The xylitol dehydrogenase(XDH)is a crucial enzyme involved in the xylose utilization in pentose⁃catabolizing yeasts and fungi.In addition to producing xylulose,XDH can also be employed to develop a biosensor for monit...The xylitol dehydrogenase(XDH)is a crucial enzyme involved in the xylose utilization in pentose⁃catabolizing yeasts and fungi.In addition to producing xylulose,XDH can also be employed to develop a biosensor for monitoring xylitol concentration.In this study,the gene encoding the thermophilic fungus Talaromyces emersonii XDH(TeXDH)was heterologously expressed in Escherichia coli BL21(DE3)at 16℃in the soluble form.Recombinant TeXDH with high purity was purified by using a Ni⁃NTA affinity column.Size⁃exclusion chromatography and SDS⁃PAGE analysis demonstrated that the puri⁃fied recombinant TeXDH exists as a native trimer with a molecular mass of approximately 116 kD,and is composed of three identical subunits,each with a molecular weight of around 39 kD.The TeXDH strictly preferred NAD^(+)as a coenzyme to NADP^(+).The optimal temperature and pH of the TeXDH were 40℃and 10.0,respectively.After EDTA treatment,the enzyme activity of TeXDH decreased to 43.26%of the initial enzyme activity,while the divalent metal ions Mg^(2+)or Ca^(2+)could recover the enzyme activity of TeXDH,reaching 103.32%and 110.69%of the initial enzyme activity,respectively,making them the optimal divalent metal ion cofactors for TeXDH enzyme.However,the divalent metal ions of Mn^(2+),Ni^(2+),Cu^(2+),Zn^(2+),Co^(2+),and Cd^(2+)significantly inhibited the activity of TeXDH.ICP⁃MS and molecular doc⁃king studies revealed that 1 mol/L of TeXDH bound 2 mol/L Zn^(2+)ions and 1 mol/L Mg^(2+)ion.Further⁃more,TeXDH exhibited a high specificity for xylitol,laying the foundation for the development of future xylitol biosensors.展开更多
Late embryogenesis abundant(LEA)proteins play an important role in plant growth and development,as well as in the plant response to various abiotic stresses.In this study,CsLEA1,a novel gene encoding a LEA_3 subfamily...Late embryogenesis abundant(LEA)proteins play an important role in plant growth and development,as well as in the plant response to various abiotic stresses.In this study,CsLEA1,a novel gene encoding a LEA_3 subfamily protein,was successfully cloned froma tea plant[Camellia sinensis(L.)O.Kuntze].Bioinformatics analysis and prokaryotic expression assays showed that CsLEA1 is a typical hydrophilic protein with a molecular weight of approximately 10.4 kD.Expression analyses revealed that the transcription of CsLEA1 in C.sinensis leaves was significantly induced by cold stress.In addition,the heterologous expression of CsLEA1 increased the tolerance of Escherichia coli and yeast to cold stress,which might be closely related to the low molecular weight and high hydrophilicity of the CsLEA1.Taken together,our results suggest that CsLEA1 might have an important function in the tolerance of C.sinensis to cold stress,thus providing a potential application in molecular breeding to enhance the cold stress tolerance of tea plants.展开更多
Lysobacter harbors a plethora of cryptic biosynthetic gene clusters(BGCs),albeit only a limited number have been analyzed to date.In this study,we described the activation of a cryptic polyketide synthase(PKS)/nonribo...Lysobacter harbors a plethora of cryptic biosynthetic gene clusters(BGCs),albeit only a limited number have been analyzed to date.In this study,we described the activation of a cryptic polyketide synthase(PKS)/nonribosomal peptide synthetase(NRPS)gene cluster(lsh)in Lysobacter sp.DSM 3655 through promoter engineering and heterologous expression in Streptomyces sp.S001.As a result of this methodology,we were able to isolate two novel linear lipopeptides,lysohexaenetides A(1)and B(2),from the recombinant strain S001-lsh.Furthermore,we proposed the biosynthetic pathway for lysohexaenetides and identified LshA as another example of entirely iterative bacterial PKSs.This study highlights the potential of heterologous expression systems in uncovering cryptic biosynthetic pathways in Lysobacter genomes,particularly in the absence of genetic manipulation tools.展开更多
Natural cyclohexapeptide AFN A_(1) from Streptomyces alboflavus 313 has moderate antibacterial and antitumor activities.An artificial designed AFN A_(1) homodimer,di-AFN A_(1),is an antibiotic exhibiting 10 to 150 fol...Natural cyclohexapeptide AFN A_(1) from Streptomyces alboflavus 313 has moderate antibacterial and antitumor activities.An artificial designed AFN A_(1) homodimer,di-AFN A_(1),is an antibiotic exhibiting 10 to 150 fold higher biological activities,compared with the monomer.Unfortunately,the yield of di-AFN A_(1) is very low(0.09±0.03 mg·L^(−1))in the engineered strain Streptomyces alboflavus 313_hmtS(S.albo/313_hmtS),which is not friendly to be genetically engineered for titer improvement of di-AFN A_(1) production.In this study,we constructed a biosynthetic gene cluster for di-AFN A_(1) and increased its production through heterologous expression.During the collection of di-AFN A_(1) biosynthetic genes,the afn genes were located at three sites of S.alboflavus 313 genome.The di-AFN A_(1) biosynthetic gene cluster(BGC)was first assembled on one plasmid and introduced into the model strain Streptomyces lividans TK24,which produced di-AFN A_(1) at a titer of 0.43±0.01 mg·L^(−1).To further increase the yield of di-AFN A_(1),the di-AFN A_(1) BGC was multiplied and split to mimic the natural afn biosynthetic genes,and the production of di-AFN A_(1) increased to 0.62±0.11 mg·L^(−1) in S.lividans TK24 by the later strategy.Finally,different Streptomyces hosts were tested and the titer of di-AFN A_(1) increased to 0.81±0.17 mg·L^(−1),about 8.0-fold higher than that in S.albo/313_hmtS.Successful heterologous expression of di-AFN A_(1) with a remarkable increased titer will greatly facilitate the following synthetic biological study and drug development of this dimeric cyclohexapeptide.展开更多
The use of antimicrobial peptides(AMPs)synthesized by bacteria(bacteriocins)is an alternative for combating multidrug resistant bacterial strains and their production by recombinant route is a viable option for their ...The use of antimicrobial peptides(AMPs)synthesized by bacteria(bacteriocins)is an alternative for combating multidrug resistant bacterial strains and their production by recombinant route is a viable option for their mass production.The bacteriocin E-760 isolated from the genus Enterococcus sp.has been shown to possess inhibitory activity against Gram-negative and Gram-positive bacteria.In this study,the expression of a chimeric protein coding for E-760 in the nucleus of C.reinhardtii was evaluated,as well as,its antibacterial activity.The synthetic gene E-760S was inserted into the genome of C.reinhardtii using Agrobacterium tumefaciens.A transgenic line was identified in TAP medium with hygromycin and also by PCR.The increment in the culture medium temperature of the transgenic strain at 35°C for 10 minutes,increased the production level of the recombinant protein from 0.14(Noninduced culture,NIC)to 0.36%(Induced culture,IC)of total soluble proteins(TSP);this was quantified by an ELISA assay.Recombinant E-760 possesses activity against Staphylococcus aureus in 0.34 U log,Streptococcus agalactiae in 0.48 U log,Enterococcus faecium in 0.36 U log,Pseudomonas aeruginosa in 2 U log and for Klebsiella pneumoniae,the activity was 0.07 U log.These results demonstrate that the nucleus transformation of C.reinhardtii can function as a stable expression platform for the production of the synthetic gene E-760 and it can potentially be used as an antibacterial agent.展开更多
Objective To characterize a possible retention function of unique sequence in the 5＇end of rat testis GABAA receptor β3t splicing variant Methods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and t...Objective To characterize a possible retention function of unique sequence in the 5＇end of rat testis GABAA receptor β3t splicing variant Methods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and two eukaryotic expression recombinant plasmids of pEGFP-N1 and pEGFP-C1 were constructed respectively by fusing green fluorescent protein to the N or C-terminus of β3t isoform. The recombinant plasmids were transfected into CHO cells by calcium phosphate co-precipitation method Fluorescence microscope and laser confocal microscope were used to analyze localization of β3t in the transfected cells. ConA-Texas-Red was used to label cell ER and the localization of rat testis β3t splicing variant in CHO cells was determined. Results When rat testis β3t splicing variant was expressed in CHO cells, two expression patterns were delineated, the distributions of uniform and mainly discrete intracellular compartments respectively, The chimera product failed to be translocated into the cell surface when expressed in ClIO cells; whereas the β3 subunit of rat brain was incorporated into the plasma membrane. Conclusion The inability of β3t to target into the ER may be a consequence of the unique 25 specific amino acid segments in the N terminus.展开更多
Unspecific peroxygenases(UPOs)are glycosylated enzymes that provide an efficient method for oxyfunctionalizing a variety of substrates using only hydrogen peroxide(H2O2)as the oxygen donor.However,their poor heterolog...Unspecific peroxygenases(UPOs)are glycosylated enzymes that provide an efficient method for oxyfunctionalizing a variety of substrates using only hydrogen peroxide(H2O2)as the oxygen donor.However,their poor heterologous expression has hindered their practical application.Here,a novel UPO from Marasmius fiardii PR910(MfiUPO)was identified and heterologously expressed in Pichia pastoris.By employing a two-copy expression cassette,the protein titer reached 1.18 g L−1 in a 5 L bioreactor,marking the highest record.The glycoprotein rMfiUPO exhib-ited a smeared band in the 40 to 55 kDa range and demonstrated hydroxylation,epoxidation and alcohol oxidation.Moreover,the peroxidative activity was enhanced by 150%after exposure to 50%(v/v)acetone for 40 h.A semipreparative production of 4-OH-β-ionone on a 100 mL scale resulted in a 54.2%isolated yield with 95%purity.With its high expression level,rMfiUPO is a promising candidate as an excellent parental template for enhancing desirable traits such as increased stability and selectivity through directed evolution,thereby meeting the necessary criteria for practical application.展开更多
Dear Editor,Gene duplication and loss are widespread in the plant kingdom and contribute substantially to phenotypic diversification and environmental adaptation.Polyploid species,particularly allopolyploids,combine d...Dear Editor,Gene duplication and loss are widespread in the plant kingdom and contribute substantially to phenotypic diversification and environmental adaptation.Polyploid species,particularly allopolyploids,combine divergent yet compatible progenitor genomes,a process that often triggers extensive gene loss during evolution(Chen et al.,2007).The Brassica"triangle of U"provides a classical model for investigating gene loss patterns.In this system,the diploid species Brassica rapa(B.rapa,AA,2n=20),Brassica nigra(B.nigra,BB,2n=16),and Brassica oleracea(B.oleracea,CC,2n=18)hybridized pairwise to give rise to the allopolyploids Brassica juncea(B.juncea,AABB,2n=36),Brassica carinata(B.carinata,BBCC,2n=34),and Brassica napus(B.napus,AACC,2n=38)(Yim et al.,2022).展开更多
The low-expression level of lactoferrin(LF)in the production process poses a significant challenge.This study aimed to effi-ciently express bovine lactoferrin(BLF)using Pichia pastoris GS115 as the expression host and...The low-expression level of lactoferrin(LF)in the production process poses a significant challenge.This study aimed to effi-ciently express bovine lactoferrin(BLF)using Pichia pastoris GS115 as the expression host and PIC9K as the recombinant vector.Optimization strategies included codon usage,promoter selection,and fermentation conditions.The blf gene was optimized for P.pastoris GS115 bias,resulting in the construction of the recombinant vector pPIC9K-UBLF1-3 controlled by the AOX1 promoter.SDS-PAGE analysis revealed soluble and efficient expression of ublf3 in P.pastoris GS115,with a molecular mass of approximately 76 kDa.The transformant P.pastoris GS115/pGAP9K-UBLF3-4 resistant at 4 mg·mL^(−1)G418,exhibited a ublf3 gene copy number of 5.88 through high-copy screening.Optimal expression conditions of recombi-nant UBLF were determined as 24℃,pH 5.0 and 220 r·min^(−1)through fermentation condition optimization.Under these con-ditions,recombinant UBLF production reached 40.62 mg·L^(−1).The yield of recombinant UBLF was reached 824.93 mg·L^(−1)through high-density fermentation.Antibacterial assay demonstrated the efficacy of recombinant UBLF against Escherichia coli JM109 and Staphylococcus aureus CGMCC 1.282.This study successfully achieved the efficient heterologous expression of recombinant UBLF in P.pastoris GS115,providing valuable insight for industrial production and the potential develop-ment of natural antibacterial agents.展开更多
Insect chitinases are involved in degradation of chitin from the exoskeleton or peritrophic metrix ofmidgut. In Locusta migratoria, two duplicated Cht5s （LmCht5-1 and LmCht5-2） have been shown to have distinct molec...Insect chitinases are involved in degradation of chitin from the exoskeleton or peritrophic metrix ofmidgut. In Locusta migratoria, two duplicated Cht5s （LmCht5-1 and LmCht5-2） have been shown to have distinct molecular characteristics and biological roles. To explore the protein properties of the two LmCht5s, we heterologously expressed both enzymes using baculovirus expression system in SF9 cells, and characterized kinetic and carbohydrate-binding properties of purified enzymes. LmCht5-1 and LmCht5-2 exhibited similar pH and temperature optimums. LmCht5-1 has lower Km value for the oligomeric substrate （4MU-（GlcNAe）3）, and higher Km value for the longer substrate （CM-Chitin- RBV） compared with LmCht5-2. A comparison of amino acids and homology modeling of catalytic domain presented similar TIM barrel structures and differentiated amino acids between two proteins. LmCht5-1 has a chitin-binding domain （CBD） tightly bound to colloidal chitin, but LmCht5-2 does not have a CBD for binding to colloidal chitin. Our results suggested both LmCht5- I and LmCht5-2, which have the critical glutamate residue in region II of catalytic domain, exhibited chitinolytic activity cleaving both polymeric and oligomeric substrates. LmCht5-1 had relatively higher activity against the oligomeric substrate, 4MU-（GlcNAc）3, whereas LmCht5-2 exhibited higher activity toward the longer substrate, CM-Chitin-RBE These findings are helpful for further research to clarit}g their different roles in insect growth and development.展开更多
Streptothricins (STs) are used commercially to treat bacterial and fungal diseases in agriculture. Mining of the sequenced microbial genomes uncovered two cryptic ST clusters from Streptomyces sp. C and Streptomyces s...Streptothricins (STs) are used commercially to treat bacterial and fungal diseases in agriculture. Mining of the sequenced microbial genomes uncovered two cryptic ST clusters from Streptomyces sp. C and Streptomyces sp. TP-A0356. The ST cluster from S. sp. TP-A0356 was verified by successful heterologous expression in Streptomyces coelicolor M145. Two new ST analogs were produced together with streptothricin F and streptothricin D in the heterologous host. The ST cluster was further confirmed by inactivation of gene stnO, which was proposed encoding an aminomutase supplying -lysines for the poly-β-Lys chain formation. A putative biosynthetic pathway for STs is proposed based on bioinformatics analyses of the ST genes and experimental evidence.展开更多
Avermectins,a group of polyketide natural products,are widely used as anthelmintics in agriculture.Metabolic engineering and combinatorial biosynthesis were extensively employed to improve Avermectins production and c...Avermectins,a group of polyketide natural products,are widely used as anthelmintics in agriculture.Metabolic engineering and combinatorial biosynthesis were extensively employed to improve Avermectins production and create novel Avermectin derivatives,including Ivermectin and Doramectin.It is labor intensive and time cost to genetically manipulate Avermectins producer Streptomyces avermitilis in vivo.Cloning and heterologous expression of Avermectins biosynthetic gene cluster will make it possible to tailor the cluster in vitro.We constructed a Bacterial Artificial Chromosome(BAC)library of S.avermitilis ATCC 31267 with inserted DNA fragments ranged from 100 to 130 Kb.Five recombinant BAC clones which carried the Avermectins biosynthetic gene cluster ave(81 Kb in size)were screened out from the library.Then,ave was hetero-expressed in S.lividans.Three Avermectin components,A2a,B1a and A1a were detected from the cell extracts of recombinant strains.It will facilitate the development of Avermectin derivatives by polyketide synthase domain swapping and provide functional element for Avermectins synthetic biology study.展开更多
Basic helix-loop-helix(bHLH)transcription factors are widely distributed in eukaryotes,and in plants,they regulate many biological processes,such as cell differentiation,development,metabolism,and stress responses.Few...Basic helix-loop-helix(bHLH)transcription factors are widely distributed in eukaryotes,and in plants,they regulate many biological processes,such as cell differentiation,development,metabolism,and stress responses.Few studies have focused on the roles of bHLH transcription factors in regulating growth,development,and stress responses in maize(Zea mays),even though such information would greatly benefit maize breeding programs.In this study,we cloned the maize transcription factor gene ZmbHLH36(Gene ID:100193615,GRMZM2G008691).ZmbHLH36 possesses conserved domains characteristic of the bHLH family.RT-qPCR analysis revealed that ZmbHLH36 was expressed at the highest level in maize roots and exhibited different expression patterns under various abiotic stress conditions.Transgenic Arabidopsis(Arabidopsis thaliana)plants heterologously expressing ZmbHLH36 had significantly longer roots than the corresponding non-transgenic plants under 0.1 and 0.15 mol L^(−1) NaCl treatment as well as 0.2 mol L^(−1) mannitol treatment.Phenotypic analysis of soil-grown plants under stress showed that transgenic Arabidopsis plants harboring ZmbHLH36 exhibited significantly enhanced drought tolerance and salt tolerance compared to the corresponding non-transgenic plants.Malondialdehyde contents were lower and peroxidase activity was higher in ZmbHLH36-expressing Arabidopsis plants than in the corresponding non-transgenic plants.ZmbHLH36 localized to the nucleus when expressed in maize protoplasts.This study provides a systematic analysis of the effects of ZmbHLH36 on root growth,development,and stress responses in transgenic Arabidopsis,laying a foundation for further analysis of its roles and molecular mechanisms in maize.展开更多
Globomycin is a cyclic lipodepsipeptide originally isolated from several Streptomyces species which displays strong and selective antibacterial activity against Gram-negative pathogens.Its mode of action is based on t...Globomycin is a cyclic lipodepsipeptide originally isolated from several Streptomyces species which displays strong and selective antibacterial activity against Gram-negative pathogens.Its mode of action is based on the competitive inhibition of the lipoprotein signal peptidase II(LspA),which is absent in eukaryotes and considered an attractive target for the development of new antibiotics.Despite its interesting biological properties,the gene cluster encoding its biosynthesis has not yet been identified.In this study we employed a genome-mining approach in the globomycin-producing Streptomyces sp.CA-278952 to identify a candidate gene cluster responsible for its biosynthesis.A null mutant was constructed using CRISPR base editing where production was abolished,strongly suggesting its involvement in the biosynthesis.The putative gene cluster was then cloned and heterologously expressed in Streptomyces albus J1074 and Streptomyces coelicolor M1146,therefore unambigu-ously linking globomycin and its biosynthetic gene cluster.Our work paves the way for the biosynthesis of new globomycin derivatives with improved pharmacological properties.展开更多
Microbial natural products and their derivatives have been developed as a considerable part of clinical drugs and agricultural chemicals.Marine microbial natural products exhibit diverse chemical structures and bioact...Microbial natural products and their derivatives have been developed as a considerable part of clinical drugs and agricultural chemicals.Marine microbial natural products exhibit diverse chemical structures and bioactivities with substantial potential for the development of novel pharmaceuticals.However,discovering compounds with new skeletons from marine microbes remains challenging.In recent decades,multiple approaches have been de-veloped to discover novel marine microbial natural products,among which heterologous expression has proven to be an effective method.Facilitated by large DNA cloning and comparative metabolomic technologies,a few novel bioactive natural products from marine microorganisms have been identified by the expression of their biosynthetic gene clusters(BGCs)in heterologous hosts.Heterologous expression is advantageous for character-izing gene functions and elucidating the biosynthetic mechanisms of natural products.This review provides an overview of recent progress in heterologous expression-guided discovery,biosynthetic mechanism elucidation,and yield optimization of natural products from marine microorganisms and discusses the future directions of the heterologous expression strategy in facilitating novel natural product exploitation.展开更多
Phospholipase A1(PLA_(1))is a member of the hydrolase family with applications in various fields,especially in the food industry.A calcium-independent PLA_(1) from Streptomyces albidoflavus was expressed in E.coli BL2...Phospholipase A1(PLA_(1))is a member of the hydrolase family with applications in various fields,especially in the food industry.A calcium-independent PLA_(1) from Streptomyces albidoflavus was expressed in E.coli BL21(DE3)in this study.The results indicated that the soluble expression of PLA_(1) was at low level,which was possibly due to the toxicity of PLA_(1) to the host.In contrast,the expression of the enzyme as inclusion bodies exhibited a high-level expression and 0.3 mg inclusion bodies protein could be derived from 1 mL culture medium.Furthermore,the renaturation of PLA_(1) was achieved through a direct dilution method,yielding 29.6 U/mL PLA_(1) activity after 16 h of renaturation at 4℃.For improving the efficiency of the dilution refolding process,a continuous refolding strategy was established,and 155 U/mL PLA_(1) activity was derived from the continuous refolding process.With soybean lecithin as the substrate,the specific activity of purified renatured PLA_(1) was 1380 U/mg and the optimal temperature and pH was found to be 60℃ and 6.5.In addition,the renatured PLA_(1) was observed with better activity towards phosphatidyl inositol,whilst lipase activity was detected when the catalyzing temperature was below 55℃.Overall,this study provides a possible solution to obtain calcium-independent PLA_(1) with high yield by heterologous expression in E.coli and hence to promote its further application in the field of food industry.展开更多
Small heat shock proteins(sHSPs)act as molecular chaperones that can prevent the accumulation of damaged proteins during abiotic stress,especially heat shock,but the mechanism is not clear.To study the function of sHS...Small heat shock proteins(sHSPs)act as molecular chaperones that can prevent the accumulation of damaged proteins during abiotic stress,especially heat shock,but the mechanism is not clear.To study the function of sHSPs in Lenzites gibbosa,a common polypore in northern temperate forests that causes spongy white rot of broadleaf trees,under temperature stress,L.gibbosa mycelia were grown at 25℃ for 9 d,treated at 33℃ for 15,30,60,and 120 min before sequencing the transcriptomes.From among 32 heat shock protein(HSP)genes found in the screen of the transcriptome data,a highly expressed gene was cloned and named Lghsp17.4.RT-qPCR was used to analyze the expression of the gene Lghsp17.4 under heat shock and dye stress.Both treatments induced higher expression of Lghsp17.4 at the transcriptional level,indicating that Lghsp17.4 might function in the response to heat stress and dye degradation.We previously found that L.gibbosa generally had a heat shock reaction(HSR)during degradation of aromatic compounds,and HSPs were always produced with manganese peroxidases(MnPs)and other lignin-degrading enzymes.Therefore,we measured the activity of MnPs in L.gibbosa after 33℃ heat shock to analyze the relationship between MnPs expression and Lghsp17.4 expression.Heat shocks of 0–30 min increased MnPs activity,and the change in MnPs activity were closely positively correlated with the expression levels of Lghsp17.4 over time,indicating a potential connection and interaction between LgHSP17.4 and MnPs during the HSR in L.gibbosa.Thus,LgHSP17.4 might have a positive regulatory effect on the HSR in L.gibbosa and be a critical component of a stress resistance mechanism.展开更多
Mangicol-type sesterterpenoids possess potent anti-inflammatory activity,characterized by a 5-5-6-5tetracyclic carbon skeleton formed by mangicdiene synthase Fg MS.Two proposed mechanisms for mangicdiene formation inv...Mangicol-type sesterterpenoids possess potent anti-inflammatory activity,characterized by a 5-5-6-5tetracyclic carbon skeleton formed by mangicdiene synthase Fg MS.Two proposed mechanisms for mangicdiene formation involve either C6-C10 cyclization(path a) or C2-C10 cyclization(path b) after the C10carbocation formation,but neither has been experimentally validated.Here,we have identified a second mangicdiene synthase Man D,which is derived from Fusarium sp.JNU-XJ070152-01 and shares high amino acid sequence identity with Fg MS.Through heterologous expression of man D in Aspergillus oryzae NSAR1,we observed production not only of mangicdiene(1) and variecoltetraene(2),previously identified by expression of Fg MS in Escherichia coli,but also two novel sesterterpene skeletons fusadiene(3)and fusatriene(4).The identification of fusadiene and fusatriene supports the occurrence of two key carbocation intermediates in path b,thus experimentally confirming that mangicdiene is built via path b for the first time,consistent with previous density functional theory(DFT) calculation results.展开更多
The lipH2 gene,encoding the expression of lignin peroxidase,was cloned from Phanerochaete chrysosporium BKM-F-1767 and expressed in Pichia pastoris X-33,a yeast.The cDNA of LiPH2 was generated from total RNA extracted...The lipH2 gene,encoding the expression of lignin peroxidase,was cloned from Phanerochaete chrysosporium BKM-F-1767 and expressed in Pichia pastoris X-33,a yeast.The cDNA of LiPH2 was generated from total RNA extracted from P chrysosporium by PCR with primers that do not contain a P.chrysosporium lignin peroxidase secretion signal.The gene was then successfully inserted into the expression vector pPICZα,and resulted in the recombinant vector pPICZα-lipH2.The transformation was conducted in two ways.One was using the wild Pichia pastoris as the recipients,which results in the recombinant P.pastoris with single or low lipH2 gene copy.The second was using P.pastoris and single or low lipH2 gene copy as the recipients,which results in the recombinant P.pastoris with multi-copies of lipH2 genes.This study firstly expressed the gene lipH2 in P.pastoris and achieved the successful expression of the lipH2 depending upon the generation of a recombinant strain that contained multiple copies.The lignin peroxidase activity reached a maximum of 15 U/L after 12 h induction.展开更多
Mammalian cells express two isoforms of Cu- and Zn-containing superoxide dismutases（SODs）, CuZn-SOD and extracellular SOD（EC-SOD）, involved in the defense system against reactive oxygen species（ROS）. The two SOD...Mammalian cells express two isoforms of Cu- and Zn-containing superoxide dismutases（SODs）, CuZn-SOD and extracellular SOD（EC-SOD）, involved in the defense system against reactive oxygen species（ROS）. The two SODs have structurally homologous centre domain with distinct N- and C-terminuses, resulting in the different characteristics of the structure and function of the two molecules. We generated a hybrid SOD molecule（namely hy- SOD） via replacing the N- and C-terminuses of hCuZnSOD with the counterparts of hEC-SOD. The hySOD was expressed in host Pichia pastoris and the purified protein was a dimer with a molecular weight of about 34000. A series of activity analyses indicates that the hySOD is similar to hEC-SOD in heat-stability, and has the activity of protecting the host cell against heat shock and oxidative stress. Our results show evidence for the study on the compound activity of multiple SOD molecules, and may be important for understanding the relationship between structure and function of hEC-SOD and hCuZnSOD.展开更多
基金湖南省教育厅基金优秀青年项目(No.22B0482)湖南科技大学博士启动基金(No.E51992 and E51993)资助。
文摘The xylitol dehydrogenase(XDH)is a crucial enzyme involved in the xylose utilization in pentose⁃catabolizing yeasts and fungi.In addition to producing xylulose,XDH can also be employed to develop a biosensor for monitoring xylitol concentration.In this study,the gene encoding the thermophilic fungus Talaromyces emersonii XDH(TeXDH)was heterologously expressed in Escherichia coli BL21(DE3)at 16℃in the soluble form.Recombinant TeXDH with high purity was purified by using a Ni⁃NTA affinity column.Size⁃exclusion chromatography and SDS⁃PAGE analysis demonstrated that the puri⁃fied recombinant TeXDH exists as a native trimer with a molecular mass of approximately 116 kD,and is composed of three identical subunits,each with a molecular weight of around 39 kD.The TeXDH strictly preferred NAD^(+)as a coenzyme to NADP^(+).The optimal temperature and pH of the TeXDH were 40℃and 10.0,respectively.After EDTA treatment,the enzyme activity of TeXDH decreased to 43.26%of the initial enzyme activity,while the divalent metal ions Mg^(2+)or Ca^(2+)could recover the enzyme activity of TeXDH,reaching 103.32%and 110.69%of the initial enzyme activity,respectively,making them the optimal divalent metal ion cofactors for TeXDH enzyme.However,the divalent metal ions of Mn^(2+),Ni^(2+),Cu^(2+),Zn^(2+),Co^(2+),and Cd^(2+)significantly inhibited the activity of TeXDH.ICP⁃MS and molecular doc⁃king studies revealed that 1 mol/L of TeXDH bound 2 mol/L Zn^(2+)ions and 1 mol/L Mg^(2+)ion.Further⁃more,TeXDH exhibited a high specificity for xylitol,laying the foundation for the development of future xylitol biosensors.
基金This work was supported by the China Postdoctoral Science Foundation(Grant No.2016M602873)the Fundamental Research Funds for the Central Universities(Grant No.2452016182,2452017074)+1 种基金the earmarked fund for Modern Agro-industry Technology Research System(Grant No.CARS-19)the special fund for University-Supported Extension Model(Grant No.TGZX2018-39).
文摘Late embryogenesis abundant(LEA)proteins play an important role in plant growth and development,as well as in the plant response to various abiotic stresses.In this study,CsLEA1,a novel gene encoding a LEA_3 subfamily protein,was successfully cloned froma tea plant[Camellia sinensis(L.)O.Kuntze].Bioinformatics analysis and prokaryotic expression assays showed that CsLEA1 is a typical hydrophilic protein with a molecular weight of approximately 10.4 kD.Expression analyses revealed that the transcription of CsLEA1 in C.sinensis leaves was significantly induced by cold stress.In addition,the heterologous expression of CsLEA1 increased the tolerance of Escherichia coli and yeast to cold stress,which might be closely related to the low molecular weight and high hydrophilicity of the CsLEA1.Taken together,our results suggest that CsLEA1 might have an important function in the tolerance of C.sinensis to cold stress,thus providing a potential application in molecular breeding to enhance the cold stress tolerance of tea plants.
基金supported by the National Natural Science Foundation of China(Nos.81573311 and 82173702).
文摘Lysobacter harbors a plethora of cryptic biosynthetic gene clusters(BGCs),albeit only a limited number have been analyzed to date.In this study,we described the activation of a cryptic polyketide synthase(PKS)/nonribosomal peptide synthetase(NRPS)gene cluster(lsh)in Lysobacter sp.DSM 3655 through promoter engineering and heterologous expression in Streptomyces sp.S001.As a result of this methodology,we were able to isolate two novel linear lipopeptides,lysohexaenetides A(1)and B(2),from the recombinant strain S001-lsh.Furthermore,we proposed the biosynthetic pathway for lysohexaenetides and identified LshA as another example of entirely iterative bacterial PKSs.This study highlights the potential of heterologous expression systems in uncovering cryptic biosynthetic pathways in Lysobacter genomes,particularly in the absence of genetic manipulation tools.
基金supported by the Ministry of Science and Technology of the People’s Republic of China(No.2020YFA0907703)the National Natural Science Foundation of China(Nos.32070044,32025002)the Biological Resources Programme,Chinese Academy of Sciences(No.KFJ-BRP-009)。
文摘Natural cyclohexapeptide AFN A_(1) from Streptomyces alboflavus 313 has moderate antibacterial and antitumor activities.An artificial designed AFN A_(1) homodimer,di-AFN A_(1),is an antibiotic exhibiting 10 to 150 fold higher biological activities,compared with the monomer.Unfortunately,the yield of di-AFN A_(1) is very low(0.09±0.03 mg·L^(−1))in the engineered strain Streptomyces alboflavus 313_hmtS(S.albo/313_hmtS),which is not friendly to be genetically engineered for titer improvement of di-AFN A_(1) production.In this study,we constructed a biosynthetic gene cluster for di-AFN A_(1) and increased its production through heterologous expression.During the collection of di-AFN A_(1) biosynthetic genes,the afn genes were located at three sites of S.alboflavus 313 genome.The di-AFN A_(1) biosynthetic gene cluster(BGC)was first assembled on one plasmid and introduced into the model strain Streptomyces lividans TK24,which produced di-AFN A_(1) at a titer of 0.43±0.01 mg·L^(−1).To further increase the yield of di-AFN A_(1),the di-AFN A_(1) BGC was multiplied and split to mimic the natural afn biosynthetic genes,and the production of di-AFN A_(1) increased to 0.62±0.11 mg·L^(−1) in S.lividans TK24 by the later strategy.Finally,different Streptomyces hosts were tested and the titer of di-AFN A_(1) increased to 0.81±0.17 mg·L^(−1),about 8.0-fold higher than that in S.albo/313_hmtS.Successful heterologous expression of di-AFN A_(1) with a remarkable increased titer will greatly facilitate the following synthetic biological study and drug development of this dimeric cyclohexapeptide.
文摘The use of antimicrobial peptides(AMPs)synthesized by bacteria(bacteriocins)is an alternative for combating multidrug resistant bacterial strains and their production by recombinant route is a viable option for their mass production.The bacteriocin E-760 isolated from the genus Enterococcus sp.has been shown to possess inhibitory activity against Gram-negative and Gram-positive bacteria.In this study,the expression of a chimeric protein coding for E-760 in the nucleus of C.reinhardtii was evaluated,as well as,its antibacterial activity.The synthetic gene E-760S was inserted into the genome of C.reinhardtii using Agrobacterium tumefaciens.A transgenic line was identified in TAP medium with hygromycin and also by PCR.The increment in the culture medium temperature of the transgenic strain at 35°C for 10 minutes,increased the production level of the recombinant protein from 0.14(Noninduced culture,NIC)to 0.36%(Induced culture,IC)of total soluble proteins(TSP);this was quantified by an ELISA assay.Recombinant E-760 possesses activity against Staphylococcus aureus in 0.34 U log,Streptococcus agalactiae in 0.48 U log,Enterococcus faecium in 0.36 U log,Pseudomonas aeruginosa in 2 U log and for Klebsiella pneumoniae,the activity was 0.07 U log.These results demonstrate that the nucleus transformation of C.reinhardtii can function as a stable expression platform for the production of the synthetic gene E-760 and it can potentially be used as an antibacterial agent.
基金The study was supported by National Basic Science Key Infrastructure Development Program (973 program), National Ministry of Science and Technology (Grant No: G19990559) and in part by E-institutes fund of Shanghai Municipal Education Commission. (Grant No:E03003)
文摘Objective To characterize a possible retention function of unique sequence in the 5＇end of rat testis GABAA receptor β3t splicing variant Methods Rat testis GABAA receptor β3t splicing variant cDNA was cloned and two eukaryotic expression recombinant plasmids of pEGFP-N1 and pEGFP-C1 were constructed respectively by fusing green fluorescent protein to the N or C-terminus of β3t isoform. The recombinant plasmids were transfected into CHO cells by calcium phosphate co-precipitation method Fluorescence microscope and laser confocal microscope were used to analyze localization of β3t in the transfected cells. ConA-Texas-Red was used to label cell ER and the localization of rat testis β3t splicing variant in CHO cells was determined. Results When rat testis β3t splicing variant was expressed in CHO cells, two expression patterns were delineated, the distributions of uniform and mainly discrete intracellular compartments respectively, The chimera product failed to be translocated into the cell surface when expressed in ClIO cells; whereas the β3 subunit of rat brain was incorporated into the plasma membrane. Conclusion The inability of β3t to target into the ER may be a consequence of the unique 25 specific amino acid segments in the N terminus.
基金funded by the National Key Research and Development Program of China(2021YFC2102700)was Supported by the Key Science and Technology Innovation Project of Hubei Province(2021BAD001)as well as the Research Program of State Key Laboratory of Biocatalysis and Enzyme Engineering。
文摘Unspecific peroxygenases(UPOs)are glycosylated enzymes that provide an efficient method for oxyfunctionalizing a variety of substrates using only hydrogen peroxide(H2O2)as the oxygen donor.However,their poor heterologous expression has hindered their practical application.Here,a novel UPO from Marasmius fiardii PR910(MfiUPO)was identified and heterologously expressed in Pichia pastoris.By employing a two-copy expression cassette,the protein titer reached 1.18 g L−1 in a 5 L bioreactor,marking the highest record.The glycoprotein rMfiUPO exhib-ited a smeared band in the 40 to 55 kDa range and demonstrated hydroxylation,epoxidation and alcohol oxidation.Moreover,the peroxidative activity was enhanced by 150%after exposure to 50%(v/v)acetone for 40 h.A semipreparative production of 4-OH-β-ionone on a 100 mL scale resulted in a 54.2%isolated yield with 95%purity.With its high expression level,rMfiUPO is a promising candidate as an excellent parental template for enhancing desirable traits such as increased stability and selectivity through directed evolution,thereby meeting the necessary criteria for practical application.
基金supported by the National Key Research and Development Program of China(2023YFF1000703)the Biological Breeding-National Science and Technology Major Project(2022ZD04008)the National Natural Science Foundation of China(32200265 and 32372184).
文摘Dear Editor,Gene duplication and loss are widespread in the plant kingdom and contribute substantially to phenotypic diversification and environmental adaptation.Polyploid species,particularly allopolyploids,combine divergent yet compatible progenitor genomes,a process that often triggers extensive gene loss during evolution(Chen et al.,2007).The Brassica"triangle of U"provides a classical model for investigating gene loss patterns.In this system,the diploid species Brassica rapa(B.rapa,AA,2n=20),Brassica nigra(B.nigra,BB,2n=16),and Brassica oleracea(B.oleracea,CC,2n=18)hybridized pairwise to give rise to the allopolyploids Brassica juncea(B.juncea,AABB,2n=36),Brassica carinata(B.carinata,BBCC,2n=34),and Brassica napus(B.napus,AACC,2n=38)(Yim et al.,2022).
基金supported by the National Key Research and Development Program of China(2023YFA0914500)the National Science Foundation of China(32271487)+1 种基金the National First-class Discipline Program of Light Industry Technology and Engineering(LITE2018-12)the Program of Introducing Talents of Discipline to Universities(111-2-06).
文摘The low-expression level of lactoferrin(LF)in the production process poses a significant challenge.This study aimed to effi-ciently express bovine lactoferrin(BLF)using Pichia pastoris GS115 as the expression host and PIC9K as the recombinant vector.Optimization strategies included codon usage,promoter selection,and fermentation conditions.The blf gene was optimized for P.pastoris GS115 bias,resulting in the construction of the recombinant vector pPIC9K-UBLF1-3 controlled by the AOX1 promoter.SDS-PAGE analysis revealed soluble and efficient expression of ublf3 in P.pastoris GS115,with a molecular mass of approximately 76 kDa.The transformant P.pastoris GS115/pGAP9K-UBLF3-4 resistant at 4 mg·mL^(−1)G418,exhibited a ublf3 gene copy number of 5.88 through high-copy screening.Optimal expression conditions of recombi-nant UBLF were determined as 24℃,pH 5.0 and 220 r·min^(−1)through fermentation condition optimization.Under these con-ditions,recombinant UBLF production reached 40.62 mg·L^(−1).The yield of recombinant UBLF was reached 824.93 mg·L^(−1)through high-density fermentation.Antibacterial assay demonstrated the efficacy of recombinant UBLF against Escherichia coli JM109 and Staphylococcus aureus CGMCC 1.282.This study successfully achieved the efficient heterologous expression of recombinant UBLF in P.pastoris GS115,providing valuable insight for industrial production and the potential develop-ment of natural antibacterial agents.
基金Acknowledgments This work was supported by the National Basic Research Program of China （2012CB114102）, National Natural Science Foundation of China （31272380）.
文摘Insect chitinases are involved in degradation of chitin from the exoskeleton or peritrophic metrix ofmidgut. In Locusta migratoria, two duplicated Cht5s （LmCht5-1 and LmCht5-2） have been shown to have distinct molecular characteristics and biological roles. To explore the protein properties of the two LmCht5s, we heterologously expressed both enzymes using baculovirus expression system in SF9 cells, and characterized kinetic and carbohydrate-binding properties of purified enzymes. LmCht5-1 and LmCht5-2 exhibited similar pH and temperature optimums. LmCht5-1 has lower Km value for the oligomeric substrate （4MU-（GlcNAe）3）, and higher Km value for the longer substrate （CM-Chitin- RBV） compared with LmCht5-2. A comparison of amino acids and homology modeling of catalytic domain presented similar TIM barrel structures and differentiated amino acids between two proteins. LmCht5-1 has a chitin-binding domain （CBD） tightly bound to colloidal chitin, but LmCht5-2 does not have a CBD for binding to colloidal chitin. Our results suggested both LmCht5- I and LmCht5-2, which have the critical glutamate residue in region II of catalytic domain, exhibited chitinolytic activity cleaving both polymeric and oligomeric substrates. LmCht5-1 had relatively higher activity against the oligomeric substrate, 4MU-（GlcNAc）3, whereas LmCht5-2 exhibited higher activity toward the longer substrate, CM-Chitin-RBE These findings are helpful for further research to clarit}g their different roles in insect growth and development.
基金supported in part by the National Natural Science Foundation of China (31170037)Ministry of Science and Technology of China (2013CB734003)the China Postdoctoral Science Foundation (2013M530755)
文摘Streptothricins (STs) are used commercially to treat bacterial and fungal diseases in agriculture. Mining of the sequenced microbial genomes uncovered two cryptic ST clusters from Streptomyces sp. C and Streptomyces sp. TP-A0356. The ST cluster from S. sp. TP-A0356 was verified by successful heterologous expression in Streptomyces coelicolor M145. Two new ST analogs were produced together with streptothricin F and streptothricin D in the heterologous host. The ST cluster was further confirmed by inactivation of gene stnO, which was proposed encoding an aminomutase supplying -lysines for the poly-β-Lys chain formation. A putative biosynthetic pathway for STs is proposed based on bioinformatics analyses of the ST genes and experimental evidence.
基金This work was financially supported by the Ministry of Science and Technology of China(“973”Program 2013CB734003)the National Science Foundation of China(31670030).
文摘Avermectins,a group of polyketide natural products,are widely used as anthelmintics in agriculture.Metabolic engineering and combinatorial biosynthesis were extensively employed to improve Avermectins production and create novel Avermectin derivatives,including Ivermectin and Doramectin.It is labor intensive and time cost to genetically manipulate Avermectins producer Streptomyces avermitilis in vivo.Cloning and heterologous expression of Avermectins biosynthetic gene cluster will make it possible to tailor the cluster in vitro.We constructed a Bacterial Artificial Chromosome(BAC)library of S.avermitilis ATCC 31267 with inserted DNA fragments ranged from 100 to 130 Kb.Five recombinant BAC clones which carried the Avermectins biosynthetic gene cluster ave(81 Kb in size)were screened out from the library.Then,ave was hetero-expressed in S.lividans.Three Avermectin components,A2a,B1a and A1a were detected from the cell extracts of recombinant strains.It will facilitate the development of Avermectin derivatives by polyketide synthase domain swapping and provide functional element for Avermectins synthetic biology study.
基金supported by the National Natural Science Foundation of China(Grant Nos.32001430,32171952,31971839).
文摘Basic helix-loop-helix(bHLH)transcription factors are widely distributed in eukaryotes,and in plants,they regulate many biological processes,such as cell differentiation,development,metabolism,and stress responses.Few studies have focused on the roles of bHLH transcription factors in regulating growth,development,and stress responses in maize(Zea mays),even though such information would greatly benefit maize breeding programs.In this study,we cloned the maize transcription factor gene ZmbHLH36(Gene ID:100193615,GRMZM2G008691).ZmbHLH36 possesses conserved domains characteristic of the bHLH family.RT-qPCR analysis revealed that ZmbHLH36 was expressed at the highest level in maize roots and exhibited different expression patterns under various abiotic stress conditions.Transgenic Arabidopsis(Arabidopsis thaliana)plants heterologously expressing ZmbHLH36 had significantly longer roots than the corresponding non-transgenic plants under 0.1 and 0.15 mol L^(−1) NaCl treatment as well as 0.2 mol L^(−1) mannitol treatment.Phenotypic analysis of soil-grown plants under stress showed that transgenic Arabidopsis plants harboring ZmbHLH36 exhibited significantly enhanced drought tolerance and salt tolerance compared to the corresponding non-transgenic plants.Malondialdehyde contents were lower and peroxidase activity was higher in ZmbHLH36-expressing Arabidopsis plants than in the corresponding non-transgenic plants.ZmbHLH36 localized to the nucleus when expressed in maize protoplasts.This study provides a systematic analysis of the effects of ZmbHLH36 on root growth,development,and stress responses in transgenic Arabidopsis,laying a foundation for further analysis of its roles and molecular mechanisms in maize.
基金funded by grants of the Novo Nordisk Foundation,Denmark(NNF16OC0021746 to O.G.,T.W.,NNF20CC0035580 to T.W.).
文摘Globomycin is a cyclic lipodepsipeptide originally isolated from several Streptomyces species which displays strong and selective antibacterial activity against Gram-negative pathogens.Its mode of action is based on the competitive inhibition of the lipoprotein signal peptidase II(LspA),which is absent in eukaryotes and considered an attractive target for the development of new antibiotics.Despite its interesting biological properties,the gene cluster encoding its biosynthesis has not yet been identified.In this study we employed a genome-mining approach in the globomycin-producing Streptomyces sp.CA-278952 to identify a candidate gene cluster responsible for its biosynthesis.A null mutant was constructed using CRISPR base editing where production was abolished,strongly suggesting its involvement in the biosynthesis.The putative gene cluster was then cloned and heterologously expressed in Streptomyces albus J1074 and Streptomyces coelicolor M1146,therefore unambigu-ously linking globomycin and its biosynthetic gene cluster.Our work paves the way for the biosynthesis of new globomycin derivatives with improved pharmacological properties.
基金supported by the National Natural Science Foundation of China (82003639)Taishan Scholars Program of Shandong Province (tsqn201909049)Qilu Youth Scholar Startup Funding of Shandong University.
文摘Microbial natural products and their derivatives have been developed as a considerable part of clinical drugs and agricultural chemicals.Marine microbial natural products exhibit diverse chemical structures and bioactivities with substantial potential for the development of novel pharmaceuticals.However,discovering compounds with new skeletons from marine microbes remains challenging.In recent decades,multiple approaches have been de-veloped to discover novel marine microbial natural products,among which heterologous expression has proven to be an effective method.Facilitated by large DNA cloning and comparative metabolomic technologies,a few novel bioactive natural products from marine microorganisms have been identified by the expression of their biosynthetic gene clusters(BGCs)in heterologous hosts.Heterologous expression is advantageous for character-izing gene functions and elucidating the biosynthetic mechanisms of natural products.This review provides an overview of recent progress in heterologous expression-guided discovery,biosynthetic mechanism elucidation,and yield optimization of natural products from marine microorganisms and discusses the future directions of the heterologous expression strategy in facilitating novel natural product exploitation.
基金supported by the National Key Research&Developmental Program of China(2021YFC2100303,2018YFA0900300).
文摘Phospholipase A1(PLA_(1))is a member of the hydrolase family with applications in various fields,especially in the food industry.A calcium-independent PLA_(1) from Streptomyces albidoflavus was expressed in E.coli BL21(DE3)in this study.The results indicated that the soluble expression of PLA_(1) was at low level,which was possibly due to the toxicity of PLA_(1) to the host.In contrast,the expression of the enzyme as inclusion bodies exhibited a high-level expression and 0.3 mg inclusion bodies protein could be derived from 1 mL culture medium.Furthermore,the renaturation of PLA_(1) was achieved through a direct dilution method,yielding 29.6 U/mL PLA_(1) activity after 16 h of renaturation at 4℃.For improving the efficiency of the dilution refolding process,a continuous refolding strategy was established,and 155 U/mL PLA_(1) activity was derived from the continuous refolding process.With soybean lecithin as the substrate,the specific activity of purified renatured PLA_(1) was 1380 U/mg and the optimal temperature and pH was found to be 60℃ and 6.5.In addition,the renatured PLA_(1) was observed with better activity towards phosphatidyl inositol,whilst lipase activity was detected when the catalyzing temperature was below 55℃.Overall,this study provides a possible solution to obtain calcium-independent PLA_(1) with high yield by heterologous expression in E.coli and hence to promote its further application in the field of food industry.
基金supported by the Fundamental Research Funds for the Central Universities(Grant No.:2572016AA04)Northeast Asia Biodiversity Research Center Double First class Funds(Grant No.:411146030416 and No.:411147021003).
文摘Small heat shock proteins(sHSPs)act as molecular chaperones that can prevent the accumulation of damaged proteins during abiotic stress,especially heat shock,but the mechanism is not clear.To study the function of sHSPs in Lenzites gibbosa,a common polypore in northern temperate forests that causes spongy white rot of broadleaf trees,under temperature stress,L.gibbosa mycelia were grown at 25℃ for 9 d,treated at 33℃ for 15,30,60,and 120 min before sequencing the transcriptomes.From among 32 heat shock protein(HSP)genes found in the screen of the transcriptome data,a highly expressed gene was cloned and named Lghsp17.4.RT-qPCR was used to analyze the expression of the gene Lghsp17.4 under heat shock and dye stress.Both treatments induced higher expression of Lghsp17.4 at the transcriptional level,indicating that Lghsp17.4 might function in the response to heat stress and dye degradation.We previously found that L.gibbosa generally had a heat shock reaction(HSR)during degradation of aromatic compounds,and HSPs were always produced with manganese peroxidases(MnPs)and other lignin-degrading enzymes.Therefore,we measured the activity of MnPs in L.gibbosa after 33℃ heat shock to analyze the relationship between MnPs expression and Lghsp17.4 expression.Heat shocks of 0–30 min increased MnPs activity,and the change in MnPs activity were closely positively correlated with the expression levels of Lghsp17.4 over time,indicating a potential connection and interaction between LgHSP17.4 and MnPs during the HSR in L.gibbosa.Thus,LgHSP17.4 might have a positive regulatory effect on the HSR in L.gibbosa and be a critical component of a stress resistance mechanism.
基金financially supported by grants from the National Key Research and Development Program of China (No.2024YFE0102000)the National Natural Science Foundation of China (Nos.81925037,82321004,U22A20371,U24A20782,32170060,22177037,22207039,22307045)+6 种基金the Guangdong Major Project of Basic and Applied Basic Research (No.2023B0303000026)the Guangdong Natural Science Funds for Distinguished Young Scholars (No.2022B1515020028,China)the Guangdong International Science and Technology Cooperation Base (No.2021A0505020015,China)the Guangdong Basic and Applied Basic Research Foundation (Nos.2023B1515040016,2023A1515110388)the Innovative and Research Teams Project of Guangdong Higher Education Institution (No.2021KCXTD001,China)the Guangzhou Science and Technology Project (Nos.202206010020,2024A04J6241,2023A04J0080,China)the Fundamental Research Funds for the Central Universities (Nos.21623105,21624210)。
文摘Mangicol-type sesterterpenoids possess potent anti-inflammatory activity,characterized by a 5-5-6-5tetracyclic carbon skeleton formed by mangicdiene synthase Fg MS.Two proposed mechanisms for mangicdiene formation involve either C6-C10 cyclization(path a) or C2-C10 cyclization(path b) after the C10carbocation formation,but neither has been experimentally validated.Here,we have identified a second mangicdiene synthase Man D,which is derived from Fusarium sp.JNU-XJ070152-01 and shares high amino acid sequence identity with Fg MS.Through heterologous expression of man D in Aspergillus oryzae NSAR1,we observed production not only of mangicdiene(1) and variecoltetraene(2),previously identified by expression of Fg MS in Escherichia coli,but also two novel sesterterpene skeletons fusadiene(3)and fusatriene(4).The identification of fusadiene and fusatriene supports the occurrence of two key carbocation intermediates in path b,thus experimentally confirming that mangicdiene is built via path b for the first time,consistent with previous density functional theory(DFT) calculation results.
基金supported by the National Natural Science Foundation of China(No.20577028).
文摘The lipH2 gene,encoding the expression of lignin peroxidase,was cloned from Phanerochaete chrysosporium BKM-F-1767 and expressed in Pichia pastoris X-33,a yeast.The cDNA of LiPH2 was generated from total RNA extracted from P chrysosporium by PCR with primers that do not contain a P.chrysosporium lignin peroxidase secretion signal.The gene was then successfully inserted into the expression vector pPICZα,and resulted in the recombinant vector pPICZα-lipH2.The transformation was conducted in two ways.One was using the wild Pichia pastoris as the recipients,which results in the recombinant P.pastoris with single or low lipH2 gene copy.The second was using P.pastoris and single or low lipH2 gene copy as the recipients,which results in the recombinant P.pastoris with multi-copies of lipH2 genes.This study firstly expressed the gene lipH2 in P.pastoris and achieved the successful expression of the lipH2 depending upon the generation of a recombinant strain that contained multiple copies.The lignin peroxidase activity reached a maximum of 15 U/L after 12 h induction.
文摘Mammalian cells express two isoforms of Cu- and Zn-containing superoxide dismutases（SODs）, CuZn-SOD and extracellular SOD（EC-SOD）, involved in the defense system against reactive oxygen species（ROS）. The two SODs have structurally homologous centre domain with distinct N- and C-terminuses, resulting in the different characteristics of the structure and function of the two molecules. We generated a hybrid SOD molecule（namely hy- SOD） via replacing the N- and C-terminuses of hCuZnSOD with the counterparts of hEC-SOD. The hySOD was expressed in host Pichia pastoris and the purified protein was a dimer with a molecular weight of about 34000. A series of activity analyses indicates that the hySOD is similar to hEC-SOD in heat-stability, and has the activity of protecting the host cell against heat shock and oxidative stress. Our results show evidence for the study on the compound activity of multiple SOD molecules, and may be important for understanding the relationship between structure and function of hEC-SOD and hCuZnSOD.
