Hematopoietic stem and progenitor cells (HSPCs) give rise to the blood system and maintain hematopoiesis throughout the human lifespan. Here, we report a transcriptional census of human bone-marrow-derived HSPCs from the neonate, infant, child, adult, and aging stages, showing two subpopulations of multipotent progenitors separated by CD52 expression. From birth to the adult stage, stem and multipotent progenitors shared similar transcriptional alterations, and erythroid potential was enhanced after the infant stage. By integrating transcriptome, chromatin accessibility, and functional data, we further showed that aging hematopoietic stem cells (HSCs) exhibited a bias toward megakaryocytic differentiation. Finally, in comparison with the HSCs from the cord blood, neonate bone-marrow-derived HSCs were more quiescent and had higher long-term regeneration capability and durable self-renewal. Taken together, this work provides an integral transcriptome landscape of HSPCs and identifies their dynamics in post-natal steady-state hemopoiesis, thereby helping explore hematopoiesis in development and diseases.

Abstract Artabotrys hexapetalus is used as traditional Chinese medicine for the treatment of malaria. Its antimalarial activity has been shown to be the result of the sesquiterpene designated as yingzhaosu A (YZSA), which mainly occurs in the roots of A. hexapetalus. Despite its medicinal significance, the genetic basis and molecular mechanisms underlying the biosynthesis of its active terpenoids remain unclear. In this study, a high-quality chromosome-scale genome of A. hexapetalus was first reported. In total, 915.15 Mb of genome sequences were assembled, of which 911.58 Mb was sorted into 8 chromosomes. Phylogenetic analysis indicated that the divergence between the genera Artabotrys and Annona happened at approximately 45.77 million years ago (Mya) and the Annonaceae family diverged from other Magnolia species about 103.58 Mya. An ancient whole genome duplication (WGD) event was discovered in the A. hexapetalus genome, earlier than the divergence between Annonaceae and Magnoliaceae families. It correlates with a significant expansion of gene families related to secondary metabolism, especially the terpene synthase (TPS) and cytochrome P450 (P450) families. The analysis of TPS, P450, and 2-oxoglutarate/Fe(II)-dependent dioxygenase (2-ODD) gene families provides a foundation for future functional studies. Additionally, three putative terpene biosynthetic gene clusters were identified within the genome. Our work provides a foundation for elucidating the complete biosynthetic pathway of terpenoids in A. hexapetalus in the future.

7-β-xylosyl-10-deacetyltaxol (XDT) is much more abundant than the anticancer drug Taxol in Taxus species and is usually regarded as the by-product of Taxol. It could be enzymatically transformed into 10-deacetyltaxol (DT), and the latter could be further converted into Taxol. The enzyme 10-deacetylbaccatin III-10-β-O-acetyltransferase (DBAT) can acetylate the non-natural substrate DT into Taxol, but the conversion efficiency was extremely low. Herein, we globally redesigned DBATcus from Taxus cuspidata to improve its efficiency in DT acetylation through combinatorial protein engineering strategies including virtual saturation mutagenesis, in silico screening, DNA shuffling, and iterative combinatorial mutagenesis. Several more active DBAT mutants against DT were obtained, among which the ICM9-6 exhibited 16.4 times higher activity than DBATcus. The transient expression system of Nicotiana benthamiana was then established, and the ICM9-6 was functionally expressed in the system, with yield of 8.2 μg g-1 FW (129.3 μg g-1 DW) Taxol when the system was fed with DT. Specifically, the fungal glycoside hydrolase LXYL-P1-2 that was responsible for converting XDT into DT was also functionally expressed in the system, and upon feeding XDT, the co-expression of LXYL-P1-2 and ICM9-6 yielded 3.6 μg g-1 FW (55.4 μg g-1 DW) Taxol. These results represent the highest reported Taxol productivity in the tobacco system to date and lay a foundation for the construction of the stable transgenic cell lines of tobacco and more efficiently converting DT or XDT into Taxol for the large-scale pharmaceutical manufacturing.

The unnatural ginsenoside 3β-O-Glc-DM exhibits potent anti-colon cancer activity. An engineered yeast strain expressing the dammarenediol-II synthase (DS) and glycosyltransferase (PgUGT74AE2) genes from Panax ginseng was previously constructed to produce 3β-O-Glc-DM. However, the titer of 3β-O-Glc-DM was insufficient for industrial-scale production. To overcome this limitation, we employed a semi-rational design approach to engineer PgUGT74AE2, integrating enzyme optimization with fermentation enhancement to boost 3β-O-Glc-DM production. Molecular docking identified 12 key residues near the active pocket as mutation hotspots. Alanine scanning at these positions revealed that substitutions at K276 and H185 improved catalytic activity. Subsequent semi-saturation mutagenesis specifically at K276 yielded mutants K276A and K276V, both exhibiting a 3.4-fold increase in catalytic activity relative to wild-type PgUGT74AE2. This improved activity resulted from reduced steric hindrance and enhanced hydrophobic interactions. The chassis strain Y-ΔHXK2 was optimized for enhanced 2,3-oxidosqualene production by overexpressing key upstream biosynthetic enzymes, down-regulating competitive branch pathways, and overexpressing the transcriptional activator HAC1, thereby generating the strain Y13. Subsequently, the mutant PgUGT74AE2-K276A and DS genes were integrated into Y13 via the CRISPR/Cas9 system to generate the strain Y13-A9. Further optimization of the shake-flask culture conditions increased the titer of 3β-O-Glc-DM produced by the strain to 425 mg/L. We conducted fed-batch fermentation using a feedback-controlled feeding method in a 3-L bioreactor. The titer of 3β-O-Glc-DM reached 3.4 g/L, representing a 42

Single-cell technologies have transformed our view of normal and malignant hematopoiesis, yet a framework linking lifelong homeostatic hematopoiesis to hematological diseases in blood ecosystem remains incomplete. Here, we constructed a high-resolution landscape of the entire hematopoietic system by integrating transcriptomes of ∼1 million cells from 207 healthy samples spanning five developmental stages (fetal, neonatal, childhood, adult, elderly), and identified 96 blood cell clusters, including a VNN2-marked monocyte subset in the fetus and TSHZ2-marked T cell subsets exhibiting distinct age-dependent dynamics. We uncovered a reversal in hematopoietic stem cell/multipotent progenitor (HSC/MPP) stemness around childhood and a postnatal lymphoid bias shift from reduced early B/T lineage potential to enhanced natural killer (NK) cytotoxicity, coordinately orchestrated by intracellular transcription factor activity and intercellular interactions. Projecting 116 pan-malignancy samples (9 hematological malignancies) to our refined blood cell reference, we identified the upregulation of MYC and MHC II signaling as relatively conserved features correlating with poor prognosis, while hematopoiesis-related modules exhibited subtype-specific distributions with divergent clinical implications. Importantly, we developed a prognostic framework incorporating two core signatures shared across hematological malignancies and subtype-specific features. We further revealed an antagonism between inflammatory and cytotoxic programs across malignancies. Together, we established a panoramic landscape of the human hematopoietic system and elaborated its lifelong behaviors, in which a steady-state reference enabled the delineation of pathological hallmarks across hematological malignancies.

ABSTRACT:Hematopoietic stem cells (HSCs) are heterogeneous, and the quality of HSCs-that is, transplantability-is a key determinant for posttransplant hematopoietic reconstitution. However, molecular modalities of high-potency HSCs with superior transplantability still remain poorly understood. Here, we conducted large-scale single-clone serial-transplant experiments and tracked descendant cells of 288 HSC clones to quantify their intrinsic capability for hematopoietic reconstitution. Using integrated single-cell transcriptional, immunophenotypical, and Bayesian dynamic analyses, we uncovered 3 classes of HSC clones ("Super," "Flash," and "Trickle") that had higher output in the first generation but exhibited markedly different behavior in later generations. The Super-class HSC clones comprised 4% of the HSCs and manifested persistent superior transplantability and balanced myeloid/lymphoid-lineage outputs across generations in serial transplants. The Super-class HSCs had a unique molecular signature, including low expression of CD27, that was distinct from previously known classical HSC signatures. Validation experiments indicated that CD27- HSCs had superior transplantability compared with CD27+ HSCs. Our study asserted an operational definition for Super transplantability of HSCs, defined its molecular program, and suggested new directions for enriching high-potency HSCs in grafts.

Abstract Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder originating from somatic mutations in PIGA within hematopoietic stem cells (HSCs). PNH appears to be related to aplastic anemia (AA). Immune evasion has been proposed as a key contributor to the clonal advantage of PNH cells. However, the intrinsic and extrinsic stress on HSCs, and the regulatory impact of wild-type (WT) or mutant mature blood cells on hematopoietic stem and progenitor cell (HSPC) homeostasis in PNH, remain to be fully elucidated. Methods We performed multiparameter immunophenotyping on fresh bone marrow samples from individuals with PNH, AA/PNH, AA, and healthy donors (HD). Single-cell RNA sequencing (scRNA-seq) using the 10x Genomics platform was conducted on sorted mutant HSPCs and WT/mutant mature blood cells from four classic PNH patients. Additionally, peripheral blood (PB) samples from three classic PNH patients before and after 2 months of complement inhibitor treatment were analyzed via 10x scRNA-seq. Results Immunophenotyping revealed an increased T cell proportion across patients with classic PNH, AA/PNH, and AA. Despite shared features of reduced HSPCs in all patient groups, lineage composition differed across diseases. In AA and AA/PNH, both WT and mutant hematopoietic stem cell / multipotent progenitor (HSC/MPP) subsets were decreased. In classic PNH and AA/PNH, lymphoid progenitors were markedly reduced regardless of genotype, while they remained largely preserved in AA. WT megakaryocyte-erythroid progenitors (MEPs) decreased in PNH and AA, whereas mutant MEPs increased in classic PNH, suggesting a shift towards erythro-myeloid differentiation. In vitro assays showed impaired expansion and differentiation potential of both WT and mutant HSPCs from PNH patients. Nonetheless, colony-forming cell assays showed higher erythroid/myeloid colony numbers in PNH, indicating a compensatory erythro-myeloid bias. Conversely, B/NK colony formation was significantly reduced, suggesting lymphoid impairment. scRNA-seq confirmed increased myeloid gene expression and reduced lymphoid genes in mutant MLPs. Mutant HSC/MPPs displayed diminished stemness, enhanced inflammatory signaling, and enrichment of erythro-myeloid-skewed subsets, indicating early lineage bias. WT T cells in PNH exhibited elevated expression of genes involved in activation, proliferation, and cytokine responses across multiple subsets, along with reduced co-inhibitory molecule expression in Tregs. Functional assays confirmed broad activation of WT T cells. Mutant neutrophils also showed pro-inflammatory activation. Correlation analysis of flow cytometry data revealed negative associations between T cell frequency and both WT and mutant HSC/MPP proportions, suggesting immune-mediated suppression. ScRNA-seq further showed up-regulation of pro-inflammatory ligands (e.g., TNF, IFNG) in activated T cells, implicating them in targeting mutant HSPCs. To test this hypothesis, CSA rescue experiments were conducted. CSA treatment improved HSPC viability and differentiation while suppressing T cell activation and proliferation. A small retrospective clinical analysis also demonstrated higher HSPC proportions in classic PNH patients who received CSA, supporting its role in mitigating hematopoietic exhaustion. Furthermore, scRNA-seq of PB samples before and after complement inhibition revealed down-regulation of inflammatory and cytokine pathways in myeloid cells, decreased T cell activation/proliferation scores, and restored expression of co-inhibitory markers in Tregs. These findings suggest that complement blockade may help alleviate immune activation and indirectly stabilize HSPC function. Conclusions This study reveals early lineage bias and functional exhaustion of HSPCs in classic PNH. WT T cells are aberrantly activated and may contribute to the suppression of mutant HSPCs. CSA restores HSPC number and function by dampening T cell activation, and complement inhibitors also alleviate immune activation, suggesting that immune microenvironment remodeling may represent a viable strategy to alleviate hematopoietic impairment in PNH.

Pediatric acute myeloid leukemia (pAML) is a clonal disease with recurrent genetic alterations that affect epigenetic states. However, the implications of epigenetic dysregulation in disease progression remain unclear. Here, we interrogated single-cell and clonal level chromatin accessibility of bone marrow samples from 28 pAML patients representing multiple subtypes using mtscATAC-seq, which revealed distinct differentiation hierarchies and abnormal chromatin accessibility in a subtype-specific manner. Innate immune signaling was commonly enhanced across subtypes and related to improved advantage of clonal competition and unfavorable prognosis, with further reinforcement in a relapse-associated leukemia stem cell-like population. We identified a panel of 31 innate immunity related genes to improve the risk classification of pAML patients. By comparing paired diagnosis and post-chemotherapy relapse samples, we showed that primitive cells significantly reduced MHC class II signaling, suggesting an immune evasion mechanism to facilitate their expansion at relapse. Key regulators orchestrating cell cycle dysregulation were identified to contribute to pAML relapse in drug-resistant clones. Our work establishes the single-cell chromatin accessibility landscape at clonal resolution and reveals the critical involvement of epigenetic disruption, offering insights into classification and targeted therapies of pAML patients.

BACKGROUND:Immune cells are closely associated with all processes of cardiac repair after myocardial infarction (MI), including the initiation, development, and resolution of inflammation. Spleen extramedullary hematopoiesis (EMH) serves as a crucial source of emergency mature blood cells that are generated through the self-renewal and differentiation of hematopoietic stem/progenitor cells (HSPCs). However, how EMH responds to MI and the role of EMH in cardiac repair after MI remains unclear. METHODS:To assess the role of spleen EMH in MI, a Tcf21CreER Scfflox/flox MI mouse model with inhibited EMH was constructed. GFP+ (green fluorescent protein) hematopoietic stem cells were sorted from eGFP (enhanced GFP) mouse spleen by flow cytometry and injected into Tcf21CreER Scfflox/flox mice to test the sources of local inflammatory cells during MI. Using highly specific liquid chromatography-tandem mass spectrometry and single-cell RNA sequencing, we analyzed the lipidomic profile of arachidonic acid metabolites and the transcriptomes of HSPCs in the spleen after MI. RESULTS:We found that MI enhanced EMH, as reflected by the increase in spleen weight and volume and the number of HSPCs in the spleen. The lack of EMH in Scf-deficient mice exacerbated tissue injury after MI. Analysis of the transcriptome of spleen HSPCs after MI revealed that the type 1 interferon pathway was substantially inhibited in hematopoietic stem cell /multipotent progenitor subclusters, and the absence of type 1 interferon signaling enhanced the MI-induced spleen EMH. Lipidomics analysis revealed that prostaglandin I2 (PGI2) was markedly reduced in the spleen. PGI2 suppressed MI-induced EMH through a PGI2 receptor (IP)-cyclic adenosine monophosphate-453p-SP1 cascade in spleen HSPCs. Hematopoietic cell-specific IP-deficient mice exhibited enhanced EMH and improved cardiac recovery after MI. CONCLUSIONS:Together, our findings revealed that a PGI2-IFN axis was involved in spleen EMH after MI, providing new mechanistic insights into spleen EMH after MI and offering a new therapeutic target for treating ischemic cardiac injury.

Ergopeptines or their derivatives are widely used for treating neurodegenerative and cerebrovascular diseases. The nonribosomal peptide synthetase—d-lysergyl peptide synthetase A (LPSA) determines ergopeptine formation but the detailed mechanism remains to be elucidated. Here, we characterized two LPSAs from Claviceps purpurea Cp-1 strain through heterologous expression in Aspergillus nidulans feeding with d-lysergic acid. We proved that Cp-LPSA1 catalyzed the formation of ergocornine, α-ergocryptine, and β-ergocryptine, precisely controlled by the substrate specificity of its three modules. Cp-LPSA2 was initially inactive but could be restored to catalyze α-ergosine formation. Using this platform, we validated that P1-LPSA1 and P1-LPSA2 from the reported C. purpurea P1 strain catalyzed ergotamine and α-ergocryptine formation, respectively. Typically, the non-ribosomal peptide codes implicated in every module of the LPSAs were defined and elucidated, in which certain key residues could play a switched role for substrate specificity and product interconversion. By constructing chimeric LPSAs through module assembly, the production of the desired ergopeptines was achieved. Notably, 1.46 mg/L of α-ergocryptine and 1.09 mg/L of ergotamine were produced respectively by mixed-culture of C. paspali No. 24 (fermentation supernatant) and the recombinants of A. nidulans. Our findings provide insights into the biosynthetic mechanism of ergopeptines and lay a foundation for directed ergopeptine biosynthesis.

Hypoxia imposes notable stress on organisms and even causes tissue damage; however, the cellular and molecular mechanisms underlying hypoxic adaptation and maladaptation are elusive. Here, we performed single-cell RNA sequencing to analyze hematopoietic stem and progenitor cells (HSPCs) and erythroid cells in a mouse model of high-altitude polycythemia (HAPC) mimicking long-term high-altitude hypoxia exposure. We identified a distinct erythroid-biased multipotent progenitor subset, FOShi MPP, characterized by a unique responsiveness to interferon (IFN) signaling, which expands under hypoxia conditions. This subset rapidly responds to hypoxia during re-ascent by sustaining low methylation of erythroid-priming genes, suggesting a memory function in HSPCs for faster acclimatization. Additionally, erythroid cells in HAPC mice had active metabolic and autophagic activity, as well as abundant CD47 expression that prevented the phagocytosis of erythrocytes. Finally, CD47 blockade and/or IFNα treatments alleviated erythrocytosis in HAPC mice. These approaches might constitute promising therapeutic strategies for HAPC.

Background Left ventricular diastolic dysfunction (LVDD) is a manifestation of heart failure, with both its incidence and prevalence increasing annually. Currently, no pharmacological treatments are available for LVDD, highlighting the urgent need for new therapeutic discoveries. Ginsenosides are commonly used in cardiovascular therapy. Previous research has synthesized the ginsenoside precursor molecule, 20S-O-Glc-DM (C20DM), through biosynthesis. C20DM shows greater bioavailability, eco-friendliness, and cost-effectiveness compared to traditional ginsenosides, positioning it as a promising option for treating LVDD. Purpose This study firstly documents the therapeutic activity of C20DM against LVDD and unveils its potential mechanisms of action. It provides a pharmacological basis for C20DM as a new cardiovascular therapeutic agent. Methods In this study, models of LVDD in mice and ISO-induced H9C2 cell damage were developed. Cell viability, ROS and Ca2+ levels, mitochondrial membrane potential, and proteins associated with mitochondrial biogenesis and autophagy were evaluated in the in vitro experiments. Animal experiments involved administering medication for 3 weeks to validate the therapeutic effects of C20DM and its impact on mitochondria and autophagy. Results Research has shown that C20DM is more effective than Metoprolol in treating LVDD, significantly lowering the E/A ratio, e'/a' ratio, and IVRT, and ameliorating myocardial inflammation and fibrosis. C20DM influences the activity of PGC-1α, downregulates PINK1 and Parkin, thereby enhancing mitochondrial quality control, and restoring mitochondrial oxidative respiration and membrane potential. Furthermore, C20DM reduces excessive autophagy in cardiomyocytes via the AMPK-mTOR-ULK1 pathway, diminishing cardiomyocyte hypertrophy and damage. Conclusions Overall, our research indicates that C20DM has the potential to enhance LVDD through the regulation of mitochondrial quality control and cellular autophagy, making it a promising option for heart failure therapy.

Sesquiterpene synthases (STPSs) catalyze carbocation-driven cyclization reactions that can generate structurally diverse hydrocarbons. The deprotonation-reprotonation process is widely used in STPSs to promote structural diversity, largely attributable to the distinct regio/stereoselective reprotonations. However, the molecular basis for reprotonation regioselectivity remains largely understudied. Herein, we analyzed two highly paralogous STPSs, Artabotrys hexapetalus (−)-cyperene synthase (AhCS) and ishwarane synthase (AhIS), which catalyze reactions that are distinct from the regioselective protonation of germacrene A (GA), resulting in distinct skeletons of 5/5/6 tricyclic (−)-cyperene and 6/6/5/3 tetracyclic ishwarane, respectively. Isotopic labeling experiments demonstrated that these protonations occur at C3 and C6 of GA in AhCS and AhIS, respectively. The cryo-electron microscopy-derived AhCS complex structure provided the structural basis for identifying different key active site residues that may govern their functional disparity. The structure-guided mutagenesis of these residues resulted in successful functional interconversion between AhCS and AhIS, thus targeting the three active site residues [L311-S419-C458]/[M311-V419-A458] that may act as a C3/C6 reprotonation switch for GA. These findings facilitate the rational design or directed evolution of STPSs with structurally diverse skeletons.

Hydroxylation of steroid core is critical to the synthesis of steroid drugs. Direct sp3 C–H hydroxylation is challenging through chemical catalysis, alternatively, fungal biotransformation offers a possible solution to this problem. However, mining and metabolic engineering of cytochrome P450 monooxygenases (CYPs) is usually regarded as a more eco-friendly and efficient strategy. Herein, we report the mining and identification of a new steroid CYP (CYP68BE1) from Beauveria bassiana by transcriptomics, heterologous expression, in vivo and in vitro functional characterization. The catalytic promiscuity of CYP68BE1 was explored, and CYP68BE1 showed promiscuously and catalytically versatile, which is qualified for monohydroxylation on C11α, C1α, C6β and dihydroxylation on C1β,11α and C6β,11α of six steroids, leading to the production of key steroid intermediates required in the industrial synthesis of some indispensable steroid drugs. Molecular dynamics simulations were performed, revealing the molecular basis of different binding orientations of CYP68BE1 with different substrates. The discovery of CYP68BE1 offers a promising biocatalyst for enriching the steroid structural and functional diversity, which also can be applied to biosynthesize valuable steroid drug intermediates.

Hematopoietic stem cells (HSCs) have been considered to progressively lose their self-renewal and differentiation potentials prior to the commitment to each blood lineage. However, recent studies have suggested that megakaryocyte progenitors (MkPs) are generated at the level of HSCs. In this study, we newly identified early megakaryocyte lineage-committed progenitors (MgPs) mainly in CD201-CD48- cells and CD48+ cells separated from the CD150+CD34-Kit+Sca-1+Lin- HSC population of the bone marrow in adult mice. Single-cell colony assay and single-cell transplantation showed that MgPs, unlike platelet-biased HSCs, had little repopulating potential in vivo, but formed larger megakaryocyte colonies in vitro (on average 8 megakaryocytes per colony) than did previously reported MkPs. Single-cell RNA sequencing supported that HSCs give rise to MkPs through MgPs along a Mk differentiation pathway. Single-cell reverse transcription polymerase chain reaction (RT-PCR) analysis showed that MgPs expressed Mk-related genes, but were transcriptionally heterogenous. Clonal culture of HSCs suggested that MgPs are not direct progeny of HSCs. We propose a differentiation model in which HSCs give rise to MgPs which then give rise to MkPs, supporting a classic model in which Mk-lineage commitment takes place at a late stage of differentiation.

Hematopoietic differentiation is controlled by intrinsic regulators and the extrinsic hematopoietic niche. Activating transcription factor 4 (ATF4) plays a crucial role in the function of fetal and adult hematopoietic stem cell maintenance; however, the precise function of ATF4 in the bone marrow niche and the mechanism by which ATF4 regulates adult hematopoiesis remain largely unknown. Here, we employ four cell-type-specific mouse Cre lines to achieve conditional knockout of Atf4 in Cdh5+ endothelial cells, Prx1+ bone marrow stromal cells, Osx+ osteo-progenitor cells, and Mx1+ hematopoietic cells, and uncover the role of Atf4 in niche cells and hematopoiesis. Intriguingly, depletion of Atf4 in niche cells does not affect hematopoiesis; however, Atf4-deficient hematopoietic cells exhibit erythroid differentiation defects, leading to hypoplastic anemia. Mechanistically, ATF4 mediates direct regulation of Rps19bp1 transcription, which is, in turn, involved in 40S ribosomal subunit assembly to coordinate ribosome biogenesis and promote erythropoiesis. Finally, we demonstrate that under conditions of 5-fluorouracil-induced stress, Atf4 depletion impedes the recovery of hematopoietic lineages, which requires efficient ribosome biogenesis. Taken together, our findings highlight the indispensable role of the ATF4-RPS19BP1 axis in the regulation of erythropoiesis.

Ginsenosides are major active components of Panax ginseng, which are generally glycosylated at C3-OH and/or C20-OH of protopanaxadiol (PPD) and C6-OH and/or C20-OH of protopanaxatriol. However, the glucosides of dammarenediol-II (DM), which is the direct precursor of PPD, have scarcely been separated from P. ginseng. Because different positions and numbers of the hydroxyl and glycosyl groups lead to a diversity of structure and function of the ginsenosides, it can be inferred that DM glucosides may have different pharmacological activities compared with natural ginsenosides. Herein, we first constructed the cell factory for de novo biosynthesis of 3-O-(β-D-glucopyranosyl-(1→2)-β-D-glucopyranosyl)-dammar-24-ene-3β,20S-diol (3β-O-Glc2-DM) by introducing the codon-optimized genes encoding dammarenediol-II synthase, two UDP-glycosyltransferases (UGTs) including UGT74AC1-M7 from Siraitia grosvenorii and UGTPg29 from P. ginseng in Saccharomyces cerevisiae via the CRISPR/Cas9 system. The titer of 3β-O-Glc2-DM was then increased from 18.9 to 148.0 mg/L by several metabolic engineering strategies including overexpressing the rate-limiting enzymes of triterpenoid biosynthesis, balancing carbon flux of biosynthetic pathways of triterpenoid and ergosterol, and engineering endoplasmic reticulum. Furthermore, the 3β-O-Glc2-DM titer of 766.3 mg/L was achieved through fed-batch fermentation in a 3-L bioreactor. Finally, in vitro assays demonstrated that 3β-O-Glc2-DM exhibited a protective effect on H/R-induced cardiomyocyte damage. This work provides a feasible approach for production of 3β-O-Glc2-DM as a potential cardioprotective drug candidate.

Mutation Status of Selected Splicing Factors in T-ALL patients

KEGG Pathway Enrichment Analysis of Differential Expressed Genes Between Thymus and T-ALL

KEGG Enrichment Analysis of Differential Expressed Genes Between Thymus and T-ALL