Celiac disease(CD)is a globally prevalent autoimmune disorder,affecting 1.4%of the population with 7.5%annual incidence increase.In genetically predisposed individuals,gliadin antigen can trigger an immune response in...Celiac disease(CD)is a globally prevalent autoimmune disorder,affecting 1.4%of the population with 7.5%annual incidence increase.In genetically predisposed individuals,gliadin antigen can trigger an immune response in the small intestine,causing villous atrophy that leads to severe symptoms.Current treatment of CD relies on strict gluten-free diets.Neprosin from Nepenthes species is one of the most promising enzymes for the detoxification of gliadin antigen.However,the potential of neprosin in gluten-safe food production has yet to be explored.In this study,neprosin from Nepenthes rafflesiana(NrNpr1)was recombinantly expressed with chicken lysozyme signal peptide in the baker's yeast Saccharomyces cerevisiae expression host.The culture media of the bioengineered S.cerevisiae can degrade pure gliadin substrate and gliadin in wheat flour.The live yeast culture was demonstrated to degrade gliadin in wheat flour dough.The purified recombinant NrNpr1(rNrNpr1)enzyme was successfully characterized to have an optimal pH of 2.5,optimal temperature of 50℃,thermal tolerance of 0-65℃,and can digest all immunogenic epitopes inα-gliadin 33-mer when tested in vitro.ELISA test proved that rNrNpr1 can detoxify 750 mg/L gliadin to a gluten-free level(<20 mg/L)after 6 h of incubation.This study characterized neprosin from N.rafflesiana and report the bioengineering of S.cerevisiae with recombinant neprosin.We demonstrated that rNrNpr1 has promising potential applications in revolutionary gluten-safe food production,which needs further optimization and formulation in bakery.展开更多
Lacto-N-neotetraose(LNn T)is a crucial neutral core human milk oligosaccharide(HMO).In this study,we established a LNn T-producing Saccharomyces cerevisiae cell factory through comprehensive metabolic engineering.Spec...Lacto-N-neotetraose(LNn T)is a crucial neutral core human milk oligosaccharide(HMO).In this study,we established a LNn T-producing Saccharomyces cerevisiae cell factory through comprehensive metabolic engineering.Specifically,the de novo biosynthetic pathway of LNn T was assembled by heterologously expressing the lactose permease(lac12)from Kluyveromyces lactis and the glycosyltransferase from Neisseria meningitidis in S.cerevisiae.Subsequently,carbon source regulation based on the glucose-sensitive GAL regulatory system was employed to optimize the expression time of heterologous genes,achieving a production of 15.61 mg/L of LNn T in shake-flask fermentation.In addition,the key rate-limiting steps involved in LNn T synthesis pathway were identified and the corresponding genes were overexpressed to enhance LNn T production,resulting in an 8-fold increase in LNn T titer compared to that of parental strain.To our knowledge,this is the first report on LNn T biosynthesis in S.cerevisiae,opening up the possibility of green production of LNn T using food-safe microorganisms.展开更多
Saccharomyces cerevisiae is not naturally capable of efficiently utilizing xylose as a carbon source.When cultured with lignocellulosic hydrolysates containing pretreatment-derived inhibitors,S.cerevisiae suffers from...Saccharomyces cerevisiae is not naturally capable of efficiently utilizing xylose as a carbon source.When cultured with lignocellulosic hydrolysates containing pretreatment-derived inhibitors,S.cerevisiae suffers from much lower sugar uptake,ethanol yield and fermentation efficiency.Thus,considering efficient xylose conversion into ethanol during non-detoxified hydrolysate culture,genetic engineering and adaptive evolution of S.cerevisiae might be a promising joint strategy for improving xylose uptake and ethanol production.In this study,an inhibitor-tolerant strain S.cerevisiae SPSC01-TAF94 was genetically engineered by overexpressing both xylose transport-and metabolism-related genes(N360F,Ru-xyl A,TAL1,TKL1,RKI1 and RPE1),yielding the xylose-utilizing strain TAF94-X,followed by three-stage adaptation in non-detoxified corn stover hydrolysate containing 5 g·L^(-1)acetic acid,0.32 g·L^(-1)furfural,0.17 g·L(-1)HMF and 0.19 g·L^(-1)vanillin as the major inhibitors as well as 20,40 and 60 g·L^(-1)xylose adjusted as the major carbon source,respectively.Finally,an active xylose-utilizing and ethanolproducing strain TAF94-X60 was obtained,which achieved 44.9 g·L^(-1)ethanol with yield of0.41 g·g^(-1),productivity of 0.62 g·L^(-1)·h^(-1)and xylose consumption rate of 0.42 g·L^(-1)·h^(-1)during hydrolysate culture,compared to those of 36.5 g·L^(-1),0.38 g·g^(-1),0.50 g·L^(-1)·h^(-1)and 0.20 g·L^(-1)·h^(-1)obtained with the control strain TAF94-X.The proposed joint strategy effectively utilizes hydrolyzed sugars while eliminating the need for conventional detoxification or water washing processes,thus enhancing the economic feasibility of large-scale lignocellulosic ethanol production.展开更多
MCM10 protein is an essential replication factor involved in the initiation of DNA replication. A mcm10 mutant (mcm10-1) of budding yeast shows a growth arrest at 37 degrees C. In the present work, we have isolated a ...MCM10 protein is an essential replication factor involved in the initiation of DNA replication. A mcm10 mutant (mcm10-1) of budding yeast shows a growth arrest at 37 degrees C. In the present work, we have isolated a mcm10-1 suppressor strain, which grows at 37 degrees C. Interestingly, this mcm10-1 suppressor undergoes cell cycle arrest at 14 degrees C. A novel gene, YLR003c, is identified by high-copy complementation of this suppressor. We called it as Cms1 (Complementation of Mcm 10 Suppressor). Furthermore, the experiments of transformation show that cells of mcm10-1 suppressor with high-copy plasmid but not low-copy plasmid grow at 14 degrees C, indicating that overexpression of Cms1 can rescue the growth arrest of this mcm10 suppressor at non-permissive temperature. These results suggest that CMS1 protein may functionally interact with MCM10 protein and play a role in the regulation of DNA replication and cell cycle control.展开更多
AIM： Anti-Saccharomyces anti-nuclear associated cerevisiae antibodies （ASCA）, anti-neutrophil antibodies （NANA） and antibodies to exocrine pancreas （PAB）, are serological tools for discriminating Crohn＇s disea...AIM： Anti-Saccharomyces anti-nuclear associated cerevisiae antibodies （ASCA）, anti-neutrophil antibodies （NANA） and antibodies to exocrine pancreas （PAB）, are serological tools for discriminating Crohn＇s disease （CrD） and ulcerative colitis （UC）. Like CrD, coeliac disease （COD） is an inflammatory bowel disease （IBD） associated with （auto） antibodies. Performing a multicenter study we primarily aimed to determine the performance of ASCA, NANA and PAB tests for IBD diagnosis in children and adults, and secondarily to evaluate the prevalence of these markers in CoD. METHODS： Sera of 109 patients with CrD, 78 with UC, 45 with CoD and 50 healthy blood donors were retrospectively included. ASCA, NANA and PAB were detected by indirect immunofluorescence （IIF）. RESULTS： ASCA＋/NANA- profile displayed a positive predictive value of 94.2% for CrD. Detection of ASCA was correlated with a more severe clinical profile of CrD and treatment of the disease did not influence their serum levels. ASCA positivity was found in 37.9% of active CoD.PAB were found in 36.7% CrD and 13.3% CoD patients and were not correlated with clinical features of CrD, except with an early onset of the disease. Fifteen CrD patients were ASCA negative and PAB positive. CONCLUSION： ASCA and PAB detected by IIF are specific markers for CrD although their presence does not rule out a possible active CoD. The combination of ASCA, NANA and PAB tests improves the sensitivity of immunological markers for CrD. Repeating ASCA, NANA, and PAB testing during the course of CrD has no clinical value.展开更多
The present study was conducted to determine effects of different forms of yeast （Saccharomyces cerevisiae, strain Y200007） on the growth performance, intestinal development, and systemic immunity in early-weaned pi...The present study was conducted to determine effects of different forms of yeast （Saccharomyces cerevisiae, strain Y200007） on the growth performance, intestinal development, and systemic immunity in early-weaned piglets. A total of 96 piglets （14-d old, initial average body weight of 4.5 kg） were assigned to 4 dietary treatments： （1） basa diet without yeast （Control）; （2） basal diet supplemented with 3.00 g/kg live yeast （LY）; （3） basal diet supplemented with 2.66 g/kg heat-killed whole yeast （HKY）; and （4） basal diet supplemented with 3.00 g/kg superfine yeast powders （SFY）. Diets and water were provided ad libitum to the piglets during 3-week experiment. Growth performance of piglets was measured weekly. Samples of blood and small intestine were collected at days 7 and 21 of experiment. Dietary supplementation with LY and SFY improved G：F of piglets at days ]-21 of the experiment （P 〈 0.05） compared to Control group. Serum concentrations of growth hormone （GH）, triiodothyronine （T3）, tetraiodothyronine （T4）, and insulin growth factor 1 （iGF-1） in piglets at day 21 of the experiment were higher when fed diets supplemented with LY and SFY than those in Control group （P 〈 0.05）. Compared to Control group, contents of serum urea nitrogen of piglets were reduced by the 3 yeast-supplemented diets （P 〈 0.05）. Diets supplemented with LY increased villus height and villus-to-crypt ratio in duodenum and jejunum of piglets （P 〈 0.05） compared to other two groups at day 7 of the experiment. Feeding diets supplemented with LY and SFY increased （P 〈 0.05） serum concentrations of IgA, IL-2, and IL-6 levels in piglets compared to Control. The CD4＋/CD8＋ ratio and proliferation of T-lymphocytes in piglets fed diets supplemented with LY were increased compared to that of Control group at day 7 of the experiment （P 〈 0.05）. In conclusion, dietary supplementation with both LY and SFY enhanced feed conversion, small intestinal development, and systemic immunity in early-weaned piglets, with better improvement in feed conversion by dietary supplementation with LY, while dietary supplementation with SFY was more effective in increasing systemic immune functions in early-weaned piglets.展开更多
Engineering the biosynthesis of plant-derived natural products in microbes presents several challenges, especially when the expression and activation of the plant cytochrome P450 enzyme is required. By recruiting two ...Engineering the biosynthesis of plant-derived natural products in microbes presents several challenges, especially when the expression and activation of the plant cytochrome P450 enzyme is required. By recruiting two enzymes—HpaB and HpaC—from several bacteria, we constructed functional 4- hydroxyphenylacetate 3-hydroxylase (4HPA3H) in Saccharomyces cerevisiae to take on a role similar to that of the plant-derived cytochrome P450 enzyme and produce caffeic acid. Along with a common tyrosine ammonia lyase (TAL), the different combinations of HpaB and HpaC presented varied capabilities in producing the target product, caffeic acid, from the substrate, L-tyrosine. The highest production of caffeic acid was obtained with the enzyme combination of HpaB from Pseudomonas aeruginosa and HpaC from Salmonella enterica, which yielded up to (289.4 ± 4.6) mg-L1 in shake-flask cultivation. The compatibility of heterologous enzymes within a yeast chassis was effectively improved, as the caffeic acid production was increased by 40 times from the initial yield. Six key amino acid residues around the flavin adenine dinucleotide (FAD) binding domain in HpaB from Pseudomonas aeruginosa were differentiate from those other HpaBs, and might play critical roles in affecting enzyme activity. We have thus established an effective approach to construct a highly efficient yeast system to synthesize non-native hydroxylated phenylpropanoids.展开更多
Background:We aimed to characterize the protective effects and the molecular mechanisms of action of a Saccharomyces cerevisiae fermentation product(NTK)in response to a mastitis challenge.Eighteen mid-lactation multi...Background:We aimed to characterize the protective effects and the molecular mechanisms of action of a Saccharomyces cerevisiae fermentation product(NTK)in response to a mastitis challenge.Eighteen mid-lactation multiparous Holstein cows(n=9/group)were fed the control diet(CON)or CON supplemented with 19 g/d NTK for 45 d(phase 1,P1)and then infected in the right rear quarter with 2500 CFU of Streptococcus uberis(phase 2,P2).After 36-h,mammary gland and liver biopsies were collected and antibiotic treatment started until the end of P2(9 d post challenge).Cows were then followed until day 75(phase 3,P3).Milk yield(MY)and dry matter intake(DMI)were recorded daily.Milk samples for somatic cell score were collected,and rectal and udder temperature,heart and respiration rate were recorded during the challenge period(P2)together with blood samples for metabolite and immune function analyses.Data were analyzed by phase using the PROC MIXED procedure in SAS.Biopsies were used for transcriptomic analysis via RNA-sequencing,followed by pathway analysis.Results:DMI and MY were not affected by diet in P1,but an interaction with time was recorded in P2 indicating a better recovery from the challenge in NTK compared with CON.NTK reduced rectal temperature,somatic cell score,and temperature of the infected quarter during the challenge.Transcriptome data supported these findings,as NTK supplementation upregulated mammary genes related to immune cell antibacterial function(e.g.,CATHL4,NOS2),epithelial tissue protection(e.g.IL17C),and anti-inflammatory activity(e.g.,ATF3,BAG3,IER3,G-CSF,GRO1,ZFAND2A).Pathway analysis indicated upregulation of tumor necrosis factorα,heat shock protein response,and p21 related pathways in the response to mastitis in NTK cows.Other pathways for detoxification and cytoprotection functions along with the tight junction pathway were also upregulated in NTK-fed cows.Conclusions:Overall,results highlighted molecular networks involved in the protective effect of NTK prophylactic supplementation on udder health during a subclinical mastitic event.展开更多
The kinetics of asymmetric production of R-（-）-mandelic acid （R-MA） from phenylglyoxylic acid （PGA） catalyzed by Saccharomyces cerevisiae sp. strain FD11b was studied by fed-batch cultures. The concentrations of...The kinetics of asymmetric production of R-（-）-mandelic acid （R-MA） from phenylglyoxylic acid （PGA） catalyzed by Saccharomyces cerevisiae sp. strain FD11b was studied by fed-batch cultures. The concentrations of glucose and PGA were controlled respectively with a dual feeding system. When the electron donor glucose was supplied at the rate of 0.0833mmol·gdw^-1·h^-1, the specific production rate （qp） and the enantiomeric excess of R-MA reached the maximum 0.353mmol·gdw^-1·h^-1 and 97.1%, respectively. The apparent reduction activity of yeast FD 11 b was obviously affected by both substrate PGA and product MA. The qp value reached the maximum 0.36-0.38mmol·gdw^-1·h^-1 when the PGA concentration was controlled between 25 and 35mmol·L^-1. The obvious substrate inhibition of bioconversion was observed at the PGA concentrations higher than 40mmol·L^-1. The accumulation of product MA also caused a severe feed-back inhibition for its production when the product concentration was above 60mmol·L^-1. The kinetic model with the inhibition effect of both substrate and product was simulated by a computer-based least-square arithrnatic. The established kinetic model was in good agreement with the experimental data.展开更多
African swine fever virus(ASFV)infects domestic pigs and European wild boars with strong,hemorrhagic and high mortality.The primary cellular targets of ASFV is the porcine macrophages.Up to now,no commercial vaccine o...African swine fever virus(ASFV)infects domestic pigs and European wild boars with strong,hemorrhagic and high mortality.The primary cellular targets of ASFV is the porcine macrophages.Up to now,no commercial vaccine or effective treatment available to control the disease.In this study,three recombinant Saccharomyces cerevisiae(S.cerevisiae)strains expressing fused ASFV proteins-porcine Ig heavy chains were constructed and the immunogenicity of the S.cerevisiae-vectored cocktail ASFV feeding vaccine was further evaluated.To be specific,the P30-Fcγand P54-Fcαfusion proteins displaying on surface of S.cerevisiae cells were produced by fusing the Fc fragment of porcine immunoglobulin IgG1 or IgA1 with p30 or p54 gene of ASFV respectively.The recombinant P30-Fcγand P54-Fcαfusion proteins expressed by S.cerevisiae were verified by Western blotting,flow cytometry and immunofluorescence assay.Porcine immunoglobulin Fc fragment fused P30/P54 proteins elicited P30/P54-specific antibody production and induced higher mucosal immunity in swine.The absorption and phagocytosis of recombinant S.cerevisiae strains in IPEC-J2 cells or porcine alveolar macrophage(PAM)cells were significantly enhanced,too.Here,we introduce a kind of cheap and safe oral S.cerevisiae-vectored vaccine,which could activate the specific mucosal immunity for controlling ASFV infection.展开更多
The bglS gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtil＆ was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeas...The bglS gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtil＆ was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone a-factor （MFals）, and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction （PCR）-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae （SC-βG） was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A （PrA） activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-1,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-1,3-1,4-β-glucanase expression level with the maximum of 69.3 U/（h·ml） after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-1,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.展开更多
Background Subacute ruminal acidosis(SARA)is a common metabolic disorder of high yielding dairy cows,and it is associated with dysbiosis of the rumen and gut microbiome and host inflammation.This study evaluated the i...Background Subacute ruminal acidosis(SARA)is a common metabolic disorder of high yielding dairy cows,and it is associated with dysbiosis of the rumen and gut microbiome and host inflammation.This study evaluated the impact of two postbiotics from Saccharomyces cerevisiae fermentation products(SCFP)on rumen liquid associated microbiota of lactating dairy cows subjected to repeated grain-based SARA challenges.A total of 32 rumen cannulated cows were randomly assigned to 4 treatments from 4 weeks before until 12 weeks after parturition.Treatment groups included a Control diet or diets supplemented with postbiotics(SCFPa,14 g/d Original XPC;SCFPb-1X,19 g/d Nutri Tek;SCFPb-2X,38 g/d Nutri Tek,Diamond V,Cedar Rapids,IA,USA).Grain-based SARA challenges were conducted during week 5(SARA1)and week 8(SARA2)after parturition by replacing 20%DM of the base total mixed ration(TMR)with pellets containing 50%ground barley and 50%ground wheat.Total DNA from rumen liquid samples was subjected to V3–V416S r RNA gene amplicon sequencing.Characteristics of rumen microbiota were compared among treatments and SARA stages.Results Both SARA challenges reduced the diversity and richness of rumen liquid microbiota,altered the overall composition(β-diversity),and its predicted functionality including carbohydrates and amino acids metabolic pathways.The SARA challenges also reduced the number of significant associations among different taxa,number of hub taxa and their composition in the microbial co-occurrence networks.Supplementation with SCFP postbiotics,in particular SCFPb-2X,enhanced the robustness of the rumen microbiota.The SCFP supplemented cows had less fluctuation in relative abundances of community members when exposed to SARA challenges.The SCFP supplementation promoted the populations of lactate utilizing and fibrolytic bacteria,including members of Ruminococcaceae and Lachnospiraceae,and also increased the numbers of hub taxa during non-SARA and SARA stages.Supplementation with SCFPb-2X prevented the fluctuations in the abundances of hub taxa that were positively correlated with the acetate concentration,andα-andβ-diversity metrics in rumen liquid digesta.Conclusions Induction of SARA challenges reduced microbiota richness and diversity and caused fluctuations in major bacterial phyla in rumen liquid microbiota in lactating dairy cows.Supplementation of SCFP postbiotics could attenuate adverse effects of SARA on rumen liquid microbiota.展开更多
As a new biofuel, isobutanol has received more attentions in recent years. Because of its high tolerance to higher alcohols, Saccharomyces cerevisiae has potential advantages as a platform microbe to produce isobutano...As a new biofuel, isobutanol has received more attentions in recent years. Because of its high tolerance to higher alcohols, Saccharomyces cerevisiae has potential advantages as a platform microbe to produce isobutanol. In this study, we investigated integration effects of enhancing valine biosynthesis by overexpression of ILV2 and BAT2 with eliminating ethanol formation by deletion of PDC6 and decreasing acetyl-Co A biosynthesis by deletion of LPD1 on isobutanol titers. Our results showed that deletion of LPD1 in strains overexpressing BAT2 and ILV2 increased isobutanol titer by 5.3-fold compared with control strain. Additional deletion of PDC6 in lpd1Δ strains carrying overexpressed BAT2 and ILV2 further increased isobutanol titer by 1.5 fold. Overexpression of BAT2 and ILV2 in lpd1Δ strains and pdc6Δ strains decreased ethanol titers. Glycerol titers of the engineered strains did not have greater changes than that of control strain, while their acetic acid titers were higher, perhaps due to the imbalance of cofactors in isobutanol synthesis. Our researches suggest that double-gene deletion of PDC6 and LPD1 in strains overexpressing BAT2 and ILV2 could increase isobutanol production dramatically than single-gene deletion of PDC6 or LPD1. This study reveals the integration effects of overexpression of ILV2/BAT2 and double-gene deletion of LPD1 and PDC6 on isobutanol production, and helps understanding future developments of engineered strains for producing isobutanol.展开更多
AIMTo confirm previous conclusions on Saccharomyces cerevisiae(S.cerevisiae)CNCM I-3856 for irritable bowel syndrome(IBS)management.METHODSAn individual patient data meta-analysis was performed on two randomized clini...AIMTo confirm previous conclusions on Saccharomyces cerevisiae(S.cerevisiae)CNCM I-3856 for irritable bowel syndrome(IBS)management.METHODSAn individual patient data meta-analysis was performed on two randomized clinical trials studying the effect of S.cerevisiae CNCM I-3856 supplementation on gastrointestinal(GI)symptoms in IBS subjects.A total of 579 IBS subjects were included.Outcomes were the daily Likert scale scores of abdominal pain/discomfort and bloating[area under the curve(AUC)and weekly means],responder status,and bowel movements(stool frequency and consistency).Statistical analyses were conducted in Intent to Treat(ITT)population,IBS-C subjects and IBS-C subjects with an abdominal pain/discomfort score higher than or equal to 2 at baseline(“IBS-C≥2 subpopulation”).RESULTSS.cerevisiae CNCM I-3856 significantly improved abdominal pain/discomfort and bloating during the second month of supplementation[AUC(W5-W8)]with improvement up to the minimal clinically relevant threshold of 10%:a 12.3%reduction of abdominal pain/discomfort in the ITT population compared to the Placebo group(P=0.0134)has been observed.In the IBS-C≥2 subpopulation,there were a 13.1%reduction of abdominal pain/discomfort and a 14.9%reduction of bloating compared to the Placebo group(P=0.0194 and P=0.0145,respectively).GI symptoms significantly decreased during supplementation but no statistical differences were reported between groups at the end of the supplementation period.Responder status was defined as a subject who experienced a decrease of 1 arbitrary unit(a.u.)or 50%of the abdominal discomfort score from baseline for at least 2 wk out of the last 4 wk of the study.A significant difference between groups was reported in the ITT population,when considering the first definition:subjects in the Active group had 1.510 higher odds to be a responder(reduction of 1 a.u.of abdominal pain/discomfort)compared with subjects in the Placebo group(P=0.0240).At the end of supplementation period,stool consistency in the Active group of the ITT population was significantly improved and classified as“normal”compared to Placebo(respectively 3.13±1.197 a.u.vs 2.58±1.020 a.u.,P=0.0003).Similar results were seen in the IBS-C≥2 subpopulation(Active group:3.14±1.219 a.u.vs Placebo group:2.59±1.017 a.u.,P=0.0009).CONCLUSIONThis meta-analysis supports previous data linking S.cerevisiae I-3856 and improvement of GI symptoms,in IBS overall population and in the IBS-C and IBS-C≥2 subpopulations.展开更多
基金supported by the Malaysian Ministry of Higher Education Fundamental Research Grant Scheme(FRGS/1/2024/STG01/UKM/02/5)Universiti Kebangsaan Malaysia Research University Grant(GUP-2024-092)awarded to Hoe-Han Gohsupported by Universiti Kebangsaan Malaysia postdoctoral research scheme。
文摘Celiac disease(CD)is a globally prevalent autoimmune disorder,affecting 1.4%of the population with 7.5%annual incidence increase.In genetically predisposed individuals,gliadin antigen can trigger an immune response in the small intestine,causing villous atrophy that leads to severe symptoms.Current treatment of CD relies on strict gluten-free diets.Neprosin from Nepenthes species is one of the most promising enzymes for the detoxification of gliadin antigen.However,the potential of neprosin in gluten-safe food production has yet to be explored.In this study,neprosin from Nepenthes rafflesiana(NrNpr1)was recombinantly expressed with chicken lysozyme signal peptide in the baker's yeast Saccharomyces cerevisiae expression host.The culture media of the bioengineered S.cerevisiae can degrade pure gliadin substrate and gliadin in wheat flour.The live yeast culture was demonstrated to degrade gliadin in wheat flour dough.The purified recombinant NrNpr1(rNrNpr1)enzyme was successfully characterized to have an optimal pH of 2.5,optimal temperature of 50℃,thermal tolerance of 0-65℃,and can digest all immunogenic epitopes inα-gliadin 33-mer when tested in vitro.ELISA test proved that rNrNpr1 can detoxify 750 mg/L gliadin to a gluten-free level(<20 mg/L)after 6 h of incubation.This study characterized neprosin from N.rafflesiana and report the bioengineering of S.cerevisiae with recombinant neprosin.We demonstrated that rNrNpr1 has promising potential applications in revolutionary gluten-safe food production,which needs further optimization and formulation in bakery.
基金funded by the National Key Research and Development Program of China(2022YFF1100300)National Natural Science Foundation of China(22108097)+2 种基金Key Research and Development Program of Jiangsu Province(BE2022850)Taihu Innovation-Leading Talent of Wuxi City(1026010241230040)Cross-Integration Innovation Funding of SFST(SFST2023-KY-10).
文摘Lacto-N-neotetraose(LNn T)is a crucial neutral core human milk oligosaccharide(HMO).In this study,we established a LNn T-producing Saccharomyces cerevisiae cell factory through comprehensive metabolic engineering.Specifically,the de novo biosynthetic pathway of LNn T was assembled by heterologously expressing the lactose permease(lac12)from Kluyveromyces lactis and the glycosyltransferase from Neisseria meningitidis in S.cerevisiae.Subsequently,carbon source regulation based on the glucose-sensitive GAL regulatory system was employed to optimize the expression time of heterologous genes,achieving a production of 15.61 mg/L of LNn T in shake-flask fermentation.In addition,the key rate-limiting steps involved in LNn T synthesis pathway were identified and the corresponding genes were overexpressed to enhance LNn T production,resulting in an 8-fold increase in LNn T titer compared to that of parental strain.To our knowledge,this is the first report on LNn T biosynthesis in S.cerevisiae,opening up the possibility of green production of LNn T using food-safe microorganisms.
基金supported by the National Key Research and Development Program of China(2021YFC2101303)the National Natural Science Foundation of China(U22A20424 and 22378048)+5 种基金the Major scientific and technological projects of Sinopecthe Dalian Technology Talents Project for Distinguished Young Scholars(2021RJ03)the Fundamental Research Funds for the Central Universities(DUT25LAB104)the Liaoning Revitalization Talents Program(XLYC2202049)the Ningbo Natural Science Foundation(2022J013)the Ningbo Municipal Public Welfare Science and Technology Foundation(2024S004)。
文摘Saccharomyces cerevisiae is not naturally capable of efficiently utilizing xylose as a carbon source.When cultured with lignocellulosic hydrolysates containing pretreatment-derived inhibitors,S.cerevisiae suffers from much lower sugar uptake,ethanol yield and fermentation efficiency.Thus,considering efficient xylose conversion into ethanol during non-detoxified hydrolysate culture,genetic engineering and adaptive evolution of S.cerevisiae might be a promising joint strategy for improving xylose uptake and ethanol production.In this study,an inhibitor-tolerant strain S.cerevisiae SPSC01-TAF94 was genetically engineered by overexpressing both xylose transport-and metabolism-related genes(N360F,Ru-xyl A,TAL1,TKL1,RKI1 and RPE1),yielding the xylose-utilizing strain TAF94-X,followed by three-stage adaptation in non-detoxified corn stover hydrolysate containing 5 g·L^(-1)acetic acid,0.32 g·L^(-1)furfural,0.17 g·L(-1)HMF and 0.19 g·L^(-1)vanillin as the major inhibitors as well as 20,40 and 60 g·L^(-1)xylose adjusted as the major carbon source,respectively.Finally,an active xylose-utilizing and ethanolproducing strain TAF94-X60 was obtained,which achieved 44.9 g·L^(-1)ethanol with yield of0.41 g·g^(-1),productivity of 0.62 g·L^(-1)·h^(-1)and xylose consumption rate of 0.42 g·L^(-1)·h^(-1)during hydrolysate culture,compared to those of 36.5 g·L^(-1),0.38 g·g^(-1),0.50 g·L^(-1)·h^(-1)and 0.20 g·L^(-1)·h^(-1)obtained with the control strain TAF94-X.The proposed joint strategy effectively utilizes hydrolyzed sugars while eliminating the need for conventional detoxification or water washing processes,thus enhancing the economic feasibility of large-scale lignocellulosic ethanol production.
文摘MCM10 protein is an essential replication factor involved in the initiation of DNA replication. A mcm10 mutant (mcm10-1) of budding yeast shows a growth arrest at 37 degrees C. In the present work, we have isolated a mcm10-1 suppressor strain, which grows at 37 degrees C. Interestingly, this mcm10-1 suppressor undergoes cell cycle arrest at 14 degrees C. A novel gene, YLR003c, is identified by high-copy complementation of this suppressor. We called it as Cms1 (Complementation of Mcm 10 Suppressor). Furthermore, the experiments of transformation show that cells of mcm10-1 suppressor with high-copy plasmid but not low-copy plasmid grow at 14 degrees C, indicating that overexpression of Cms1 can rescue the growth arrest of this mcm10 suppressor at non-permissive temperature. These results suggest that CMS1 protein may functionally interact with MCM10 protein and play a role in the regulation of DNA replication and cell cycle control.
文摘AIM： Anti-Saccharomyces anti-nuclear associated cerevisiae antibodies （ASCA）, anti-neutrophil antibodies （NANA） and antibodies to exocrine pancreas （PAB）, are serological tools for discriminating Crohn＇s disease （CrD） and ulcerative colitis （UC）. Like CrD, coeliac disease （COD） is an inflammatory bowel disease （IBD） associated with （auto） antibodies. Performing a multicenter study we primarily aimed to determine the performance of ASCA, NANA and PAB tests for IBD diagnosis in children and adults, and secondarily to evaluate the prevalence of these markers in CoD. METHODS： Sera of 109 patients with CrD, 78 with UC, 45 with CoD and 50 healthy blood donors were retrospectively included. ASCA, NANA and PAB were detected by indirect immunofluorescence （IIF）. RESULTS： ASCA＋/NANA- profile displayed a positive predictive value of 94.2% for CrD. Detection of ASCA was correlated with a more severe clinical profile of CrD and treatment of the disease did not influence their serum levels. ASCA positivity was found in 37.9% of active CoD.PAB were found in 36.7% CrD and 13.3% CoD patients and were not correlated with clinical features of CrD, except with an early onset of the disease. Fifteen CrD patients were ASCA negative and PAB positive. CONCLUSION： ASCA and PAB detected by IIF are specific markers for CrD although their presence does not rule out a possible active CoD. The combination of ASCA, NANA and PAB tests improves the sensitivity of immunological markers for CrD. Repeating ASCA, NANA, and PAB testing during the course of CrD has no clinical value.
基金financially supported by grants from China Agriculture Research System(CARS-36)the Special Fund for Agro-scientific Research in the Public Interest(No.201403047)+1 种基金National Basic Research Program of China(2013CB127301 and 2013CB127304)Presidential Foundation of Guangdong Academy of Agricultural Sciences(201312)
文摘The present study was conducted to determine effects of different forms of yeast （Saccharomyces cerevisiae, strain Y200007） on the growth performance, intestinal development, and systemic immunity in early-weaned piglets. A total of 96 piglets （14-d old, initial average body weight of 4.5 kg） were assigned to 4 dietary treatments： （1） basa diet without yeast （Control）; （2） basal diet supplemented with 3.00 g/kg live yeast （LY）; （3） basal diet supplemented with 2.66 g/kg heat-killed whole yeast （HKY）; and （4） basal diet supplemented with 3.00 g/kg superfine yeast powders （SFY）. Diets and water were provided ad libitum to the piglets during 3-week experiment. Growth performance of piglets was measured weekly. Samples of blood and small intestine were collected at days 7 and 21 of experiment. Dietary supplementation with LY and SFY improved G：F of piglets at days ]-21 of the experiment （P 〈 0.05） compared to Control group. Serum concentrations of growth hormone （GH）, triiodothyronine （T3）, tetraiodothyronine （T4）, and insulin growth factor 1 （iGF-1） in piglets at day 21 of the experiment were higher when fed diets supplemented with LY and SFY than those in Control group （P 〈 0.05）. Compared to Control group, contents of serum urea nitrogen of piglets were reduced by the 3 yeast-supplemented diets （P 〈 0.05）. Diets supplemented with LY increased villus height and villus-to-crypt ratio in duodenum and jejunum of piglets （P 〈 0.05） compared to other two groups at day 7 of the experiment. Feeding diets supplemented with LY and SFY increased （P 〈 0.05） serum concentrations of IgA, IL-2, and IL-6 levels in piglets compared to Control. The CD4＋/CD8＋ ratio and proliferation of T-lymphocytes in piglets fed diets supplemented with LY were increased compared to that of Control group at day 7 of the experiment （P 〈 0.05）. In conclusion, dietary supplementation with both LY and SFY enhanced feed conversion, small intestinal development, and systemic immunity in early-weaned piglets, with better improvement in feed conversion by dietary supplementation with LY, while dietary supplementation with SFY was more effective in increasing systemic immune functions in early-weaned piglets.
基金the Ministry of Science and Technology of China (2014CB745100)the National Natural Science Foundation of China (21390203 and 21706186).
文摘Engineering the biosynthesis of plant-derived natural products in microbes presents several challenges, especially when the expression and activation of the plant cytochrome P450 enzyme is required. By recruiting two enzymes—HpaB and HpaC—from several bacteria, we constructed functional 4- hydroxyphenylacetate 3-hydroxylase (4HPA3H) in Saccharomyces cerevisiae to take on a role similar to that of the plant-derived cytochrome P450 enzyme and produce caffeic acid. Along with a common tyrosine ammonia lyase (TAL), the different combinations of HpaB and HpaC presented varied capabilities in producing the target product, caffeic acid, from the substrate, L-tyrosine. The highest production of caffeic acid was obtained with the enzyme combination of HpaB from Pseudomonas aeruginosa and HpaC from Salmonella enterica, which yielded up to (289.4 ± 4.6) mg-L1 in shake-flask cultivation. The compatibility of heterologous enzymes within a yeast chassis was effectively improved, as the caffeic acid production was increased by 40 times from the initial yield. Six key amino acid residues around the flavin adenine dinucleotide (FAD) binding domain in HpaB from Pseudomonas aeruginosa were differentiate from those other HpaBs, and might play critical roles in affecting enzyme activity. We have thus established an effective approach to construct a highly efficient yeast system to synthesize non-native hydroxylated phenylpropanoids.
文摘Background:We aimed to characterize the protective effects and the molecular mechanisms of action of a Saccharomyces cerevisiae fermentation product(NTK)in response to a mastitis challenge.Eighteen mid-lactation multiparous Holstein cows(n=9/group)were fed the control diet(CON)or CON supplemented with 19 g/d NTK for 45 d(phase 1,P1)and then infected in the right rear quarter with 2500 CFU of Streptococcus uberis(phase 2,P2).After 36-h,mammary gland and liver biopsies were collected and antibiotic treatment started until the end of P2(9 d post challenge).Cows were then followed until day 75(phase 3,P3).Milk yield(MY)and dry matter intake(DMI)were recorded daily.Milk samples for somatic cell score were collected,and rectal and udder temperature,heart and respiration rate were recorded during the challenge period(P2)together with blood samples for metabolite and immune function analyses.Data were analyzed by phase using the PROC MIXED procedure in SAS.Biopsies were used for transcriptomic analysis via RNA-sequencing,followed by pathway analysis.Results:DMI and MY were not affected by diet in P1,but an interaction with time was recorded in P2 indicating a better recovery from the challenge in NTK compared with CON.NTK reduced rectal temperature,somatic cell score,and temperature of the infected quarter during the challenge.Transcriptome data supported these findings,as NTK supplementation upregulated mammary genes related to immune cell antibacterial function(e.g.,CATHL4,NOS2),epithelial tissue protection(e.g.IL17C),and anti-inflammatory activity(e.g.,ATF3,BAG3,IER3,G-CSF,GRO1,ZFAND2A).Pathway analysis indicated upregulation of tumor necrosis factorα,heat shock protein response,and p21 related pathways in the response to mastitis in NTK cows.Other pathways for detoxification and cytoprotection functions along with the tight junction pathway were also upregulated in NTK-fed cows.Conclusions:Overall,results highlighted molecular networks involved in the protective effect of NTK prophylactic supplementation on udder health during a subclinical mastitic event.
基金Supported by the Natural Science Foundation of Fujian Province (No.E0310019) and Key Project of Science and Technology of Fujian Province (No.2003H023).
文摘The kinetics of asymmetric production of R-（-）-mandelic acid （R-MA） from phenylglyoxylic acid （PGA） catalyzed by Saccharomyces cerevisiae sp. strain FD11b was studied by fed-batch cultures. The concentrations of glucose and PGA were controlled respectively with a dual feeding system. When the electron donor glucose was supplied at the rate of 0.0833mmol·gdw^-1·h^-1, the specific production rate （qp） and the enantiomeric excess of R-MA reached the maximum 0.353mmol·gdw^-1·h^-1 and 97.1%, respectively. The apparent reduction activity of yeast FD 11 b was obviously affected by both substrate PGA and product MA. The qp value reached the maximum 0.36-0.38mmol·gdw^-1·h^-1 when the PGA concentration was controlled between 25 and 35mmol·L^-1. The obvious substrate inhibition of bioconversion was observed at the PGA concentrations higher than 40mmol·L^-1. The accumulation of product MA also caused a severe feed-back inhibition for its production when the product concentration was above 60mmol·L^-1. The kinetic model with the inhibition effect of both substrate and product was simulated by a computer-based least-square arithrnatic. The established kinetic model was in good agreement with the experimental data.
基金supported by the National Key Research and Development Program of China (2018YFD0500500)。
文摘African swine fever virus(ASFV)infects domestic pigs and European wild boars with strong,hemorrhagic and high mortality.The primary cellular targets of ASFV is the porcine macrophages.Up to now,no commercial vaccine or effective treatment available to control the disease.In this study,three recombinant Saccharomyces cerevisiae(S.cerevisiae)strains expressing fused ASFV proteins-porcine Ig heavy chains were constructed and the immunogenicity of the S.cerevisiae-vectored cocktail ASFV feeding vaccine was further evaluated.To be specific,the P30-Fcγand P54-Fcαfusion proteins displaying on surface of S.cerevisiae cells were produced by fusing the Fc fragment of porcine immunoglobulin IgG1 or IgA1 with p30 or p54 gene of ASFV respectively.The recombinant P30-Fcγand P54-Fcαfusion proteins expressed by S.cerevisiae were verified by Western blotting,flow cytometry and immunofluorescence assay.Porcine immunoglobulin Fc fragment fused P30/P54 proteins elicited P30/P54-specific antibody production and induced higher mucosal immunity in swine.The absorption and phagocytosis of recombinant S.cerevisiae strains in IPEC-J2 cells or porcine alveolar macrophage(PAM)cells were significantly enhanced,too.Here,we introduce a kind of cheap and safe oral S.cerevisiae-vectored vaccine,which could activate the specific mucosal immunity for controlling ASFV infection.
基金the National Hi-Tech Research and Develop-ment Program (863) of China (No. 2007AA10Z315)the Natural Science Foundation of Zhejiang Province, China (No. Z304076)
文摘The bglS gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtil＆ was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone a-factor （MFals）, and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction （PCR）-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae （SC-βG） was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A （PrA） activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-1,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-1,3-1,4-β-glucanase expression level with the maximum of 69.3 U/（h·ml） after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-1,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.
基金supported by grants from Natural Science and Engineering Research Council(NSERC)of Canada Collaborative Research and Development(CRD)programNSERC Discovery program,Dairy Farmers of Manitoba,and Diamond V,USA,to EK and JCP。
文摘Background Subacute ruminal acidosis(SARA)is a common metabolic disorder of high yielding dairy cows,and it is associated with dysbiosis of the rumen and gut microbiome and host inflammation.This study evaluated the impact of two postbiotics from Saccharomyces cerevisiae fermentation products(SCFP)on rumen liquid associated microbiota of lactating dairy cows subjected to repeated grain-based SARA challenges.A total of 32 rumen cannulated cows were randomly assigned to 4 treatments from 4 weeks before until 12 weeks after parturition.Treatment groups included a Control diet or diets supplemented with postbiotics(SCFPa,14 g/d Original XPC;SCFPb-1X,19 g/d Nutri Tek;SCFPb-2X,38 g/d Nutri Tek,Diamond V,Cedar Rapids,IA,USA).Grain-based SARA challenges were conducted during week 5(SARA1)and week 8(SARA2)after parturition by replacing 20%DM of the base total mixed ration(TMR)with pellets containing 50%ground barley and 50%ground wheat.Total DNA from rumen liquid samples was subjected to V3–V416S r RNA gene amplicon sequencing.Characteristics of rumen microbiota were compared among treatments and SARA stages.Results Both SARA challenges reduced the diversity and richness of rumen liquid microbiota,altered the overall composition(β-diversity),and its predicted functionality including carbohydrates and amino acids metabolic pathways.The SARA challenges also reduced the number of significant associations among different taxa,number of hub taxa and their composition in the microbial co-occurrence networks.Supplementation with SCFP postbiotics,in particular SCFPb-2X,enhanced the robustness of the rumen microbiota.The SCFP supplemented cows had less fluctuation in relative abundances of community members when exposed to SARA challenges.The SCFP supplementation promoted the populations of lactate utilizing and fibrolytic bacteria,including members of Ruminococcaceae and Lachnospiraceae,and also increased the numbers of hub taxa during non-SARA and SARA stages.Supplementation with SCFPb-2X prevented the fluctuations in the abundances of hub taxa that were positively correlated with the acetate concentration,andα-andβ-diversity metrics in rumen liquid digesta.Conclusions Induction of SARA challenges reduced microbiota richness and diversity and caused fluctuations in major bacterial phyla in rumen liquid microbiota in lactating dairy cows.Supplementation of SCFP postbiotics could attenuate adverse effects of SARA on rumen liquid microbiota.
基金Supported by the National Natural Science Foundation of China(No.21206028)the Doctoral Fund of Ministry of Education of China(No.20121317120014)+3 种基金the Natural Science Foundation of Heibei Province(No.B2013202288)the Hebei Provincial Office of Education Science and Technology Research Projects(No.q2012024)the Hebei University of Technology Outstanding Youth Science and Technology Innovation Fund(No.2012009)the Open Fund of Key Laboratory of System Bioengineering of Ministry of Education of China(Tianjin University)(No.20130315)
文摘As a new biofuel, isobutanol has received more attentions in recent years. Because of its high tolerance to higher alcohols, Saccharomyces cerevisiae has potential advantages as a platform microbe to produce isobutanol. In this study, we investigated integration effects of enhancing valine biosynthesis by overexpression of ILV2 and BAT2 with eliminating ethanol formation by deletion of PDC6 and decreasing acetyl-Co A biosynthesis by deletion of LPD1 on isobutanol titers. Our results showed that deletion of LPD1 in strains overexpressing BAT2 and ILV2 increased isobutanol titer by 5.3-fold compared with control strain. Additional deletion of PDC6 in lpd1Δ strains carrying overexpressed BAT2 and ILV2 further increased isobutanol titer by 1.5 fold. Overexpression of BAT2 and ILV2 in lpd1Δ strains and pdc6Δ strains decreased ethanol titers. Glycerol titers of the engineered strains did not have greater changes than that of control strain, while their acetic acid titers were higher, perhaps due to the imbalance of cofactors in isobutanol synthesis. Our researches suggest that double-gene deletion of PDC6 and LPD1 in strains overexpressing BAT2 and ILV2 could increase isobutanol production dramatically than single-gene deletion of PDC6 or LPD1. This study reveals the integration effects of overexpression of ILV2/BAT2 and double-gene deletion of LPD1 and PDC6 on isobutanol production, and helps understanding future developments of engineered strains for producing isobutanol.
文摘AIMTo confirm previous conclusions on Saccharomyces cerevisiae(S.cerevisiae)CNCM I-3856 for irritable bowel syndrome(IBS)management.METHODSAn individual patient data meta-analysis was performed on two randomized clinical trials studying the effect of S.cerevisiae CNCM I-3856 supplementation on gastrointestinal(GI)symptoms in IBS subjects.A total of 579 IBS subjects were included.Outcomes were the daily Likert scale scores of abdominal pain/discomfort and bloating[area under the curve(AUC)and weekly means],responder status,and bowel movements(stool frequency and consistency).Statistical analyses were conducted in Intent to Treat(ITT)population,IBS-C subjects and IBS-C subjects with an abdominal pain/discomfort score higher than or equal to 2 at baseline(“IBS-C≥2 subpopulation”).RESULTSS.cerevisiae CNCM I-3856 significantly improved abdominal pain/discomfort and bloating during the second month of supplementation[AUC(W5-W8)]with improvement up to the minimal clinically relevant threshold of 10%:a 12.3%reduction of abdominal pain/discomfort in the ITT population compared to the Placebo group(P=0.0134)has been observed.In the IBS-C≥2 subpopulation,there were a 13.1%reduction of abdominal pain/discomfort and a 14.9%reduction of bloating compared to the Placebo group(P=0.0194 and P=0.0145,respectively).GI symptoms significantly decreased during supplementation but no statistical differences were reported between groups at the end of the supplementation period.Responder status was defined as a subject who experienced a decrease of 1 arbitrary unit(a.u.)or 50%of the abdominal discomfort score from baseline for at least 2 wk out of the last 4 wk of the study.A significant difference between groups was reported in the ITT population,when considering the first definition:subjects in the Active group had 1.510 higher odds to be a responder(reduction of 1 a.u.of abdominal pain/discomfort)compared with subjects in the Placebo group(P=0.0240).At the end of supplementation period,stool consistency in the Active group of the ITT population was significantly improved and classified as“normal”compared to Placebo(respectively 3.13±1.197 a.u.vs 2.58±1.020 a.u.,P=0.0003).Similar results were seen in the IBS-C≥2 subpopulation(Active group:3.14±1.219 a.u.vs Placebo group:2.59±1.017 a.u.,P=0.0009).CONCLUSIONThis meta-analysis supports previous data linking S.cerevisiae I-3856 and improvement of GI symptoms,in IBS overall population and in the IBS-C and IBS-C≥2 subpopulations.
