Single-cell multi-Omics(SCM-Omics)and spatial multi-Omics(SM-Omics)technologies provide state-of-the-art methods for exploring the composition and function of cell types in tissues/organs.Since its emergence in 2009,s...Single-cell multi-Omics(SCM-Omics)and spatial multi-Omics(SM-Omics)technologies provide state-of-the-art methods for exploring the composition and function of cell types in tissues/organs.Since its emergence in 2009,single-cell RNA sequencing(scRNA-seq)has yielded many groundbreaking new discoveries.The combination of this method with the emergence and development of SM-Omics techniques has been a pioneering strategy in neuroscience,developmental biology,and cancer research,especially for assessing tumor heterogeneity and T-cell infiltration.In recent years,the application of these methods in the study of metabolic diseases has also increased.The emerging SCM-Omics and SM-Omics approaches allow the molecular and spatial analysis of cells to explore regulatory states and determine cell fate,and thus provide promising tools for unraveling heterogeneous metabolic processes and making them amenable to intervention.Here,we review the evolution of SCM-Omics and SM-Omics technologies,and describe the progress in the application of SCM-Omics and SM-Omics in metabolism-related diseases,including obesity,diabetes,nonalcoholic fatty liver disease(NAFLD)and cardiovascular disease(CVD).We also conclude that the application of SCM-Omics and SM-Omics approaches can help resolve the molecular mechanisms underlying the pathogenesis of metabolic diseases in the body and facilitate therapeutic measures for metabolism-related diseases.This review concludes with an overview of the current status of this emerging field and the outlook for its future.展开更多
Capillary electrophoresis with many advantages plays an important role in pharmaceutical analysis and drug screening. This review gives an overview on the recent advances in the developments and applications of capill...Capillary electrophoresis with many advantages plays an important role in pharmaceutical analysis and drug screening. This review gives an overview on the recent advances in the developments and applications of capillary electrophoresis in the field of enzyme inhibitor screening. The period covers 2013 to 2017. Both the pre-capillary enzyme assays and in-capillary enzyme assays which include electrophoretically mediated microanalysis(EMMA) and immobilized enzyme microreactor(IMER) are summarized in this article.展开更多
A novel method for analysis of three active components curcumin, demethoxycurcumin and bisdemethoxycurcumin in Curcuma longa L. was developed by HPLC coupled with electrochemical detection. Three curcuminoids were wel...A novel method for analysis of three active components curcumin, demethoxycurcumin and bisdemethoxycurcumin in Curcuma longa L. was developed by HPLC coupled with electrochemical detection. Three curcuminoids were well separated on a C18 column and detected with high sensitivity. A mobile phase containing acetonitrile and 10 mM Na2HPO4-H3PO4 (pH 5.0) (50:50, v/v) was used. Good linearity was obtained in the range of 0.208-41.6, 0.197-39.4, and 0.227-114μM for curcumin, demethoxycurcumin and bisdemethoxycurcumin respectively. The limit of detection reached up to 10 ? 8 M, which was lower than that by UV detection. The relative standard deviations (RSDs) ranged from 1.06%to 1.88%for intra-day precision and from 4.30%to 5.79%for inter-day precision, respectively. The proposed method has been applied in real herb sample and recoveries ranging from 86.3%to 111%were obtained.展开更多
Natural long-chain alkanol and alkyl carboxylic acid were used to prepare novel hydrophobic deep eutectic solvents(HDESs).These HDESs are liquid at room temperature and have low viscosity(<12.26 mPa·s),low pol...Natural long-chain alkanol and alkyl carboxylic acid were used to prepare novel hydrophobic deep eutectic solvents(HDESs).These HDESs are liquid at room temperature and have low viscosity(<12.26 mPa·s),low polarity(lower than that of methanol,ChCl-based deep eutectic solvents and other reported HDESs),and low density(<0.928 g/mL).A simple one-pot method based on a novel HDES-water two-phase extraction system was constructed for the extraction of weak-polarity bioactive components,anthraquinones,from Rhei Radix et Rhizoma.This HDES-based new extraction method does not consume hazardous organic solvents and can obtain a total anthraquinone yield of 21.52 mg/g,which is close to that obtained by the Chinese pharmacopoeia method(21.22 mg/g)and considerably higher than those by other reported HDESs-based extraction methods(14.20-20.09 mg/g,p<0.01).The high extraction yield can be mainly attributed to the severe destruction of the RRR cell walls by the extraction system and the excellent dissolving ability of novel HDESs for anthraquinones.展开更多
A simple, sensitive and selective method of high-performance liquid chromatography (HPLC) has been successfully developed for separation of bavachinin enantiomers in Fructus Psoraleae and rat plasma. The separation ...A simple, sensitive and selective method of high-performance liquid chromatography (HPLC) has been successfully developed for separation of bavachinin enantiomers in Fructus Psoraleae and rat plasma. The separation and detection conditions of HPLC were optimized. Chiral bavachinin were separated with the mobile phase of methanol and water (70:30, v/v) at a flow rate of 1.0 mL/min. The linear ranges were in the range of 20-1000 μg/mL. The detection limits were tested as 4 ng/mL and 6 ng/mL for (+)-bavachinin and (-)-bavachinin, respectively. The method has been applied to analyze chiral bavachinin in rat plasma. HPLC-MS method was used to test the accuracy.展开更多
The cyanobacterium Arthrospira platensis,spirulina,is a source of pigments such as phycobiliprotein and phycocyanin.Phycocyanin is used in the food,cosmetics,and pharmaceutical industries because of its antioxidant,an...The cyanobacterium Arthrospira platensis,spirulina,is a source of pigments such as phycobiliprotein and phycocyanin.Phycocyanin is used in the food,cosmetics,and pharmaceutical industries because of its antioxidant,anti-inflammatory,and anticancer properties.The different steps involved in extraction and purification of this protein can alter the final properties.In this review,the stability of phycocyanin(pH,temperature,and light)is discussed,considering the physicochemical parameters of kinetic modeling.The optimal working pH range for phycocyanin is between 5.5 and 6.0 and it remains stable up to 45℃;however,exposure to relatively high temperatures or acidic pH decreases its half-life and increases the degradation kinetic constant.Phycobiliproteins are sensitive to light;preservatives such as mono-and disaccharides,citric acid,or sodium chloride appear to be effective stabilizing agents.Encapsulation within nano-or micro-structured materials such as nanofibers,microparticles,or nanoparticles,can also preserve or enhance its stability.展开更多
Covalent organic frameworks(COFs)are a novel type of crystalline porous organic polymer materials recently developed.It has several advantages in chromatographic separation field,such as high thermal stability,porosit...Covalent organic frameworks(COFs)are a novel type of crystalline porous organic polymer materials recently developed.It has several advantages in chromatographic separation field,such as high thermal stability,porosity,structural regularity,and large specific surface area.Here,a novel spherical COF 1,3,5-tris(4-aminophenyl)benzene(TAPB)and 2,5-bis(2-propyn-1-yloxy)-1,4-benzenedicarboxaldehyde(BPTA)was developed as an electrochromatographic stationary phase for capillary electrochromatography separation.The COF TAPB-BPTA modified capillary column was fabricated via a facile in situ growth method at room temperature.The characterization results of scanning electron microscopy(SEM),Fourier transform infrared(FT-IR)spectroscopy,and X-ray diffraction(XRD)confirmed that COF TAPB-BPTA were successfully modified onto the capillary inner surface.The electrochromatography separation performance of the COF TAPB-BPTA modified capillary was investigated.The prepared column demonstrated outstanding separation performance toward alkylbenzenes,phenols,and chlorobenzenes compounds.Furthermore,the baseline separations of non-steroidal anti-inflammatory drugs(NSAIDs)and parabens with good efficiency and high resolution were achieved.Also,the prepared column possessed satisfactory precision of the intra-day runs(n=5),inter-day runs(n=3),and parallel columns(n=3),and the relative standard deviations(RSDs)of the retention times of tested alkylbenzenes were all less than 2.58%.Thus,this new COF-based stationary phase shows tremendous application potential in chromatographic separation field.展开更多
Here,a styrene-based polymer monolithic column poly(VBS-co-TAT-co-AHM)with reversed-phase/hydrophilic interaction liquid chromatography(RPLC/HILIC)bifunctional separation mode was success-fully prepared for capillary ...Here,a styrene-based polymer monolithic column poly(VBS-co-TAT-co-AHM)with reversed-phase/hydrophilic interaction liquid chromatography(RPLC/HILIC)bifunctional separation mode was success-fully prepared for capillary electrochromatography by the in situ polymerization of sodium p-styrene sulfonate(VBS)with cross-linkers 3-(acryloyloxy)-2-hydroxypropyl methacrylate(AHM)and 1,3,5-triacryloylhexahydro-1,3,5-triazine(TAT).The preparation conditions of the monolith were optimized.The morphology and formation of the poly(VBS-co-TAT-co-AHM)monolith were confirmed by scanning electron microscopy(SEM)and Fourier transform infrared spectroscopy(FT-IR).The separation perfor-mances of the monolith were evaluated systematically.It should be noted that the incorporation of VBS functional monomer can provideπ-πinteractions,hydrophilic interactions,and ion-exchange in-teractions.Hence,the prepared poly(VBS-co-TAT-co-AHM)monolith can achieve efficient separation of thiourea compounds,benzene series,phenol compounds,aniline compounds and sulfonamides in RPLC or HILIC separation mode.The largest theoretical plate number for N,N0-dimethylthiourea reached 1.7×10^(5)plates/m.In addition,the poly(VBS-co-TAT-co-AHM)monolithic column showed excellent reproducibility and stability.This novel monolithic column has great application value and potential in capillary electrochromatography(CEC).展开更多
The central nervous system is susceptible to the modulation of various neurophysiological processes by the cytochrome P450 enzyme(CYP),which plays a crucial role in the metabolism of neurosteroids.The antiepileptic dr...The central nervous system is susceptible to the modulation of various neurophysiological processes by the cytochrome P450 enzyme(CYP),which plays a crucial role in the metabolism of neurosteroids.The antiepileptic drug phenytoin(PHT)has been observed to induce neuronal side effects in patients,which could be attributed to its induction of CYP expression and testosterone(TES)metabolism in the hippocampus.While pregnane X receptor(PXR)is widely known for its regulatory function of CYPs in the liver,we have discovered that the treatment of mice with pregnenolone 16α-carbonitrile(PCN),a PXR agonist,has differential effects on CYP expression in the liver and hippocampus.Specifically,the PCN treatment resulted in the induction of cytochrome P450,family 3,subfamily a,polypeptide 11(CYP3A11),and CYP2B10 expression in the liver,while suppressing their expression in the hippocampus.Functionally,the PCN treatment protected mice from PHT-induced hippocampal nerve injury,which was accompanied by the inhibition of TES metabolism in the hippocampus.Mechanistically,we found that the inhibition of hippocampal CYP expression and attenuation of PHT-induced neurotoxicity by PCN were glucocorticoid receptor dependent,rather than PXR independent,as demonstrated by genetic and pharmacological models.In conclusion,our study provides evidence that PCN can negatively regulate hippocampal CYP expression and attenuate PHT-induced hippocampal neurotoxicity independently of PXR.Our findings suggest that glucocorticoids may be a potential therapeutic strategy for managing the neuronal side effects of PHT.展开更多
Sulfation is a crucial and prevalent conjugation reaction involved in cellular processes and mammalian physiology.3’-Phosphoadenosine 5’-phosphosulfate(PAPS)synthase 2(PAPSS2)is the primary enzyme to generate the un...Sulfation is a crucial and prevalent conjugation reaction involved in cellular processes and mammalian physiology.3’-Phosphoadenosine 5’-phosphosulfate(PAPS)synthase 2(PAPSS2)is the primary enzyme to generate the universal sulfonate donor PAPS.The involvement of PAPSS2-mediated sulfation in adenomatous polyposis coli(APC)mutation-promoted colonic carcinogenesis has not been reported.Here,we showed that the expression of PAPSS2 was decreased in human colon tumors along with cancer stages,and the lower expression of PAPSS2 was correlated with poor prognosis in advanced colon cancer.Gut epithelial-specific heterozygous Apc deficient and Papss2-knockout(Apc^(Δgut-Het)Papss2^(Δgut))mice were created,and the phenotypes were compared to the spontaneous intestinal tumorigenesis of Apc^(Δgut-Het)mice.Apc^(Δgut-Het)Papss2^(Δgutmice) were more sensitive to gut tumorigenesis,which was mechanistically accounted for by the activation of Wnt/β-catenin signaling pathway due to the suppression of chondroitin sulfation and inhibition of the farnesoid X receptor(FXR)-transducin-like enhancer of split 3(TLE3)gene regulatory axis.Chondroitin sulfate supplementation in Apc^(Δgut-Het)Papss2^(Δgutmice) alleviated intestinal tumorigenesis.In summary,we have uncovered the protective role of PAPSS2-mediated chondroitin sulfation and bile acids-FXR-TLE3 activation in the prevention of gut carcinogenesis via the antagonization of Wnt/β-catenin signaling.Chondroitin sulfate may be explored as a therapeutic agent for Papss2 deficiency-associated colonic carcinogenesis.展开更多
Capillary electrochromatography (CEC) is a micro-scale separation technique which is a hybrid between capillary electrophoresis (CE) and liquid chromatography (LC). CEC can be performed in packed, monolithic and open-...Capillary electrochromatography (CEC) is a micro-scale separation technique which is a hybrid between capillary electrophoresis (CE) and liquid chromatography (LC). CEC can be performed in packed, monolithic and open-tubular columns. In recent three years (from 2016 to 2018), enormous attention for CEC has been the development of novel stationary phases. This review mainly covers the development of novel stationary phases for open-tubular and monolithic columns. In particular, some biomaterials attracted increasing interest. There are no significant breakthroughs in technology and principles in CEC. The typical CEC applications, especially chiral separations are described.展开更多
Endocrine-resistance remains a major challenge in estrogen receptorαpositive(ERα^(+))breast cancer(BC)treatment and constitutively active somatic mutations in ERαare a common mechanism.There is an urgent need to de...Endocrine-resistance remains a major challenge in estrogen receptorαpositive(ERα^(+))breast cancer(BC)treatment and constitutively active somatic mutations in ERαare a common mechanism.There is an urgent need to develop novel drugs with new mode of mechanism to fight endocrineresistance.Given aberrant ERαactivity,we herein report the identification of novel covalent selective estrogen receptor degraders(cSERDs)possessing the advantages of both covalent and degradation strategies.A highly potent cSERD 29c was identified with superior anti-proliferative activity than fulvestrant against a panel of ERa+breast cancer cell lines including mutant ERα.Crystal structure of ERα-29c complex alongside intact mass spectrometry revealed that 29c disrupted ERa protein homeostasis through covalent targeting C530 and strong hydrophobic interaction collied on H11,thus enforcing a unique antagonist conformation and driving the ERαdegradation.These significant effects of the cSERD on ERαhomeostasis,unlike typical ERαdegraders that occur directly via long side chains perturbing the morphology of H12,demonstrating a distinct mechanism of action(MoA).In vivo,29c showed potent antitumor activity in MCF-7 tumor xenograft models and low toxicity.This proof-of-principle study verifies that novel cSERDs offering new opportunities for the development of innovative therapies for endocrine-resistant BC.展开更多
Testis specific serine/threonine protein kinase 4(TSSK4) belongs to the TSSK family, and its members play an important role in spermatogenesis and/or spermiogenesis. Mouse TSSK4 has been reported to be expressed exc...Testis specific serine/threonine protein kinase 4(TSSK4) belongs to the TSSK family, and its members play an important role in spermatogenesis and/or spermiogenesis. Mouse TSSK4 has been reported to be expressed exclusively in the testis and can maintain its kinase activity through autophosphorylation at Thr-197. However, its biological function remains poorly understood. Here we found that GFP-TSSK4-overexpressed He La cells showed apoptotic bodies, indicating TSSK4 can lead to apoptosis in vitro. Furthermore, TSSK4 induced apoptosis in different cell lines including He La, Cos-7 and H1299 tested by flow cytometry but not its kinase-dead mutant TSSK4-K54 M. TSSK4 knockout mice showed increased testes weight and decreased apoptotic spermatogonia and spermatocytes at 21 st day after birth tested by TUNEL technology. So TSSK4 was able to induce cell apoptosis in vitro depending on its kinase activity, which leads to abnormal testes weight and apoptosis, shedding light on its function in the process of spermatogenesis and/or spermiogenesis.展开更多
Melanoma is the most aggressive and malignant form of skin cancer.Current melanoma treatment methods generally suffer from frequent drug administration as well as difficulty in direct monitoring of drug release.Here,a...Melanoma is the most aggressive and malignant form of skin cancer.Current melanoma treatment methods generally suffer from frequent drug administration as well as difficulty in direct monitoring of drug release.Here,a self-monitoring microneedle(MN)-based drug delivery system,which integrates a dissolving MN patch with aggregation-induced emission(AIE)-active PATC microparticles,is designed to achieve light-controlled pulsatile chemo-photothermal synergistic therapy of melanoma.The PATC polymeric particles,termed D/I@PATC,encapsulate both of chemotherapeutic drug doxorubicin(DOX)and the photothermal agent indocyanine green(ICG).Upon light illumination,PATC gradually dissociates into smaller particles,causing the release of encapsulated DOX and subsequent fluorescence intensity change of PATC particles,thereby not only enabling direct observation of the drug release process under light stimuli,but also facilitating verification of drug release by fluorescence recovery after light trigger.Moreover,encapsulation of ICG in PATC particles displays significant improvement of its photothermal stability both in vitro and in vivo.In a tumor-bearing mouse,the application of one D/I@PATC MN patch combining with two cycles of light irradiation showed excellent controllable chemo-photothermal efficacy and exhibited~97%melanoma inhibition rate without inducing any evident systemic toxicity,suggesting a great potential for skin cancer treatment in clinics.展开更多
The diabetic wounds remain to be unsettled clinically,with chronic wounds characterized by drug-resistant bacterial infections,compromised angiogenesis and oxidative damage to the microenvironment.To ameliorate oxidat...The diabetic wounds remain to be unsettled clinically,with chronic wounds characterized by drug-resistant bacterial infections,compromised angiogenesis and oxidative damage to the microenvironment.To ameliorate oxidative stress and applying antioxidant treatment in the wound site,we explore the function of folliculininteracting protein 1(FNIP1),a mitochondrial gatekeeper protein works to alter mitochondrial morphology,reduce oxidative phosphorylation and protect cells from unwarranted ROS accumulation.And our in vitro experiments showed the effects of FNIP1 in ameliorating oxidative stress and rescued impaired angiogenesis of HUVECs in high glucose environment.To realize the drug delivery and local regulation of FNIP1 in diabetic wound sites,a novel designed glucose-responsive HA-PBA-FA/EN106 hydrogel is introduced for improving diabetic wound healing.Due to the dynamic phenylboronate ester structure with a phenylboronic acid group between hyaluronic acid(HA)and phenylboronic acid(PBA),the hydrogel is able to realize a glucose-responsive release of drugs.Fulvic acid(FA)is added in the hydrogel,which not only severs as crosslinking agent but also provides antibacterial and anti-inflammatory abilities.Moreover,the release of FEM1b-FNIP1 axis inhibitor EN106 ameliorated oxidative stress and stimulated angiogenesis through FEM1b-FNIP1 axis regulation.These in vivo and in vitro results demonstrated that accelerated diabetic wounds repair with the use of the HA-PBA-FA/EN106 hydrogel,which may provide a promising strategy for chronic diabetic wound repair.展开更多
A series of novel gossypol derivatives were synthesized and screened for their in vitro anti-HIV- 1I activity. The results showed that replacing the aldehyde groups of gossypol with certain oligopeptides and Dglucosam...A series of novel gossypol derivatives were synthesized and screened for their in vitro anti-HIV- 1I activity. The results showed that replacing the aldehyde groups of gossypol with certain oligopeptides and Dglucosamine not only reduced the cytotoxicity of gossypol derivatives but also enhanced their antiviral activity against HIV-1. Interestingly, D-glucosamine derivative of gossypol that lacked the COONa group also exhibited the same potent anti-HIV-1 activity as oligopeptide derivatives with the COONa group. These compounds blocked the entry of HIV-1ⅢB into target cell. which was similar to T20. Furthermore, the molecular docking analysis rationalized their anti-HIV-1 activity. The results also implied that certain oligopeptides and D-glucosamine were important moities to prepare gossypol derivatives as HIV- 1 entry inhibitors besides certain amino acids.展开更多
Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection can cause acute respiratory distress syndrome,hypercoagulability,hypertension,and multiorgan dysfunction.Effective antivirals with safe clinical pro...Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection can cause acute respiratory distress syndrome,hypercoagulability,hypertension,and multiorgan dysfunction.Effective antivirals with safe clinical profile are urgently needed to improve the overall prognosis.In an analysis of a randomly collected cohort of 124 patients with COVID-19,we found that hypercoagulability as indicated by elevated concentrations of D-dimers was associated with disease severity.By virtual screening of a U.S.FDA approved drug library,we identified an anticoagulation agent dipyridamole(DIP)in silico,which suppressed SARS-CoV-2 replication in vitro.In a proof-of-concept trial involving 31 patients with COVID-19,DIP supplementation was associated with significantly decreased concentrations of D-dimers(P<0.05),increased lymphocyte and platelet recovery in the circulation,and markedly improved clinical outcomes in comparison to the control patients.In particular,all 8 of the DIP-treated severely ill patients showed remarkable improvement:7 patients(87.5%)achieved clinical cure and were discharged from the hospitals while the remaining 1 patient(12.5%)was in clinical remission.展开更多
With the developments in metabolic engineering and the emergence of synthetic biology,many breakthroughs in medicinal,biological and chemical products as well as biofuels have been achieved in recent decades.As an imp...With the developments in metabolic engineering and the emergence of synthetic biology,many breakthroughs in medicinal,biological and chemical products as well as biofuels have been achieved in recent decades.As an important barrier to traditional metabolic engineering,however,the identification of ratelimiting step(s)for the improvement of specific cellular functions is often difficult.Meanwhile,in the case of synthetic biology,more and more BioBricks could be constructed for targeted purposes,but the optimized assembly or engineering of these components for high-efficiency cell factories is still a challenge.Owing to the lack of steady-state kinetic data for overall flux,balancing many multistep biosynthetic pathways is time-consuming and needs vast resources of labor and materials.A strategy called targeted engineering is proposed in an effort to solve this problem.Briefly,a targeted biosynthetic pathway is to be reconstituted in vitro and then the contribution of cofactors,substrates and each enzyme will be analyzed systematically.Next is in vivo engineering or de novo pathway assembly with the guidance of information gained from in vitro assays.To demonstrate its practical application,biosynthesis pathways for the production of important products,e.g.chemicals,nutraceuticals and drug precursors,have been engineered in Escherichia coli and Saccharomyces cerevisiae.These cases can be regarded as concept proofs indicating targeted engineering might help to create high-efficiency cell factories based upon constructed biological components.展开更多
Spinosyns are natural broad-spectrum biological insecticides with a double glycosylated polyketide structure that are produced by aerobic fermentation of the actinomycete,Saccharopolyspora spinosa.However,their large-...Spinosyns are natural broad-spectrum biological insecticides with a double glycosylated polyketide structure that are produced by aerobic fermentation of the actinomycete,Saccharopolyspora spinosa.However,their large-scale overproduction is hindered by poorly understood bottlenecks in optimizing the original strain,and poor adaptability of the heterologous strain to the production of spinosyn.In this study,we genetically engineered heterologous spinosyn-producer Streptomyces albus J1074 and optimized the fermentation to improve the production of spinosad(spinosyn A and spinosyn D)based on our previous work.We systematically investigated the result of overexpressing polyketide synthase genes(spnA,B,C,D,E)using a constitutive promoter on the spinosad titer in S.albus J1074.The supply of polyketide synthase precursors was then increased to further improve spinosad production.Finally,increasing or replacing the carbon source of the culture medium resulted in a final spinosad titer of~70 mg/L,which is the highest titer of spinosad achieved in heterologous Streptomyces species.This research provides useful strategies for efficient heterologous production of natural products.展开更多
Microbial-derived natural products are important in both the pharmaceutical industry and academic research.As the metabolic potential of original producer especially Streptomyces is often limited by slow growth rate,c...Microbial-derived natural products are important in both the pharmaceutical industry and academic research.As the metabolic potential of original producer especially Streptomyces is often limited by slow growth rate,complicated cultivation profile,and unfeasible genetic manipulation,so exploring a Streptomyces as a super industrial chassis is valuable and urgent.Streptomyces sp.FR-008 is a fast-growing microorganism and can also produce a considerable amount of macrolide candicidin via modular polyketide synthase.In this study,we evaluated Streptomyces sp.FR-008 as a potential industrial-production chassis.First,PacBio sequencing and transcriptome analyses indicated that the Streptomyces sp.FR-008 genome size is 7.26 Mb,which represents one of the smallest of currently sequenced Streptomyces genomes.In addition,we simplified the conjugation procedure without heat-shock and pre-germination treatments but with high conjugation efficiency,suggesting it is inherently capable of accepting heterologous DNA.In addition,a series of promoters selected from literatures was assessed based on GusA activity in Streptomyces sp.FR-008.Compared with the common used promoter ermE*-p,the strength of these promoters comprise a library with a constitutive range of 60e860%,thus providing the useful regulatory elements for future genetic engineering purpose.In order to minimum the genome,we also target deleted three endogenous polyketide synthase(PKS)gene clusters to generate a mutant LQ3.LQ3 is thus an“updated”version of Streptomyces sp.FR-008,producing fewer secondary metabolites profiles than Streptomyces sp.FR-008.We believe this work could facilitate further development of Streptomyces sp.FR-008 for use in biotechnological applications.展开更多
基金supported by the National Key Research and Development Program of China(Program No.:2019YFA0904300)Shenzhen Science and Technology Program(Program No.:JCYJ20220530150404009)the Guangdong Basic and Applied Basic Research Foundation(Grant No.:2022A1515110608).
文摘Single-cell multi-Omics(SCM-Omics)and spatial multi-Omics(SM-Omics)technologies provide state-of-the-art methods for exploring the composition and function of cell types in tissues/organs.Since its emergence in 2009,single-cell RNA sequencing(scRNA-seq)has yielded many groundbreaking new discoveries.The combination of this method with the emergence and development of SM-Omics techniques has been a pioneering strategy in neuroscience,developmental biology,and cancer research,especially for assessing tumor heterogeneity and T-cell infiltration.In recent years,the application of these methods in the study of metabolic diseases has also increased.The emerging SCM-Omics and SM-Omics approaches allow the molecular and spatial analysis of cells to explore regulatory states and determine cell fate,and thus provide promising tools for unraveling heterogeneous metabolic processes and making them amenable to intervention.Here,we review the evolution of SCM-Omics and SM-Omics technologies,and describe the progress in the application of SCM-Omics and SM-Omics in metabolism-related diseases,including obesity,diabetes,nonalcoholic fatty liver disease(NAFLD)and cardiovascular disease(CVD).We also conclude that the application of SCM-Omics and SM-Omics approaches can help resolve the molecular mechanisms underlying the pathogenesis of metabolic diseases in the body and facilitate therapeutic measures for metabolism-related diseases.This review concludes with an overview of the current status of this emerging field and the outlook for its future.
基金financial support from the National Natural Science Foundation of China (Grant nos. 81573384 and 21375101)
文摘Capillary electrophoresis with many advantages plays an important role in pharmaceutical analysis and drug screening. This review gives an overview on the recent advances in the developments and applications of capillary electrophoresis in the field of enzyme inhibitor screening. The period covers 2013 to 2017. Both the pre-capillary enzyme assays and in-capillary enzyme assays which include electrophoretically mediated microanalysis(EMMA) and immobilized enzyme microreactor(IMER) are summarized in this article.
基金supported by the National Scientific Foundation of China (Grant nos.21375101,90817103,and 30973672)Doctroral Fund of Ministry of Education of China (No.20110141110024)Innovation Seed Fund and Translational Medical Research Fund of Wuhan University School of Medicine
文摘A novel method for analysis of three active components curcumin, demethoxycurcumin and bisdemethoxycurcumin in Curcuma longa L. was developed by HPLC coupled with electrochemical detection. Three curcuminoids were well separated on a C18 column and detected with high sensitivity. A mobile phase containing acetonitrile and 10 mM Na2HPO4-H3PO4 (pH 5.0) (50:50, v/v) was used. Good linearity was obtained in the range of 0.208-41.6, 0.197-39.4, and 0.227-114μM for curcumin, demethoxycurcumin and bisdemethoxycurcumin respectively. The limit of detection reached up to 10 ? 8 M, which was lower than that by UV detection. The relative standard deviations (RSDs) ranged from 1.06%to 1.88%for intra-day precision and from 4.30%to 5.79%for inter-day precision, respectively. The proposed method has been applied in real herb sample and recoveries ranging from 86.3%to 111%were obtained.
基金the National Natural Science Foundation of China (Grant Nos.: 81673394 and 82073811)the Fundamental Research Funds for the Central Universities (Grant No.: 2042020kf1010)the Large-scale Instrument and Equipment Sharing Foundation of Wuhan University (Grant No.: LF20170838)
文摘Natural long-chain alkanol and alkyl carboxylic acid were used to prepare novel hydrophobic deep eutectic solvents(HDESs).These HDESs are liquid at room temperature and have low viscosity(<12.26 mPa·s),low polarity(lower than that of methanol,ChCl-based deep eutectic solvents and other reported HDESs),and low density(<0.928 g/mL).A simple one-pot method based on a novel HDES-water two-phase extraction system was constructed for the extraction of weak-polarity bioactive components,anthraquinones,from Rhei Radix et Rhizoma.This HDES-based new extraction method does not consume hazardous organic solvents and can obtain a total anthraquinone yield of 21.52 mg/g,which is close to that obtained by the Chinese pharmacopoeia method(21.22 mg/g)and considerably higher than those by other reported HDESs-based extraction methods(14.20-20.09 mg/g,p<0.01).The high extraction yield can be mainly attributed to the severe destruction of the RRR cell walls by the extraction system and the excellent dissolving ability of novel HDESs for anthraquinones.
基金supported by the National Scientifc Foundation of China(NSFC nos.9081710330973672 and 20775055)+2 种基金Doctoral Fund of Ministry of Education of China(No 20110141110024)Hubei Provincial Scientifc Foundation(No 2011CDB475)the Fundamental Research Funds for the Central Universities
文摘A simple, sensitive and selective method of high-performance liquid chromatography (HPLC) has been successfully developed for separation of bavachinin enantiomers in Fructus Psoraleae and rat plasma. The separation and detection conditions of HPLC were optimized. Chiral bavachinin were separated with the mobile phase of methanol and water (70:30, v/v) at a flow rate of 1.0 mL/min. The linear ranges were in the range of 20-1000 μg/mL. The detection limits were tested as 4 ng/mL and 6 ng/mL for (+)-bavachinin and (-)-bavachinin, respectively. The method has been applied to analyze chiral bavachinin in rat plasma. HPLC-MS method was used to test the accuracy.
文摘The cyanobacterium Arthrospira platensis,spirulina,is a source of pigments such as phycobiliprotein and phycocyanin.Phycocyanin is used in the food,cosmetics,and pharmaceutical industries because of its antioxidant,anti-inflammatory,and anticancer properties.The different steps involved in extraction and purification of this protein can alter the final properties.In this review,the stability of phycocyanin(pH,temperature,and light)is discussed,considering the physicochemical parameters of kinetic modeling.The optimal working pH range for phycocyanin is between 5.5 and 6.0 and it remains stable up to 45℃;however,exposure to relatively high temperatures or acidic pH decreases its half-life and increases the degradation kinetic constant.Phycobiliproteins are sensitive to light;preservatives such as mono-and disaccharides,citric acid,or sodium chloride appear to be effective stabilizing agents.Encapsulation within nano-or micro-structured materials such as nanofibers,microparticles,or nanoparticles,can also preserve or enhance its stability.
基金supported by the National Natural Science Foundation of China(Grant Nos.:82073808,81872828,and 81573384).
文摘Covalent organic frameworks(COFs)are a novel type of crystalline porous organic polymer materials recently developed.It has several advantages in chromatographic separation field,such as high thermal stability,porosity,structural regularity,and large specific surface area.Here,a novel spherical COF 1,3,5-tris(4-aminophenyl)benzene(TAPB)and 2,5-bis(2-propyn-1-yloxy)-1,4-benzenedicarboxaldehyde(BPTA)was developed as an electrochromatographic stationary phase for capillary electrochromatography separation.The COF TAPB-BPTA modified capillary column was fabricated via a facile in situ growth method at room temperature.The characterization results of scanning electron microscopy(SEM),Fourier transform infrared(FT-IR)spectroscopy,and X-ray diffraction(XRD)confirmed that COF TAPB-BPTA were successfully modified onto the capillary inner surface.The electrochromatography separation performance of the COF TAPB-BPTA modified capillary was investigated.The prepared column demonstrated outstanding separation performance toward alkylbenzenes,phenols,and chlorobenzenes compounds.Furthermore,the baseline separations of non-steroidal anti-inflammatory drugs(NSAIDs)and parabens with good efficiency and high resolution were achieved.Also,the prepared column possessed satisfactory precision of the intra-day runs(n=5),inter-day runs(n=3),and parallel columns(n=3),and the relative standard deviations(RSDs)of the retention times of tested alkylbenzenes were all less than 2.58%.Thus,this new COF-based stationary phase shows tremendous application potential in chromatographic separation field.
基金the National Natural Science Foundation of China(Grant Nos.:82273885,82073808 and 81872828).
文摘Here,a styrene-based polymer monolithic column poly(VBS-co-TAT-co-AHM)with reversed-phase/hydrophilic interaction liquid chromatography(RPLC/HILIC)bifunctional separation mode was success-fully prepared for capillary electrochromatography by the in situ polymerization of sodium p-styrene sulfonate(VBS)with cross-linkers 3-(acryloyloxy)-2-hydroxypropyl methacrylate(AHM)and 1,3,5-triacryloylhexahydro-1,3,5-triazine(TAT).The preparation conditions of the monolith were optimized.The morphology and formation of the poly(VBS-co-TAT-co-AHM)monolith were confirmed by scanning electron microscopy(SEM)and Fourier transform infrared spectroscopy(FT-IR).The separation perfor-mances of the monolith were evaluated systematically.It should be noted that the incorporation of VBS functional monomer can provideπ-πinteractions,hydrophilic interactions,and ion-exchange in-teractions.Hence,the prepared poly(VBS-co-TAT-co-AHM)monolith can achieve efficient separation of thiourea compounds,benzene series,phenol compounds,aniline compounds and sulfonamides in RPLC or HILIC separation mode.The largest theoretical plate number for N,N0-dimethylthiourea reached 1.7×10^(5)plates/m.In addition,the poly(VBS-co-TAT-co-AHM)monolithic column showed excellent reproducibility and stability.This novel monolithic column has great application value and potential in capillary electrochromatography(CEC).
基金supported in part by grants from the National Natural Science Foundation of China(Grant Nos.:81973405,82122071,and 82030111)to Dan XuHui Wang,the National Key R&D Program of China(Grant No.:2020YFA0803900)to Hui Wangthe Hubei Provincial Natural Science Foundation Outstanding Youth Found,China(Grant No.:2022CFA083).
文摘The central nervous system is susceptible to the modulation of various neurophysiological processes by the cytochrome P450 enzyme(CYP),which plays a crucial role in the metabolism of neurosteroids.The antiepileptic drug phenytoin(PHT)has been observed to induce neuronal side effects in patients,which could be attributed to its induction of CYP expression and testosterone(TES)metabolism in the hippocampus.While pregnane X receptor(PXR)is widely known for its regulatory function of CYPs in the liver,we have discovered that the treatment of mice with pregnenolone 16α-carbonitrile(PCN),a PXR agonist,has differential effects on CYP expression in the liver and hippocampus.Specifically,the PCN treatment resulted in the induction of cytochrome P450,family 3,subfamily a,polypeptide 11(CYP3A11),and CYP2B10 expression in the liver,while suppressing their expression in the hippocampus.Functionally,the PCN treatment protected mice from PHT-induced hippocampal nerve injury,which was accompanied by the inhibition of TES metabolism in the hippocampus.Mechanistically,we found that the inhibition of hippocampal CYP expression and attenuation of PHT-induced neurotoxicity by PCN were glucocorticoid receptor dependent,rather than PXR independent,as demonstrated by genetic and pharmacological models.In conclusion,our study provides evidence that PCN can negatively regulate hippocampal CYP expression and attenuate PHT-induced hippocampal neurotoxicity independently of PXR.Our findings suggest that glucocorticoids may be a potential therapeutic strategy for managing the neuronal side effects of PHT.
基金supported by NIH grants DK117370,DK135538,and ES030429(to Wen Xie,US)a Pilot&Feasibility grant(to Pengfei Xu,US)from the Pittsburgh Liver Research Center funded by NIH grant P30DK120531NIH shared instrumentation grant:Olympus FV3000 Confocal Microscope SIG:NIH S10OD030254-01A1。
文摘Sulfation is a crucial and prevalent conjugation reaction involved in cellular processes and mammalian physiology.3’-Phosphoadenosine 5’-phosphosulfate(PAPS)synthase 2(PAPSS2)is the primary enzyme to generate the universal sulfonate donor PAPS.The involvement of PAPSS2-mediated sulfation in adenomatous polyposis coli(APC)mutation-promoted colonic carcinogenesis has not been reported.Here,we showed that the expression of PAPSS2 was decreased in human colon tumors along with cancer stages,and the lower expression of PAPSS2 was correlated with poor prognosis in advanced colon cancer.Gut epithelial-specific heterozygous Apc deficient and Papss2-knockout(Apc^(Δgut-Het)Papss2^(Δgut))mice were created,and the phenotypes were compared to the spontaneous intestinal tumorigenesis of Apc^(Δgut-Het)mice.Apc^(Δgut-Het)Papss2^(Δgutmice) were more sensitive to gut tumorigenesis,which was mechanistically accounted for by the activation of Wnt/β-catenin signaling pathway due to the suppression of chondroitin sulfation and inhibition of the farnesoid X receptor(FXR)-transducin-like enhancer of split 3(TLE3)gene regulatory axis.Chondroitin sulfate supplementation in Apc^(Δgut-Het)Papss2^(Δgutmice) alleviated intestinal tumorigenesis.In summary,we have uncovered the protective role of PAPSS2-mediated chondroitin sulfation and bile acids-FXR-TLE3 activation in the prevention of gut carcinogenesis via the antagonization of Wnt/β-catenin signaling.Chondroitin sulfate may be explored as a therapeutic agent for Papss2 deficiency-associated colonic carcinogenesis.
基金supported by the National Natural Science Foundation of China(Grant Nos.81872828 and 81573384)
文摘Capillary electrochromatography (CEC) is a micro-scale separation technique which is a hybrid between capillary electrophoresis (CE) and liquid chromatography (LC). CEC can be performed in packed, monolithic and open-tubular columns. In recent three years (from 2016 to 2018), enormous attention for CEC has been the development of novel stationary phases. This review mainly covers the development of novel stationary phases for open-tubular and monolithic columns. In particular, some biomaterials attracted increasing interest. There are no significant breakthroughs in technology and principles in CEC. The typical CEC applications, especially chiral separations are described.
基金supported by National Key R&D Program of China(2020YFA0908800,2021YFC2100300)National Natural Science Foundation of China(82273774,82073690,81773557,82173676,82103994)+1 种基金the Fundamental Research Funds for the Central Universities of China(2042022kf0056)the China Postdoctoral Science Foundation(2020M672435).
文摘Endocrine-resistance remains a major challenge in estrogen receptorαpositive(ERα^(+))breast cancer(BC)treatment and constitutively active somatic mutations in ERαare a common mechanism.There is an urgent need to develop novel drugs with new mode of mechanism to fight endocrineresistance.Given aberrant ERαactivity,we herein report the identification of novel covalent selective estrogen receptor degraders(cSERDs)possessing the advantages of both covalent and degradation strategies.A highly potent cSERD 29c was identified with superior anti-proliferative activity than fulvestrant against a panel of ERa+breast cancer cell lines including mutant ERα.Crystal structure of ERα-29c complex alongside intact mass spectrometry revealed that 29c disrupted ERa protein homeostasis through covalent targeting C530 and strong hydrophobic interaction collied on H11,thus enforcing a unique antagonist conformation and driving the ERαdegradation.These significant effects of the cSERD on ERαhomeostasis,unlike typical ERαdegraders that occur directly via long side chains perturbing the morphology of H12,demonstrating a distinct mechanism of action(MoA).In vivo,29c showed potent antitumor activity in MCF-7 tumor xenograft models and low toxicity.This proof-of-principle study verifies that novel cSERDs offering new opportunities for the development of innovative therapies for endocrine-resistant BC.
基金supported by National Natural Science Foundation of China(No.31301202)
文摘Testis specific serine/threonine protein kinase 4(TSSK4) belongs to the TSSK family, and its members play an important role in spermatogenesis and/or spermiogenesis. Mouse TSSK4 has been reported to be expressed exclusively in the testis and can maintain its kinase activity through autophosphorylation at Thr-197. However, its biological function remains poorly understood. Here we found that GFP-TSSK4-overexpressed He La cells showed apoptotic bodies, indicating TSSK4 can lead to apoptosis in vitro. Furthermore, TSSK4 induced apoptosis in different cell lines including He La, Cos-7 and H1299 tested by flow cytometry but not its kinase-dead mutant TSSK4-K54 M. TSSK4 knockout mice showed increased testes weight and decreased apoptotic spermatogonia and spermatocytes at 21 st day after birth tested by TUNEL technology. So TSSK4 was able to induce cell apoptosis in vitro depending on its kinase activity, which leads to abnormal testes weight and apoptosis, shedding light on its function in the process of spermatogenesis and/or spermiogenesis.
基金support from the following fundings:National Natural Science Foundation of China(NSFC,No.52103182,21704026,22075087)Natural Science Foundation of Hubei Province(No.2021CFB103)the Fundamental Research Funds for the Central Universities(No.2042021kf0073).
文摘Melanoma is the most aggressive and malignant form of skin cancer.Current melanoma treatment methods generally suffer from frequent drug administration as well as difficulty in direct monitoring of drug release.Here,a self-monitoring microneedle(MN)-based drug delivery system,which integrates a dissolving MN patch with aggregation-induced emission(AIE)-active PATC microparticles,is designed to achieve light-controlled pulsatile chemo-photothermal synergistic therapy of melanoma.The PATC polymeric particles,termed D/I@PATC,encapsulate both of chemotherapeutic drug doxorubicin(DOX)and the photothermal agent indocyanine green(ICG).Upon light illumination,PATC gradually dissociates into smaller particles,causing the release of encapsulated DOX and subsequent fluorescence intensity change of PATC particles,thereby not only enabling direct observation of the drug release process under light stimuli,but also facilitating verification of drug release by fluorescence recovery after light trigger.Moreover,encapsulation of ICG in PATC particles displays significant improvement of its photothermal stability both in vitro and in vivo.In a tumor-bearing mouse,the application of one D/I@PATC MN patch combining with two cycles of light irradiation showed excellent controllable chemo-photothermal efficacy and exhibited~97%melanoma inhibition rate without inducing any evident systemic toxicity,suggesting a great potential for skin cancer treatment in clinics.
基金the National Science Foundation of China(No.82272491,No.82072444)the Wuhan Science and Technology Bureau(2022020801020464)+2 种基金the Department of Science and Technology of Hubei Province(No.2021CFB425)Chinese Pharmaceutical Association Hospital Phamacy department(No.CPA-Z05-ZC-2022-002)Grants from Hubei Province Unveiling Science and Technology Projects(No.2022-35).
文摘The diabetic wounds remain to be unsettled clinically,with chronic wounds characterized by drug-resistant bacterial infections,compromised angiogenesis and oxidative damage to the microenvironment.To ameliorate oxidative stress and applying antioxidant treatment in the wound site,we explore the function of folliculininteracting protein 1(FNIP1),a mitochondrial gatekeeper protein works to alter mitochondrial morphology,reduce oxidative phosphorylation and protect cells from unwarranted ROS accumulation.And our in vitro experiments showed the effects of FNIP1 in ameliorating oxidative stress and rescued impaired angiogenesis of HUVECs in high glucose environment.To realize the drug delivery and local regulation of FNIP1 in diabetic wound sites,a novel designed glucose-responsive HA-PBA-FA/EN106 hydrogel is introduced for improving diabetic wound healing.Due to the dynamic phenylboronate ester structure with a phenylboronic acid group between hyaluronic acid(HA)and phenylboronic acid(PBA),the hydrogel is able to realize a glucose-responsive release of drugs.Fulvic acid(FA)is added in the hydrogel,which not only severs as crosslinking agent but also provides antibacterial and anti-inflammatory abilities.Moreover,the release of FEM1b-FNIP1 axis inhibitor EN106 ameliorated oxidative stress and stimulated angiogenesis through FEM1b-FNIP1 axis regulation.These in vivo and in vitro results demonstrated that accelerated diabetic wounds repair with the use of the HA-PBA-FA/EN106 hydrogel,which may provide a promising strategy for chronic diabetic wound repair.
基金the National Natural Science Foundation of China(No.30770228)for financial support
文摘A series of novel gossypol derivatives were synthesized and screened for their in vitro anti-HIV- 1I activity. The results showed that replacing the aldehyde groups of gossypol with certain oligopeptides and Dglucosamine not only reduced the cytotoxicity of gossypol derivatives but also enhanced their antiviral activity against HIV-1. Interestingly, D-glucosamine derivative of gossypol that lacked the COONa group also exhibited the same potent anti-HIV-1 activity as oligopeptide derivatives with the COONa group. These compounds blocked the entry of HIV-1ⅢB into target cell. which was similar to T20. Furthermore, the molecular docking analysis rationalized their anti-HIV-1 activity. The results also implied that certain oligopeptides and D-glucosamine were important moities to prepare gossypol derivatives as HIV- 1 entry inhibitors besides certain amino acids.
基金National Key R&D Program of China(2017YFB0202600 and 2020YFC0841400)National Natural Science Foundation of China(91742109,8152204,31770978,81773674,and 21877134)+8 种基金National Health&Medical Research of Australia(1080321,1143976 and 1150425)Science Foundation of Guangzhou City(201904020023,China)Guangdong Province Higher Vocational Colleges and Schools Pearl River Scholar Funded Scheme(2016 and 2019,China)Guangdong Provincial Key Laboratory of Construction Foundation(2017B030314030,China)Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program(2017BT01Y093,China)Zhejiang University special scientific research fund for COVID-19 prevention and control(China)National Health&Medical Research of Australia(1080321,1143976,and 1150425)Taikang Insurance Group Co.,Ltd.Beijing Taikang Yicai Foundation(Beijing,China)
文摘Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)infection can cause acute respiratory distress syndrome,hypercoagulability,hypertension,and multiorgan dysfunction.Effective antivirals with safe clinical profile are urgently needed to improve the overall prognosis.In an analysis of a randomly collected cohort of 124 patients with COVID-19,we found that hypercoagulability as indicated by elevated concentrations of D-dimers was associated with disease severity.By virtual screening of a U.S.FDA approved drug library,we identified an anticoagulation agent dipyridamole(DIP)in silico,which suppressed SARS-CoV-2 replication in vitro.In a proof-of-concept trial involving 31 patients with COVID-19,DIP supplementation was associated with significantly decreased concentrations of D-dimers(P<0.05),increased lymphocyte and platelet recovery in the circulation,and markedly improved clinical outcomes in comparison to the control patients.In particular,all 8 of the DIP-treated severely ill patients showed remarkable improvement:7 patients(87.5%)achieved clinical cure and were discharged from the hospitals while the remaining 1 patient(12.5%)was in clinical remission.
基金the National 973 Program of China(No.2011CBA00800 and 2012CB721000)National 863 Program of China(No.2012AA02A701)+2 种基金National Natural Science Foundation of China(No.31170096,312220170 and 31500072)the Natural Science Foundation of Hubei Province(No.2015CFB415)China Postdoctoral Science Foundation Grant(No.2014M562052).
文摘With the developments in metabolic engineering and the emergence of synthetic biology,many breakthroughs in medicinal,biological and chemical products as well as biofuels have been achieved in recent decades.As an important barrier to traditional metabolic engineering,however,the identification of ratelimiting step(s)for the improvement of specific cellular functions is often difficult.Meanwhile,in the case of synthetic biology,more and more BioBricks could be constructed for targeted purposes,but the optimized assembly or engineering of these components for high-efficiency cell factories is still a challenge.Owing to the lack of steady-state kinetic data for overall flux,balancing many multistep biosynthetic pathways is time-consuming and needs vast resources of labor and materials.A strategy called targeted engineering is proposed in an effort to solve this problem.Briefly,a targeted biosynthetic pathway is to be reconstituted in vitro and then the contribution of cofactors,substrates and each enzyme will be analyzed systematically.Next is in vivo engineering or de novo pathway assembly with the guidance of information gained from in vitro assays.To demonstrate its practical application,biosynthesis pathways for the production of important products,e.g.chemicals,nutraceuticals and drug precursors,have been engineered in Escherichia coli and Saccharomyces cerevisiae.These cases can be regarded as concept proofs indicating targeted engineering might help to create high-efficiency cell factories based upon constructed biological components.
基金This work was supported by the National Key R&D Program of China[grant number 2018YFA0900400]the National Natural Science Foundation of China[grant number 31670090],and J1 Biotech Co.,Ltd.
文摘Spinosyns are natural broad-spectrum biological insecticides with a double glycosylated polyketide structure that are produced by aerobic fermentation of the actinomycete,Saccharopolyspora spinosa.However,their large-scale overproduction is hindered by poorly understood bottlenecks in optimizing the original strain,and poor adaptability of the heterologous strain to the production of spinosyn.In this study,we genetically engineered heterologous spinosyn-producer Streptomyces albus J1074 and optimized the fermentation to improve the production of spinosad(spinosyn A and spinosyn D)based on our previous work.We systematically investigated the result of overexpressing polyketide synthase genes(spnA,B,C,D,E)using a constitutive promoter on the spinosad titer in S.albus J1074.The supply of polyketide synthase precursors was then increased to further improve spinosad production.Finally,increasing or replacing the carbon source of the culture medium resulted in a final spinosad titer of~70 mg/L,which is the highest titer of spinosad achieved in heterologous Streptomyces species.This research provides useful strategies for efficient heterologous production of natural products.
基金grants from J1 Biotech Co.Ltd.,the 973 Project(2011CBA00800,2012CB721000)the 863 Project(2012AA02A701)the Ministry of Science and Technology of China,and from the Natural Science Foundation of Hubei Province(2015CFB415).
文摘Microbial-derived natural products are important in both the pharmaceutical industry and academic research.As the metabolic potential of original producer especially Streptomyces is often limited by slow growth rate,complicated cultivation profile,and unfeasible genetic manipulation,so exploring a Streptomyces as a super industrial chassis is valuable and urgent.Streptomyces sp.FR-008 is a fast-growing microorganism and can also produce a considerable amount of macrolide candicidin via modular polyketide synthase.In this study,we evaluated Streptomyces sp.FR-008 as a potential industrial-production chassis.First,PacBio sequencing and transcriptome analyses indicated that the Streptomyces sp.FR-008 genome size is 7.26 Mb,which represents one of the smallest of currently sequenced Streptomyces genomes.In addition,we simplified the conjugation procedure without heat-shock and pre-germination treatments but with high conjugation efficiency,suggesting it is inherently capable of accepting heterologous DNA.In addition,a series of promoters selected from literatures was assessed based on GusA activity in Streptomyces sp.FR-008.Compared with the common used promoter ermE*-p,the strength of these promoters comprise a library with a constitutive range of 60e860%,thus providing the useful regulatory elements for future genetic engineering purpose.In order to minimum the genome,we also target deleted three endogenous polyketide synthase(PKS)gene clusters to generate a mutant LQ3.LQ3 is thus an“updated”version of Streptomyces sp.FR-008,producing fewer secondary metabolites profiles than Streptomyces sp.FR-008.We believe this work could facilitate further development of Streptomyces sp.FR-008 for use in biotechnological applications.