Enhanced extracellular production of alpha-lactalbumin from Bacillus subtilis through signal peptide and promoter screening

Alpha-lactalbumin(α-LA)is a major whey protein found in breast milk and plays a crucial role in the growth and development of infants.In this study,Bacillus subtilis RIK1285 harboring AprE signal peptide(SP)was selected as the original strain for the production ofα-LA.It was found thatα-LA was identified in the pellet after ultrasonic disruption and centrifugation instead of in the fermentation supernatant.The original strain most likely only producedα-LA intracellular,but not extracellular.To improve the expression and secretion ofα-LA in RIK1285,a library of 173 homologous SPs from the B.subtilis 168 genome was fused with target LALBA gene in the pBE-S vector and expressed extracellularly in RIK1285.SP YjcN was determined to be the best signal peptide.Bands in supernatant were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and purified by nickel column to calculate the highest yield signal peptide.In addition,different promoters(P_(aprE),P_(43),and P_(glv))were compared and applied.The results indicated that the strain RIK1285-pBE-P_(glv)-YjcN-LALBA had the highestα-LA yield,reaching 122.04μg/mL.This study demonstrates successful expression and secretion of humanα-LA in B.subtilis and establishes a foundation for simulating breast milk for infant formulas and developing bioengineered milk.

The specificity of ten non-digestible carbohydrates to enhance butyrate-producing bacteria and butyrate production in vitro fermentation

Butyrate and butyrate-producing bacteria are important indicators of gut microbial metabolism in human health.Ten non-digestible carbohydrates(NDCs),including inulin,fructooligosaccharide(FOS),oatsβ-glucans(OGS),oatsβ-glucan oligosaccharides(OGOS),Astragalus polysaccharides(APS),Astragalus oligosaccharides(AOS),xanthan gum oligosaccharides(XGOS),gellan gum oligosaccharides(GGOS),curdlan oligosaccharides(COS),and pullulan oligosaccharides(POS)were used to investigate NDC specifi city in modulating butyrate-producing bacteria and butyrate production in 48 h in vitro fermentation studies in combination with fecal inocula from 7 healthy donors and 11 patients with type 2 diabetes(T2D).We observed that the amount of these ten NDCs utilized depended on NDC structure and inter-individual gut microbial differences.XGOS and GGOS fermentations signifi cantly increased butyrate-producing bacteria(especially f_Lachnospiraceae)and butyric acid production.Furthermore,XGOS and GGOS fermentations showed a better ability to consistently modulate gut microbiota composition and metabolic properties between individuals of healthy donors or T2D patients when compared to inulin,FOS,APS,AOS,OGS,OGOS,COS and POS fermentation.This research indicated that xanthan gum and gellan gum oligosaccharides have strong specifi city to enhance butyrate-producing bacteria and butyrate production.

Composition and immuno-stimulatory properties of extracellular DNA from mouse gut flora

AIM To demonstrate that specific bacteria might release bacterial extracellular DNA(e DNA) to exert immunomodulatory functions in the mouse small intestine.METHODS Extracellular DNA was extracted using phosphate buffered saline with 0.5 mmol/L dithiothreitol combined with two phenol extractions. TOTO-1 iodide, a cell-impermeant and high-affinity nucleic acid stain, was used to confirm the existence of e DNA in the mucus layers of the small intestineand colon in healthy Male C57 BL/6 mice. Composition difference of e DNA and intracellular DNA(i DNA) of the small intestinal mucus was studied by Illumina sequencing and terminal restriction fragment length polymorphism(T-RFLP). Stimulation of cytokine production by e DNA was studied in RAW264.7 cells in vitro.RESULTS TOTO-1 iodide staining confirmed existence of e DNA in loose mucus layer of the mouse colon and thin surface mucus layer of the small intestine. Illumina sequencing analysis and T-RFLP revealed that the composition of the e DNA in the small intestinal mucus was significantly different from that of the i DNA of the small intestinal mucus bacteria. Illumina Miseq sequencing showed that the e DNA sequences came mainly from Gram-negative bacteria of Bacteroidales S24-7. By contrast, predominant bacteria of the small intestinal flora comprised Grampositive bacteria. Both e DNA and i DNA were added to native or lipopolysaccharide-stimulated Raw267.4 macrophages, respectively. The e DNA induced significantly lower tumor necrosis factor-α/interleukin-10(IL-10) and IL-6/IL-10 ratios than i DNA, suggesting the predominance for maintaining immune homeostasis of the gut.CONCLUSION Our results indicated that degraded bacterial genomic DNA was mainly released by Gram-negative bacteria, especially Bacteroidales-S24-7 and Stenotrophomonas genus in gut mucus of mice. They decreased pro-inflammatory activity compared to total gut flora genomic DNA.

A review on current conventional and biotechnical approaches to enhance biosynthesis of steviol glycosides in Stevia rebaudiana

Stevia rebaudiana Bertoni is commonly called stevia and mostly found in the north east regions of South America.It is an herbaceous and shrubby plant belonging to the Asteraceae family.Stevia is considered as a natural sweetener and a commercially important plant worldwide.The leaves of S.rebaudiana contain steviol glycosides(SGs)which are highly potent and non-caloric sweeteners.The sweetening property of S.rebaudiana is contributed to the presence of these high potency,calorie free steviol glycosides.SGs are considerably suitable for replacing sucrose and other artificial sweetening agents which are used in different industries and pharmaceuticals.SGs amount in the plant mostly varies from 8%to 10%,and the enhancement of SGs is always in demand.These glycosides have the potential to become healthier alternatives to other table sugars for having desirable taste and zero calories.SGs are almost 300 times sweeter than sucrose.Being used as alternative sugar intensifier the commercial value of this plant in biopharmaceutical,food and beverages industries and in international market is increasing day by day.SGs have made stevia an important part of the medicinal world as well as the food and beverage industry,but the limited production of plant material is not fulfilling the higher global market demand.Therefore,researchers are working worldwide to increase the production of important SGs through the intercession of different biotechnological approaches in S.rebaudiana.This review aims to describe the emerging biotechnological strategies and approaches to understand,stimulate and enhance biosynthesis of secondary metabolites in stevia.Conventional and biotechnological methods for the production of steviol glycosides have been briefly reviewed and discussed.

Dynamic shielding of bacterial outer membrane vesicles for safe and efficient chemo-immunotherapy against tumors

Bacterial outer membrane vesicles(OMVs)are potent immunostimulants of regulating the tumor microenvironment(TME)for immunotherapy,and can be used to deliver drugs.However,the severe systemic inflammatory response triggered by OMVs upon intravenous(i.v.)injection has limited their application.Here,we developed a safe and effective strategy by conjugating doxorubicin-loaded serum albumin(SA-DOX,AD)onto the surface of OMVs using a matrix metalloproteinase(MMP)-cleavable peptide linker(cL).This approach enabled the dynamic shielding of OMVs to reduce the systemic side effects while simultaneously enhancing the anti-tumor effects through chemo-immunotherapy.Specifically,the resulting OMV-cL-AD formulation exhibited significantly enhanced accumulation at the tumor site after i.v.administration,facilitated by the SA decoration on the OMVs surface.Subsequently,the shield on the OMV-cL-AD was cleaved by the over-expressed MMP in the TME,leading to the release of both OMVs and AD.This process provided OMV-induced immunotherapy and DOX-induced chemotherapy,resulting in synergistic tumor inhibition.In conclusion,our work demonstrated the potential of OMV-cL-AD as an effective immunochemotherapy strategy that can prolong the survival time of mice without inducing side effects.

Fabrication of agarose hydrogel with patterned silver nanowiresfor motion sensor

In this work, a facile strategy is proposed to construct stretchable electronics based on agarose hydrogels. The hot agarose solution is casted onto a template with patterned Ag nanowires, endowing agarose hydrogel with patterned conductive surface. After further heating treatment, Ag nanowires can be embedded into the agarose hydrogel, which improves the stability of Ag pattern and has no obvious e ffect on the conductivity of hydrogels. The agarose hydrogel with patterned Ag nanowires is certi fied to be an e ffective stretchable electrode to record the motion of joints, which has great potential applications in the field of wearable devices.

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.

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.

Systematic bio‑fabrication of aptamers and their applications in engineering biology

Aptamers are single-stranded DNA or RNA molecules that have high affinity and selectivity to bind to specific targets.Compared to antibodies,aptamers are easy to in vitro synthesize with low cost,and exhibit excellent thermal stability and programmability.With these features,aptamers have been widely used in biology and medicine-related fields.In the meantime,a variety of systematic evolution of ligands by exponential enrichment(SELEX)technologies have been developed to screen aptamers for various targets.According to the characteristics of targets,customizing appropriate SELEX technology and post-SELEX optimization helps to obtain ideal aptamers with high affinity and specificity.In this review,we first summarize the latest research on the systematic bio-fabrication of aptamers,including various SELEX technologies,post-SELEX optimization,and aptamer modification technology.These procedures not only help to gain the aptamer sequences but also provide insights into the relationship between structure and function of the aptamers.The latter provides a new perspective for the systems bio-fabrication of aptamers.Furthermore,on this basis,we review the applications of aptamers,particularly in the fields of engineering biology,including industrial biotechnology,medical and health engineering,and environmental and food safety monitoring.And the encountered challenges and prospects are discussed,providing an outlook for the future development of aptamers.

Current advances in the biotechnological synthesis of betulinic acid:new findings and practical applications

Betulinic acid(BA),a penta-cyclic triterpenoid found as a ubiquitous secondary metabolite throughout the plant kingdom,has aroused tremendous interests due to its different pharmacological properties,which lead to large market demand.However,the content of BA in plant is low for phytoextraction.Although chemical semi-synthesis or biotransformation of BA from betulin with high conversion efficiency is achieved,it still relies on phytoextraction from the bark of medicinal trees.To circumvent this issue,the biotechnological synthesis of BA in engineered yeasts has been developed.In this review,the pharmacological properties of BA are first summarized,including antitumor,anti-HIV,antiprotozoal,anti-inflammatory,apoptosis activator and anti-metabolic syndrome.Then,the traditional phytoextraction,semi-synthesis and biotechnological synthesis of BA are discussed.Particularly,current advances in its biotechnological synthesis and strategies to improve BA production are focused.Moreover,potential strategies for further promotion of BA yield,including the introduction of artificial isopentenol utilization pathway,semi-rational mutagenesis of lupeol synthase and cytochrome P450,and subcellular morphology and compartmentalization,are discussed.

Advances and applications of machine learning and intelligent optimization algorithms in genome‑scale metabolic network models

Due to the increasing demand for microbially manufactured products in various industries,it has become important to find optimal designs for microbial cell factories by changing the direction of metabolic flow and its flux size by means of metabolic engineering such as knocking out competing pathways and introducing exogenous pathways to increase the yield of desired products.Recently,with the gradual cross-fertilization between computer science and bioinformatics fields,machine learning and intelligent optimization-based approaches have received much attention in Genome-scale metabolic network models(GSMMs)based on constrained optimization methods,and many high-quality related works have been published.Therefore,this paper focuses on the advances and applications of machine learning and intelligent optimization algorithms in metabolic engineering,with special emphasis on GSMMs.Specifically,the development history of GSMMs is first reviewed.Then,the analysis methods of GSMMs based on constraint optimization are presented.Next,this paper mainly reviews the development and application of machine learning and intelligent optimization algorithms in genome-scale metabolic models.In addition,the research gaps and future research potential in machine learning and intelligent optimization methods applied in GSMMs are discussed.

Enhanced catalytic efficiency and substrate specificity of Streptomyces griseus trypsin by evolution‑guided mutagenesis

Streptomyces griseus trypsin(SGT)is a bacteria-sourced trypsin that could be potentially applied to industrial insulin productions.However,SGT produced by microbial hosts displayed low catalytic efficiency and undesired preference to lysine residue.In this study,by engineering theαsignal peptide in Pichia pastoris,we increased the SGT amidase activity to 67.91 U mL^(−1)in shake flask cultures.Afterwards,we engineered SGT by evolution-guided mutagenesis and obtained three variants A45S,V177I and E180M with increased catalytic efficiencies.On this basis,we performed iterative combinatorial mutagenesis and constructed a mutant A45S/V177I/E180M which the amidase activity reached 98 U mL^(−1)in shake flasks and 2506 U mL^(−1)in 3-L fed-batch cultures.Moreover,single mutation T190 to S190 increased the substrate catalytic preference of R to K and the R/K value was improved to 7.5,which was 2 times better than the animal-sourced trypsin.

Construction and application of carbohydrate microarrays to detect foodborne bacteria

The rapid detection of pathogenic bacteria is vital for the prevention of outbreaks of infectious diseases, including infections by the common foodborne bacteria E.coli and Salmonella Carbohydrate microarrays have been developed as a powerful method to investigate carbohydrate-protein interaction with only very small amounts of glycans, which show great potential for detect the carbohydrate mediated interaction with pathogens. Here, different mannose-coated microarrays were constructed and tested with E.coli(K-12 and BL-21) and Salmonella enterica strains(ATCC9184 and ATCC31685) exhibiting different mannose binding affinities. The optimized carbohydrate microarray was then applied to test the binding of 12 Salmonella enterica and 9 E.coli isolates from local patients for the first time and showed strong binding with certain serovars or subtypes. The results showed that microarray probed with the single mannose structure is not enough for the detection of bacteria with various serovars or subtypes, which contain a high degree of allelic variation in adhesin. We suggest that a complex carbohydrate microarray containing different glycan conformation may be needed for detection of different bacteria isolates.

Yeast surface display of leech hyaluronidase for the industrial production of hyaluronic acid oligosaccharides

Leech hyaluronidase(LHyal)is a hyperactive hyaluronic acid(HA)hydrolase that belongs to the hyaluronoglu-curonidase family.Traditionally,LHyal is extracted from the heads of leeches,but the recent development of the Pichia pastoris recombinant LHyal expression method permitted the industrial production of size-specific HA oligosaccharides.However,at present LHyal expressed by recombinant yeast strains requires laborious protein purification steps.Moreover,the enzyme is deactivated and removed after single use.To solve this problem,we developed a recyclable LHyal biocatalyst using a yeast surface display(YSD)system.After screening and charac-terization,we found that the cell wall protein Sed1p displayed stronger anchoring to the P.pastoris cell wall than other cell wall proteins.By optimizing the type and length of the linkers between LHyal and Sed1p,we increased the activity of enzymes displayed on the P.pastoris cell wall by 50.34%in flask cultures.LHyal-(GGGS)6-Sed1p activity further increased to 3.58×105 U mL−1 in fed-batch cultivation in a 5 L bioreactor.Enzymatic prop-erty analysis results revealed that the displayed LHyal-(GGGS)6-Sed1p generated the same oligosaccharides but exhibited higher thermal stability than free LHyal enzyme.Moreover,displayed LHyal-(GGGS)6-Sed1p could be recovered easily from HA hydrolysis solutions via low-speed centrifugation and could be reused at least 5 times.YSD of LHyal not only increased the utilization efficiency of the enzyme but also simplified the purification pro-cess for HA oligosaccharides.Thus,this study provides an alternative approach for the industrial preparation of LHyal and HA oligosaccharides.

Synthesis and biological evaluation of nitric oxide(NO)-hydrogen sulfide(H2S) releasing derivatives of(S)-3-n-butylphthalide as potential antiplatelet agents

In the present study, a series of novel nitric oxide-hydrogen sulfide releasing derivatives of(S)-3-n-butylphthalide((S)-NBP) were designed, synthesized, and evaluated as potential antiplatelet agents. Compound NOSH-NBP-5 displayed the strongest activity in inhibiting the arachidonic acid(AA)- and adenosine diphosphate(ADP)-induced platelet aggregation in vitro, with 3.8- and 7.0-fold more effectiveness than(S)-NBP, respectively. Furthermore, NOSH-NBP-5 could release moderate levels of NO and H2 S, which would be beneficial in improving cardiovascular and cerebral circulation. Moreover, NOSH-NBP-5 could release(S)-NBP when incubated with rat brain homogenate. In conclusion, these findings may provide new insights into the development of novel antiplatelet agents for the treatment of thrombosis-related ischemic stroke.

Synthesis of 2-Amino-2-deoxy-1,3-dithioidoglycosides via Organocatalytic Relay Glycosylation of 3-O-Acetyl-2-nitrogalactals

Idose-type glycosides have numerous biological activities and have been widely used as anticoagulant drugs and anti-infection drugs.Thioglycosides have enhanced stability for acid-mediated or enzymatic hydrolysis,and have a wide range of applications in glycobiology and drug development.Herein,we describe an efficient method for site-selective and stereoselective synthesis of potential bioactive 2-amino-2-deoxy-1,3-dithioidoglycosides via organocatalysis sequential C3-Ferrier rearrangement and Michael addition of 3-O-acetyl-2-nitrogalactals.Both stepwise and one-pot protocols were carried out and work well.This unique thio-glycosylation protocol highlighted the various advantages,including(i)mild reaction conditions;(ii)excellent site-selectivity and stereoselectivity,good to excellent yields;(iii)broad substrate scopes;(iv)being atom-economic and environmentally friendly;(v)the reactions can be scaled up.

Design of artificial small regulatory trans-RNA for gene knockdown in Bacillus subtilis

Bacillus subtilis as the Gram-positive model bacterium has been widely used in synthetic biology and biotechnology while the regulatory RNA tools for B.subtilis are still not fully explored.Here,a bottom-up approach is proposed for designing artificial trans-acting sRNAs.By engineering the intrinsic sRNA SR6,a minimized core scaffold structure consisting of an 8 bp stem,a 4 nt loop,and a 9 nt polyU tail was generated and proven to be sufficient for constructing sRNAs with strong repression activity(83%).Moreover,we demonstrate this artificial sRNA system functions well in an hfq-independent manner and also achieves strong repression efficiency in Escherichia coli(above 80%).A structure-based sRNA design principle was further developed for the automatic generation of custom sRNAs with this core scaffold but various sequences,which facilitates the manipulation and avoids structure disruption when fusing any base-pairing sequence.By applying these auto-designed sRNAs,we rapidly modified the cell morphology and biofilm formation,and regulated metabolic flux toward acetoin biosynthesis.This sRNA system with cross-species regulatory activities not only enriched the gene regulation toolkit in synthetic biology for B.subtilis and E.coli but also enhanced our understanding of trans-acting sRNAs.

Total synthesis of D-glycero-D-mannno-heptose 1β, 7-bisphosphate with 3-O-amyl amine linker and its monophosphate derivative

D-Glycero-D-mannno-heptose 1β,7-bisphosphate(HBPβ)is an important intermediate for constructing the core structure of Gram-negative bacterial lipopolysaccharides and was reported as a pathogen-associated molecular pattern(PAMP)that regulates immune responses.HBPβwith 3-O-amyl amine linker and its monophosphate derivative D-glycero-D-mannno-heptose 7-phosphate(HP)with 1α-amyl amine linker have been synthesized as candidates for immunity study of HBPβ.The O3-amyl amine linker of heptose was installed by dibutyltin oxide-mediated regioselective alkylation under fine-tuned protecting condition.The stereoselective installation of 1β-phosphate ester was achieved by NIS-mediated phosphorylation at low temperature.The strategy for installation of 3-O-amyl amine linker onto HBP derivative can be expanded to the syntheses of other conjugation-ready carbohydrates bearing anomeric phosphoester.

Remodeling Isoprene Pyrophosphate Metabolism for Promoting Terpenoids Bioproduction

Terpenoids are the largest family of natural products.They are made from the building block isoprene pyrophosphate(IPP),and their bioproduction using engineered cell factories has received a great deal of attention.To date,the insufficient metabolic supply of IPP remains a great challenge for the efficient synthesis of terpenoids.In this work,we discover that the imbalanced metabolic flux distribution between the central metabolism and the IPP supply hinders IPP accumulation in Bacillus subtilis(B.subtilis).Therefore,we remodel the IPP metabolism using a series of genetically encoded two-input-multioutput(TIMO)circuits that are responsive to pyruvate or/and malonyl-CoA,resulting in an IPP pool that is significantly increased by up to four-fold.As a proof-of-concept validation,we design an IPP metabolism remodeling strategy to improve the production of three valuable terpenoids,including menaquinone-7(MK-7,4.1-fold),lycopene(9-fold),andβ-carotene(0.9-fold).In particular,the titer of MK-7 in a 50-L bioreactor reached 1549.6 mg·L^(-1),representing the highest titer reported so far.Thus,we propose a TIMO genetic circuits-assisted IPP metabolism remodeling framework that can be generally used for the synergistic fine-tuning of complicated metabolic modules to achieve the efficient bioproduction of terpenoids.

Chemical glycobiology drives the discovery of carbohydrate-based drugs

As one of the four major classes of biomolecules(carbo-hydrate,protein,nuclcotide,and lipid),carbohydrates are themost prominent features of the cell's exterior.Carbohydratesand their conjugates(glycoproteins.glycolipids)play import-ant roles in various biological processes,including inflamma-tion and immune response,angiogenesis and tumor cell meta-stasis,viral and bacterial infection,and many other cell-cellcommunications[1,2].Glycoscicnce has gained significant in-tcrest in biomedical research owing to their critical physiolo-gical functions.