Ferroptosis is involved in deoxynivalenol-induced intestinal damage in pigs

Background Deoxynivalenol(DON)is a widespread issue for feed and food safety,leading to animal and human health risks.The objective of this study was to determine whether ferroptosis is involved in DON-induced intestinal injury in piglets.Three groups of 21-day-old male weanling piglets(n 4,serum and small intestines were=7/group)were fed a control diet,or diet adding 1.0 or 3.0 mg DON/kg.At week collected to assay for biochemistry,histology,redox status and ferroptosis-related genes expression.In addition,the involvement of ferroptosis and the role of FTL gene in DON-induced cell death were further verified in the IPEC-J2 cells.Results Compared to the control,dietary supplementation of DON at 1.0 and 3.0 mg/kg induced different degrees of damage in the duodenum,jejunum and ileum,and increased(P<0.05)serum lipopolysaccharide concentration by 46.2%-51.4%.Dietary DON supplementation at 1.0 and(or)3.0 mg/kg increased(P<0.05)concentrations of malondialdehyde(17.4%-86.5%)and protein carbonyl by 33.1%-92.3%in the duodenum,jejunum and ileum.In addition,dietary supplemented with DON upregulated(P<0.05)ferroptotic gene(DMT1)and anti-ferroptotic genes(FTL and FTH1),while downregulated(P<0.05)anti-ferroptotic genes(FPN,FSP1 and CISD1)in the duodenum of the porcine.Furthermore,the in vitro study has demonstrated that deferiprone,a potent ferroptotic inhibitor,mitigated(P<0.05)DON-induced cytotoxicity in porcine small intestinal IPEC-J2 cells.Additionally,deferiprone prevented or alleviated(P<0.05)the dysregulation of ferroptosis-related genes(ACSL4 and FTL)by DON in IPEC-J2 cells.Moreover,specific siRNA knockdown FTL gene expression compromised the DON-induced cell death in IPEC-J2 cells.Conclusions In conclusion,this study revealed that ferroptosis is involved in DON-induced intestinal damage in porcine,and sheds a new light on the toxicity of DON to piglets.

Alleviation of mycotoxin biodegradation agent on zearalenone and deoxynivalenol toxicosis in immature gilts

Background: The current study was carried out to evaluate the effects of mycotoxin biodegradation agent(MBA, composed of Bacillus subtilis ANSB01 G and Devosia sp. ANSB714) on relieving zearalenone(ZEA) and deoxynivalenol(DON) toxicosis in immature gilts.Methods: A total of forty pre-pubertal female gilts(61.42 ± 1.18 kg) were randomly allocated to four diet treatments: CO(positive control); MO(negative control, ZEA 596.86 μg/kg feed and DON 796 μg/kg feed);COA(CO + 2 g MBA/kg feed); MOA(MO + 2 g MBA/kg feed). Each treatment contained 10 replicates with 1 gilt per replicate. Gilts were housed in an environmentally controlled room with the partially slatted floor.Results: During the entire experimental period of 28 d, average daily gain(ADG) and average daily feed intake(ADFI)of gilts in MO group was significantly reduced compared with those in CO group. The vulva size of gilts was significantly higher in MO group than CO group. In addition, significant increases in the plasma levels of Ig A,Ig G, IL-8, IL-10 and PRL were determined in MO group compared with that in CO group. ZEA and DON in the diet upregulated apoptotic caspase-3 in ovaries and uteri, along with down-regulated the anti-apoptotic protein Bcl-2 in ovaries. The supplementation of MBA into diets co-contaminated with ZEA and DON significantly increased ADG, decreased the vulva sizes, reduced the levels of Ig G, IL-8 and PRL in plasma, and regulated apoptosis in ovaries and uteri of gilts.Conclusions: The present results indicated that feeding diet contaminated with ZEA and DON simultaneously(596.86 μg/kg + 796 μg/kg) had detrimental effects on growth performance, plasma immune function and reproductive status of gilts. And MBA could reduce the negative impacts of these two toxins, believed as a promising feed additive for mitigating toxicosis of ZEA and DON at low levels in gilts.

Dietary resveratrol attenuation of intestinal inflammation and oxidative damage is linked to the alteration of gut microbiota and butyrate in piglets challenged with deoxynivalenol

Background:Deoxynivalenol(DON)is a widespread mycotoxin that induces intestinal inflammation and oxidative stress in humans and animals.Resveratrol(RES)effectively exerts anti-inflammatory and antioxidant effects.However,the protective effects of RES on alleviating DON toxicity in piglets and the underlying mechanism remain unclear.Therefore,this study aimed to investigate the effect of RES on growth performance,gut health and the gut microbiota in DON-challenged piglets.A total of 64 weaned piglets[Duroc×(Landrace×Yorkshire),21-d-old,6.97±0.10 kg body weight(BW)]were randomly allocated to 4 treatment groups(8 replicate pens per treatment,each pen containing 2 males;n=16 per treatment)for 28 d.The piglets were fed a control diet(CON)or the CON diet supplemented with 300 mg RES/kg diet(RES group),3.8 mg DON/kg diet(DON)or both(DON+RES)in a 2×2 factorial design.Results:DON-challenged piglets fed the RES-supplemented diet had significantly decreased D-lactate concentrations and tumor necrosis factor alpha(TNF-α)and interleukin 1 beta(IL-1β)mRNA and protein expression,and increased zonula occludens-1(ZO-1)mRNA and protein expression compared with those of DON-challenged piglets fed the unsupplemented diet(P<0.05).Compared with unsupplemented DON-challenged piglets,infected piglets fed a diet with RES showed significantly decreased malondialdehyde(MDA)levelsand increased mRNA expression of antioxidant enzymes and antioxidant genes(i.e.,GCLC,GCLM,HO-1,SOD1 and NQO-1)and glutamatecysteine-ligase modulatory subunit(GCLM)protein expression(P<0.05).Moreover,RES supplementation significantly abrogated the increase in the proportion of TUNEL-positive cells and the protein expression of caspase3 in DON-challenged piglets(P<0.05).Finally,RES supplementation significantly increased the abundance of Roseburia and butyrate concentrations,while decreasing the abundances of Bacteroides and unidentified-Enterobacteriaceae in DON-challenged piglets compared with DON-challenged piglets alone(P<0.05).Conclusions:RES supplementation improved gut health in DON-challenged piglets by strengthening intestinal barrier function,alleviating intestinal inflammation and oxidative damage,and positively modulating the gut microbiota.The protective effects of RES on gut health may be linked to increased Roseburia and butyrate concentrations,and decreased levels of Bacteroides and unidentified-Enterobacteriaceae.

Intestinal toxicity of deoxynivalenol is limited by supplementation with Lactobacillus plantarum JM113 and consequentially altered gut microbiota in broiler chickens

Background: Limited research has focused on the effect of Lactobacillus on the intestinal toxicity of deoxynivalenol(DON).The present study was conducted to investigate the role of Lactobacillus plantarum(L.plantarum) JM113 in protecting against the intestinal toxicity caused by DON.Methods: A total of 144 one-day-old healthy Arbor Acres broilers were randomly distributed into 3 treatments,including the CON(basal diet),the DON(extra 10 mg/kg deoxynivalenol),and the DL(extra 1 × 109 CFU/kg L.plantarum JM113 based on DON group) treatments.The growth performance,organ indexes,intestinal morphology,pancreatic digestive enzymes,intestinal secreted immunoglobulin A(sIgA),jejunal transcriptome,and intestinal microbiota were evaluated.Results: Compared with the CON and DL groups,the DON supplementation altered intestinal morphology,especially in duodenum and jejunum,where villi were shorter and crypts were deeper(P < 0.05).Meanwhile,the significantly decreased mRNA expression of jejunal claudin-1 and occludin(P < 0.05),ileal rBAT and jejunal GLUT1 of 21-day-old broilers(P < 0.05),as well as duodenal PepT1 and ileal rBAT of 42-day-old broilers were identified in the DON group.Moreover,supplementation with L.plantarum JM113 could increase duodenal expression of IL-10 and IL-12 of 21-dayold broilers,ileal s IgA of 42-day-old broilers,and the bursa of Fabricius index of 21-day-old broilers.Further jejunal transcriptome proved that the genes related to the intestinal absorption and metabolism were significantly reduced in the DON group but a significant increase when supplemented with extra L.plantarum JM113.Furthermore,the bacteria related to nutrient utilization,including the Proteobacteria,Escherichia,Cc-115(P < 0.05),Lactobacillus and Prevotella(P < 0.1) were all decreased in the DON group.By contrast,supplementation with L.plantarum JM113 increased the relative abundance of beneficial bacterium,including the Bacteroidetes,Roseburia,Anaerofustis,Anaerostipe,and Ruminococcus bromi(P < 0.05).Specifically,the increased abundance of bacteria in the DL group could be proved by the significantly increased caecal content of propionic acid,n-Butyric acid,and total short-chain fatty acid.Conclusions: L.plantarum JM113 enhanced the digestion,absorption,and metabolic functions of the gut when challenged with DON by reducing the injury to intestinal barriers and by increasing the abundance of beneficial bacterium.

Effects of Sterigmatocystin, Deoxynivalenol and Aflatoxin G_1 on Apoptosis of Human Peripheral Blood Lymphocytes in vitro

Objective To explore the effects of Sterigmatocystin (ST), Deoxynivalenol (DON) and Aflatoxin G1 (AFG1) on apoptosis of human peripheral blood lymphocytes (HPBLs) in vitro and thus to further elucidate the putative roles of these three mycotoxins on human immunosystem. Methods The effects of ST, DON and AFG1 on apoptosis of HPBLs were studied with cell culture, flow cytometric (FCM) DNA analysis and DNA agarose gel electrophoresis. Results DNA agarose gel electrophoresis results showed the characteristic 'ladder' pattern of apoptosis in HPBLs treated with ST, DON and AFG1. Flow cytometric DNA analysis revealed that typical subdiploid peaks of apoptosis in DNA histogram could be seen in all groups treated with the three mycotoxins. Significant time-effect and dose-effect relationships were found between the apoptosis rates and treatment time as well as concentrations of the three mycotoxins. Conclusion ST, DON and AFG1 can induce apoptosis of HPBLs in vitro and may have some negative effects on human immunosystem.

Occurrence of Aflatoxin B_(1),deoxynivalenol and zearalenone in feeds in China during 2018-2020

Background:The current study was conducted to investigate the individual and combined occurrence of aflatoxin B_(1)(AFB_(1)),deoxynivalenol(DON)and zearalenone(ZEN)in feeds from various Provinces of China during 2018 to 2020.A total of 3,507 feed samples,including 2,090 feed ingredients and 1,417 complete feed samples,were collected from different areas of China for mycotoxins analysis.Results:The individual contamination of AFB_(1),DON and ZEN were present in more than 81.9%,96.4% and 96.9% of feed samples,respectively,with average concentration ranges of AFB_(1) between 1.2-27.4μg/kg,DON between 458.0-1,925.4μg/kg and ZEN between 48.1-326.8μg/kg.Notably,0.9%,0.5% and 0.1% of feed ingredients,and 1.2-12.8%,0.9-2.9% and 0-8.9% of complete feeds for pigs,poultry and ruminants with AFB_(1),ZEN and DON that exceeded China’s safety standards,respectively.Moreover,more than 81.5%of feed ingredients and 95.7% of complete feeds were co-contaminated with various combinations of these mycotoxins.Conclusion:This study indicates that the feeds in China were universally contaminated with AFB_(1),DON and ZEN during the past 3 years.These findings highlight the significance of monitoring mycotoxin contaminant levels in the domestic animal feed,and the importance of carrying out feed administration and remediation strategies for mycotoxin control.

Aflatoxin B1, zearalenone and deoxynivalenol in feed ingredients and complete feed from different Province in China

Background： The current study was carried out to provide a reference for monitory of aflatoxin B_1（AFB_1）,zearalenone（ZEN） and deoxynivalenol（DON） contamination in feed ingredients and complete feeds were collected from different Province in China from 2013 to 2015.Methods： A total of 443 feed ingredients, including 220 corn, 24 wheat, 24 domestic distillers dried grains with soluble（DDGS）, 55 bran, 20 wheat shorts and red dog, 37 imported DDGS, 34 corn germ meal and 29 soybean meal as well as 127 complete feeds including 25 pig complete feed（powder）, 90 pig complete feed（pellet）, six duck complete feed and six cattle complete feed were randomly collected from different Province in China,respectively, by high-performance chromatography in combined with UV or fluorescence analysis.Results： The incidence rates of AFB_1, ZEN and DON contamination of feed ingredients and complete feeds were80.8, 92.3 and 93.9 %, respectively. The percentage of positive samples for DON ranged from 66.7 to 100 %.Domestic DDGS and imported DDGS presented the most serious contamination AFB_1, ZEN and DON contamination levels of feeds ranged from 61.5 to 100 %, indicated that serious contamination over the studied 3-year period.Conclusion： The current data provide clear evidence that AFB_1, ZEN and DON contamination of feed ingredients and complete feeds in different Province in China is serious and differs over past 3-year. The use of corn, domestic DDGS, imported DDGS and corn germ meal, which may be contaminated with these three mycotoxins, as animal feed may triggered a health risk for animal. Feeds are most contaminated with DON followed by ZEN and AFB_1.Mycotoxins contamination in feed ingredients and complete feeds should be monitored routinely in China.

Separation and purification of deoxynivalenol(DON) mycotoxin from wheat culture using a simple two-step silica gel column chromatography

Deoxynivalenol（DON） is a type B trichothecenes mycotoxin produced by several Fusarium species, often found in foodstuffs for humans and animals. DON is in great demand for the toxicological researches both in vivo and in vitro. In this work, wheat culture was inoculated with a Fusarium graminearum PH-1 strain for DON production. The solvent system for crude extraction was acetonitrile-water（84：16, v/v）. A simple two-step silica gel column chromatography was employed to separate the DON mycotoxin from wheat culture, combined with preparative high performance liquid chromatography（preparative HPLC） to purify the compound. The solvent system for the second silica gel column chromatography was methylene chloride-methanol（17：1, v/v）, which provided a good elution effect selected on thin layer chromatography（TLC）. The target compound was identified by HPLC, and the chemical structure was confirmed by mass spectrometry（MS） and ~1H and ^（13）C nuclear magnetic resonance（NMR） spectroscopy. A total of 433 mg of purified DON was obtained from 1 kg of wheat culture, with a purity of 99.01%. The study had provided an easy-operating and cost-effective method to isolate an expensive compound in a simple way.

Regulation of the phytotoxic response of Arabidopsis thaliana to the Fusarium mycotoxin deoxynivalenol

Phytopathogenic fungi,such as Fusarium spp.,synthesize trichothecene family phytotoxins.The type B trichothecene,namely deoxynivalenol(DON),is highly prevalent in small-grain cereals,such as wheat,corn and barley.DON is thought to be a virulence factor allowing plant infections and has an elicitor activity.We used the model plant Arabidopsis thaliana to evaluate the phytotoxic effects of DON in host plants.The growth of A.thaliana on media was significantly inhibited by DON.Moreover,DON induced cell death in detached leaves was observed by trypan blue staining.This is consistent with the phenomenon of organelle changes observed at the ultrastructural level.In our study,DON exposure stimulated oxidative bursts in the leaves,resulting in the concomitant down-regulation of antioxidant enzyme defense responses and up-regulation of lipid peroxidation.In addition,a real-time PCR analysis revealed that the DON treatment rapidly induced the transcription of defense genes,like AtrbohC and AtrbohD,and up-regulated the transcriptional level of the ascorbic acid peroxidase gene.These results suggested that DON phytotoxicity might result from reactive oxygen species pathways,and that DON production by the plant pathogen Fusarium graminearum can act as an elicitor influencing plant cell fate.

THE EFFECT ON PROTEOGLYCAN METABOLISM OF DEOXYNIVALENOL AND SELENIUM IN THE CULTURED HUMAN FETAL CHONDROCYTES IN VITRO

Objective To investigate the effect of deoxynivalenol (DON) and selenium (Se) on the morphology of chondrocytes and the metabolism of cartilage matrix, and the expression of aggrecanase-1, 2 mRNA in monolayer cultured chondrocytes in vitro. Methods To plant human fetal chondrocytes on the BMG, the expression of Aggrecanase-1, 2 mRNA were analyzed by RT-PCR, the immunohistochemistry of NITEGE epitope was quantitativly analyzed by the image collection and analysis system. Results With the increase of the concentration of DON, the damage of cultured chondrocytes was more and more severe; the expression of NITEGE epitope showed an increasing trend and the fluorescent bands of aggrecanase-1, 2 mRNA were more and more obvious. After adding Se, the damage was relieved, and there was a decreasing trend of NITEGE epitope expressed in matrix. Conclusion DON can enhance transcription of aggrecanase gene and increase the expression of NITEGE epitope which eventually lead to the metabolic disorder of cartilage proteoglycan. It suggested that Se can partially alleviate the damage of DON on cartilage, but can not completely prevent the occurrence of these changes.

Tissue distribution of deoxynivalenol in piglets following intravenous administration

Contamination of deoxynivalenol（DON） in grains is common worldwide and pigs are particularly susceptible to this mycotoxin. The distribution of DON in porcine tissues following intravenous administration was investigated in this study. Fifteen pigs were randomly divided into three groups. Animals in groups A and B were administrated with DON at the dose of 250 and 750 μg kg–1 body weight, respectively, while group C served as blank control. Plasma, bile and 27 tissues were collected at 30 min post-administration. DON concentrations in all samples were tested using high-performance liquid chromatography-tandem mass spectrometry（HPLC-MS/MS）. To observe the distribution of DON in tissues, these samples were further subjected to the immunohistochemical analyses. Totally, the bile and 13 tissues were sampled for DON-based detection, including kidney, mesenteric lymph nodes, muscle, stomach, jejunum, colon, plasma, spleen, rectum, cecum, liver, ileum, and duodenum. No significant difference was observed for the concentrations of DON in duodenum, ileum and liver samples between groups A and B; while the DON concentrations in cecum and rectum of group B were significantly higher（P-value 〈0.05） than those in group A. In addition, the DON concentrations in stomach, jejunum, colon, mesenteric lymph nodes, muscle, kidney, spleen, bile, and plasma of group B were remarkably higher than those of group A（P-value〈0.01）. Levels of DON in other 14 tissues including medulla oblongata, midbrain, diencephalon, pons, tip and tongue body, tongue, soft palate, tonsils, pharyngeal mucosa, oral buccal mucosa, thymus, thyroid, esophagus and adrenal gland were all below the limit of detection. The results of immunohistochemistry showed that 11 tissue samples（medullaoblongata, tonsil, adrenal medulla, thyroid gland, thyroid, stomach, duodenum, jejunum, kidney, spleen, and mesenteric lymph nodes） were positive and DON was mainly distributed around blood vessels in these tissues. Therefore, we believed that concentrations of DON in tissues differ when pigs are in exposure to various dosages and DON causes lesions in many pig tissues.

Establishment of a chicken intestinal organoid culture system to assess deoxynivalenol‑induced damage of the intestinal barrier function

Background Deoxynivalenol(DON)is a mycotoxin that has received recognition worldwide because of its ability to cause growth delay,nutrient malabsorption,weight loss,emesis,and a reduction of feed intake in livestock.Since DON-contaminated feedstuff is absorbed in the gastrointestinal tract,we used chicken organoids to assess the DON-induced dysfunction of the small intestine.Results We established a culture system using chicken organoids and characterized the organoids at passages 1 and 10.We confirmed the mRNA expression levels of various cell markers in the organoids,such as KI67,leucine-rich repeat containing G protein-coupled receptor 5(Lgr5),mucin 2(MUC2),chromogranin A(CHGA),cytokeratin 19(CK19),lysozyme(LYZ),and microtubule-associated doublecortin-like kinase 1(DCLK1),and compared the results to those of the small intestine.Our results showed that the organoids displayed functional similarities in permeability compared to the small intestine.DON damaged the tight junctions of the organoids,which resulted in increased permeability.Conclusions Our organoid culture displayed topological,genetic,and functional similarities with the small intes-tine cells.Based on these similarities,we confirmed that DON causes small intestine dysfunction.Chicken organoids offer a practical model for the research of harmful substances.

丙硫菌唑与丙环唑混剂对小麦赤霉病菌的联合毒力及田间防效

为研发防治小麦赤霉病的新药剂,采用菌丝生长速率法分别检测了丙硫菌唑、丙环唑及其7种配比混剂对赤霉病菌的室内毒力,并进行了优选配比制剂对赤霉病、籽粒DON毒素、白粉病和叶锈病的田间药效试验。结果表明,丙硫菌唑与丙环唑配比为1:1时增效系数为2.4375,增效最显著。田间药效试验中,40%丙硫菌唑·丙环唑可分散油悬浮剂(OD)600 g/hm^(2)对赤霉病病指防效和籽粒DON防效分别为93.46%和91.26%,2次药后21 d对白粉病和叶锈病病指防效分别为93.50%和93.19%,均高于常规药剂戊唑醇和多菌灵,并且对小麦生长安全。40%丙硫菌唑·丙环唑OD对赤霉病和籽粒DON毒素防效优良,同时对白粉病和叶锈病兼治效果优良,具有较好的开发潜力。

基于电子鼻技术的小麦籽粒DON含量检测

为探究利用电子鼻技术对小麦籽粒中DON含量定量检测的可行性,本研究在25℃和40℃平衡温度下对DON含量不同的80个小麦籽粒样品的顶空气体进行电子鼻检测,并结合由UPLC-MS/MS测得的各样品DON含量进行统计分析,结果发现,除4号和5号传感器外,其余8个传感器的响应值均与样品DON含量具有显著或极显著的相关性,其中1号传感器的响应值及其参数与样品DON含量的相关性最强,表明1号传感器可以作为检测小麦籽粒样品DON含量的关键气体传感器。以1号传感器的参数为主,分别建立了在两平衡温度下样品DON含量的回归模型。对各模型的拟合效果分析发现,在40℃平衡温度下基于X_(17)(1号传感器21~60 s响应值的和)所建一元回归模型最好;在25℃平衡温度下,基于X_(1)(1号传感器20~40 s响应值的平均值)和X_(12)(8号传感器1~5 s响应值的和)所建二元回归模型拟合效果最好,其次为基于X_(1)所建的一元回归模型。该研究可为电子鼻技术在小麦籽粒中DON含量检测中的应用提供理论依据和技术支撑。

啤酒中呕吐毒素的污染、变化及生产过程中的控制进展

啤酒产业是我国饮料酒行业中发展最为迅速的产业之一,啤酒的原料生产和加工酿造环节与产品品质和产业发展密切相关。其中,啤酒原料污染或加工过程不当而产生的呕吐毒素(Deoxynivalenol,DON)控制是最关键、最受关注的研究热点之一。本文针对影响啤酒品质和安全的重要真菌毒素DON的污染情况进行分析,结合啤酒酿造关键工艺分析DON迁移及转化情况,从物理、化学和生物这三大方法分别探讨了啤酒酿造过程中DON污染的控制措施,并提出DON控制潜在发展方向,旨在为啤酒中DON污染控制、啤酒品质进一步提升提供理论支撑。

农产品检测用脱氧雪腐镰刀菌烯醇纯度标准物质的研制

[目的]本文旨在研制脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)国家级纯度标准物质,为农产品中DON的检测等工作提供物质基础。[方法]利用质量平衡法和定量核磁共振法对DON纯度标准物质进行纯度定值。质量平衡法包括采用液相色谱面积归一化法测定DON主成分含量,采用卡尔费休库仑法测定纯度标准物质中水分含量,采用顶空气相色谱-质谱法测定挥发性杂质含量,以及采用电感耦合等离子质谱法测定无机离子杂质含量。定量核磁共振法采用苯甲酸为内标,选择合适的共振峰,内标法定量标准物质的纯度,并对标准物质进行均匀性、稳定性检测和不确定度评估。[结果]质量平衡法的纯度定值结果为99.47%,定量核磁共振法定值结果为99.45%,最终所研制的DON纯度标准物质的纯度为99.5%,扩展不确定度为0.4%,均匀性良好,且满足12个月以上的稳定性要求。[结论]本试验所研制的DON纯度标准物质符合国家标准物质技术规范,目前已被批准为国家一级标准物质,并已被用于小麦、玉米等农产品实际检测过程中。

基于表达元件优化提高脱氧雪腐镰刀菌烯醇解毒酶DepB在枯草芽孢杆菌中的表达水平

本研究实现了脱氧雪腐镰刀菌烯醇解毒酶DepB在枯草芽孢杆菌(Bacillus subtilis)RIK 1285中的异源表达,但DepB较低的发酵水平限制了其在食品加工和饲料中的应用,可采用转录和翻译相结合的策略提高DepB在枯草芽孢杆菌中的表达水平。首先,选择9个单启动子替换原始启动子P43,其中单启动子P_(spoVG)介导的重组菌经发酵后酶活力最高,酶活力为29.59 U/mL。其次,选择4个具有较高DepB表达水平的启动子(P43、PsacB、P_(spoVG)和P_(aprE))构建16个双启动子系统,其中,DepB在双启动子P_(aprE^(-))P_(spoVG)的介导下活力达到最高,为48.87 U/mL。此外,通过对启动子P_(aprE^(-))P_(spoVG)的核心区域(-35和-10区)进行优化,Mutant-5酶活力高达69.17 U/mL,是原始菌株(14.45 U/mL)的4.79倍。在此基础上,通过优化启动子P_(spoVG)的核糖体结合位点序列,进一步提高了DepB的表达水平,RBS15酶活力为115.15 U/mL,是原始菌株的7.97倍。研究结果表明,转录和翻译相结合策略是提高重组蛋白发酵水平的有效手段。