(12)-diene-5 α -ol is achieved in an engineered yeast. These systematic studies lay the foundation for the complete elucidation of the biosynthetic pathway of Taxol.

Protein methylation is a commonly posttranslational modification of transcriptional regulators to fine-tune protein function, however, whether this regulation strategy participates in the regulation of lignocellulase synthesis and secretion in Trichoderma reesei remains unexplored. Here, a putative protein methyltransferase (TrSAM) is screened from a T. reesei mutant with the ability to express heterologous β-glucosidase efficiently even under glucose repression. The deletion of its encoding gene trsam causes a significant increase of cellulase activities in all tested T. reesei strains, including transformants of expressing heterologous genes using cbh1 promotor. Further investigation confirms that TrSAM interacts with the cellulase negative regulator ACE1 via its amino acid residue Arg383, which causes a decrease in the ACE1-DNA binding affinity. The enzyme activity of a T. reesei strain harboring ACE1R383Q increases by 85.8%, whereas that of the strains with trsam or ace1 deletion increases by more than 100%. By contrast, the strain with ACE1R383K shows no difference to the parent strain. Taken together, our results demonstrate that TrSAM plays an important role in regulating the expression of cellulase and heterologous proteins initiated by cbh1 promotor through interacting with ACE1R383. Elimination and mutation of TrSAM and its downstream ACE1 alleviate the carbon catabolite repression (CCR) in expressing cellulase and heterologous protein in varying degrees. This provides a new solution for the exquisite modification of T. reesei chassis.

Background:The 27th Meeting(PC27)of the Plants Committee of the Convention on International Trade in Endangered Species of Wild Fauna and Flora(CITES)was held from 8 to 13 July 2024 in Geneva,Switzerland,attended by 190 representatives of 46 Parties and 30 observer organizations from around the world. Progress:This meeting included 42 agenda items,mainly discussing the progress in the implementation,and approving some revised drafts of resolutions and decisions for submission to the 20th Conference of the Parties to the CITES(CoP 20).Specifically,the Review of Significant Trade in specimens of Appendix Ⅱ species such as Aquilaria spp.,Gyrinops spp.,and Pterocarpus erinaceus was decided;the regulation and protection of products containing specimens of Appendix Ⅱ Orchidaceae spp.was discussed;the new resolution of"Trade in medicinal and aromatic plant species"was postponed;the resolution of"Implementation of the Convention for agarwood-producing taxa"and the new"CITES Non-Detriment Findings Guidance"and"Guidance on the term artificially propagated"were approved for submission in this meeting. Perspectives:PC27 reflected the concerted efforts made by all parties to ensure the sustainability of international trade in wild plants,promotes international cooperation and capacity building on CITES science and technology issues,and was also characterized by increased collaboration between CITES and the Convention on Biological Diversity,expansion of its mandate,and weakening of the science.A new trend of the CITES Plants Committee appeared during PC27,from the establishment of a regulatory system for ornamental plants two decades ago,with a focus on the conservation and management of tropical and subtropical tree species a decade ago,to the current exploration of sustainable trade in medicinal and aromatic plants that are vital to health and livelihoods. Recommendations:To implement the CITES more effectively,our country should consolidate the scientific foundation of CITES,improve the research capacity on Non-Detriment Findings,establish a talent team for CITES implementation,and promote the conservation and sustainable use of wild plants.

Silk fibroin (SF) from the cocoon of silkworm has exceptional mechanical properties and biocompatibility and is used as a biomaterial in a variety of fields. Sustainable, affordable, and scalable manufacturing of SF would enable its large-scale use. We report for the first time the high-level secretory production of recombinant SF peptides in engineered Pichia pastoris cell factories and the processing thereof to nanomaterials. Two SF peptides (BmSPR3 and BmSPR4) were synthesized and secreted by P. pastoris using signal peptides and appropriate spacing between hydrophilic sequences. By strain engineering to reduce protein degradation, increase glycyltRNA supply, and improve protein secretion, we created the optimized P. pastoris chassis PPGSP-8 to produce BmSPR3 and BmSPR4. The SF fed-batch fermentation titers of the resulting two P. pastoris cell factories were 11.39 and 9.48 g/L, respectively. Protein self-assembly was inhibited by adding Tween 80 to the medium. Recombinant SF peptides were processed to nanoparticles (NPs) and nanofibrils. The physicochemical properties of nanoparticles R3NPs and R4NPs from the recombinant SFs synthesized in P. pastoris cell factories were similar or superior to those of RSFNPs (Regenerated Silk Fibroin NanoParticles) originating from commercially available SF. Our work will facilitate the production by microbial fermentation of functional SF for use as a biomaterial.

Phytase efficiently hydrolyzes phytate to phosphate; thus, it is widely used to increase phosphorus availability in animal feeds and reduce phosphorus pollution through excretion. Phytase is easily inactivated during feed pelleting at high temperature, and sufficient thermostability of phytase is essential for industrial applications. In this study, directed evolution was performed to enhance phytase thermostability. Variants were initially expressed in Escherichia coli BL21 for screening, then in Pichia pastoris for characterization. Over 19,000 clones were generated from an error-prone Polymerase Chain Reaction (epPCR) library; 5 mutants (G10, D7, E3, F8, and F9) were obtained with approximately 9.6%, 10.6%, 11.5%, 11.6%, and 12.2% higher residual activities than the parent after treatment at 99°C for 60 min. Three of these mutants, D7, E3, and F8, exhibited 79.8%, 73.2%, and 92.6% increases in catalytic efficiency (kcat/Km), respectively. In addition, the specific activities of D7, E3, and F8 were 2.33-, 1.98-, and 2.02-fold higher than parental phytase; they were also higher than the activities of all known thermostable phytases. Sequence analysis revealed that all mutants were substituted at residue 75 and was confirmed that the substitution of cysteine at position 75 was the main contribution to the improvement of thermostability of mutants by saturation mutagenesis, indicating that this amino acid is crucial for the stability and catalytic efficiency of phytase. Docking structure analysis revealed that substitution of the C75 residue allowed the mutants to form additional hydrogen bonds in the active pocket, thereby facilitating binding to the substrate. In addition, we confirmed that the intrinsic C77-C108 disulfide bond in E. coli phytase is detrimental to its stability.

β-glucosidases play an important role in the synthesis of cellulase in fungi, but their molecular functions and mechanisms remain unknown. We found that the 10 putative β-glucosidases investigated in Trichoderma reesei facilitate cellulase production, with cel3j being the most crucial. Transcriptional analysis revealed that the most affected biological processes in △cel3j strain were cellulase synthesis, ribosome biogenesis, and RNA polymerases. Moreover, CEL3J was unconventionally transported through the endoplasmic reticulum, bypassing the Golgi apparatus, whereas cel3j overexpression altered cellulase secretion from conventional to unconventional, likely owing to the activated unconventional protein secretion pathway (UPS), as indicated by the upregulation of genes related to UPS. The mTORC1-GRASP55 signaling axis may modulate the unconventional secretion of CEL3J and cellulase. The transcriptional levels of genes associated with DNA replication, the cell cycle, and meiosis were noticeably affected by overexpressing cel3j. These data give new clues for exploring the roles of β-glucosidases and the molecular mechanisms of their unconventional secretion in fungi.

Osthole is a coumarin compound found in the traditional Chinese medicine Cnidium monnieri. Extensive studies have shown that osthole exhibits many medicinal properties, and recently, researchers have found that it possesses potent airway-relaxation activity by inhibiting phosphodiesterase 4D activity, making it a potential novel bronchodilator that does not target β2-adrenoceptors for asthma treatment. Here, we report the complete biosynthesis of osthole in engineered yeast. We created an umbelliferone (UMB)-producing strain by reconstituting the complete UMB pathway in yeast. We found that coumarin synthase (COSY) is essential for the conversion of 2′,4′-dihydroxycinnamoyl-CoA into UMB in yeast; this conversion has been treated as a spontaneous step in previously reported UMB-producing microbials. By introducing downstream prenyltransferase and methyltransferase genes and addressing problems such as protein expression and cofactor supply to fulfill the downstream steps, complete biosynthesis of osthole was achieved. Finally, through metabolic engineering, to ensure precursor supply, and the debugging of rate-limited steps, the osthole titer reached 108.10 mg/L in shake flasks and 255.1 mg/L in fed-batch fermentation. Our study is the first to produce osthole using engineered microbes, providing a blueprint for the supply of plant-derived osthole via microbial fermentation, which will remove the barriers of resource limitations for osthole-based drug development.

Claviceps purpurea produces many pharmacologically important ergot alkaloids (EAS), which are widely used to treat migraine and hypertension and to aid childbirth. Although an EAS biosynthetic cluster of C. purpurea has been discovered more than 20 years ago, the complete biosynthetic pathway of EAS has not been fully characterized until now. The main obstacle to elucidating this pathway and strain modification is the lack of efficient genome-editing tools for C. purpurea. The conventional gene manipulation method for C. purpurea relies on homologous recombination (HR), although the efficiency of HR in C. purpurea is very low (∼1-5%). Consequently, the disruption of target genes is laborious and time-consuming. Although CRISPR/Cas9 genome-editing methods based on in vivo Cas9 expression and gRNA transcription have been reported recently, their gene-disruption efficiency is still very low. Here, we developed an efficient genome-editing system in C. purpurea based on in vitro assembled CRISPR/Cas9 gRNA ribonucleoprotein complexes. As proof of principle, three target genes were efficiently knocked out using this CRISPR/Cas9 ribonucleoprotein complex-mediated HR system, with editing efficiencies ranging from 50% to 100%. Inactivation of the three genes, which are closely related to uridine biosynthesis (ura5), hypha morphology (rac), and EAS production (easA), resulted in a uridine auxotrophic mutant, a mutant with a drastically different phenotype in axenic culture, and a mutant that did not produce EAS, respectively. Our ribonucleoprotein-based genome-editing system has a great advantage over conventional and in vivo CRISPR/Cas9 methods for genome editing in C. purpurea, which will greatly facilitate elucidation of the EAS biosynthetic pathway and other future basic and applied research on C. purpurea.

Rg2 and Re are both rhamnose-containing ginsenosides isolated exclusively from Panax plants, which exhibit broad spectrum of pharmacological activities. However, limitations of current plant-relied manufacturing methods have largely hampered their medical applications. Here, we report elucidation of the complete biosynthetic pathway of these two ginsenosides by the identification of a rhamnosyltransferase PgURT94 from Panax ginseng . We then achieve de novo bio-production of Rg2 and Re from glucose by reconstituting their biosynthetic pathways in yeast. Through stepwise strain engineering and fed-batch fermentation, the maximum yield of Rg2 and Re reach 1.3 and 3.6 g/L, respectively. Our work completes the identification of the last missing enzyme for Rg2 and Re biosynthesis and achieves their high-level production by engineered yeasts. Once scaled, this microbial biosynthesis platform will enable a robust and stable supply of Rg2 and Re and facilitate their food and medical applications.

Gut microbiota plays an important role in metabolic homeostasis. Previous studies demonstrated that ginsenoside Rb1 might improve obesity-induced metabolic disorders through regulating glucose and lipid metabolism in the liver and adipose tissues. Due to low bioavailability and enrichment in the intestinal tract of Rb1, we hypothesized that modulation of the gut microbiota might account for its pharmacological effects as well. Here, we show that oral administration of Rb1 significantly decreased serum LDL-c, TG, insulin, and insulin resistance index (HOMA-IR) in mice with a high-fat diet (HFD). Dynamic profiling of the gut microbiota showed that this metabolic improvement was accompanied by restoring of relative abundance of some key bacterial genera. In addition, the free fatty acids profiles in feces were significantly different between the HFD-fed mice with or without Rb1. The content of eight long-chain fatty acids (LCFAs) was significantly increased in mice with Rb1, which was positively correlated with the increase of Akkermansia and Parasuttereller, and negatively correlated with the decrease of Oscillibacter and Intestinimonas. Among these eight increased LCFAs, eicosapentaenoic acid (EPA), octadecenoic acids, and myristic acid were positively correlated with metabolic improvement. Furthermore, the colonic expression of the free fatty acid receptors 4 (Ffar4) gene was significantly upregulated after Rb1 treatment, in response to a notable increase of LCFA in feces. These findings suggested that Rb1 likely modulated the gut microbiota and intestinal free fatty acids profiles, which should be beneficial for the improvement of metabolic disorders in HFD-fed mice. This study provides a novel mechanism of Rb1 for the treatment of metabolic disorders induced by obesity, which may provide a therapeutic avenue for the development of new nutraceutical-based remedies for treating metabolic diseases, such as hyperlipidemia, insulin resistance, and type 2 diabetes.