PCH-1) was further evaluated for its effect on cell cycle distribution, apoptosis, migration, epithelial–mesenchymal transition, and oxidative stress. Furthermore, studies on the mechanism of action revealed that PCH-1 disrupts microtubule assembly, leading to cancer cell death. Molecular modeling studies confirmed the potent interaction of PCH-1 with the vinblastine binding site on tubulin. Overall, this study provides novel opportunities to identify anticancer agents in the pyrazole series.Lung cancer is considered to account for approximately one-fifth of all malignant tumor-related deaths worldwide and is therefore one of the most lethal malignancies. Pyrazole scaffold possesses a wide range of biological and pharmacological activities, which play important roles in medicinal chemistry. The present study reports the synthesis and in vitro biological characterization of nine pyrazoles derived from chalcones as potential anticancer agents for non-small cell lung cancer A-549, H226, and H460 cell lines. Most of the compounds efficiently inhibited the growth of all the tested cancer cell lines at micromolar concentrations. One of the most active compounds ( Chalcone family members have often been used in traditional medicine and possess a large number of biological properties, including anticancer, antibacterial, antifungal, antimalarial, antidiabetic, anti-inflammatory, and antioxidant activities2. Several chalcones are currently being used in clinical practice or are undergoing clinical trials, exemplified by the selected analogs shown in Table 3.Chalcones are an important class of natural compounds from the flavonoids family and are present in many edible plants such as fruits (tomatoes and citrus), nuts, vegetables, and teatrans or cis isomerism with two aromatic rings linked by a three-carbon α,β-unsaturated carbonyl system12. The predominant configuration is trans-isomer that is more stable thermodynamically4. Structural modification mainly focuses on both phenyl rings. Similar to naturally occurring chalcones, methoxy and hydroxy groups in positions on the phenyl rings are important for anticancer activities of chalcone derivatives13. Marquina et al. reported that 2′-hydroxy-4′-alkoxy chalcone derivatives exhibit strong cytotoxic activity against PC-3 cells by inducing a G2/M phase arrest and mitochondrial apoptotic pathway14. Wang et al. found that chalcones bearing the 3-hydroxyl-4-methoxy phenyl moiety demonstrated high cytotoxic activity against the MCF-7 breast cancer cell line and displayed potent tubulin polymerization inhibitory activity15. Moreover, Pawlak et al. established that 2′-hydroxy-2″,5″-dimethoxychalcone exerted a potent antitumor activity on canine lymphoma and leukemia cells and that the addition of two methoxy groups increased the proapoptotic activities of chalcones16.Chemically, chalcones have a scaffold of 1,3-diphenylprop-2-en-1-one either in 17. Similar to combretastatin and colchicine, methoxylated chalcones bind to tubulin and prevent its polymerization, leading to cell cycle arrest by interruption of mitotic spindle assembly and resulting in cell death18. The methoxy substituent in A ring of chalcones appeared to be the crucial pharmacophoric group for the inhibition of tubulin polymerization20. Moreover, structural manipulations that vary in the basic structural framework or substitutions pattern have also been reported for antitumor properties, such as phenoxy moiety in the ortho position of the phenyl ring B21, methyl substitution in α position8, pyrazole hybrids22, and many others23.Structurally, methoxylated chalcones are related to natural combretastatin A-4 and colchicine because of congruous spatial orientation between two aromatic rings25. Because many studies have reported the importance of the heterocyclic moiety of the pyrazole ring in medicinal chemistry, skeletons of pyrazoles are often modified to ameliorate their biological activity. Among others, pyrazoles derived from chalcones display an increase in the potency and spectrum of activity26.Pyrazoles are heterocyclic compounds that possess two adjacent nitrogen atoms of the 5-membered ring of three carbon atoms. The pyrazole nucleus has diverse biological properties and therapeutic applications, such as analgesic, anticancer, anti-inflammatory, antimicrobial, antiviral, antidiabetic, anticonvulsant, antipyretic, and antidepressant propertiesPCH-1 exhibited the strongest cytotoxic activity through interaction with tubulin, leading to cell cycle arrest and induction of apoptosis. To determine the molecular mechanism of action of these compounds, we performed molecular modeling studies, namely molecular docking. Our studies confirmed the potent interaction of PCH-1 with the vinblastine binding site on tubulin. Overall, we have demonstrated that PCH-1 has a potent antitumor potential for the treatment of lung cancer and could be a lead for new drug development.On the basis of discussed studies and considering a constant need to develop new potent and selective anticancer compounds, we have designed and synthesized a series of derivatives of chalcone-derived pyrazoles with various combinations of substituents in the phenyl rings. Novel compounds were evaluated for their antitumor activity against various lung cancer cell lines, including studies on cytotoxic, antiproliferative, prooxidant, and proapoptotic effects. Among these compounds, 27 and are presented in Table The chalcone-derived pyrazoles were synthesized according to the protocols described by Yin Luo et al.N-acylated pyrazole ring. PCH-1, PCH-2, PCH-3, PCH-4, PCH-5, PCH-6, PCH-8 have at least one 1,3-benzodioxole moiety which is described as the structural element of compounds exhibiting an anticancer potential28. In PCH-7 and PCH-9 the oxygen atom in the benzodioxole ring is bioisosterically replaced by –CH2– group and sulfur atom, respectively. PCH-1, PCH-2, PCH-4, PCH-7, PCH-8, and PCH-9 have from one to three methoxy groups substituting aromatic rings. In case of PCH-3 the methoxy group is changed into methyl group. Fluorine atoms have been incorporated into the methoxy group in PCH-6 derivative.Compounds PCH contain two differently substituted aromatic rings and 50 concentration that inhibits 50% growth of cells. As depicted in Table PCH-1 was found to be the most potent cytotoxic compound against A-549 cells among all the tested pyrazoles, with the IC50 value of 4.32 ± 0.28 µM. H226 and H460 cells showed similar sensitivity to PCH-1 treatment, with IC50 values of 4.69 ± 0.43 µM and 8.40 ± 1.10 µM, respectively. Other compounds exhibited moderate activity against cancer cells, besides PCH-2, PCH-3, PCH-4, PCH-7, and PCH-8, which were active only against H226 and H460 cells. Following treatment with PCH-1, the viability of all the tested cancer cell lines decreased in a concentration-dependent manner. In contrast, the nonmalignant human embryonic kidney cells (HEK293) exhibited less reduction of cell viability after PCH-1 treatment. This finding indicates that compound PCH-1 inhibited the cell viability of cancer cells without significantly affecting normal kidney cells. Additionally, PCH-1 showed superior activity to the reference compound Cisplatin by approximately 6.7-fold, 3.7-fold, and 2.6-fold more for A-549, H226, and H460 cells, respectively.All the synthesized chalcone-derived pyrazoles were evaluated for their antiproliferative activity against three human non-small cell lung cancer cell lines: A-549, H226, and H460, and a nonmalignant human embryonic kidney cell line HEK293. Cisplatin and 5-fluorouracil (5-FU) were used as a reference. Cells were exposed to a range of PCH concentrations (0.5–100 µM) for 72 h and analyzed by 3--2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The results of the studies are expressed as the ICPCH-1 was selected to test its antiproliferative property by using a colony formation assay. As depicted in Fig. PCH-1 treatment reduced the number of colonies in all the tested cell lines in a dose-dependent manner.The most potent compound PCH-1 led us to further investigate the effect of this compound on cell cycle distribution. Therefore, cell cycle progression was analyzed by quantifying DNA content using flow cytometry. As shown in Fig. PCH-1 after 24 h significantly (**p < 0.001) induced cell cycle arrest in the G2/M phase, and this effect was followed by a decreasing trend at 48 h as compared to control. Concomitantly, we observed a significant increase in the sub-G1 population of cells with the highest peak at 48 h after PCH-1 treatment, thus suggesting apoptotic cell death. To thoroughly analyze the course of the cycle, A-549 cells were synchronized with a double-thymidine block in the G1 phase and treated with IC90 concentration of PCH-1 for the indicated time. DNA content was quantified by the flow cytometric method and presented in Fig. PCH-1 resulted in the accumulation of cells at the G2/M phase, resulting in a 50.12% increase in population as compared to that of the control. After 10 h of thymidine release, the DMSO control group showed a typical pattern of the cell cycle in the G1/G0, S, and G2/M phases. The results indicated that PCH-1 arrests cells in the G2/M checkpoint and halts cell mitosis in a time-dependent manner, leading to the inhibition of the growth of A-549 cells.Promising antiproliferative properties of PCH-1, in vitro microtubule polymerization assays were performed using the microtubule protein with Paclitaxel and Vincristine as a reference. Paclitaxel and Vincristine are antimitotic drugs that have been commonly used in conventional chemotherapy for decades29. In 1979, it was shown that Paclitaxel accelerates the initiation of tubulin fiber polymerization and stabilizes the formed tubulin fibers in vitro, even in the absence of GTP or MAP in the solution30. Vincristine, on the other hand, binds to tubulin and inhibits its polymerization31. As shown in Fig. PCH-1 remarkably reduced the total mass of tubulin polymer, without a significant increase in nucleation time. As expected, Paclitaxel accelerated the nucleation phase and increased the total mass of tubulin polymer, in contrast to Vincristine which extended the nucleation time and reduced the total mass of tubulin polymer compared to the vehicle. These results indicated that compound PCH-1 interferes with microtubule polymerization by blocking microtubule assembly.To further evaluate whether the tubulin–microtubule system was the target site of PCH-1 on the organization of the tubulin cytoskeleton in A-549 cells, immunofluorescence was performed using antibodies for detecting β-tubulin and Aurora B. Aurora B is a protein kinase involved in the condensation and cohesion of chromosomes as well as in the process of attachment of tubulin spindles to the kinetochore and passing through the checkpoint of spindle assembly32. The expression of Aurora B is cell cycle-dependent and reaches a maximum at the G2/M transition, with the highest kinase activity during mitosis. Specifically, Aurora B localizes at the prophase in chromosomes, in the prometaphase and metaphase at centromeres, and is then transferred to the central mitotic spindle in the anaphase34. Visualization of the intracellular localization of Aurora B enables to determine whether the tubulin fibers forming the spindle are properly attached to the kinetochores and whether the cytokinesis process is proceeding properly.To confirm the influence of PCH-1 treatment groups, several mitotic cells could be seen in one field of view. As shown in Fig. PCH-1 treatment were nearly identical to those that occurred after Vincristine treatment and were the result of a prolonged metaphase arrest characterized by uncoordinated loss of chromatid cohesion  double staining using flow cytometry /PI(−)) and late (Annexin V(+)/PI(+)) apoptotic fractions was observed. Later exposure (48 h) to PCH-1 demonstrated a potent increase in the late apoptotic phase, from approximately 5% to 30–60%, depending on the cell line for vehicle and PCH-1-treated cells, respectively. Simultaneously, under the same conditions, a constant number of Annexin V(−)/PI(+) cells was observed for all cell lines. PI can enter only those cells with damaged plasma membrane, which enables to differentiate early apoptotic cells from late apoptotic and necrotic cells. Overall, biochemical changes involved in apoptotic cell death were prevailing, whereas necrotic changes were not observed. Furthermore, typical phenotypic features of cells undergoing apoptosis were visualized by fluorescent microscopy in Supplementary Fig. To examine whether the effect of PCH-1, the ability of the compound to activate caspases 3–7 was determined by flow cytometry. As depicted in Supplementary Fig. PCH-1 activates executioner caspase-3/7 that executes apoptosis, as revealed by a 2.6-fold, 2.9-fold, and 3.15-fold increase in apoptosis for A-549, H226, and H460 cells, respectively, as compared to control. Later exposure to PCH-1 resulted in a massive increase in subpopulation cells with activated caspase 3/7, sustaining at a level greater than 5.8 times in A-549 cells and 4.7 times in H226 cells than in the control, while remaining approximately constant in H460 cells. The most pronounced activation of caspase 3 occurred equally in A-549 and H226 cell lines after 48 h of treatment and was sevenfold greater than that in the vehicle.To explain the type of cell death that cancer cells undergo after treatment with PCH-1-induced apoptosis in A-549 cells, we measured the expression of apoptosis-related proteins by western blot analysis. As shown in Fig. PCH-1 treatment downregulated the expression of full-length caspase 3, caspase 8, and caspase 9, accompanied by an increase in the levels of their cleaved forms. The activation of the caspase cascade by the test compound leads to proteolytic cleavage of poly(ADP-ribose) polymerase-1 (PARP-1), as revealed by a decrease in its full-length expression. Next, we determined the role of the antiapoptotic protein Bcl-2 as well as the proapoptotic proteins Bax and Bid in PCH-1-induced cell death in A-549 cells. As shown in Fig. PCH-1 treatment led to a significant increase in the Bax protein level , whereas the level of Bcl-2 was not changed. Additionally, we observed Bid cleavage (48 h: *p < 0.01), which was noted as a reduction in full-length Bid protein.To investigate the potential molecular mechanisms responsible for PCH-1 dysregulated both biomarkers N-cadherin and vimentin of mesenchymal cells. The activity of N-cadherin in A-549 cells was reduced by 45% at 24 h (p = 0.0002) and by 50% at 48 h (p = 0.0002), while vimentin activity was reduced by 43% at 24 h (p = 0.005) and by 46% at 48 h (p = 0.004). Additional PCH-1 inhibited Transforming Growth Factor β1 (TGF-β1) induced mesenchymal-like phenotype in A-549 cells, indicating downstream of N-cadherin by 66% at 48 h (p = 0.0003), and another EMT effector β-catenin, which was suppressed by 31% at 24 h (p = 0.0275) and by 66% at 48 h (p = 0.0019). Moreover, 24 and 48 h exposure to PCH-1 significantly decreased (p = 0.001) the expression of the catalytic subunit of the telomerase reverse transcriptase hTERT, which is not only responsible for EMT but also plays an essential role in the maintenance of chromosomal telomere length, stemness, and resistance.Epithelial-mesenchymal transition (EMT) plays a crucial role in tumorigenesis by promoting cell motility, migration, and metastasis. Thus, we examined the expression of proteins involved in the EMT process by western blot assays. Figure PCH-1, the wound healing assay was performed on A-549 cells. As shown in Fig. PCH-1, except for 30–36 h, where the ability of cells to repopulate the wounded area was temporarily attenuated as compared to that in the vehicle. In contrast, 10 and 15 µM concentrations of PCH-1 significantly reduced the migration of the cells. This effect was retained during the entire exposure time, with the highest difference during 18–24 h compared to that in the control group. Finally, the anti-migratory properties of PCH-1 were studied by co-treatment A-549 cells with TGF-β1. As shown in Fig. PCH-1 inhibited TGF-β1—induced cell motility, and as a result, the wound remained opened after 20 h of treatment with 5 µM and 10 µM of PCH-1. 2.5 µM of PCH-1 also resulted in a decrease in the rate of cell migration, but after 20 h of exposure, the wound closed similarly to the control-treated with TGF-β1 alone. A noteworthy aspect is that we did not observe detached cells at any point of treatment. Moreover, studied concentrations of PCH-1 were only slightly cytotoxic as evidenced by the IC50 values determined after 24 and 48 h of treatments, which were 41.27 ± 0.75 µM, and 25.58 ± 0.97 µM, respectively. This suggests that the observed antimigratory properties are not affected by the cytotoxic effect of the tested compound.To further confirm the antimigratory potential of 35. In the present study, the effect of PCH-1 on ROS generation in A-549 cells was investigated by flow cytometry. As an indicator of ROS generation, the fluorescent probe 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate (H2DCFDA) was used, which undergoes oxidation by ROS to form a highly fluorescent compound 2ʹ,7ʹ-dichlorofluorescein (DCF). As shown in Supplementary Fig. PCH-1 induced a massive amount of ROS in A-549 cells in a time-dependent manner. During 3, 6, and 24 h was observed accordingly a significant 15% (****p < 0.00001), 56.9% (****p < 0.00001), and 68.1% (****p < 0.00001) increase of ROS towards vehicle control-treated cells. Moreover, the induction of ROS was equally high for PCH-1 and the reference compound H2O2 in all the tested time points.The generation of intracellular reactive oxygen species (ROS) causes an imbalance of cellular redox homeostasis, followed by damage of cellular components and cell death. Therefore, compounds with the ability to produce high levels of ROS and/or inhibit antioxidant processes are often used in anticancer therapies36. To estimate ligand–receptor affinity, an empirical scoring function inspired by the X-score function was used in Vina37. Docking was performed on 3 ligands, namely PCH-1, vinblastine (VLB), and vincristine (VNC), to investigate their binding mode in the vinblastine site of tubulin. The latter two ligands are already known for their effectiveness toward the tubulin target and served here as a reference31. Currently, there are a number of tubulin structures in the PDB database . However, we decided that the most appropriate one for our study would be the 5JT2 structure from 2016 because it is a complex of protein with one of the ligands. Thus, we used the bound docking approach, for example, the structure of the target was used from the ligand–target complex. Both the receptor and the VLB ligand used in the docking simulation originated from the abovementioned 5J2T PDB file38. The VNC ligand was derived from PDB 7A6939. PCH-1 is a new molecule; therefore, its structure was built and optimized using the Biovia software40. The maximum energy difference between the worst and the best docking modes was set to 5 kcal/mol. The grid center of Vina docking was selected as the center of mass of the ligand VLB from 5JT2, which was obtained using the experimental pose. The grid size in docking was set to 20 Å × 20 Å × 20 Å, which was adequate to cover the entire vinblastine site. Additionally, the rigid receptor and flexible ligands were parametrized by AutodockTools 1.5.641. The parameterized systems were recorded in the PDBQT file. In particular, both receptor and ligand were presented using a united atom model, which involves the nonpolar hydrogen atoms. Atomic charges were estimated through the Gasteiger–Marsili method42.In addition to the abovementioned biological tests, a series of docking simulations were performed using the Autodock Vina programPCH-1 had scores similar to those of VLB, where the scores of VNC were lower by almost 2 kcal/mol, even with different spatial configurations. This finding agrees with our biological test results that PCH-1 is biologically slightly more active than VLB or VNC.The performed simulations were narrowed down to 10 best results based on the score function; after careful visual inspection, the best ones were selected based on their spatial arrangement and docking score Table . The doc43. Figure These results were also visually checked by the VMD program44. The incidence of lung cancer is increasing dramatically each year, and this increase is associated with aging of the human population which has been exposed to several factors that promote mutations leading to cancer, such as smoking tobacco, unhealthy diet, or air pollution60. Treatment approaches for lung cancer mainly depend on tumor staging and type of cancer. Patients with small cell lung cancer show a good response to chemotherapy, while the progress in radiation therapy ensures their long-term survival45. Current pharmacological treatments for non-small cell lung cancer are not effective, but surgical resection offers substantial cure rates in early-stage cases. Patients with advanced, metastatic cancers are receiving combined chemotherapy and radiation46.According to the World Health Organization (WHO), cancer is the leading cause of death worldwide and caused 10 million deaths in 2020, and lung cancer had the highest mortality rate and accounted for 1.8 million deaths48. In our present study, a series of pyrazoles derived from chalcones were reported based on the consideration of commonly used ring substitution modifications. Initially, the compounds were evaluated for cytotoxicity in the selected cell line models. Almost all derivatives exhibited moderate activity against all the three non-small cell lung cancer cell lines, except for the dimethoxy analogue PCH-1, which displayed potent cytotoxic properties and thus was chosen to evaluate its molecular mechanism of action. The substantial objective of cancer therapy is to kill cancer cells, but without affecting healthy cells. The toxicity studies on normal human embryonic kidney cells revealed that PCH-1 is less toxic than the reference compound Cisplatin, and therefore, it is suitable for further evaluation of anticancer properties.Many studies have proved that nitrogen-containing heterocyclic compounds such as pyrazole possess significant biological potency for cancer treatmentPCH-1 induces G2/M cell cycle arrest after 24 h of incubation in all the tested cancer cell lines.The determination of the mechanism responsible for cancer cell death is extremely important for drug efficacy and development. The antiproliferative effects of anticancer drugs are often linked with the suppression of the cell cycle. Studies on cell cycle distribution showed that 49. Many existing microtubule inhibitors are registered as drugs, but their clinical use is often limited due to the chemoresistance mechanisms50. PCH-1 inhibited tubulin polymerization in the cell-free tubulin assembly assay, but the effect was less pronounced similar to that observed for the reference compounds. In cell-based assays, we observed similar depolymerization of the tubulin network and impaired spindle assembly in the test compound and Vincristine groups. These results were supported by molecular docking.Antimitotic properties of chalcones are known from 1990 and are associated with the inhibition of microtubule assembly51. Apoptosis, also known as programmed cell death, is a natural mechanism for the regulation of physiological growth and homeostasis. This process is characterized by several morphological and biochemical hallmarks, including chromatin condensation, cell shrinkage, and phosphatidylserine externalization. There are also many ways in which cells may undergo apoptosis. The two main pathways linked to the activation of apoptosis signal transduction are intrinsic and extrinsic pathways52. Microscopic observations and flow cytometry analysis revealed that PCH-1 induces apoptosis in all the tested cancer cell lines in a time-dependent manner. Therefore, one of the assumptions of our studies was to obtain information about the exact mechanism by which PCH-1 induces apoptosis. For this purpose, the A-549 cell line was selected as the most sensitive cell line to the test compound. Western blot analyses revealed full activation of the initiation caspase-9 and execution caspase-3, with a significant increase in their cleaved forms in a time-dependent manner. Caspase-9 is activated by translocation from the cytosol into the intermembrane space and partially by recruitment into the Apaf-1 apoptosome complex following the efflux of cytochrome c from the mitochondria53. Once activated, caspase-9 cleaves and activates caspase-3 and caspase-7 to induce apoptosis54. Next, the activated caspases cleave cellular proteins such as nuclear enzyme PARP to dismantle the apoptotic cells55. Cleavage and inactivation of PARP by PCH-1 lead to loss of genomic integrity, thereby depleting the cell of NAD and ATP and thus contributing to cell death. Moreover, the activation of caspase-8 was also observed in PCH-1-treated cells, which is closely related to apoptosis signaling by the extrinsic pathway56. Caspase-8 mediates the mitochondrial pathways through the cleavage of the proapoptotic Bcl-2 family members such as Bid to tBid. Subsequently, tBid switches the conformation of Bak, resulting in its oligomerization and allosteric activation and releasing cytochrome c57. PCH-1 treatment leads to both cleavage of Bid and increased expression of Bax, thus suggesting the activation of cell surface death receptors and therefore the extrinsic apoptosis pathway.Current cancer chemotherapies mostly exert an antitumor effect by triggering apoptosis in cells58. One of the earliest events in cancer metastasis is EMT, a process that converts epithelial cells to mesenchymal cells that undergo cytoskeletal rearrangements to promote cancer motility and invasiveness59. Intermediate filaments (IFs) play an important role in EMT progression by maintaining cellular polarity and stiffness60. The most abundant member of the IF family is Vimentin (a type III protein) that is responsible for forming associated parallel arrays with microtubules and modulating prosurvival functions61. The disassembling of the microtubule network leads to a collapse of Vimentin IFs to the perinuclear region62. Recent studies have shown the significance of Vimentin in lung cancer, wherein a high expression of Vimentin was found to be related to a high frequency of metastases and therefore a poor prognosis in patients with resected non-small cell lung cancer63. On the basis of these findings, we decided to test the effect of PCH-1 on the suppression of the EMT process. PCH-1 effectively downregulated Vimentin and significantly promoted the suppression of another mesenchymal marker, N-cadherin, in A-549 cells. In order to show retrieving the epithelial phenotypes capacity by tested compound, A-549 cells were stimulated with well-known EMT inducer TGF-β1, and then exposed to PCH-164. Treatment with TGF-β1 alone activated N-cadherin, and β-catenin, whereas co-treatment with PCH-1 abrogated their expression. This suggests that PCH-1 is a therapeutic agent for cancer treatment controlling TGF-β1-mediated EMT. Moreover, we studied the expression of telomerase reverse transcriptase, which plays a key role in ensuring chromosomal stability by maintaining telomere length, and therefore assert the uncontrolled proliferation of the cells65. Besides, telomerase exhibit many multiple non-canonical biological functions, like protecting mitochondrial DNA, regulating the expression of Wnt target genes , and promoting EMT through the NF-kB signaling pathway68. Additionally, Liu et al. shows that depletion of hTERT leads to abolished TGF-β1 and β-catenin-mediated EMT. β-catenin in cooperation with hTERT regulates transcription of Vimentin, so deregulation of all those proteins leads to preventing cancer progression69. We demonstrated that PCH-1 significantly decreased the level of hTERT, which suggests that it not only suppresses EMT but also inhibits tumor proliferation and induces cell mortality.Metastasis involves the spread of cancer cells from the primary tumor site to other tissues or organs of the body and is the major cause of cancer-related deaths71. Many microtubule-targeting agents inhibit cell migration, by preventing activation of Rac1/Cdc4273. During microtubule polymerization, Rac1 is activated in lamellipodia and stimulates actin stress fiber formation. Whereas, Cdc42 regulates polarization of cells, and proper orientation of the centrosome. Consequently, interference with microtubule dynamics inhibit Rac1 and Cdc42, and therefore decrease cell motility. PCH-1 efficiently decreased cell migration in concentration-dependent manner in TGF-β1-stimulated, and unstimulated cells, which was observed under time-lapse microscopy by using a wound healing assay. However, the exact mechanism of inhibition cell migration by PCH-1 should be further explored, such as confirming the involvement of Rac1 and Cdc42 in this process.Microtubule dynamics are essential in the cell migration by providing protrusive and contractile forces, a front-rear polarity allowing movement of organelle and protein through the cells, and controlling signaling during motility75. Similar to its analogs, PCH-1 robustly induces the generation of ROS in A-549 cells. This accumulation can activate both the intrinsic and extrinsic apoptotic cell death, by activating the downstream caspase cascades76.Several studies have shown that many compounds derived from chalcone possess oxidative propertiesPCH-1 reduces cell viability and colony formation of non-small lung cancer cells in vitro. Our study demonstrated that PCH-1 induces G2/M phase arrest by stabilizing tubulin polymers and cellular apoptosis through extrinsic and intrinsic pathways. In addition to its proapoptotic properties, PCH-1 shows potent antimetastatic and prooxidant potential in A-549 cells. The molecular mechanism of action of this compound is shown in Fig. PCH-1 is a promising antitumor compound that warrants further preclinical development.In conclusion, this study provides evidence that 1H-NMR spectra were acquired on a Bruker Avance III spectrometer at 500 MHz. The NMR data were processed and analyzed by TopSpin . Chemical shifts were expressed at δ value to the internal standard (TMS). IR spectra were measured on an IF S66 spectrometer  in KBr pellets at an absorption range of 400–4000 cm−1. Thin layer chromatography (TLC) was performed on Merck Kieselgel 60 F254 aluminum plates and visualized with UV and iodine.Liquid chromatography-mass spectrometry (LCMS) spectra were registered on an LCMS IT-TOF  with an electrospray ionization (ESI) source in the positive ion mode. Each compound was synthesized in two stages: condensation of the appropriately substituted benzaldehydes and acetophenones, and reaction with hydrazine hydrate to yield chalcone-derived pyrazoles.First, 10 mmol of the appropriately substituted benzaldehyde and acetophenone were dissolved in 20 ml of ethanol. The mixture was then stirred at 5 °C for few minutes. Next, 10 ml of 40% aqueous KOH was added, and the solution was stirred at room temperature (RT) for approximately 4 h. The formed precipitate was collected by filtration, characterized by HPLC and LCMS, and subjected to further reaction.2CO3, poured into crushed ice, and allowed to stand overnight at RT. The product was collected by filtration and purified by flash chromatography or reversed-phase high-performance liquid chromatography (RP-HPLC). The product was then characterized by LCMS, 1H-NMR, and IR.Hydrazine hydrate (20 mmol) was added to a solution of chalcone (5 mmol) in acetic acid (10 ml). The reaction mixture was refluxed for approximately 2 h. The progress of the reaction was monitored by TLC. After the completion of the reaction, the mixture was cooled, adjusted to pH 7 with 10% Nam/z): 369.139 [M + H]+; 1H-NMR , δ (ppm): 2.39 , 3.30 , 3.78 , 3.79 , 5.49 , 6.00 , 6.54 , 6.85 , 7.19 , 7.48 ; IR (KBr), ν (cm−1): 1650.92, 1468.94.ESI–MS: (m/z): 399.125 [M + H]+; 1H-NMR , δ (ppm): 2.45 , 3.14 , 3.73 , 3.91 , 3.93 , 5.54 , 5.94 , 6.73 , 6.97 ; IR (KBr), ν (cm−1): 1665.00, 1417.26.ESI–MS: (m/z): 323.098 [M + H]+; 1H-NMR , δ (ppm): 2.42 , 2.44 , 3.15 , 3.73 , 5.52 , 5.94 , 6.70 , 6.75 , 7.25 , 7.65 ; IR (KBr), ν (cm−1): 1649.10, 1443.97.ESI–MS: (m/z): 339.101 [M + H]+; 1H-NMR , δ (ppm): 2.42 , 3.31 , 3.85 , 3.86 , 5.47 , 5.93 , 6.73 , 6.76 , 6.96 , 7.04 , 7.41 , 7.94 ; IR (KBr), ν (cm−1): 1659.49, 1419.84.ESI–MS: (m/z): 353.068 [M + H]+; 1H-NMR , δ (ppm): 2.42 , 3.09 , 3.68 , 5.51 , 5.94 , 6.05 , 6.69 , 6.76 , 6.85 , 7.11 , 7.39 ; IR (KBr), ν (cm−1): 1653.92, 1458.82.ESI–MS: (m/z): 393.056 [M + H]+; 1H-NMR , δ (ppm): 2.44 , 3.14 , 3.73 , 5.55 , 5.94 , 6.73 , 7.29 , 7.79 ; IR (KBr), ν (cm−1): 1652.13, 1447.62.ESI–MS: (m/z): 337.108 [M + H]+; 1H-NMR , δ (ppm): 2.43 , 3.16 , 3.71 , 3.88 , 4.55 , 5.53 , 6.73 , 6.96 , 7.01 , 7.07 , 7.72 ; IR (KBr), ν (cm−1): 1661.06, 1493.03.ESI–MS: (m/z): 369.092 [M + H]+; 1H-NMR , δ (ppm): 2.46 , 2.98 , 3.67 , 3.82 , 3.86 , 5.77 , 5.88 , 6.54 , 6.93 , 7.68 ; IR (KBr), ν (cm−1): 1661.13, 1483.86.ESI–MS: (m/z): 371.045 [M + H]+; 1H-NMR , δ (ppm): 2.32 , 3.18 , 3.69 , 3.73 , 5.37 , 5.67 , 6.42 , 6.55 , 6.94 , 7.69 ; IR (KBr), ν (cm−1): 1638.59, 1468.15.ESI–MS: (l-glutamine (Sigma-Aldrich), and antibiotics . The cells were incubated in a humidified 5% CO2 atmosphere at 37 °C and routinely screened for Mycoplasma contamination.HEK293, A-549, H226, and H460 cells were obtained from the American Type Culture Collection  and cultured in DMEM (HEK293) or RPMI medium (Corning) supplemented with 10% fetal bovine serum (Corning), 2 mM 2, the MTT solution (0.4 mg/ml) was added to each well. After 2–4 h of incubation, the medium was removed, and the formazan crystals were dissolved in 100 μl of DMSO. The absorption was measured at 450 nm with an ASYS UVM340 microplate reader (Biochrom Ltd.). The 50% inhibitory concentration (IC50) was defined as the concentration that reduced the absorbance of the DMSO-treated wells by 50% of the vehicle. The IC50 values were generated from GraphPad Prism 9 software by plotting the survival curve as a function of dose from 3 independent experiments.The cell viability assay was performed for A-549, H226, H460, and HEK293 cells after treatment with the test compounds by using the 3--2,5-diphenyltetrazolium bromide  assay. Briefly, cells were seeded onto 96-well culture plates for attachment. After 24 h, triplicate wells were treated with DMSO (1%) and the agents. After 72 h of incubation at 37 °C in 5% COCells were seeded onto 6-well plates (500 cells/well) and allowed to attach for 24 h in a fresh RPMI medium. Next, the cells were pretreated with DMSO (1%) solution of the examined compound  for 24 h. The medium containing compound was then changed, and the cells were cultured for additional 9 days. After incubation, the cells were washed twice with phosphate-buffered saline (PBS), fixed with 100% methanol for 30 min, and stained with 0.5% crystal violet for 15 min. The colonies were photographed and counted by ImageJ software.PCH-1 or DMSO. After incubation, the cells were rinsed with PBS, fixed in 3.7% paraformaldehyde (Sigma-Aldrich) for 10 min at RT and stained with 4′,6-diamidino-2-phenylindole . The stained nuclei were visualized using a fluorescence microscope (Olympus BX60) with an appropriate filter.Cells were seeded onto tissue culture plates with a glass slide and allowed to attach for 24 h. Next, the cells were treated for the indicated time with PCH-1 or Etoposide at IC90 concentration for 24 and 48 h, while synchronized cells were treated with PCH-1 or Etoposide at IC90 concentration for 2, 4, 6, 8, and 10 h. Synchronization of the cells was performed as described previously by Chen et al.77. Next, the cells were trypsinized, fixed in ice-cold 75% ethanol, and stored overnight at − 20 °C. After centrifugation, the cells were rinsed with PBS and stained with 20 μg/μl PI (Sigma-Aldrich) and 50 μg/μl RNaseA (Thermo Fisher) in PBS for 30 min at RT. The distribution of the cells in different phases of the cell cycle was determined by flow cytometry  and FlowJo software v10. Each experiment was repeated independently three times.Cells were seeded onto tissue culture plates and allowed to attach for 24 h. Unsynchronized cells were treated with The assay was performed according to the manufacturer’s instructions. Briefly, compounds were diluted in DMSO to a concentration of 2 mM and then in GTB buffer to a concentration of 50 µM. In the wells of a 96-well plate  previously heated to 37 °C, 10 µl of dilutions of the test compounds were placed in duplicate. The plate was incubated for 2 min at 37 °C, and 100 µl of freshly prepared tubulin solution (4 mg/ml) in GTB-GTP buffer was then added. The plate was immediately placed in a Tecan SPARK 10 M plate reader, and the reaction kinetics were measured with the following settings: absorbance reading: 340 nm, 37 °C, interval: 45 s, number of cycles: 160.Cells were seeded onto tissue culture plates with a glass slide and allowed to attach for 24 h. After the incubation period, the cells were washed in PBS, fixed for 15 min at RT with 4% paraformaldehyde (Sigma-Aldrich) in PBS, and permeabilized for 15 min in 0.25% Triton-X100 (Sigma-Aldrich) in PBS. The cells were then washed twice with PBS, blocked with 3% bovine serum albumin  in PBS for 1 h at RT, and incubated for 1 h at 37 °C in a humidified chamber with the following primary antibodies diluted in 3% BSA in PBS-T (PBS with 0.1% (v/v) Triton X-100): rabbit anti-Aurora B antibody  1:500 and mouse anti-Tubulin 1:200 . The slides were washed three times with PBS-T and incubated for 1 h at 37 °C in a humidified chamber with the following secondary antibodies diluted in 3% BSA in PBS-T: goat anti-mouse IgG antibody conjugated with Fluorochrome DyLight-488  and goat anti-rabbit IgG antibody conjugated to Alexa Fluor 594 . The slides were then washed twice in PBS-T and stained with 0.25 µg/ml DAPI. Images were acquired with an LSM 800 inverted laser scanning confocal microscope (Carl Zeiss) with an Airyscan detector using a × 63 1.4 NA Plan Apochromat objective (Carl Zeiss).PCH-1 or Etoposide at IC90 concentration for 6, 24, and 48 h. After incubation, the cells were harvested by trypsinization, rinsed twice with PBS, and stained with Annexin V Alexa Fluor™ 488 conjugate  for apoptosis assay and with CellEvent™ Caspase-3/7 Green Flow Cytometry Assay Kit  for caspase-3/7 activation according to the manufacturer’s protocols. The analysis was performed with Guava EasyCyte 8 cell sorter (Merck Millipore) and FlowJo software v10. Each experiment was repeated independently three times.Cells were seeded onto tissue culture plates and allowed to attach for 24 h. Next, the cells were treated with 6) were cultured in tissue culture plates for overnight attachment. The cells were then incubated with IC90 of PCH-1 or 1% DMSO for 6, 24, and 48 h, or were co-treatment with 2.5 ng/ml TGF-β1 (Sigma-Aldrich), and IC90 of PCH-1 or 1% DMSO for 24, and 48 h. Subsequently, the cells were collected and lysed in Laemmli buffer with a protease inhibitor cocktail (Roche) and phosphatase inhibitors . The cells were then sonicated in triplicate  and centrifuged at 16,000×g at 12 °C for 15 min. Protein concentrations were determined using the DC Protein Assay Kit (Bio-Rad). For determining total protein content, the cellular extract was separated by 10% or 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a microporous polyvinylidene difluoride (PVDF) membrane (Bio-Rad). After blocking with 5% (w/v) BSA (Sigma-Aldrich) in 1 × TBST (pH 7.4 and 0.1% Tween-20) for 1 h, the membranes were incubated overnight with primary antibodies at 4 °C, followed by horseradish peroxidase-conjugated secondary antibodies for 1 h. All the used antibodies are listed in Table Cells  in an imaging chamber  at 37 °C with 5% CO2. Images were captured every 10 min for 20 or 42 h under × 10 magnification using a fluorescence microscope  connected to an XC50 digital color camera (Olympus). The percentage of wound closure was quantified with ImageJ software.To analyze cell motility, A-549 cells were seeded onto the Ibidi-silicone insert on a cover glass-bottom 24-well plate for live cell imaging and incubated for 24 h. Subsequently, the inserts were dislodged, the cellular debris was removed by washing with RPMI, and the cells were incubated with different concentrations of PCH-1, H2O2, or 1% DMSO for 3, 6, and 24 h. Next, a 1 µM CM-H2DCFDA  probe was added to the cells in each plate 30 min before the end of the drug treatment. After incubation cells were collected by trypsinization, stained with 7-Aminoactinomycin D , and measured. The analysis was performed with Guava EasyCyte 8 cell sorter (Merck Millipore) and FlowJo software v10. Each experiment was repeated independently three times.For determination the ROS generation, cells were treated with IC90 of Supplementary Information.


Fusarium fujikuroi that produces a mix of several GAs. We explored the potential of the oleaginous yeast Yarrowia lipolytica to produce specific profiles of GAs. Firstly, the production of the GA-precursor ent-kaurenoic acid (KA) at 3.75 mg/L was achieved by expression of biosynthetic enzymes from the plant Arabidopsis thaliana and upregulation of the mevalonate (MVA) pathway.The application of small amounts of natural plant growth hormones, such as gibberellins (GAs), can increase the productivity and quality of many vegetable and fruit crops. However, gibberellin growth hormones usage is limited by the high cost of their production, which is currently based on fermentation of a natural fungal producer 4-producing strain by extending the GA-biosynthetic pathway and upregulating the MVA-pathway further, resulting in 17.29 mg/L GA4. Additional expression of the F. fujikoroi GA-biosynthetic enzymes resulted in the production of GA7 (trace amounts) and GA3 (2.93 mg/L). Lastly, through protein engineering and the expression of additional KA-biosynthetic genes, we increased the GA3-production 4.4-fold resulting in 12.81 mg/L. The developed system presents a promising resource for the recombinant production of specific gibberellins, identifying bottlenecks in GA biosynthesis, and discovering new GA biosynthetic genes.We then built a GABiological Sciences, Applied Biological Sciences. •A complete biosynthetic pathway towards gibberellins was reconstructed in a microbial host•ent-kaurenoic acid consisted of Arabidopsis thaliana enzymesThe pathway towards •ent-kaurenoic acid to gibberellins GA3, GA4 and GA7 consisted of Fusarium fujikuroi enzymesThe pathway from •Y. lipolytica expressed 14 heterologous genes for gibberellins biosynthesis and had 5 genome edits for improved mevalonate flux•3 and up to 17 mg/L GA4 in small-scale cultivationsThe strains produced up to 12 mg/L of GA Of the mixtures . Sprayinmixtures , as wellmixtures . Valent mixtures . Gibberemixtures . Accordimixtures .12-aldehyde are similar in fungi and plants. Cyclization of GGPP into ent-copalyl diphosphate and then into ent-kaurene is catalyzed by the copalyl diphosphate synthase (CPSp) and the ent-kaurene synthase (KSp) in plants. In fungi, one bi-functional synthase (CPS/KSp) forms ent-kaurene  by the ent-kaurene oxidase (KOp) (Ent-kaurenoic acid oxidase (KAOp) then catalyzes the formation of GA12-aldehyde via the intermediate ent-7α-hydroxykarenoic acid  (4 is converted to GA7 by the GA desaturase (DESp) and then to GA3 by another monooxygenase (P450-3p) , while in fungi, they are produced through the mevalonate pathway (MVA) . The ini-kaurene  (Sun and-kaurene . Ent-kause (KOp) . Ent-kauoic acid . In planplastids . In fungnto GA14 . In plantive GA4 . In fungP450-2p) . SubsequP450-3p) .Fig. 1ThSphaceloma manihoticola or Phaeosphaeria sp. and bacteria species like Rhizobium phaseoli or Azospirillum lipoferum can produce gibberellins, the industrially used microbe for gibberellin production is the fungus Fusarium fujikuroi (formerly Gibberella fujikuroi)  . Submergjikuroi) . Titers terature . While Gmilarity . Since Gsome GA7 .F. fujikuroi that are able to produce predominately GA7 at 234 mg/L or solely GA4 at 420 mg/L during ten days of fermentation, it is unclear whether these strains can be used industrially , nugm-Htg2, ndh2i, lys11−, leu2−, ura3−, MatB) was a gift from Volker Zickermann  (Y. lipolytica strains were grown on Yeast extract Peptone Dextrose (YPD) or synthetic drop-out media (SC) medium at 30°C. YPD medium contained 20 g/L peptone, 10 g/L yeast extract, 20 g/L glucose. For growth on solid media, YPD and SC medium were supplemented with 20 g/L agar. When necessary, the YPD medium was supplemented with antibiotics hygromycin (50 mg/L) or nourseothricin (250 mg/L). Cultivation of the recombinant strains for isoprenoid production was performed in Yeast extract Peptone medium containing 80 g/L glucose instead of 20 g/L glucose (YP+8%D).Germany) . Y. lipoEscherichia coli strain DH5α was used for DNA manipulations. Cells were cultivated at 37°C on Lysogeny Broth (LB) medium supplemented with 100 mg/L ampicillin for plasmid selection.The chemicals were obtained, if not indicated otherwise, from Sigma-Aldrich. Nourseothricin was purchased from Jena BioScience GmbH (Germany).2.2E. coli and correct assemblies were verified by sequencing. Genes encoding Arabidopsis thaliana AtCPS (GenBank: AAA53632.1), AtKS (GenBank: AAC39443.1), AtKO (GenBank: AAC39507.1), AtKAO2 (GenBank: ABJ17103.1), AtC3ox1 (GenBank: AAO64762.1), AtC20ox1 (GenBank: AEE36106.1), AtATR2 (GenBank: AEE85737.1), Fusarium (Gibberella) fujikuroi GfCPS/KS (GenBank: BAA84917.1), GfP450-1 (GenBank: CAA75565.1), GfP450-2 (GenBank: CAA75566.1), GfP450-3 (GenBank: CAA75567.1), GfP450-4 , GfDES (GenBank: CAD10289.1), GfCPR (GenBank: CAE09055.1), GfCyb5 (GenBank: KLO84088.1), GfCybRed (GenBank: KLO80170.1), and Synechococcus sp. SsGGPPs7 (NCBI Reference Sequence: WP_011429285.1) were codon-optimized for Y. lipolytica and synthesized as GeneArt String DNA fragments or Synthetic Genes by Thermo Fisher Scientific (see Supplementary sequences for full DNA sequences). The software ChloroP v1.1 was used to predict chloroplast targeting protein sequences  was resuspended in 100 μL of a transformation mix containing 43.8% poly-ethylene glycol (PEG 3350), 100 mM dithiothreitol, 0.25 g/L single-stranded DNA and 0.1 M LiAc. DNA was then added (a minimum of 500 ng). After a heat-shock at 39°C for 1 h, cells were pelleted for 5 min at 3000 rpm. The transformation mix was removed, and cell pellets were resuspended in 1 mL YPD and allowed to recover for 2 h at 30°C, 250 rpm agitation. Finally, cells were pelleted for 5 min at 3000 rpm, resuspended in 100 μL sterile water, and plated on selective medium. Plates were incubated at 30°C for 2–5 days until colonies appeared.All strains used in this study are listed in protocol . In detaAll strains were verified by PCR using insert-specific oligos in combination with oligos specific to the regions outside the different recombination sites. The CreA-loxP-mediated selection marker loop-out was performed as described previously . The eli2.4600) of 1.0. The shake flask cultures were cultivated for 72 h at 30°C with 300 rpm agitation.For KA-quantification, single colonies were inoculated from fresh plates into 3 mL YPD in 24-well plates with air-penetrable lids  and grown for 24 h at 30°C and 300 rpm agitation as pre-cultures. For cultivation, the required volume of the pre-culture was transferred to 50 ml YP+8%D in 250 mL baffled shake flasks for an initial optical density at 600 nm (OD600) of 0.1. The plates were cultivated for 72 h at 30°C with 300 rpm agitation. By the end of the cultivation, either cell dry weight, OD600, or both were measured. OD600 was measured using a NanoPhotometer . All cultivations were done in biological triplicates.For GA-quantification, single colonies were inoculated from fresh plates into 2.5 or 3 mL YPD in 24-well plates with air-penetrable lids and grown for 16–24 h at 30°C and 300 rpm agitation as pre-cultures. For cultivation, the required volume of the pre-culture was transferred to 2.5 or 3 mL YP+8%D in 24-well plates for an initial optical density at 600 nm . The stock solutions of GA9, GA4, and GA7 were 5 mg/mL in absolute ethanol, which yielded a final ethanol concentration of 2% in the media.For the feeding experiments with GAFor growth profiling of selected strains, pre-culture preparations from single colonies were inoculated from fresh plates into 2.5 YPD in 24-well plates with air-penetrable lids and grown for ~16 h at 30°C and 300 rpm agitation. Then the required volume of pre-culture was transferred to 1 mL of YP+8%D in 24-roundwell plates  with air-penetrable lids. The plates were cultivated, and their growth continuously measured in a Growth Profiler 960 (Enzyscreen) at 30°C with 225 rpm agitation for 72 h. All growth profiling experiments were conducted in biological triplicates.2.512-detection, GA-analysis of ST6513 and ST6514, and feeding experiments, the supernatant was sampled directly in the case of cell-free media or extracted by the following protocol. 300 μL of cultivation broth was transferred into a 2 ml microtube (Sarstedt). 500 μL of 0.5–0.75 mm acid-washed glass beads and 1.2 mL of acetonitrile were added to each tube. Thereafter, the cells were disrupted with a Precellys®24 homogenizer (Bertin Corp.) four times at 5500 rpm for 10 s, with samples being kept on ice in between rounds of breaking. The samples were then shaken for 10 min at room temperature. Lastly, the samples were centrifuged, and the supernatant fractions were sampled for analysis.For the KA-quantification, GA9, GA4, GA7, and GA3 for all other strains, 1 mL of broth were transferred to a 2 mL microtube. The samples were centrifuged, and the supernatant was extracted for analysis. Samples were diluted in water prior to analysis.For quantification of GA9, GA4, GA7, and GA3 in ST8580 cell pellets, 1 mL of broth was transferred 2 mL microtube. The cells were pelleted by centrifugation, and the supernatant was removed. The cell pellets were washed twice by resuspension in 1 mL of water, centrifugation, and removal of the liquid phase. Thereafter, 500 μl of 0.5–0.75 mm acid-washed glass beads and 1 mL of acetonitrile were added to each tube. Subsequently, the cells were disrupted with a Precellys®24 homogenizer four times at 5500 rpm for 10 s, with samples being kept on ice in between rounds of breaking. The samples were then shaken for 10 min in a DVX-2500 Multi-tube vortexer at room temperature. Lastly, the tubes were centrifuged, and the acetonitrile fractions were sampled for analysis.Furthermore, to investigate the presence of GA12 mass confirmation can be seen in the supplementary methods.The quantitative LC-MS data for the feeding experiments, KA-quantification, GA-quantification for ST6513 and ST6514, and GA3 (012 2503), GA4 (012 2532), GA7 (012 2543), and GA9 (012 2663) standards were acquired from OlChemIm s.r.o. (Czech Republic) and the detection limits of all GAs were 0.05 mg/L.The LC-MS/MS analysis of GAs for all other strains was performed on a Vanquish Duo UHPLC binary system  coupled to IDX-Orbitrap Mass Spectrometer . The chromatographic separation was achieved using a Waters ACQUITY BEH C18  equipped with an ACQUITY BEH C18 guard column kept at 40 C and using a flow rate of 0.35 mL/min. The mobile phases consisted of MilliQ© water + O.1% formic acid (A) and acetonitrile + 0.1% formic acid (B). The initial composition was 2%B held for 0.8 min, followed by a linear gradient till 5% in 3.3 min, and afterward, 100%B was reached in 10 min and held for 1 min before going back to initial conditions. Re-equilibration time was 2.7 min. The MS measurement was done in negative-heated electrospray ionization (HESI) mode with a voltage of 2500 V acquiring in full MS/MS spectra (Data dependent Acquisition-driven MS/MS) in the mass range of 70–1000 Da. The authentic GA33.1ent-kaurenoic acid production in Y. lipolytica. All genes that were not native to Y. lipolytica were codon-optimized. To produce the GA-precursor ent-kaurenoic acid via the plant pathway, we expressed the genes encoding copalyl diphosphate synthase (AtCPS), ent-kaurene synthase (AtKS), ent-kaurene oxidase (AtKO), NADPH cytochrome P450 reductase (AtATR2) from A. thaliana, and the native Y. lipolytica cytochrome b5 (YlCyb5) to generate strain ST6687 , and the native geranylgeranyl diphosphate synthase (GGPPS), while the endogenous squalene synthase gene was downregulated by truncating its promoter down to 50 base pairs (SQS↓)  . The HMGs (SQS↓) . Furtheractivity .Fig. 2OvY. lipolytica and gibberellins in F. fujikuroi , F. fujikuroi ent-kaurene oxidase (GfKO), F. fujikuroi NADPH cytochrome P450 reductase (GfCPR) and F. fujikuroi cytochrome b5 (GfCyb5), while tHMG and GGPPS were overexpressed, and the squalene promoter truncated (SQS↓), which generated ST6521.To produce .The MVA-optimized strain ST6349 expressing the plant KA-biosynthetic pathway produced 3.75 mg/L KA, while no KA was detected for the non-optimized strain ST6687 A and B. 3.212 was constructed based on the ent-kaurene producer ST6295 and further expression of AtKO, A. thaliana ent-kaurenoic acid oxidase (AtKAO), YlCyb5, and AtATR2. After cultivation, a compound identified as GA12 could be detected for ST6439 based on targeted ion mass and retention time  and GA C3-oxidase (AtC3ox) in the GA12-producing strain ST6439 to generate ST6513 that should be able to produce GA4 from GA12 via the intermediates GA15, GA24, and GA9. However, the cultivation of ST6513 did not result in detectable amounts of GA4. However, we still attempted to further extend the pathway from GA4 to GA7 and to GA3 by expressing fungal enzymes GfP450-3p and GfDESp generating ST6514.As the next step, we wanted to extend the pathway from GA9 was detected for ST6514, neither GA4, GA7, nor GA3 were detected  note that the GA4 standard we used is ≥90% pure, which indicates the presence of impurities  was expressed in ST8257, giving strain ST8414. Expression of SsGGPPS7 was previously shown to substantially increase carotenoid production in Y. lipolytica  from F. fujikuroi were expressed to promote the electron transfer from NADPH to F. fujikuroi cytochromes P450 for the last steps towards GA3, resulting in strain ST8416. Finally, AtC20ox and AtC3ox were expressed under stronger promoters to overcome the potential rate-limitation of these steps. The genes AtC20ox and AtC3ox were expressed under the control of the promoters PrExp and PrTefintron, respectively, to generate strain ST8447 and under promoters PrTefintron and PrGpd, respectively, to generate ST8448. Both strains should be capable of producing GA4, albeit potentially at different levels due to the different promoter pairs used. Furthermore, the PrTefintron promoter is currently the strongest known constitutive promoter for Y. lipolytica  compared to without (15 g dry cell weight/L). Furthermore, GA4 was also detected when both ST3683 and cell-free media were supplemented with GA7, possibly due to product impurities  was expressed together with AtKO in ST8580, generating ST10331. Furthermore, truncated variants of the A. thaliana KA-biosynthetic enzymes with N-terminal plastidial targeting sequences removed  were expressed under individual promoters or with tAtCPS fused to tAtKS (tAtCPS/KS) alongside the individual expression of tAtKO in ST8580 generating ST10350 and ST10334, respectively. The ChloroP software and earlier reports were used to predict the plastidial targeting sequences of AtCPSp and AtKOp based on which the 2–60 and 2–28 amino acids were removed to make tAtCPSp and tAtKOp, respectively  to GA12 16, 17-dihydro-16α-ol (DHGA12), which can enhance seed germination and seedling development in A. thaliana (12 and bioactive GA12-derivatives may therefore become useful in the future.The further expression of AtKAOp resulted in the production of a compound putatively identified as GA of GA12 . This inproteins . Therefothaliana . Cell fa12 to GA3 by expression of AtC20ox, AtC3ox, GfDES, and GfP450-3 to generate ST6514, neither GA4, GA7, nor GA3 were produced by this strain. The feeding studies indicated that GfP450-3p was not active in ST6514. However, GA3 was produced when GfP450-3p was expressed in ST8580. Interestingly, ST6514 expressed only AtATR2 and YlCyb5, while ST8580 expressed the fungal redox partners GfCyb5, GfCybRed, and GfCPR in addition to the prior. Therefore, the presence of specific redox partners may be necessary to support the activity of F. fujikuroi cytochromes P450. Indeed, previous research has demonstrated that the activity of heterologous cytochromes P450 in yeast depends heavily on their co-expressed redox partners. The cytochrome P450 FoCYP53A19p from Fusarium oxysporum could convert 3-hydroxybenzoic acid to 3,4-dihydroxybenzoic acid when paired with an F. oxysporum CPR but not when paired with an S. cerevisiae or Candida albicans CPR (Y. lipolytica vary greatly in strength (While we attempted to complete the biosynthetic pathway towards GAcans CPR . Furtherstrength .3-titer of 12.81 mg/L reported herein is still very low compared to the highest reported titer of 3.9 g/L for the natural producer F. fujikuroi (The GAujikuroi . HoweverY. lipolytica is a relatively new host for recombinant terpene production, but it has already outperformed S. cerevisiae for the production of β-carotene (Y. lipolytica, which may serve to sequester and store lipophilic terpenes (Y. lipolytica holds great potential as a microbial cell factory for terpenoid production.Indeed, carotene . This isterpenes . Therefo53, GA4, and GA7 in Y. lipolytica was achieved by expressing a GA-biosynthetic pathway with both plant and fungal enzymes. The complete biosynthesis of GA3, GA4, and GA7 was done by heterologous expression of A. thaliana enzymes for KA-production and F. fujikuroi enzymes for the subsequent biosynthetic steps. We demonstrate that the GA-production could be enhanced by further engineering of the yeast chassis. This yeast-based platform holds the potential to improve the future production of bioactive GAs.Production of bioactive gibberellins GAKanchana R. Kildegaard: Conceptualization, methodology, investigation, formal analysis, supervision, writing – original draft, writing – review & editing, funding acquisition. Jonathan A. Arnesen: methodology, investigation, validation, formal analysis, visualization, writing – original draft, writing – review & editing. Belén Adiego-Pérez: Investigation, writing – review & editing. Daniela Rago: methodology, investigation, data curation, visualization. Mette Kristensen: methodology, investigation, data curation, writing – review & editing. Andreas K. Klitgaard: methodology, investigation, data curation, visualization, writing – review & editing. Esben H. Hansen: resources, methodology, writing – review & editing. Jørgen Hansen: resources, writing – review & editing. Irina Borodina: Conceptualization, methodology, supervision, writing – original draft, writing – review & editing, supervision, funding acquisition.No conflicts of interest.


T, described as Kaustia mangrovi gen. nov. sp. nov. from the sediments of a mangrove forest on the coast of the Red Sea in Saudi Arabia. This isolate is a moderately halophilic, aerobic/facultatively anaerobic Gram-stain-negative bacterium showing optimum growth at between 30 and 40 °C, at a pH of 8.5 and with 3–5 % NaCl. The genome of R1DC25T comprises a circular chromosome that is 4 630 536 bp in length, with a DNA G+C content of 67.3 mol%. Phylogenetic analyses based on the 16S rRNA gene sequence and whole-genome multilocus sequence analysis of 120 concatenated single-copy genes revealed that R1DC25T represents a distinct lineage within the family Parvibaculaceae in the order Rhizobiales within the class Alphaproteobacteria. R1DC25T showing 95.8, 95.3 and 94.5 % 16S rRNA gene sequence identity with Rhodoligotrophos appendicifer, Rhodoligotrophos jinshengii and Rhodoligotrophos defluvii, respectively. The predominant quinone was Q-10, and the polar lipids were phosphatidylglycerol, phosphatidylcholine, diphosphatidylglycerol, as well as several distinct aminolipids and lipids. The predominant cellular fatty acids were C19 : 0 cyclo ω8c, a combination of C18 : 1ω7c and/or C18 : 1ω6c and C16 : 0. On the basis of the differences in the phenotypic, physiological and biochemical characteristics from its known relatives and the results of our phylogenetic analyses, R1DC25T  is proposed to represent a novel species in a novel genus, and we propose the name Kaustia mangrovi gen. nov., sp. nov. .We isolated a novel strain, R1DC25 Bacillus, Halobacillus, Microbacterium, Novosphingobium, Paracoccus, Streptomyces, Thalassotalea and Vibrio  (Table S2). These results indicate the existence of a possible beneficial relationship between R1DC25T and mangrove plants, which is mediated via a phytohormone homeostasis mechanism that positively affects mangroves, particularly during stressful conditions, such as drought and salinity, as previously observed in other natural and induced stress conditions  at the Imaging Core Lab at KAUST. Motility was determined using semisolid 0.3 % MB agar. To determine the temperature range for growth, the strain was grown in 50 ml MB and incubated at temperatures of 10, 20, 30, 37, 40 and 50 °C (T was tested using oxidase strips (Sigma-Aldrich), and catalase activity was determined by bubble formation in 3 % (v/v) hydrogen peroxide solution .19 : 0cyclo ω8c, a combination of C18 : 1ω7c and/or C18 : 1ω6c and C16 : 0. Ubiquinone Q-10 (100%) is the major respiratory quinone. The polar lipid composition is dominated by phosphatidylglycerol, phosphatidylcholine and diphosphatidylglycerol, as well as several distinct aminolipids and lipids.The bacterial cells are Gram-stain-negative, aerobic/facultatively anaerobic, moderately halophilic, mesophilic, non-motile, non-sporulating, catalase-negative, oxidase-positive and nitrate-reduction-positive. The cellular fatty acids included significant amounts (>5%) of CKaustia mangrovi.The type species is Kaustia mangrovi (man.gro′vi. N.L. gen. n. mangrovi of a mangrove).l-arabinose, d-arabinose, d-glucosamine, d-saccharinic acid, dihydroxy acetone, l-alaninamide, l-alanine, l-asparagine, l-glutamic acid, l-glutamine, l-ornithine, l-proline, mucic acid, oxalomalic acid, pyruvic acid, 5-keto-d-gluconic acid and pyruvate.Colonies are circular and creamy in colour and measure 0.8 µm in diameter. Growth occurs at 2–14 % NaCl , 15–40 °C  and a pH of 6.5–10 . Can grow in the presence of several osmolytes and ions at different concentrations. Cells are positive for the production of indole and ammonia from tryptophan, but are negative for amylase, protease, lipase and cellulase activity. The following substrates are used: T (=KCTC 72348T=JCM 33619T=NCCB 100699T) isolated from the mangrove sediments on the coast of the Red Sea in KAUST . The genome of the type strain has a size of 4.63 Mb and a DNA G+C content of 67.3 mol%. The GenBank accession number for the 16S rRNA gene sequence of strain R1DC9T extracted from the genome DNA sequence is MT146883. The whole-genome shotgun sequence project was deposited at DDBJ/EMBL/GenBank under the following accession identification: CP028923.1.The type strain is R1DC25Click here for additional data file.


The duration of antibodies against SARS-CoV-2 in Covid-19 patients remains uncertain. Longitudinal serological studies are needed to prevent disease and transmission of the virus.In 2020, 414 blood samples were tested, obtained from 157 confirmed Covid-19 patients, in a prospective cohort study in Shanghai.The seropositive rate of IgM peaked at 40.5% (17/42) within 1 month after illness onset and then declined. The seropositive rate of IgG was 90.6% (58/64) after 2 months, remained above 85% from 2 to 9 months and was 90.9% (40/44) after 9 months. Generalized estimating equations models suggested that IgM (P < 0.001) but not IgG significantly decreased over time. Age ≥ 40 years , and cigarette smoking  were associated with IgG, and age ≥ 40 years  was associated with IgM. After seroconversion, over 90% and 75.1% of subjects were estimated to remain IgG-positive 220 and 254 days, respectively. Of 1420 self-reported symptoms questionnaires, only 5% reported symptoms 9 months after onset.In patients with a history of natural infection, anti-SARS-CoV-2 IgG is long-lived, being present for at least 9 months after illness onset. The long duration of natural immunity can mitigate and eliminate Covid-19 and the ongoing pandemic. Coronavirus disease 2019 (Covid-19), caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in December 2019 and has since progressed rapidly worldwide , 4. As oLimited pre-existing immunity to SARS-CoV-2 is considered responsible for the explosive increase in the number of cases . VaccinePreliminary studies have yielded inconclusive results about the duration of antiviral antibodies and the ability of these antibodies to protect against SARS-Cov-2 , with thThis prospective cohort study of patients in Shanghai discharged after having confirmed Covid-19 was started in March 2020. Patients were diagnosed, treated and discharged according to the Diagnosis and Treatment Guidelines for Novel Coronavirus Pneumonia issued by the National Health Commission of the People's Republic of China . A suspeThis study included all patients in Shanghai confirmed as having Covid-19 on December 10. Patients were excluded if they: (1) died before discharge, (2) had not been discharged or were discharged within 7 days, (3) were unable to be contacted, (4) were lost to follow-up, or (5) refused participation or lived outside Shanghai.Patients were contacted by telephone within 7 days after hospitalization discharge by trained healthcare workers of the corresponding community health centers. Participants who agreed to participate were interviewed within 1 month and at 1, 2, 3, 6, and 9 months after onset of illness. Patients who agreed were also followed-up at 4–5 and at 7–8 months. Follow-up consisted of sample collection, as well as a face-to-face interview, including administration of a structured questionnaire, at the outpatient clinic of each community health center or hospital. When face-to-face interviews could not be performed, questionnaires were administered over the telephone.+ T cells, CD8+ T cells and B cells) with fluorochrome-conjugated antibodies (BD Biosciences).Blood samples were collected in clinical laboratories of each outpatient clinic, stored at 4 °C and sent to Shanghai Municipal CDC within 3 days. All tests of blood samples were performed in the laboratory of Shanghai Municipal CDC. IgM and IgG antibodies against SARS-Cov-2 were assayed using colloidal gold immunoassays kits  –18, whicThe primary outcomes were IgM and IgG antibodies against SARS-CoV-2 in blood samples, as determined by to Prevention and Control Guidelines for Novel Coronavirus Pneumonia  issued b2), and overweight or obese (BMI ≥ 24.0 kg/m2) subjects. Potential factors incorporated into generalized estimating equations models included age group, gender, cigarette smoking (yes or no), alcohol drinking (yes or no), BMI group, abroad imported case (yes or no), involvement in a cluster (yes or no), duration from onset to admission (days), duration from admission to discharge (days), comorbidities (yes or no), clinical intensity , any symptoms (yes or no), fever (yes or no), pneumonia (yes or no), cough (yes or no), fatigue (yes or no), and first blood test . The clinical severity of a patient was divided into mild, normal, and severe according to clinical manifestations  days. During the follow-up period, the total seroconversion rates were 40.76% (64/157) for IgM and 88.54% (139/157) for IgG. The difference of seroconversion rates for IgM among 4 age groups was significant (P = 0.011) Leukomonocyte cell percentage during the acute phase was significantly lower (P = 0.027), while neutrophil percentage was significantly higher (P = 0.034), in patients positive than negative for antiviral IgM.Median [IQR] age was significantly higher in patients positive than negative for antiviral IgM . The percentage of cigarette smokers was significantly lower , and the percentage having fever during the acute phase significantly higher , among patients positive than negative for antiviral IgG. White blood cell (P = 0.024) and leukomonocyte cell (P = 0.038) counts during the acute phase were significantly lower in IgG-positive than in IgG-negative participants. Other characteristics, including gender, BMI, alcohol drinking, comorbidities, clinical intensity, and symptoms other than fever, did not differ significantly in patients positive and negative for antiviral antibody.Median age was also significantly higher in patients positive than negative for antiviral IgG  within 1 month after illness onset, declining to 35.1% (20/57), 25.5% (14/55), 11.5% (6/52), and 4.55% (2/44) at 1, 3, 6 and 9 months, respectively  within 1 month to 90.6% (58/64) after 2 months. This rate remained above 85% from 2 to 9 months, being 85.5% (47/55), 88.5% (46/52) and 90.9% (40/44) after 3, 6, and 9 months, respectively , cigarette smoking , fever , fatigue , white blood cell count , and leukomonocyte cell count  were significantly associated with antiviral IgG. In contrast, age ≥ 40 years  was the only factor associated with antiviral IgG on multivariate GEE analysis, including duration since onset and the above significant factors potential factors. GEE model analyses found that there was no tendency for IgG to decrease over time for up to 9 months after disease onset , found that age ≥ 40 years  was the only factor significantly associated with antiviral IgM. GEE model analyses found that IgM decreased significantly over time (P < 0.001).GEE model analyses with logit link of antiviral IgM positivity including 2 covariates , found that age ≥ 40 years  and fever  were significantly associated with IgG seroconversion time. Similarly, abroad imported case , involvement in a cluster , any symptom during the acute phase of Covid-19 , and pneumonia  were significantly associated with IgM seroconversion time. In contrast, age ≥ 40 years  was significantly associated with conversion from antiviral IgM positive to negative. No factor, however, was significantly associated with IgG duration in univariate proportional hazard model analyses  6.331, 95% CI 1.56–26.63, P = 0.0191), CD8+ T cell counts  and CD4+/CD8+ ratio  decreased significantly over time, whereas CD4+ T cell and B cell counts did not. Considering the temporal change of peripheral lymphocytes in GEE models, antiviral IgM was significantly associated with T cell , CD4+ T cell , CD8+ T cell , and B cell  counts, whereas antiviral IgG was not significantly associated with any of these subsets of peripheral lymphocytes. Cigarette smoking was significantly associated with T cell  and CD4+ T cell  counts. Age ≥ 40 years was significantly associated with CD8+ T cell  and B cell  counts and with CD4+/CD8+ T cell ratios . Time from onset to admission (days) was significantly associated with CD8+ T cell counts , and CD4+/CD8+ T cell ratio was significantly associated with male sex .GEE model analyses suggested that T cell counts .A total of 1420 questionnaires were collected from the 168 face-to-face and 259 telephone participants over the 9 month follow-up period. Among these 1420 questionnaire interviews, 20 (1.4%), 18 (1.3%), 15 (1.1%), 11 (0.8%), 9 (0.6%), 9 (0.6%), 8 (0.6%), 6 (0.4%), 6 (0.4%), 5 (0.4%), 5 (0.4%), 4 (0.3%) and 71 (5%) reported fatigue, cough, shortness of breath, chest pain, fever, sputum production, joint pain, nasal congestion, runny nose, headache, muscle soreness, throat soreness, and at least one of the above symptoms, respectively. Self-reported shortness of breath was associated with clinical intensity, with all 15 patients who reported shortness of breath having normal clinical intensity  for 2–9 months after disease onset and was 90.9% after 9 months. In addition, these IgG antibodies were present in 90.1% and 75.1% of participants 220 and 254 days, respectively, after seroconversion. These results showed that humoral immunity against SARS-CoV-2 was long-lived, with no decrease in IgG against SARS-CoV-2 for at least for 9 months after illness onset, in contrast to studies reporting a rapid reduction in antiviral antibodies immunity . Other s5% for 2–SARS-CoV-2 has been shown to induce a classic immune response pattern to viral infection , with anThe present study found that the seropositivity rate was higher in patients aged ≥ 40 than < 40 years, confirming that anti-SARS-CoV-2 antibody levels are higher in older people , 34–36. + T cell counts decreased 9 months after disease onset, whereas CD4+ T cell and B cell counts did not. The observed trend of peripheral lymphocytes was consistent with that of SARS-CoV specific lymphocytes at 6–8 months, with these lymphocytes, along with antibodies, being responsible for immunological memory [The dynamics of peripheral lymphocytes have been described in patients recovering from Covid-19. T cell and CD8l memory . Our finl memory , 41, 42.Of the patients interviewed 9 months after infection, only 5% reported symptoms. In contrast, 76% of patients reported at least one symptom 6 months after disease onset . This diColloidal gold immunoassays were used in the present study to assay for IgM and IgG antibodies against SARS-CoV-2. Although this is a point-of-care test applying lateral flow immunochromatographic methods, it has been recommended in research settings by the WHO . AdditioThis study had several limitations. First, the cohort population consisted only of participants with mild or normal clinical intensity of Covid-19, as few patients in Shanghai had severe or critical clinical intensity. Second, patients discharged within 7 days were excluded in this study, as the first follow-up visit began 7 days after the patient was discharge. This may bias our results when the assessment of short-term immune responses. And this study was more concerned with the long-term immune response. Third, antibodies against SARS-CoV-2 were evaluated qualitatively, not by assessing quantitative antibody titers. This may reduce the accuracy of temporal trends in antibody responses. Fourth, the number of participants was limited and the follow-up compliance was moderate, with 44–46 samples collected at each follow-up time from 157 participants.In conclusion, IgG antibody against SARS-CoV-2 in patients with a history of natural infection was long-lived, being detectable for at least 9 months after illness onset. In contrast, antiviral IgM antibody gradually declined after 1 month. Immune responses tended to be weaker in younger than in older patients and in smokers than in non-smokers. The long duration of natural immunity may have a positive impact on mitigation and elimination of the ongoing pandemic.


Saccharomyces cerevisiae var. boulardii is a representative probiotic yeast that has been widely used in the food and pharmaceutical industries. However, S. boulardii has not been studied as a microbial cell factory for producing useful substances. Agarose, a major component of red macroalgae, can be depolymerized into neoagarooligosaccharides (NAOSs) by an endo-type β-agarase. NAOSs, including neoagarotetraose (NeoDP4), are known to be health-benefiting substances owing to their prebiotic effect. Thus, NAOS production in the gut is required. In this study, the probiotic yeast S. boulardii was engineered to produce NAOSs by expressing an endo-type β-agarase, BpGH16A, derived from a human gut bacterium Bacteroides plebeius.S. boulardii for protein (BpGH16A) secretion for the first time. The SED1 signal peptide derived from Saccharomyces cerevisiae was selected as optimal for extracellular production of NeoDP4 from agarose. Expression of BpGH16A was performed in two ways using the plasmid vector system and the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system. The production of NeoDP4 by engineered S. boulardii was verified and quantified. NeoDP4 was produced by S. boulardii engineered using the plasmid vector system and CRISPR-Cas9 at 1.86 and 0.80 g/L in a 72-h fermentation, respectively.In total, four different signal peptides were compared in S. boulardii. Our results suggest that S. boulardii can be considered a microbial cell factory to produce health-beneficial substances in the human gut.This is the first report on NAOS production using the probiotic yeast The online version contains supplementary material available at 10.1186/s12934-021-01644-w. Saccharomyces cerevisiae, have been approved for human use and have been developed to produce therapeutic proteins [Saccharomyces cerevisiae var. boulardii, could be promising hosts as expression systems.The gut microbiota and intestinal immunity have been found to have a major impact on human health , 2. Micrproteins , 4. Thusproteins , based oS. boulardii, this yeast was washed out in the gut within 3–5 days after discontinuing oral administration [S. boulardii is considered a promising host to use as an intestinal microbial cell factory [Lactic acid bacteria or intestinal bacteria can be used as hosts for microbial cell factories, but there may be some constraints. First, from a pharmacokinetic point of view, it may be necessary to control the inhabiting population and residence time in the gut . In the stration , 8. Howestration . Therefostration . Taken t factory , 11.S. boulardii, originally isolated from lychee and mangosteen, is a generally recognized as safe (GRAS) yeast [S. boulardii is known to survive in the human gastrointestinal tract owing to its high tolerance to low pH and high temperatures [S. boulardii is the only probiotic yeast found to be effective in double-blind studies [S. boulardii and its use as a potential oral vaccine delivery vehicle were studied in mouse models [S. boulardii [S. boulardii to produce bioactive substances with prebiotic effects with the ultimate goal of developing a synbiotic system for humans.S) yeast , 13. S. eratures . Additio studies , 15. Pree models , 17. Howoulardii . In thisBifidobacterium, which is considered as a beneficial probiotic microorganism having therapeutic benefits and is one of the most commonly used probiotics in humans [BpGH16A originating from human gut bacterium Bacteroides plebeius, has been reported [B. plebeius was isolated from human gut microbes that can be considered relatively safe, BpGH16A was chosen to be expressed in S. boulardii to enzymatically produce NAOSs, primarily NeoDP4, from agarose.When non-digestible diets reach the large intestine, they are utilized by the gut microbiota. Subsequently, non-digestible diets change the intestinal microflora and affect the overall health of the host , 20. As n humans , signifin humans . NeoDP4 reported , 33. As BpGH16A in the probiotic yeast S. boulardii using CRISPR-Cas9, and the production of prebiotic NeoDP4 from agarose by the engineered S. boulardii was verified and optimized  containing 100 µg/mL ampicillin  at 37 °C and 200 rpm. S. boulardii ATCC MYA-796 was used as the parental strain for producing NeoDP4. Yeast strains were grown at 37 °C on yeast synthetic complete (YSC) medium, which contained a 6.7 g/L yeast nitrogen base without amino acids , 20 g/L glucose, and 1.92 g/L yeast synthetic drop-out medium supplements without uracil (Sigma-Aldrich), or YSC medium, which contained a 6.7 g/L yeast nitrogen base  and 20 g/L glucose. For CRISPR-Cas9-based genome editing experiments, 100 µg/mL nourseothricin  and 500 µg/mL geneticin  were added to the medium if required for selection of the transformants.HIS3, TRP1, and URA3 was created first. Based on the SB-TU strain [TRP1 and URA3 were inactivated, HIS3 was further inactivated. The gene fragment of HIS3 [HIS3  genomic DNA using PCR with different primer pairs depending on the type of signal peptide , α-mating factor signal peptide (α-MF) from S. cerevisiae, Sta1 signal peptide (STA1) from Saccharomyces diastaticus, and Sed1 signal peptide (SED1) from S. cerevisiae, were used [BpGH16A nor signal peptide, and only pRS426GPD vector, and SB-HTU_W harboring the BpGH16A gene but no signal peptide were prepared.Plasmids that were used to screen an optimal signal peptide for secretion of ere used , 37. AddBpGH16A gene into the genome of S. boulardii for the stable expression of BpGH16A, a guide RNA plasmid p42K_CS5 was constructed  was used to prevent congealing of agarose. First, strains SB-HTU_16A_C, SB-HTU_16A_A, SB-HTU_16A_S, and SB-HTU_16A_D were grown in YSC medium at 37 °C and 200 rpm. Pre-cultured cells were centrifuged at 10,170 × g for 10 min and washed twice using sterilized distilled water. The harvested cells were inoculated into 20 mL of YSC medium containing 20 g/L glucose and 2.5 g/L agarose in 50 mM potassium hydrogen phthalate (KHP) buffer (pH 5.5). The initial cell density was adjusted to an optical density at 600 nm (OD600) of 1.0. As a control, strains SB-HTU_16A_E and SB-HTU_16A_W were fermented under the same conditions.To produce NAOSs in the engineered S. boulardii SB_16A_D engineered by CRISPR-Cas9, fermentation was performed in YSC medium. Pre-cultured cells in YSC medium were inoculated in 20 mL of YSC medium containing 20 g/L glucose and 2.5 g/L agarose in 50 mM KHP buffer (pH 5.5). The initial cell density was adjusted to OD600 = 1.0. Wild-type S. boulardii ATCC MYA-796 was used as the control strain. Fermentation experiments were performed in triplicates.To examine NAOSs production by 600 using a UV-visible spectrophotometer . To analyze and quantify the reaction products of agarose by the engineered S. boulardii, including NeoDP4, glucose, acetic acid, and ethanol, high-performance liquid chromatography (HPLC) analysis was performed. The HPLC system  was equipped with a refractive index (RI) detector (Agilent Technologies) using an Aminex HPX-87H column (Bio-Rad). The column and RI detector temperatures were set to 65 and 55 °C, respectively, and the column was eluted with 0.005 M H2SO4 at a flow rate of 0.5 mL/min.Cell growth was monitored by measuring ODg for 15 min at 4 °C, a 1-µL aliquot from each supernatant was loaded onto silica gel 60 plates . After drying the TLC plates, they were visualized with 10% (v/v) H2SO4 in ethanol and 0.2% (w/v) naphthoresorcinol in ethanol, as previously described [To identify the hydrolyzed products of agarose during fermentation, thin-layer chromatography (TLC) analysis was performed. During fermentation, 1 mL of cell culture containing the fermentation products was obtained for each time point . For accurate measurements, the obtained cell culture was boiled to terminate the possible enzymatic reaction. After centrifugation at 16,609×escribed .BpGH16A, by yeast S. boulardii, was tested first. To confirm that BpGH16A is functionally expressed and secreted by S. boulardii, SB-HTU_16A_C containing CL, which has been previously proven to work in S. boulardii, was used [To produce NeoDP4 by engineered yeast, β-agarase secretion from yeast was necessary for agarose degradation. Therefore, the expression and secretion of an endo-type β-agarase, was used .BpGH16A was functionally expressed, secreted from S. boulardii, and produced NeoDP4 by hydrolyzing agarose.In the HPLC analysis of SB-HTU_16A_C fermentation products, a significant peak at an approximate retention time of 7.6 min, corresponding to NeoDP4, was detected in the sample Fig. . In contBpGH16A was confirmed to be functionally expressed and secreted by S. boulardii, the next step was to identify the optimal signal peptide to further increase NeoDP4 production. Four different signal peptides, CL, α-MF, STA1, and SED1, were tested. Each signal peptide was individually pre-fixed to the BpGH16A sequences and introduced into SB-HTU to identify the signal peptide that produces more NeoDP4. Production of NeoDP4 by the engineered yeasts was verified using TLC analysis at 72 h  Diagram for the construction of engineered S. boulardii expressing BpGH16A using the CRISPR-Cas9 system. (B) Yeast colony PCR for confirmation of the genomic integration of each mutant.