Acetic acid-and furfural-based adaptive evolution of Saccharomyces cerevisiae strains for improving stress tolerance and lignocellulosic ethanol production

Acetic acid and furfural are known as prevalent inhibitors deriving from pretreatment during lignocellulosic ethanol production.They negatively impact cell growth,glucose uptake and ethanol biosynthesis of Saccharomyces cerevisiae strains.Development of industrial S.cerevisiae strains with high tolerance towards these inhibitors is thus critical for efficient lignocellulosic ethanol production.In this study,the acetic acid or furfural tolerance of different S.cerevisiae strains could be significantly enhanced after adaptive evolution via serial cultivation for 40 generations under stress conditions.The acetic acid-based adaptive strain SPSC01-TA9 produced 30.5 g·L^(-1)ethanol with a yield of 0.46 g·g^(-1)in the presence of 9 g·L^(-1)acetic acid,while the acetic acid/furfural-based adaptive strain SPSC01-TAF94 produced more ethanol of 36.2 g·L^(-1)with increased yield up to 0.49 g·g^(-1)in the presence of both 9 g·L^(-1)acetic acid and 4 g·L^(-1)furfural.Significant improvements were also observed during non-detoxified corn stover hydrolysate culture by SPSC01-TAF94,which achieved ethanol production and yield of 29.1 g·L^(-1)and 0.49 g·g^(-1),respectively,the growth and fermentation efficiency of acetic acid/furfural-based adaptive strain in hydrolysate was 95%higher than those of wildtype strains,indicating the acetic acid-and furfural-based adaptive evolution strategy could be an effective approach for improving lignocellulosic ethanol production.The adapted strains developed in this study with enhanced tolerance against acetic acid and furfural could be potentially contribute to economically feasible and sustainable lignocellulosic biorefinery.

Postbiotics from Saccharomyces cerevisiae fermentation stabilize microbiota in rumen liquid digesta during grain-based subacute ruminal acidosis(SARA) in lactating dairy cows

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.

Saccharomyces cerevisiae prevents postoperative recurrence of Crohn's disease modeled by ileocecal resection in HLA-B27 transgenic rats

BACKGROUND Postoperative recurrence(POR)after ileocecal resection(ICR)affects most Crohn's disease patients within 3-5 years after surgery.Adherent-invasive Escherichia coli(AIEC)typified by the LF82 strain are pathobionts that are frequently detected in POR of Crohn's disease and have a potential role in the early stages of the disease pathogenesis.Saccharomyces cerevisiae CNCM I-3856 is a probiotic yeast reported to inhibit AIEC adhesion to intestinal epithelial cells and to favor their elimination from the gut.AIM To evaluate the efficacy of CNCM I-3856 in preventing POR induced by LF82 in an HLA-B27 transgenic(TgB27)rat model.METHODS Sixty-four rats[strain F344,38 TgB27,26 control non-Tg(nTg)]underwent an ICR at the 12th wk(W12)of life and were sacrificed at the 18th wk(W18)of life.TgB27 rats were challenged daily with oral administration of LF82(109 colony forming units(CFUs)/day(d),n=8),PBS(n=5),CNCM I-3856(109 CFUs/d,n=7)or a combination of LF82 and CNCM I-3856(n=18).nTg rats receiving LF82(n=5),PBS(n=5),CNCM I-3856(n=7)or CNCM I-3856 and LF82(n=9)under the same conditions were used as controls.POR was analyzed using macroscopic(from 0 to 4)and histologic(from 0 to 6)scores.Luminal LF82 quantifications were performed weekly for each animal.Adherent LF82 and inflammatory/regulatory cytokines were quantified in biopsies at W12 and W18.Data are expressed as the median with the interquartile range.RESULTS nTg animals did not develop POR.A total of 7/8(87%)of the TgB27 rats receiving LF82 alone had POR(macroscopic score≥2),which was significantly prevented by CNCM I-3856 administration[6/18(33%)TgB27 rats,P=0.01].Macroscopic lesions were located 2 cm above the anastomosis in the TgB27 rats receiving LF82 alone and consisted of ulcerations with a score of 3.5(2-4).Seven out of 18 TgB27 rats(39%)receiving CNCM I-3856 and LF82 had no macroscopic lesions.Compared to untreated TgB27 animals receiving LF82 alone,coadministration of CNCM I-3856 and LF82 significantly reduced the macroscopic[3.5(2-4)vs 1(0-3),P=0.002]and histological lesions by more than 50%[4.5(3.3-5.8)vs 2(1.3-3),P=0.003].The levels of adherent LF82 were correlated with anastomotic macroscopic scores in TgB27 rats(r=0.49,P=0.006),with a higher risk of POR in animals having high levels of luminal LF82(71.4%vs 25%,P=0.02).Administration of CNCM I-3856 significantly reduced the levels of luminal and adherent LF82,increased the production of interleukin(IL)-10 and decreased the production of IL-23 and IL-17 in TgB27 rats.CONCLUSION In a reliable model of POR induced by LF82 in TgB27 rats,CNCM I-3856 prevents macroscopic POR by decreasing LF82 infection and gut inflammation.

Reducing Yield of Fusel Alcohols by Saccharomyces cerevisiae of Compound Mutagenesis Through UV-MPMS Method

Excessive fusel alcohol contents will cause the beer to produce off-flavors and cause dizziness and headaches.Reducing the contents of fusel alcohols in beer is very important to people's health.The excessive fusel alcohol contents in beer is a common problem in the industry.How to control the contents of fusel alcohols in a reasonable range is of great significance for improving beer quality.After one round of ultraviolet(UV)and one round of multifunctional plasma mutagenesis system(MPMS)mutagenesis,the yeast strains with lower fusel oil yield and more stablility could be screened.According to the relationship between the fusel alcohol Harris metabolic pathway of brewer's yeast and lactic acid metabolism,excellent strains were obtained by triple screening with lactic acid medium,calcium carbonate medium and 2,3,5-triphenyl tetrazolium chloride upper medium.The content of fusel alcohol in the finished beer fermentation test of screened strain Z43 was 52.1±0.142 mg•L^(-1),which was 43%lower than that of the starting strain,and other fermentation properties remained unchanged.After eight passages,it was verified that the strain was stable and heritable.These results showed that strain Z43 presented promising characteristics for use in the production of beer with a potentially low contents of fusel alcohols.

伴有辅酶再生过程的Saccharomyces cerevisiae B5不对称还原2’-氯-苯乙酮的催化反应动力学

对以5%(体积比)乙醇为辅助底物的伴有辅酶再生过程的SaccharomycescerevisiaeB5不对称还原2’-氯-苯乙酮制备手性药物中间体R-2’-氯-1-苯乙醇的生物催化反应建立了描述底物消耗和产物合成的动力学模型。考察了反应过程中辅酶的种类和含量,以及底物和产物随时间的变化量。研究表明,参加反应的还原剂是辅酶Ⅰ。当底物初始浓度≤8.09mmol?L?1,可不考虑底物对微生物的毒害作用,反应可看作两个均符合顺序反应机制的氧化还原反应的耦联。通过实验数据对动力学方程式的拟合,得到动力学参数:Vm1=8.0×10?4mol?L?1?h?1,KmB1=9.0×10-4mol?L?1,KiA1=2.0×10-6mol?L?1。动力学模型模拟计算结果与实验值能较好地吻合。

利用Saccharomyces cerevisiae KD控制苹果汁中棒曲霉素的污染

研究一株食品生产用酿酒酵母Saccharomyces cerevisiae KD在培养基及市售100%苹果汁中对棒曲霉素污染的控制作用。通过高效液相色谱法对棒曲霉素进行定量,分析起始棒曲霉素浓度、菌体接种量和培养基p H对S.cerevisiae KD去除棒曲霉素活力的影响;利用酶标仪监测S.cerevisiae KD的生长状况,且通过检测可溶性固形物、酸度、总酚、黄酮含量对菌体发酵后苹果汁的品质进行了评估。结果表明:有氧条件下S.cerevisiae KD能够在28 h内完全去除培养基中的棒曲霉素,其去除机理包括物理吸附和酶解;在较低的起始棒曲霉素浓度和较高的菌体接种量条件下,S.cerevisiae KD对棒曲霉素的去除率较高,但在培养后期,不同菌体接种量下棒曲霉素的去除率接近一致;实验还发现酸性条件有利于S.cerevisiae KD去除棒曲霉素。此外,S.cerevisiae KD对棒曲霉素的耐受性较强,甚至在棒曲霉素浓度高达100 mg/L的环境中依然能较好生长。在市售100%苹果汁中,S.cerevisiae KD也能高效控制棒曲霉素的污染,且与Lactococcus lactis MG1363联合发酵2 d后,果汁中已无棒曲霉素检出,总酚含量显著高于发酵前苹果汁(p<0.05),发酵果汁的品质较好。结论:S.cerevisiae KD可有效控制食品中棒曲霉素的污染,具有潜在的应用前景。

响应面法优化高选择性羰基还原酶产生菌Saccharomyces cerevisiae B5的培养基组成

借助于SAS软件,采用部分因子实验设计(FFD)和响应面分析法(RSM),对能够产生高选择性羰基还原酶的菌株Saccharomyces cerevisiae B5进行发酵培养基的优化.部分因子实验设计结果表明葡萄糖、硫酸铵、硫酸镁及磷酸氢二钾为影响Saccharomyces cerevisiae B5产生高选择性羰基还原酶的四个显著性因素.通过响应面分析法,得到最优发酵培养基组成为:葡萄糖29.7 g/L,酵母粉3 g/L,硫酸铵4.784 g/L,无水硫酸镁0.267 2 g/L,K2HPO4.3H2O 1.026 g/L,KH2PO41g/L.在优化的培养基条件下,羰基还原酶活力最高可达1 498.2 U/g.

水/有机相两相体系中Saccharomyces cerevisiae B5不对称还原制备手性药物中间体R-2′-氯-1-苯乙醇的研究

本文对水/有机相两相体系中Saccharomyces cerevisiae B5催化2-氯-苯乙酮不对称还原制备R-氯丙那林中间体R-2-氯-1-苯乙醇进行了详细研究。结果表明,水/十二烷两相体系还原2-氯-苯乙酮可获得较高产率,R-2'-氯-1-苯乙醇的对映体过剩值可达100%ee,最佳转化时间为72h,证实了在水/有机溶剂两相系统中还原反应的活力倍数与有机溶剂的Log_(Poct)

植物乳杆菌Saccharomy cescerevisiae P13对大黄鱼的促生长作用研究

[目的]研究植物乳杆菌Saccharomyces cerevisiae P13对大黄鱼生长和抗病能力的影响.[方法]用合0、104、106、108、1010cfu/kg植物乳杆菌Saccharomyces cerevisiae P13含量的饲料喂养大黄鱼4周后,测定大黄鱼的体重增长率、饲料转化率和感染病菌后的存活率.[结果]大黄鱼后肠中的Saccharomyces cerevisiae P13菌数量在喂食过程中显著增加,含106～1010 cfu/kg Saccharomyces cerevisiae P13的配方饲料明显促进了大黄鱼的体重增长率和摄食率.喂食含106～ 1010cfu/kg Saccharomyces cerevisiae P13的配方饲料后大黄鱼对Streptococcus sp.的抵抗力明显增强.[结论]植物乳杆菌Saccharomyces cerevisiae P13能促进大黄鱼生长,并增强大黄鱼的抗病力.

Overexpression of a novel gene , Cms1, can rescue the growth arrest of a Saccharomyces cerevisiae mcm10 suppressor

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.

Saccharomyces cerevisiae谷氨酸脱羧酶的诱导纯化及其活性的研究

为了了解酿酒酵母(Saccharomyces cerevisiae,S.cerevisiae)谷氨酸脱羧酶(GAD)的情况,分别用酵母提取物及胰蛋白胨(3×YP)+6%棉子糖液体培养基、3×YP+6%半乳糖液体培养基进行诱导培养,粉碎破碎酵母,采用ProfiniaTM蛋白质纯化系统纯化,并用SDS-PAGE凝胶电泳测定分子质量,同时研究了磷酸吡哆醛(PLP)对酿酒酵母谷氨酸脱羧酶(S.cerevisiae GAD)活性的影响。结果表明:转基因酿酒酵母在28℃振动培养47 h,3×YP+6%棉子糖培养基OD600值为7.4,3×YP+6%半乳糖培养基OD600值为6.8。棉子糖诱导的酿酒酵母没有表达目的基因,没有纯化到GAD;相反,半乳糖诱导的酿酒酵母表达了目的基因,纯化到了GAD,其分子质量为67 ku。PLP对GAD的活化作用较显著。

Update on Anti-Saccharomyces cerevisiae antibodies, anti-nuclear associated anti-neutrophil antibodies and antibodies to exocrine pancreas detected by indirect immunofluorescence as biomarkers in chronic inflammatory bowel diseases: Results of a multicent

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.

Effects of different forms of yeast Saccharomyces cerevisiae on growth performance,intestinal development,and systemic immunity in early-weaned piglets

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.

Engineering the Biosynthesis of Caffeic Acid in Saccharomyces cerevisiae with Heterologous Enzyme Combinations

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.

Kinetics of Asymmetric Reduction of Phenylglyoxylic Acid to R-(-)-Mandelic Acid by Saccharomyces Cerevisiae FD11b

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.

Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination

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.

Feeding a Saccharomyces cerevisiae fermentation product improves udder health and immune response to a Streptococcus uberis mastitis challenge in mid-lactation dairy cows

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.

Increasing isobutanol yield by double-gene deletion of PDC6 and LPD1 in Saccharomyces cerevisiae

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.

Saccharomyces cerevisiae CNCM I-3856 in irritable bowel syndrome: An individual subject meta-analysis

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 (&#x0201c;IBS-C &#x02265; 2 subpopulation&#x0201d;).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 &#x02265; 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 &#x0201c;normal&#x0201d; compared to Placebo (respectively 3.13 &#x000b1; 1.197 a.u. vs 2.58 &#x000b1; 1.020 a.u., P = 0.0003). Similar results were seen in the IBS-C &#x02265; 2 subpopulation (Active group: 3.14 &#x000b1; 1.219 a.u. vs Placebo group: 2.59 &#x000b1; 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 &#x02265; 2 subpopulations.

Integrated Expression of the Oenococcus oeni mleA Gene in Saccharomyces cerevisiae

Malolactic enzyme is the function enzyme which catalyses the reaction for L-malate converting to L-lactic during malolactic fermentation （MLF）. In this paper, researches concerning the malolactic enzyme gene mleA cloned from a patent strain Oenococcus oeni SD-2a screened in Chinese wine and integrated expressing in Saccharomyces cerevisiae were performed in order to carry out both alcoholic fermentation （AF） and malolactic fermentation （MLF） during winemaking, with a view to achieving a better control of MLF in enology. To construct the expression plasmid named pYILmleA, cloned mleA gene, PGK1 promoter, and ADH1 terminator were ligated and inserted into integrating vector YIp5. Yeast transformants were screened on SD/-Ura and identified by auxotrophic test, mating type test, and colony PCR. Target protein was detected by SDS-PAGE and the targeted gene integrated to the chromosome was detected by dot bloting hybridization. After the transformant was cultured in SD/-Ura adding glucose （10%） and L-malate （5 648 mg L-1） for 4 d, the culture supernatant was collected and L-malate and L-lactic acid contents were detected by HPLC. 1 278-1 312 mg L-1 L-lactic acids were detected, while the comparative drop rates of L-malate were 20.18-20.85%. L-malate and L-lactic contents of the transformants showed extra significant difference and significant difference with the control ones by t-test respectively. The result indicated that the functional expression was achieved in recombinants S. cerevisiae.