Efficient secretory expression of phospholipase D for the high-yield production of phosphatidylserine and phospholipid derivates from soybean lecithin

Phospholipase D(PLD)is an essential biocatalyst for the biological production of phosphatidylserine and phospholipid modification.However,the efficient heterologous expression of PLD is limited by its cell toxicity.In this study,a PLD was secretory expressed efficiently in Bacillus subtilis with an activity around 100 U/mL.A secretory expression system containing the signal peptide SPEstA and the dual-promoter PHpaII-SrfA was estab-lished,and the extracellular PLD activity further reached 119.22 U/mL through scale-up fermentation,191.30-fold higher than that of the control.Under optimum reaction conditions,a 61.61%conversion ratio and 21.07 g/L of phosphatidylserine production were achieved.Finally,the synthesis system of PL derivates was established,which could efficiently synthesis novel PL derivates.The results highlight that the secretory expression system constructed in this study provides a promising PLD producing strain in industrial application,and laid the foundation for the biosynthesis of phosphatidylserine and other PL derivates.As far as we know,this work re-ports the highest level of extracellular PLD expression to date and the enzymatic production of several PL der-ivates for the first time.

Metabolic engineering strategies for microbial utilization of methanol

The increasing shortage of fossil resources and environmental pollution has renewed interest in the synthesis of value-added biochemicals from methanol.However,most of native or synthetic methylotrophs are unable to assimilate methanol at a sufficient rate to produce biochemicals.Thus,the performance of methylotrophs still needs to be optimized to meet the demands of industrial applications.In this review,we provide an in-depth discussion on the properties of natural and synthetic methylotrophs,and summarize the natural and synthetic methanol assimilation pathways.Further,we discuss metabolic engineering strategies for enabling microbial utilization of methanol for the bioproduction of value-added chemicals.Finally,we highlight the potential of microbial engineering for methanol assimilation and offer guidance for achieving a low-carbon footprint for the biosynthesis of chemicals.

Metabolic engineering strategies for microbial utilization of C1 feedstocks

The use of abundant and cheap one carbon(C1)feedstocks to produce value-added chemicals is an important approach for achieving carbon neutrality and tackling environmental problems.The conversion of C1 feedstocks to high-value chemicals is dependent on efficient C1 assimilation pathways and microbial chassis adapted for efficient incorporation.Here,we opted to summarize the natural and synthetic C1 assimilation pathways and their key factors for metabolizing C1 feedstock.Accordingly,we discussed the metabolic engineering strategies for enabling the microbial utilization of C1 feedstocks for the bioproduction of value-added chemicals.In addition,we highlighted future perspectives of C1-based biomanufacturing for achieving a low-carbon footprint for the biosynthesis of chemicals.

Advances in microbial engineering for the production of value‑added products in a biorefinery

Microbial biorefineries to produce chemicals from renewable feedstock provides attractive advantages,including mild reaction conditions and sustainable manufacturing.However,low-efficiency biorefineries always result in an uncompetitive biological process compared to the current petrochemical process.Thus,improving microbial capacity to maximize product yield,productivity,and titer has been recognized as a central goal for bioengineers and biochemists.The knowledge of cellular biochemistry has enabled the regulation of microbial physiology to couple with chemical production.The rapid development in metabolic engineering provides diverse strategies to enhance the efficiency of chemical biosynthesis pathways.New synthetic biology tools as well as novel regulatory targets also offer the opportunity to improve biorefinery environmental adaptivity.In this review,the recent advances in building efficient biorefineries were showcased.In addition,the challenges and future perspectives of microbial host engineering for increased microbial capacity of a biorefinery were discussed.

Immunological Exploration of Helicobacter pylori Serotype O_(2) O-Antigen by Using a Synthetic Glycan Library

Helicobacter pylori(H.pylori)infection is a threat to human health.The lipopolysaccharide(LPS)O-antigen holds promise for developing vaccines.It is meaningful to explore the immunological activity of oligosaccharides with different lengths and frameshifts for antigen development.Herein,a glycan library of H.pylori O2 O-antigen containing eight fragments is constructed.After screening with anti-H.pylori O2 LPS sera and patients’sera by glycan microarray,the disaccharide HPO2G-2b and trisaccharide HPO2G-3a show strong antigenicity and then are separately conjugated with carrier protein CRM197.Both glycoconjugates elicit a robust immunoglobulin G(IgG)immune response in rabbits.The anti-HPO2G-3a IgG antibodies possess a much stronger binding affinity with the LPS and bacteria of H.pylori O2 than the anti-HPO2G-2b IgG antibodies.There is no cross-reaction between both sera IgG antibodies with LPS and bacteria of H.pylori O1,O6,and E.coli.The results demonstrate the trisaccharide HPO2G-3a is a promising candidate for H.pylori vaccine development.

Aggregation-induced delayed electrochemiluminescence of organic dots in aqueous media

Full utilization of the excited species at both singlet states(1R*)and triplet states(3R*)is crucial to improving electrochemiluminescence(ECL)efficiency but is challenging for organic luminescent materials.Here,an aggregation-induced delayed ECL(AIDECL)active organic dot(OD)containing a benzophenone acceptor and dimethylacridine donor is reported,which shows high ECL efficiency via reverse intersystem crossing(RISC)of non-emissive 3R*to emissive 1R*,overcoming the spin-forbidden radiative decay from 3R*.By introducing dual donor-acceptor pairs into luminophores,it is found that nonradiative pathway could be further suppressed via enhanced intermolecular weak interactions,and multiple spin-up conversion channels could be activated.As a consequence,the obtained OD enjoys a 6.8-fold higher ECL efficiency relative to the control AIDECL-active OD.Single-crystal studies and theoretical calculations reveal that the enhanced AIDECL behaviors come from the acceleration of both radiative transition and RISC.This work represents a major step towards purely organic,high-efficiency ECL dyes and a direction for the design of next-generation ECL dyes at the molecular level.

Characterization,heterologous expression and engineering of trehalase for biotechnological applications

Trehalose is a non-reducing disaccharide connected byα-1,1-glycosidic bonds;it is widely distributed in bacteria,fungi,yeast,insects,and plant tissues and plays various roles.It can be hydrolyzed by trehalase into two glucose molecules.Trehalases from different sources have been expressed in Escherichia coli,Pichia pastoris,Saccharomyces cerevisiae,baculovirus-silkworm,and other expression systems;however,it is most common in E.coli.The structural characteristics of different glycoside hydrolase(GH)family trehalases and the sources of trehalase have been analyzed.The catalytic mechanism of GH37 trehalase has also been elucidated in detail.Moreover,the molecular modification of trehalase has mainly focused on directed evolution for improving enzyme activity.We comprehensively reviewed the current application status and adaptable transformations was comprehensively overviewed in the context of industrial performance.We suggest that the level of recombinant production is far from meeting industrial requirements,and the catalytic performance of trehalase needs to be improved urgently.Finally,we discuss developmental prospects and future trends.

The enhanced photocatalytic sterilization of MOF-Based nanohybrid for rapid and portable therapy of bacteria-infected open wounds

Open wounds are prone to infection and difficult to heal,which even threatens the life of patients because bacterial infections can induce other lethal complications without prompt treatment.The commonly used antibiotics treatment for bacterial infections has been reported to cause globally bacterial resistance and even the occurrence of superbacteria.The highly effective and antibiotic-independent therapeutic strategies are urgently needed for treating various kinds of bacteria-infected diseases.In this work,we synthesized an eco-friendly nanohybrid material(ZnDMZ)consisting of a kind of biodegradable metal organic framework(MOF,ZIF-8)combined with Zn-doped MoS_(2)(Zn-MoS_(2))nanosheets,which exhibited great ability to kill bacteria and promote the healing of bacteria-infected wounds under 660 nm light irradiation.The underlying mechanism is that besides the local hyperthermia,the nanohybrid material exhibits enhanced photocatalytic performance than single component in it,i.e.,it can also be excited by 660 nm light to produce more oxygen radical species(ROS)due to the following factors.On one hand,the Zn doping can reduce the work function and the band gap of MoS_(2),which promotes the movement of photoexcited electrons to the surface of the material.On the other hand,the combination between Zn-MoS_(2) and MOF induces the formation of a built-in electric field due to their work function difference,thus accelerating the separation of photoexcited electron-hole pairs.Because of the synergy of photocatalytic effect,photothermal effect and the released Zn ions,the synthesized ZnDMZ possessed a highly effective antibacterial efficacy of 99.9%against Staphylococcus aureus under 660 nm light irradiation for 20 min without cytotoxicity.In vivo tests showed that this nanohybrid material promoted the wound healing due to the released Zn ions.This nanohybrid will be promising for rapid and portable treatment of bacteria-infected open wounds in pathogenic bacteria contaminated environments.

Simultaneously enhancing the photocatalytic and photothermal effect of NH_(2)-MIL-125-GO-Pt ternary heterojunction for rapid therapy of bacteria-infected wounds

Infections caused by bacteria threaten human health,so how to effectively kill bacteria is an urgent problem.We therefore synthesized a NH_(2)-MIL-125-GO-Pt ternary composite heterojunction with graphene oxide(GO)and platinum(Pt)nanoparticles co-doped with metal-organic framework(NH_(2)-MIL-125)for use in photocatalytic and photothermal synergistic disinfection under white light irradiation.Due to the good conductivity of GO and the Schottky junction between Pt and MOF,the doping of GO and Pt will effectively separate and transfer the photogenerated electron-hole pairs generated by NH_(2)-MIL-125,thereby effectively improving the photocatalytic efficiency of NH_(2)-MIL-125.Meanwhile,NH_(2)-MIL-125-GO-Pt has good photothermal effect under white light irradiation.Therefore,the NH_(2)-MIL-125-GO-Pt composite can be used for effective sterilization.The antibacterial efficiency of NH_(2)-MIL-125-GO-Pt against Staphylococcus aureus and Escherichia coli were as high as 99.94%and 99.12%,respectively,within 20 min of white light irradiation.In vivo experiments showed that NH_(2)-MIL-125-GO-Pt could effectively kill bacteria and promote wound healing.This work brings new insights into the use of NH_(2)-MIL-125-based photocatalyst materials for rapid disinfection of environments with pathogenic microorganisms.

Sequence and thermodynamic characteristics of terminators revealed by FlowSeq and the discrimination of terminators strength

The intrinsic terminator in prokaryotic forms secondary RNA structure and terminates the transcription.However,leaking transcription is common due to varied terminator strength.Besides of the representative hairpin and U-tract structure,detailed sequence and thermodynamic features of terminators were not completely clear,and the effect of terminator on the upstream gene expression was unclearly.Thus,it is still challenging to use terminator to control expression with higher precision.Here,in E.Coli,we firstly determined the effect of the 3′-end sequences including spacer sequences and terminator sequences on the expression of upstream and downstream genes.Secondly,terminator mutation library was constructed,and the thermodynamic and sequence features differing in the termination efficiency were analyzed using the FlowSeq technique.The result showed that under the regulation of terminators,a negative correlation was presented between the expression of upstream and downstream genes(r=0.60),and the terminators with lower free energy corelated with higher upstream gene expression.Meanwhile,the terminator with longer stem length,more compact loop and perfect U-tract structure was benefit to the transcription termination.Finally,a terminator strength classification model was established,and the verification experiment based on 20 synthetic terminators indicated that the model can distinguish strong and weak terminators to certain extent.The results help to elucidate the role of terminators in gene expression,and the key factors identified are crucial for rational design of terminators,and the model provided a method for terminator strength prediction.

Current state and future perspectives of cytochrome P450 enzymes for C-H and C=C oxygenation

Cytochrome P450 enzymes(CYPs)catalyze a series of C-H and C=C oxygenation reactions,including hydroxylation,epoxidation,and ketonization.They are attractive biocatalysts because of their ability to selectively introduce oxygen into inert molecules under mild conditions.This review provides a comprehensive overview of the C-H and C=C oxygenation reactions catalyzed by CYPs and the various strategies for achieving higher selectivity and enzymatic activity.Furthermore,we discuss the application of C-H and C=C oxygenation catalyzed by CYPs to obtain the desired chemicals or pharmaceutical intermediates in practical production.The rapid development of protein engineering for CYPs provides excellent biocatalysts for selective C-H and C=C oxygenation reactions,thereby promoting the development of environmentally friendly and sustainable production processes.

Characterization and implications of prokaryotic ribosome-binding sites across species

The ribosome-binding site(RBS)in the 5′untranslated region is recognized by 16S rRNA to start translation and is an essential element of the gene expression system.RBSs have been widely applied in regulating gene expression in various scenarios,including Gram-negative or Gram-positive bacteria.Here,we first rationally designed and constructed an RBS mutant library containing 66 RBSs.The strength of these RBSs in E.coli and C.glutamicum was characterized individually.The RBS strength spanned about 200 and 15 times in the two species,respectively.The strength of RBSs in C.glutamicum was generally lower than that of in E.coli.A total of 18 RBSs showed similar strength(within twofold differences)between the species in our study,and the correlation analysis of the strength of RBSs between E.coli and C.glutamicum(R^(2)=0.7483)revealed that these RBSs can be used across species.The sequence analysis revealed that the RBS region with two Ts stated was beneficial for RBS to function cross-species.The RBS characterized here can be used to precisely regulate gene expression in both hosts,and the characteristics of cross-species RBSs provide basic information for RBS rational design.

Metabolic engineering of Streptomyces to enhance the synthesis of valuable natural products

The mycelial bacterium Streptomyces is a workhorse for producing natural products,serving as a key source of drugs and other valuable chemicals.However,its complicated life cycle,silent biosynthetic gene clusters(BGCs),and poorly characterized metabolic mechanisms limit efficient production of natural products.There-fore,a metabolic engineering strategy,including traditional and emerging tools from different disciplines,was developed to further enhance natural product synthesis by Streptomyces.Here,current trends in systems metabolic engineering,including tools and strategies,are reviewed.Particularly,this review focuses on recent developments in the selection of methods for regulating the Streptomyces life cycle,strategies for the activation of silent gene clusters,and the exploration of regulatory mechanisms governing antibiotic production.Finally,future challenges and prospects are discussed.

Novel thieno[2,3-b]quinoline-procaine hybrid molecules:A new class of allosteric SHP-1 activators evolved from PTP1B inhibitors

Small molecule activators could equally provide powerful tools as inhibitors do for interrogating cellular signal transduction.However,targeted protein activation is chemically challenging.Developing activators against Src homology region 2 domain-containing phosphatase-1(SHP-1)to block STAT3 pathway represents a promising strategy for DLBCL therapy.Here we reported a new class of thieno[2,3-b]quinolineprocaine hybrid molecules as SHP-1 allosteric activators.The representative hybrid compound 3b displayed SHP-1 activating effect with EC50 of 5.48±0.28μmol/L.Further investigations confirmed that 3b allosterically interacted with SHP-1,switched it from close to open conformation,blocked SHP-1/p-STAT3 pathway,induced apoptosis and inhibited ABC-DLBCL cell proliferation in vitro,and delayed tumor growth in the xenograft model of SU-DHL-2.Overall,this work offered a novel paradigm to develop SHP-1 allosteric activators through chemical space evolution of PTPs inhibitors,and firstly validated the therapeutic strategy that directly activating SHP-1 alone could be a potential therapy against ABC-DLBCL via blocking STAT3 pathway.

Five Rutaceae family ethanol extracts alleviate H2O2 and LPS-induced inflammation via NF-κB and JAK-STAT3 pathway in HaCaT cells

This study was designed to investigate the effects of five Rutaceae family ethanol extracts(FRFEE):Citrus medica Linn(CML),Citrus aurantium L.Cv.Daidai(CAD),Citrus medica Linn.var.sarcodactylis(Noot.)Swingle(CMS),Citrus sinensis L.Osbeck(CSO)and Zanthoxylum bungeanum Maxim(ZBM)on retarding the progression of H_(2)O_(2) and LPS-induced HaCaT cells.Cell inflammatory injury model was established by H_(2)O_(2) and LPS.The alleviative effects of FRFEE were evaluated by detecting the activity of superoxide dismutase(SOD),glutathione(GSH)and the generation of reactive oxygen species(ROS).The inflammatory signaling pathways of NF-κB and JAK-STAT3 were detected by Western blotting,the mRNA expression levels of inflammatory factors and skin barrier factors were detected by RT-PCR.50%ethanol extracts of five medicinal and food homologous herbs of Rutaceae family showed different levels of anti-oxidant and anti-inflammatory activities.The FRFEE effectively improved SOD and GSH content and decreased ROS levels.Meanwhile,FRFEE strongly suppressed two inflammatory signaling pathways NF-κB and JAK-STAT3.The RT-PCR examination of inflammatory factors and skin barrier factor revealed significant anti-inflammatory effects of FRFEE.It was worth noting that among the five extracts,Zanthoxylum bungeanum Maxim extract had the best anti-inflammatory and anti-oxidation effects.In addition,it could strongly inhibit the expression of psoriasis factor CCL20.In summary,these results suggested that Zanthoxylum bungeanum Maxim extract could be used as an anti-psoriatic agent in the treatment of psoriasis among FRFEE.

EGCG and ECG induce apoptosis and decrease autophagy via the AMPK/mTOR and PI3K/AKT/mTOR pathway in human melanoma cells

Catechins have been proven to exert antitumor effects in different kinds of cancers.However,the underlying mechanisms have not been completely clarified yet.This study aimed to assess the effects and mechanisms of(-)-epigallocatechin-3-gallate(EGCG)and(-)-epicatechin-3-gallate(ECG)on human melanoma skin A375 cells.Results showed that EGCG and ECG inhibited the proliferation of A375 cells and ECG showed better inhibitory effect.Flow cytometry analysis had shown that EGCG and ECG induced apoptosis and led to cell cycle arrest.EGCG and ECG decreased Bcl-2 expression and upregulated Caspase-3 protein level,indicating the development of apoptosis.Furthermore,EGCG and ECG could decreased mitochondrial membrane potential of A375 cells.In addition,the expression of Beclin-1,LC3 and Sirt3 were downregulated at protein levels,which known to be associated with autophagy.After autophagy was increased by rapamycin,the apoptotic trend was not change,indicating that apoptosis and autophagy are independent.Mechanistically,EGCG and ECG treatments decreased phosphorylated-AMPK(p-AMPK)and increased the ratios of P-PI3K,p-AKT and p-mTOR in melanoma cells.Conclusively,EGCG and ECG induced apoptosis via mitochondrial signaling pathway,downregulated autophagy through modulating the AMPK/mTOR and PI3K/AKT/mTOR signaling pathway.It indicated that EGCG and ECG may be utilized in human melanoma treatment.

Surface photodynamic ion sterilization of ITO-Cu_(2)O/ZnO preventing touch infection

Pathogenic microbial infections are threatening the people’s health and even life.The most common channel of infections can be caused by skin contact,especially hand touching facilities such as touching screen.In this work,Cu_(2)O covered with ZnO nanofilm was prepared on the surface of indium tin oxide conductive glass by electrodeposition and the followed atomic layer deposition process.This composite coating had a light transmittance of 71.5%,which met the light transmission needs of touch screen device.Electron spin resonance spectra showed that composite materials can generate more reactive oxygen species(ROS)than a single component under solar light irradiation.This was because a p-n junction with a built-in electric field was formed at the interface after Cu_(2)O contacting with ZnO.In the process of photocatalysis,photogenerated electrons and holes migrated at the interface driven by the built-in electric field,which promoted the separation of carriers.The antibacterial rate against Staphylococcus aureus reached 92.5%after 3 min of light irradiation with simulated sunlight due to the synergy of ROS and Cu ions,Zn ions.Therefore,this work may provide a potential method for antibacterial application of preventing hand touch infections.

Construction of a CHO cell line with site‑specific integration to stably express exogenous proteins using the CRISPR–Cas9 technique

Chinese hamster ovary(CHO)cells are widely used in biopharmaceuticals because of their high-density suspension culture,high safety,and high similarity between expressed exogenous proteins and natural proteins.However,the level of exogenous protein expression decreases with increasing culture time;this phenomenon occurs due to the recombination of foreign genes into chromosomes through random integration.The present study integrated the foreign genes into a specific chromosomal site for stable expression based on CRISPR–Cas9 technology.The results showed that the exogenous proteins enhanced green fluorescent protein(EGFP)and human serum albumin(HSA)were successfully integrated into the vicinity of base 1969647 on chromosome NW_003613638.1 of CHO-K1 cells.The obtained positive monoclonal cell lines expressed all the corresponding exogenous proteins after 60 consecutive passages,and no significant differences in expression levels were observed.This study might provide a feasible method to construct a CHO cell line with long-term stable expression of exogenous proteins.

Combinatorial mutagenesis of Bacillus amyloliquefaciens for efficient production of protease

As an important industrial enzyme,protease is widely used in feed,food and other fields.At present,the insufficient protease activity obtained from microorganisms cannot meet the purpose of industrial production.In this study,Bacillus amyloliquefaciens with high protease production was screened from animal feces by plate transparent circle method.To improve the production of protease,atmospheric room temperature plasma(ARTP)mutagenesis was used in the first round,protease activity reached 315.0 U/mL.Then,to enhance production of protease,^(60)Co-γirradiation was used for combined mutagenesis,leading to protease activity of B.amyloliquefaciens FMME ZK003 up to 355.0 U/mL.Furthermore,to realize the efficient production of protease,after optimization of fermentation conditions,protease activity was increased to 456.9 U/mL.Finally,protease activity of B.amyloliquefaciens FMME ZK003 reached 823.0 U/mL in a 5 L fermenter.These results indicate that B.amyloliquefaciens can efficiently produce protease,which provides a good foundation for the industrial production of protease.