Transcriptome analysis for understanding the mechanism of dark septate endophyte S16 in promoting the growth and nitrate uptake of sweet cherry

Sweet cherry is one of the most popular fresh fruits in the world.Previously,we isolated a soilborne dark septate endophyte(DSE)strain,S16,which promoted the growth of Gisela 5 sweet cherry rootstock.However,little is known about the molecular mechanism of the effect of S16 on the growth of sweet cherry.In this study,the physiological parameters and transcript profiles of sweet cherry roots were analyzed under S16 treatment compared with a control to elucidate the molecular mechanisms of the effect of this strain on sweet cherry growth.After inoculation with S16,sweet cherry seedlings exhibited more vigorous growth.Moreover,we identified 4249 differentially expressed genes(DEGs)between S16-treated plants and the control.Many of the DEGs are involved in pathways related to plant growth,such as cellular metabolic and plant hormone pathways.Additionally,some genes involved in nitrate regulation were also enriched;and these genes may be involved in the regulation of nitrate uptake in plants.Physiological index detection demonstrated that S16 could improve the nitrate assimilation of sweet cherry via NO3–transporters.This RNA-seq dataset provides comprehensive insight into the transcriptomic landscape to reveal the molecular mechanisms whereby the DSE influences the growth of sweet cherry.

Biochemical Characterization of Uracil-DNA Glycosylase from Pyrococcus furiosus

We report the characterization of a uracil-DNA glycosylase（UDG） from the hyperthermophilic archaea Pyrococcus furiosus（P, furiosus）. P. furiosus UDG（PfUDG） has high sequence similarity to the families IV and V UDGs（thermostable UDG family and PaUDG-b family）. PfUDG excises uracil from various DNA substrates with the following order： U/T=U/C〉U/G=U/AP=U/-〉U/U=U/I=U/A. The optimal temperature and pH value for uracil exci- sion by PfUDG are 70 ℃ and 9.0, respectively. The removal of U is inhibited by the divalent ions of Fe, Ca, Zn, Cu, Co, Ni and Mn, as well as a high concentration of NaC1. The phosphorothioates near uracil strongly inhibit the exci- sion of uracil by PfUDG. Interestingly, pfuDNA（Pyrococcusfuriosus DNA） polymerase, which tightly binds the ura- cil-carrying oligonucleotide, does not inhibit the excision by Pfl.IDG, suggesting PfUDG in vivo functions as the re- pair enzyme to excise uracil damage in genome.

Lamin A/C Modulate Apoptosis of Rat Vascular Smooth Muscle Cells During Cyclic Stretch Application

Objective The apoptosis of vascular smooth muscle cells(VSMCs)influenced by abnormal cyclic stretch is crucial for vascular remodeling during hypertension.We explored that the causes of mechano-responsive lamin A/C changingin aonormai cyclic stretcn and its roles in VSMC apoptosis.Methods and results Our previous vascular proteomics study revealed that LaminA/C is mechano-sensitive molecule.When VSMCs are subjected to cyclic stretch,the expression of LaminA/C is significantly changed which participates dysfunctions of VSMCs during hypertension.However,the molecular mechanism involved in regulation of LaminA/C expression and the role of LaminA/C in the VSMC apoptosis during cyclic stretch application are still unclear.In the present study,VSMCs were subjected to different amplitudes of cyclic steetch in vitro:5%cyclic stretch(physiological strain)or 15%cyclic stretch(pathological strain).The expression of 2 different selective cleavage isomers of LaminA/C,i.e.LaminA and LaminC,and the apoptosis of VSMCs were detected.The results showed that compared with 5%group,15%cyclic stretch significantly decreased the expression of LaminA and LaminC,and promoted the apoptosis of VSMCs.Using specific small interfering RNA(siRNA)transfection which targets on LMNA the encoding gene of LaminA/C,the expression of LaminA and LaminC in VSMCs was significantly decreased,and the apoptosis was significantly increased.In order to study the molecular mechanism involved in cyclic stretch regulating the expression of LaminA/C,we focused on the microRNA(miR).Bioinformatics analysis showed that the 3’untranslated region(3’UTR)of LMNA has two potential binding sites to miR-124-3p.Double luciferase reported system revealed that both sites have binding abilities to miR-124-3p.Under static condition,miR-124-3p inhibitor significantly up-regulated the expression levels of LaminA and LaminC,while the miR-124-3p mimics significantly down-regulated them.RT-PCR results showed that 15%cyclic stretch significantly up-regulated the expression of miR-124-3p compared with 5%cyclic stretch.Furthermore,in order to study the role of changeed LaminA/C in VSMC apoptosis,LMNA-specific siRNA was transfected to repress the expression of LaminA/C in VSMCs,and Protein/DNA microarray was used to detecte the activity of transcription factors.The transcription factors whose activity were changed significantly(increase or decrease more than 2 times)were analyzed by cluster analysis and ingenurity pathway analysis(IPA).Six transcription factors associated with apoptosis were screened,in which TP53 was activated by the specific siRNA transfection and the other 5 were inavtived,including TP53,CREB1,MYC,STAT1/5/6 and JUN.Using abdominal aorta coarctation hypertensive model,the change of miR-124-3p in VSMCs was explored in vivo.A marked increase of miR-124-3p in thoracic aorta was revealed compared with the sham-operated controls,and in situ FISH revealed that this increase was mainly in the VSMCs.Conclusions The present study suggest that abnormally increased cyclic stretch(15%)up-regulates the expression of miR-124-3p in VSMCs,which subsequently targets on the 3’UTR of LMNA and decreases the expression of nuclear envelope protein LaminA/C;the repressed LaminA/C may play an important role in the apoptosis of VSMCs by regulating the activity of virious transcription factors,such as TP53,CREB1,MYC,STAT1/5/6 and JUN.The present study may provide a new insight into understanding the molecular mechanisms of vascular remodeling.

Characterization of HpnG as a Purine Nucleoside Phosphorylase in Bacteriohopanepolyol Biosynthesis

Comprehensive Summary HpnG plays a crucial role in the production of ribosylhopane,a key intermediate in the biosynthesis of bacteriohopanepolyol.Despite early extensive studies,the precise function of HpnG has remained elusive.Here,we report functional characterization of HpnG as a purine nucleoside phosphorylase,which converts adenosylhopane to phosphoribosylhopane in the presence of phosphate.HpnG demonstrates broad substrate specificity and impressive stability,making it a valuable enzymatic tool for applications in nucleoside processing and related biotechnology.

Zoonotic human liver flukes,a type 1 biocarcinogen,in freshwater fishes:genetic analysis and confirmation of molluscan vectors and reservoir hosts in Bangladesh

Background Opisthorchiid flukes,particularly Opisthorchis viverrini,Opisthorchis felineus,Clonorchis sinensis,and Metorchis spp.are the most common fish-borne zoonotic human liver flukes(hLFs).Liver fluke infections are more prevalent in resource-deprived and underprivileged areas.We herein estimated the prevalence of the metacercariae(MC)of major hLFs in common large freshwater fishes(lFWF)marketed for human consumption from some selected areas of Bangladesh along with detection of their molluscan vectors and reservoirs.Methods The current status of fish-borne zoonotic hLF infections in lFWF was investigated along with their molluscan vectors and mammalian reservoir hosts in Mymensingh and Kishoreganj in Bangladesh from July 2018-June 2022 using conventional and multiple molecular techniques,such as PCR,PCR-restriction fragment length polymorphism(RFLP),sequencing,and bioinformatic analyses.The infection rate of fishes was analyzed using the Z-test and the loads of MC were compared using the chi-squared(χ^(2))test.Results The MC of C.sinensis,Opisthorchis spp.,and Metorchis spp.were detected in 11 species of common and popular lFWF.In lFWF,the estimated prevalence was 18.7%and the mean load was 137.4±149.8 MC per 100 g of fish.The prevalence was the highest(P<0.05)in spotted snakehead fishes(Channa punctata,63.6%).The highest rate of infection(P<0.05)was observed with the MC of C.sinensis(11.8%).Metacercariae were almost equally(P>0.05)distributed between the head and body of fishes.The infection rate was slightly higher in cultured(19.6%)fishes.The MC of C.sinensis,O.felineus,O.viverrini,and Metorchis orientalis in fishes were confirmed using PCR,PCR-RFLP and bioinformatics.The cercariae of opisthorchiid(Pleurolophocercus cercariae)flukes were only recovered from Bithynia spp.(3.9%,42 out of 1089).The ova of hLFs from dogs(4.3%,5 out of 116)and cats(6.0%,6 out of 100),and adult flukes(M.orientalis)from ducks(41.1%113 out of 275)were detected.Conclusions The MC of hLFs are highly prevalent in fresh water fishes in Bangladesh.Reservoir hosts,such as street dogs,cats,and ducks carried the patent infection,and residents of Bangladesh are at risk.

Inactivation of the positive LuxR-type oligomycin biosynthesis regulators OlmRI and OlmRII increases avermectin production in Streptomyces avermitilis

Oligomycins are a group of 26 macrocyclic lactones that exhibit broad biological activities,including antifungal,anticancer and nematocidal activities.Analysis of the oligomycin biosynthetic gene cluster (olm) in S.avermitilis revealed 2 tandem LuxR-type regulators,OlmRI (931 aa) and OlmRII (941 aa),with shared identity of 38%.Gene replacement of olmRI or olmRII abolished oligomycin production,and this production could be partially restored in the disruptants by introducing cloned olmRI and olmRII with their native promoters,demonstrating the essential role of OlmRI and OlmRII for oligomycin biosynthesis.Quantitative real-time RT-PCR analysis revealed that transcription of 14 olm genes was differentially affected by the deletion of olmRI and olmRII.Unexpectedly,avermectin production in both mutants was enhanced at least 4-fold.The identification of the positive cluster-situated regulators,OlmRI and OlmRII,paves the way for the transcriptional analysis of oligomycin biosynthesis and for the enhancement of oligomycin and avermectin production through regulator engineering.

A LuxR family transcriptional regulator AniF promotes the production of anisomycin and its derivatives in Streptomyces hygrospinosus var.beijingensis

The protein synthesis inhibitor anisomycin features a unique benzylpyrrolidine system and exhibits potent selective activity against pathogenic protozoa and fungi.It is one of the important effective components in Agricultural Antibiotic120,which has been widely used as naturally-originated agents for treatment of crop decay in China.The chemical synthesis of anisomycin has recently been reported,but the complex process with low productivity made the biosynthesis still to be a vital mainstay in efforts.The biosynthetic gene cluster(BGC)of anisomycin in Streptomyces hygrospinosus var.beijingensis has been identified in our previous work,while poor understanding of the regulatory mechanism limited the yield enhancement via regulation engineering of S.hygrospinosus var.beijingensis.In this study here,we characterized AniF as an indispensable LuxR family transcriptional regulator for the activation of anisomycin biosynthesis.The genetic manipulations of aniF and the real-time quantitative PCR(RT-qPCR)revealed that it positively regulated the transcription of the anisomycin BGC.Moreover,the overexpression of aniF contributed to the improvement of the production of anisomycin and its derivatives.Dissection of the mechanism underlying the function of AniF revealed that it directly activated the transcription of the genes aniR-G involved in anisomycin biosynthesis.Especially,one AniF-binding site in the promoter region of aniR was identified by DNase I footprinting assay and an inverted repeat sequence(5′-GGGC-3′)composed of two 4-nt half sites in the protected region was found.Taken together,our systematic study confirmed the positive regulatory role of AniF and might facilitate the future construction of engineering strains with high productivity of anisomycin and its derivatives.

Improving acarbose production and eliminating the by-product component C with an efficient genetic manipulation system of Actinoplanes sp.SE50/110

Theα-glucosidase inhibitor acarbose is commercially produced by Actinoplanes sp.and used as a potent drug in the treatment of type-2 diabetes.In order to improve the yield of acarbose,an efficient genetic manipulation system for Actinoplanes sp.was established.The conjugation system between E.coli carryingØC31-derived integrative plasmids and the mycelia of Actinoplanes sp.SE50/110 was optimized by adjusting the parameters of incubation time of mixed culture(mycelia and E.coli),quantity of recipient cells,donor-to-recipient ratio and the concentration of MgCl2,which resulted in a high conjugation efficiency of 29.4%.Using this integrative system,a cloned acarbose biosynthetic gene cluster was introduced into SE50/110,resulting in a 35%increase of acarbose titer from 2.35 to 3.18 g/L.Alternatively,a pIJ101-derived replicating plasmid combined with the counter-selection system CodA(sm)was constructed for gene inactivation,which has a conjugation frequency as high as 0.52%.Meanwhile,almost all 5-flucytosine-resistant colonies were sensitive to apramycin,among which 75%harbored the successful deletion of targeted genes.Using this replicating vector,the maltooligosyltrehalose synthase gene treY responsible for the accumulation of component C was inactivated,and component C was eliminated as detected by LC-MS.Based on an efficient genetic manipulation system,improved acarbose production and the elimination of component C in our work paved a way for future rational engineering of the acarbose-producing strains.

Characterization of the positive SARP family regulator PieR for improving piericidin A1 production in Streptomyces piomogeues var.Hangzhouwanensis

Piericidin A1,a member ofɑ-pyridone antibiotic,exhibits various biological activities such as antimicrobial,antifungal,and antitumor properties and possesses potent respiration-inhibitory activity against insects due to its competitive binding capacity to mitochondrial complex I.The biosynthetic pathway of piericidin A1 has been reported in Streptomyces piomogeues var.Hangzhouwanensis,while the regulatory mechanism remains poorly understood.In this study,a Streptomyces antibiotic regulatory protein(SARP)family transcriptional regulator PieR was characterized.Genetic disruption and complementation manipulations revealed that PieR positively regulated the production of piericidin A1.Moreover,the overexpression of pieR contributed to the improvement of piericidin A1 productivity.The real-time quantitative PCR(RT-qPCR)was carried out and the data showed that pieR stimulated the transcription of all the biosynthesis-related genes for piericidin A1.In order to explore the regulatory mechanism,electrophoresis mobility shift assays(EMSA)and DNase I footprinting experiments have been conducted.A protected region covering 50 nucleotides within the upstream region of pieR was identified and two 5-nt direct repeat sequences(5′-CCGGA-3′)in the protected region were found.These findings,taken together,set stage for transcriptional control engineering in the view of optimizing piericidin A1 production and thus provide a viable potent route for the construction of strains with high productivity.

Radical SAM-Dependent Demetallation of Heme

Demetallation of heme to release iron is a chemical difficult reaction and is highly rare in biochemistry,with apoferritin as the only known enzyme responsible for this process.Here we show the heme degradation enzyme Chuw catalyzes heme demetallation besides its known methyltransferase activity(which converts heme to a ring-open product anaerobilin).We show the demetallation activity of Chuw is radical SAM-dependent,and likely involves the same set of intermediates involved in the anaerobilin-producing pathway.The ChuW-catalyzed demetallation reaction does not require external reductant,and can occur on several heme analogs with different metal centers.These findings establish a brand-new chemistry in the radical SAM enzymes,highlighting the remarkable catalytic diversity of this superfamily of enzymes.

Comparative functional genomics of the acarbose producers reveals potential targets for metabolic engineering

Theα-glucosidase inhibitor acarbose is produced in large-scale by strains derived from Actinoplanes sp.SE50 and used widely for the treatment of type-2 diabetes.Compared with the wild-type SE50,a high-yield derivative Actinoplanes sp.SE50/110 shows 2-fold and 3–7-fold improvement of acarbose yield and acb cluster transcription,respectively.The genome of SE50 was fully sequenced and compared with that of SE50/110,and 11 SNVs and 4 InDels,affecting 8 CDSs,were identified in SE50/110.The 8 CDSs were individually inactivated in SE50.Deletions of ACWT_4325(encoding alcohol dehydrogenase)resulted in increases of acarbose yield by 25%from 1.87 to 2.34 g/L,acetyl-CoA concentration by 52.7%,and PEP concentration by 22.7%.Meanwhile,deletion of ACWT_7629(encoding elongation factor G)caused improvements of acarbose yield by 36%from 1.87 to 2.54 g/L,transcription of acb cluster,and ppGpp concentration to 2.2 folds.Combined deletions of ACWT_4325 and ACWT_7629 resulted in further improvement of acarbose to 2.83 g/L(i.e.76%of SE50/110),suggesting that the metabolic perturbation and improved transcription of acb cluster caused by these two mutations contribute substantially to the acarbose overproduction.Enforced application of similar strategies was performed to manipulate SE50/110,resulting in a further increase of acarbose titer from 3.73 to 4.21 g/L.Therefore,the comparative genomics approach combined with functional verification not only revealed the acarbose overproduction mechanisms,but also guided further engineering of its high-yield producers.

SecReT6 update:a comprehensive resource of bacterial TypeⅥSecretion Systems

TypeⅥSecretion System(T6SS)plays significant roles in microbial activities via injecting effectors into adjacent cells or environments.T6SS increasingly gained attention due to its important influence on pathogenesis,microbial competition,etc.T6SS-associated research is explosively expanding on numerous grounds that call for an efficient resource.The SecReT6 version3 provides comprehensive information on T6SS and the interactions between T6SS and T6SS-related proteins such as T6SS regulators and T6SS effectors.To assist T6SS researches like microbial competition and regulatory mechanisms,SecReT6 v3developed online tools for detection and analysis of T6SS and T6SS-related proteins and estimation of T6SS-dependent killing risk.We have identified a novel T6SS regulator and T6SS-dependent killing capacity in Acinetobacter baumannii clinical isolates with the aid of SecReT6 v3.17,212 T6SSs and plentiful T6SS-related proteins in 26,573 bacterial complete genomes were also detected,analyzed and incorporated into the database.The database is freely available at https://bioinfo-mml.sjtu.edu.cn/SecReT6/.

Guided by the principles of microbiome engineering:Accomplishments and perspectives for environmental use

Although the accomplishments of microbiome engineering highlight its significance for the targeted manipulation of microbial communities,knowledge and technical gaps still limit the applications of microbiome engineering in biotechnology,especially for environmental use.Addressing the environmental challenges of refractory pollutants and fluctuating environmental conditions requires an adequate understanding of the theoretical achievements and practical applications of microbiome engineering.Here,we review recent cutting-edge studies on microbiome engineering strategies and their classical applications in bioremediation.Moreover,a framework is summarized for combining both top-down and bottom-up approaches in microbiome engineering toward improved applications.A strategy to engineer microbiomes for environmental use,which avoids the build-up of toxic intermediates that pose a risk to human health,is suggested.We anticipate that the highlighted framework and strategy will be beneficial for engineering microbiomes to address difficult environmental challenges such as degrading multiple refractory pollutants and sustain the performance of engineered microbiomes in situ with indigenous microorganisms under fluctuating conditions.

Metabolic engineering of a methyltransferase for production of drug precursors demecycline and demeclocycline in Streptomyces aureofaciens

Demecycline(DMTC)and demeclocycline(DMCTC)are C6-demethylated derivatives of tetracycline(TC)and chlortetracycline(CTC),respectively.They are precursors of minocycline and tigecycline,which showed remarkable bioactivity against TC-resistant bacteria and have been used clinically for decades.In order to biosynthesize drug precursors DMTC and DMCTC,the function of a possible C-methyltransferase encoding gene ctcK was studied systematically in the CTC high-yielding industrial strain Streptomyces aureofaciens F3.TheΔctcK mutant accumulated two new products,which were turned out to be DMTC and DMCTC.Meanwhile,timecourse analysis of the fermentation products detected the epimers of DMTC and DMCTC transformed spontaneously.Finally,an engineering strain with higher productivity of DMCTC was constructed by deleting ctcK and overexpressing ctcP of three extra copies simultaneously.Construction of these two engineering strains not only served as a successful example of synthesizing required products through metabolic engineering,but also provided original strains for following elaborate engineering to synthesize more effective tetracycline derivatives.

Characterization of the tunicamycin gene cluster unveiling unique steps involved in its biosynthesis

Tunicamycin,a potent reversible translocase I inhibitor,is produced by several Actinomycetes species.The tunicamycin structure is highly unusual,and contains an 11-carbon dialdose sugar and anα,β-1″,11′-glycosidic linkage.Here we report the identification of a gene cluster essential for tunicamycin biosynthesis by high-throughput heterologous expression(HHE)strategy combined with a bioassay.Introduction of the genes into heterologous non-producing Streptomyces hosts results in production of tunicamycin by these strains,demonstrating the role of the genes for the biosynthesis of tunicamycins.Gene disruption experiments coupled with bioinformatic analysis revealed that the tunicamycin gene cluster is minimally composed of 12 genes(tunA–tunL).Amongst these is a putative radical SAM enzyme(Tun B)with a potentially unique role in biosynthetic carbon-carbon bond formation.Hence,a seven-step novel pathway is proposed for tunicamycin biosynthesis.Moreover,two gene clusters for the potential biosynthesis of tunicamycin-like antibiotics were also identified in Streptomyces clavuligerus ATCC 27064 and Actinosynnema mirums DSM 43827.These data provide clarification of the novel mechanisms for tunicamycin biosynthesis,and for the generation of new-designer tunicamycin analogs with selective/enhanced bioactivity via combinatorial biosynthesis strategies.

Genome Mining and Biosynthesis Study of a Type B Linaridin Reveals a Highly Versatileα-N-Methyltransferase

Linaridins are a small but growing family of natural products belonging to the ribosomally synthesized and post-translationally modified peptide(RiPP)superfamily.In this study,a genome mining approach led to the identification of a novel linaridin,mononaridin(MON),from Streptomyces monomycini.In-frame deletion genetic knockout studies showed that,in addition to many genes essential for MON biosynthesis,monM encodes an S-adenosyl methionine(SAM)-dependentα-N-methyltransferase that is responsible for installing two methyl groups in the MON N-terminus.Besides SAM,MonM also accepts ethyl-SAM and allyl-SAM,in which the methyl of SAM is replaced by an ethyl and an allyl,respectively.We showed that ethyl-SAM and allyl-SAM have distinct reactivities in MonM catalysis,and this observation was further investigated in detail by density functional theory(DFT)calculations.Remarkably,MonM acts efficiently on nisin,a prototypic lantibiotic that is structurally very different from the native substrate,and the ability of MonM to transfer an allyl group to the nisin N-terminus allowed production of a fluorescently labeled nisin,which can be further used in microscopic cell analysis.Our studies provide new insights into linaridin biosynthesis and demonstrate the potential of linaridin methyltransferases in bioengineering applications.

Multiple functions of the vacuole in plant growth and fruit quality

Vacuoles are organelles in plant cells that play pivotal roles in growth and developmental regulation.The main functions of vacuoles include maintaining cell acidity and turgor pressure,regulating the storage and transport of substances,controlling the transport and localization of key proteins through the endocytic and lysosomal-vacuolar transport pathways,and responding to biotic and abiotic stresses.Further,proteins localized either in the tonoplast(vacuolar membrane)or inside the vacuole lumen are critical for fruit quality.In this review,we summarize and discuss some of the emerging functions and regulatory mechanisms associated with plant vacuoles,including vacuole biogenesis,vacuole functions in plant growth and development,fruit quality,and plant-microbe interaction,as well as some innovative research technology that has driven advances in the field.Together,the functions of plant vacuoles are important for plant growth and fruit quality.The investigation of vacuole functions in plants is of great scientific significance and has potential applications in agriculture.

Changes of calcium cycling in HFrEF and HFpEF

Dysfunctions of calcium cycling occur in heart failure wi+reduced and preserved ejection fraction(HFrEF and HFpEF).HFrEF and HFpEF showed Ca^(2+)leakage at diastole.the compensation of Na^(+)/Ca^(2+)exchanger andthe decrease of T-tubule density reduces cytoplasmic Ca^(2+)concentration in HFrEF and impairs systolic function.In contrast,HFpEF hasthe increase of cytoplasmic Ca^(2+)concentration and diastolic dysfunctions.the decrease of mitochondrial Ca^(2+)concentration weakens myocardial contractility in HFrEF while the increased concentration retainsthe contractility in HFpEF.Here,the changes of calcium cycling in HFrEF and HFpEF are summarized andthe possibility of relevanttherapeutic targets is discussed.

Microbial production of cis,cis-muconic acid from aromatic compounds in engineered Pseudomonas

Industrial expansion has led to environmental pollution by xenobiotic compounds like polycyclic aromatic hydrocarbons and monoaromatic hydrocarbons.Pseudomonas spp.have broad metabolic potential for degrading aromatic compounds.The objective of this study was to develop a“biological funneling”strategy based on genetic modification to convert complex aromatic compounds into cis,cis-muconate(ccMA)using Pseudomonas putida B6-2 and P.brassicacearum MPDS as biocatalysts.The engineered strains B6-2(B6-2ΔcatBΔsalC)and MPDS(MPDSΔsalC(pUCP18k-catA))thrived with biphenyl or naphthalene as the sole carbon source and produced ccMA,attaining molar conversions of 95.3%(ccMA/biphenyl)and 100%(ccMA/naphthalene).Under mixed substrates,B6-2ΔcatBΔsalC grew on biphenyl as a carbon source and transformed ccMA from non-growth substrates benzoate or salicylate to obtain higher product concentration.Inserting exogenous clusters like bedDC1C2AB and xylCMAB allowed B6-2 recombinant strains to convert benzene and toluene to ccMA.In mixed substrates,constructed consortia of engineered strains B6-2 and MPDS specialized in catabolism of biphenyl and naphthalene;the highest molar conversion rate of ccMA from mixed substrates was 85.2%when B6-2ΔcatBΔsalC was added after 24 h of MPDSΔsalC(pUCP18k-catA)incubation with biphenyl and naphthalene.This study provides worthwhile insights into efficient production of ccMA from aromatic hydrocarbons by reusing complex pollutants.

Staged-probability strategy of processing shotgun proteomic data to discover more functionally important proteins

Biologically important proteins related to membrane receptors,signal transduction,regulation,transcription,and translation are usually low in abundance and identified with low probability in mass spectroscopy(MS)-based analyses.Most valuable proteomics information on them were hitherto discarded due to the application of excessively strict data filtering for accurate identification.In this study,we present a stagedprobability strategy for assessing proteomic data for potential functionally important protein clues.MS-based protein identifications from the second(L2)and third(L3)layers of the cascade affinity fractionation using the Trans-Proteomic Pipeline software were classified into three probability stages as 1.00–0.95,0.95–0.50,and 0.50–0.20 according to their distinctive identification correctness rates(i.e.100%–95%,95%–50%,and 50%–20%,respectively).We found large data volumes and more functionally important proteins located at the previously unacceptable lower probability stages of 0.95–0.50 and 0.50–0.20 with acceptable correctness rate.More importantly,low probability proteins in L2 were verified to exist in L3.Together with some MS spectrogram examples,comparisons of protein identifications of L2 and L3 demonstrated that the stagedprobability strategy could more adequately present both quantity and quality of proteomic information,especially for researches involving biomarker discovery and novel therapeutic target screening.