阿维拉霉素生物合成研究进展

阿维拉霉素是由绿色产色链霉菌生产的寡糖类抗生素,对革兰氏阳性致病菌具有极强抗性,是一种新型饲料添加剂,广泛应用于肉鸡、仔猪等畜禽养殖中.以阿维拉霉素最新国内外研究进展为基础,综述了阿维拉霉素的抑菌机制、结构改造、菌种选育及发酵优化等方面的研究进展,重点阐述了阿维拉霉素的生物合成基因簇、合成途径及生物合成转录调控机制,探...     

利用基因组改组技术选育阿维拉霉素高产菌

为选育阿维拉霉素高产菌株,对野生型绿色产色链霉菌(Streptomyces viridochromogenes)Tü57进行传统物理和化学诱变,并获得一系列以阿维拉霉素A的含量为指标的正向突变体,再采用基因组改组技术将不同的正向突变体进行融合杂交,获得融合子。在筛选融合子的过程中,采用传统薄层色谱(TLC)和管碟法(二剂量法)结合进行初筛、高效液相色谱法进行复筛的筛选策略。经过基因组改组,筛选得到一株高产菌株R708,其在摇瓶实验中阿维拉霉素A产量达到0.208 g/L,比野生菌株提高了20倍。基因组改组选育阿维拉霉素高产菌,能提高子代菌株的遗传多样性,比传统诱变选育更快速有效。     

微波对阿维拉霉素产生菌诱变效应的研究

阿维拉霉素产生菌SV微波诱变效应的研究发现：菌株SV对微波敏感（微波处理60s,SV菌株存活率低于10％）,菌落形态变化大。微波诱变处理最佳作用方式为培养皿不加盖、快速冰上冷却,最佳处理时间为50s（其正突变率25．3％）。通过微波诱变处理、阿维拉霉素推理筛选,最终获得一株稳定性良好,阿维拉霉素产量达到21．5mg／L,较出发菌株提高119．4％的突变株SV-15。     

氮离子注入选育阿维拉霉素高产菌株的研究

以提高产绿链霉菌(Streptomyces viridochromogenes)SV-1产阿维拉霉素(Avilamycin)产量为目的,采用低能氮离子注入技术,辅之以链霉素抗性筛选法进行诱变选育研究。结果表明,“马鞍”区域即注入剂量范围在3×10~(15)～5×10~(15)ions/cm~2诱变效果最佳,菌株的抗药性突变与产量突变密切相关,链霉素抗性筛选法具有可行性。在摇瓶条件下,最终获得稳定性良好,阿维拉霉素产量达到83．5mg/L,较出发菌株提高195%的突变株SVT-45。实验表明,离子注入技术是一种有潜力的微生物诱变育种新方法。     

有效霉素A对棘孢木霉的影响及协同防治玉米纹枯病作用

【背景】玉米纹枯病逐渐发展为制约我国玉米持续增产的主要病害,有效霉素A与生防菌木霉均对纹枯病菌具有抗性,但二者各有优缺点。【目的】研究有效霉素A与木霉菌协同作用的可行性,提高对玉米纹枯病的防治水平,实现抗生素类化学农药的减量使用,提高生态环境安全性。【方法】测定有效霉素A处理后的棘孢木霉GDSF1009的细胞壁降解酶及防御反应相关酶活性,烟草叶片验证活性氧及过敏性反应。利用转录组及气相色谱-飞行时间质谱联用分析有效霉素A对GDSF1009基因表达差异及生长代谢的影响,并进行叶片及平皿协同抑菌实验。【结果】有效霉素A对棘孢木霉GDSF1009的几丁质酶、纤维素酶及木聚糖酶的活性无影响,有效霉素A未进入到棘孢木霉菌细胞内。在转录组水平,有效霉素A处理木霉24h和48 h后,与对照相比,差异基因所占比例分别为8.932%和6.779%,有效霉素A对GDSF1009相关的糖类及脂类代谢没有显著影响,仅对氨基酸代谢相关的基因有一些影响,这些基因并不会造成氨基酸代谢的大量变化。同时验证了在二者联合使用时,可以显著地提高玉米纹枯病的防治效果。【结论】有效霉素A对棘孢木霉菌的初生代谢系统是比较安全的,二者协同作用的抗病效果显著高于单因子作用。     

哺乳动物低氧应激代谢成因的研究进展

氧气是哺乳动物机体代谢稳态维持的物质基础,若代谢过程中氧气供给不足,可造成低氧应激。目前,环境低氧、代谢性低氧和携氧细胞功能障碍是造成动物低氧应激的重要成因。目前,低氧对动物机体代谢和组织功能的影响研究主要集中于肺脏、肝脏、消化道、肌肉和乳腺等部位。若处于低氧状态的哺乳动物形成了适应低氧的代谢模式,则可维持其代谢稳态;相反,若动物无法维持低氧状态下的代谢稳态,则会导致机体氧化应激甚至病变。目前,低氧应激在家畜方面的研究主要集中于高原动物代谢适应机制;然而,泌乳期动物机体代谢速率、氧气消耗和自由基水平均较高,但氧在泌乳动物代谢应激形成中的作用及其对泌乳性能的影响,仍有待探索。综述了哺乳动物产生低氧应激的代谢成因与作用结果,旨在探讨哺乳动物低氧应激生物学基础,为进一步从低氧应激调控角度为泌乳动物的健康状况维持提供理论依据。     

纳他霉素高产菌株Streptomyces gilvosporeus F607基因组及其生物合成基因簇分析

【背景】纳他霉素(Natamycin)是一种天然、广谱、高效的多烯大环内酯类抗真菌剂,褐黄孢链霉菌(Streptomyces gilvosporeus)是一种重要的纳他霉素产生菌。目前S. gilvosporeus基因组序列分析还未有报道,限制了该菌中纳他霉素及其他次级代谢产物合成及调控的研究。【目的】解析纳他霉素高产菌株S. gilvosporeus F607的基因组序列信息,挖掘其次级代谢产物基因资源,为深入研究该菌株的纳他霉素高产机理及生物合成调控机制奠定基础。【方法】利用相关软件对F607菌株的基因组序列进行基因预测、功能注释、进化分析和共线性分析,并预测次级代谢产物合成基因簇;对纳他霉素生物合成基因簇进行注释分析,比较分析不同菌种中纳他霉素生物合成基因簇的差异;分析预测S.gilvosporeusF607中纳他霉素生物合成途径。【结果】F607菌株基因组总长度为8482298bp,(G+C)mol%为70.95%,分别在COG、GO、KEGG数据库提取到5 062、4 428、5063个基因的注释信息。同时,antiSMASH软件预测得到29个次级代谢产物合成基因簇,其中纳他霉素基因簇与S.natalensis、S. chattanoogensis等菌株的纳他霉素基因簇相似性分别为81%和77%。除2个参与调控的sngT和sgnH基因和9个未知功能的orf基因有差异外,S. gilvosporeus F607基因簇中其他纳他霉素生物合成基因及其排列顺序与已知的纳他霉素基因簇高度一致。【结论】分析了S. gilvosporeus全基因组信息,预测了S. gilvosporeus F607中纳他霉素生物合成的途径,为从基因组层面上解析S. gilvosporeus F607菌株高产纳他霉素的内在原因提供了基础数据,为揭示纳他霉素高产的机理及工业化生产和未来新药的发现奠定了良好的基础。     

代谢组学技术在水产动物疾病研究中的应用

代谢组学是定量描述生物内源性代谢物质的整体及其对内因和外因变化应答规律的的一门新学科。近年来,代谢组学技术在水产动物疾病中的研究备受关注,特别是为感染性疾病发生机制及防控研究提供了一种新的手段。本文介绍了代谢组学技术及其在水产动物研究中的应用,包括代谢组学技术在水产动物感染性疾病、细菌耐药及环境应激等方面应用进行综述,分析了代谢组学在水产动物疾病研究中面临的问题与挑战,并对未来水产动物代谢组学研究趋势进行了展望,以期为代谢组学技术在水产动物疾病发病机制和药物研发方面更深入的运用提供参考。     

Kisspeptin调控动物机体代谢的研究进展

Kisspeptin是由KISS1基因编码的蛋白产物,是下丘脑GnRH上游的主要调控因子,其不仅在中枢系统调控动物的生殖,而且也可在外周局部影响动物配子发生。然而,最近诸多研究证实Kisspeptin除了在动物繁殖方面起到重要的作用外,还对动物能量平衡、摄食、肥胖以及代谢性疾病等方面起到重要的调节作用。本文就Kisspeptin在中枢神经系统和外周组织器官调控动物机体的代谢进行了详细论述,重点阐明Kisspeptin与外周代谢激素互作对动物机体代谢影响的最新进展,并总结分析了Kisspeptin在调控动物代谢方面所面临的诸多问题,以期充分理解Kisspeptin在调节动物代谢中发挥的作用,为防治动物及人类代谢紊乱性疾病提供新策略。     

达托霉素耐药分子机制研究进展

环脂肽抗生素达托霉素抗菌活性强,致病菌不容易产生耐药性,已成为治疗革兰氏阳性菌特别是耐药菌感染的一线药物。但由于广泛使用,仍然出现了达托霉素耐药菌。细胞膜磷脂代谢和细胞壁结构动态与致病菌达托霉素耐药密切相关。文中综述了达托霉素作用机制和耐药机制,以期对药物研发和临床用药有所裨益。     

Bioengineering for the industrial production of 2,3-butanediol by the yeast,Saccharomyces cerevisiae

World Journal of Microbiology and Biotechnology - Avilamycin, an excellent growth-promoting feed additive, produced by Streptomyces viridochromogenes, was widely used to promote the growth of...     

Effect of avilamycin and tylosin on the metabolizable energy in growing and finishing pigs

In two metabolism trials with growing and finishing pigs the influence of the antibiotic feed additives Avilamycin and Tylosin on the metabolizable energy was investigated at different levels of dietary protein content. In the first experiment (growing pigs) the antibiotics were supplied at levels of 0 mg/kg, 40 mg/kg Avilamycin and 40 mg/kg Tylosin to diets containing 18.5%, 17.5%, 16.5% and 14.0% of crude protein. In the second experiment (finishing pigs) 0 mg/kg antibiotics, 20 mg/kg Avilamycin and 20 mg/kg Tylosin were used in diets containing 16.5% and 14.0% of crude protein. The body weight of the animals averaged 46 kg (growing pigs) and 68 kg (finishing pigs).

In growing pigs the supplementation of Tylosin increased the digestibility of dry matter and energy at 1 percentage unit each, while in finishing pigs no effects were observed. Since the urinary energy excretion was not affected by antibiotics, there was only in Tylosin treated growing pigs a slight rise in dietary contents of metabolizable energy by 1.6%. The reduction of the dietary protein content resulted in increased digestibility of dry matter and energy, reduced urinary energy excretions and increased dietary contents of metabolizable energy.     

Hot-water extract from mycelia of Cordyceps sinensis as a substitute for antibiotic growth promoters

Broiler chicks were orally dosed with a hot-water extract of mycelia from Cordyceps sinensis (CS-HW) to assess possible substitution of Avilamycin as an antibiotic growth promoter (AGP). The growth performance (body weight gain and survivability) and the health index (the microflora in the small intestines and the antibody titer to Newcastle disease virus) of chicks were significantly improved in the CS-HW (600 mg/kg diet) and the Avilamycin (20 mg/kg diet) fed group in comparison with the control group (p<0.05). The Avilamycin-fed group and the CS-HW-fed group had similar growth performances but the latter gave a better microbial flora in the small intestines. These results indicate that CS-HW enhances the physiological activity in chicks and can be used as a substitute for AGPs.     

Understanding the mechanisms of zinc bacitracin and avilamycin on animal production: linking gut microbiota and growth performance in chickens

Unravelling the mechanisms of how antibiotics influence growth performance through changes in gut microbiota can lead to the identification of highly productive microbiota in animal production. Here we investigated the effect of zinc bacitracin and avilamycin on growth performance and caecal microbiota in chickens and analysed associations between individual bacteria and growth performance. Two trials were undertaken; each used 96 individually caged 15-day-old Cobb broilers. Trial 1 had a control group (n = 48) and a zinc bacitracin (50 ppm) treatment group (n = 48). Trial 2 had a control group (n = 48) and an avilamycin (15 ppm) treatment group (n = 48). Chicken growth performance was evaluated over a 10-day period, and caecal microbiota was characterised by sequencing of bacterial 16S rRNA gene amplicons. Avilamycin produced no effect on growth performance and exhibited little significant disturbance of the microbiota structure. However, zinc bacitracin reduced the feed conversion ratio (FCR) in treated birds, changed the composition and increased the diversity of their caecal microbiota by reducing dominant species. Avilamycin only produced minor reductions in the abundance of two microbial taxa, whereas zinc bacitracin produced relatively large shifts in a number of taxa, primarily Lactobacillus species. Also, a number of phylotypes closely related to lactobacilli species were positively or negatively correlated with FCR values, suggesting contrasting effects of Lactobacillus spp. on chicken growth performance. By harnessing such bacteria, it may be possible to develop high-productivity strategies in poultry that rely on the use of probiotics and less on in-feed antibiotics.     

The active conformation of avilamycin A is conferred by AviX12, a radical AdoMet enzyme

The antibiotic avilamycin A is produced by Streptomyces viridochromogenes Tü57. Avilamycin belongs to the family of orthosomycins with a linear heptasaccharide chain linked to a terminal dichloroisoeverninic acid as aglycone. The gene cluster for avilamycin biosynthesis contains 54 open reading frames. Inactivation of one of these genes, namely aviX12, led to the formation of a novel avilamycin derivative named gavibamycin N1. The structure of the new metabolite was confirmed by mass spectrometry (MS) and NMR analysis. It harbors glucose as a component of the heptasaccharide chain instead of a mannose moiety in avilamycin A. Antibacterial activity tests against a spectrum of Gram-positive organisms showed that the new derivative possesses drastically decreased biological activity in comparison to avilamycin A. Thus, AviX12 seems to be implicated in converting avilamycin to its bioactive conformation by catalyzing an unusual epimerization reaction. Sequence comparisons grouped AviX12 in the radical S-adenosylmethionine protein family. AviX12 engineered with a His tag was overexpressed in Escherichia coli and purified by affinity chromatography. The iron sulfur cluster [Fe-S] present in radical AdoMet enzymes was detected in purified AviX12 by means of electron paramagnetic resonance spectroscopy.     

Genome shuffling of Streptomyces viridochromogenes for improved production of avilamycin

Avilamycin is one of EU-approved antimicrobial agents in feed industry to inhibit the growth of multidrug-resistant Gram-positive bacteria. Here, we applied a process of combining ribosome engineering and genome shuffling to achieve rapid improvement of avilamycin production in Streptomyces viridochromogenes AS 4.126. The starting mutant population was generated by ⁶⁰Co γ-irradiation treatments of the spores. After five rounds of protoplast fusion with streptomycin-resistance screening, an improved recombinant E-219 was obtained and its yield of avilamycin reached 1.4 g/L, which was increased by 4.85-fold and 36.8-fold in comparison with that of the shuffling starter Co γ-316 and the ancestor AS 4.126. Furthermore, the mechanism for the improvement of shuffled strains was investigated. Recombinants with enhanced streptomycin resistance exhibited significantly higher avilamycin production and product resistance, probably due to the mutations in the ribosome protein S12. The morphological difference between the parent mutant and shuffled recombinant was observed in conidiospore, and hyphae pellets. The presence of genetic diversity among shuffled populations with varied avilamycin productivity was confirmed by randomly amplified polymorphic DNA analysis. In summary, our results demonstrated that genome shuffling combined with ribosome engineering was a powerful approach for molecular breeding of high-yield industrial strains.     

Influence of antimicrobial feed additives on broiler commensal posthatch gut microbiota development and performance

The effects of avilamycin, zinc bacitracin, and flavophospholipol on broiler gut microbial community colonization and bird performance in the first 17 days posthatch were investigated. Significant differences in gut microbiota associated with gut section, dietary treatment, and age were identified by terminal restriction fragment length polymorphism (T-RFLP), although no performance-related differences between dietary treatments were detected. Similar age-related shifts in the gut microbiota were identified regardless of diet but varied between the ilea and ceca. Interbird variabilities in ileal bacterial communities were reduced (3 to 7 days posthatch) in chicks fed with feed containing antimicrobial agents. Avilamycin and flavophospholipol had the most consistent effect on gut microbial communities. Operational taxonomic units (OTU) linked to changes in gut microbiota in birds on antimicrobial-supplemented diets were characterized and identified. Some OTUs could be identified to the species level; however, the majority could be only tentatively classified to the genus, family, order, or domain level. OTUs 140 to 146 (Lachnospiraceae), OTU 186/188 (Lactobacillus johnsonii), OTU 220 (Lachnospiraceae), OTUs 284 to 288 (unclassified bacterial spp. or Ruminococcaceae), OTU 296/298 (unclassified bacterium or Clostridiales), and OTU 480/482 (Oxalobacteraceae) were less prevalent in the guts of chicks fed antimicrobial-supplemented diets. OTU 178/180 (Lactobacillus crispatus), OTU 152 (Lactobacillus reuteri or unclassified Clostridiales), OTU 198/200 (Subdoligranulum spp.), and OTU 490/492 (unclassified bacterium or Enterobacteriaceae) were less prevalent in the gut of chicks raised on the antimicrobial-free diet. The identification of key bacterial species influenced by antimicrobial-supplemented feed immediately posthatch may assist in the formulation of diets that facilitate beneficial gut microbial colonization and, hence, the development of alternatives to current antimicrobial agents in feed for sustainable poultry production.     

Regulation of avilamycin biosynthesis in Streptomyces viridochromogenes: effects of glucose, ammonium ion, and inorganic phosphate

Effects of glucose, ammonium ions and phosphate on avilamycin biosynthesis in Streptomyces viridochromogenes AS4.126 were investigated. Twenty grams per liter of glucose, 10 mmol/L ammonium ions, and 10 mmol/L phosphate in the basal medium stimulated avilamycin biosynthesis. When the concentrations of glucose, ammonium ions, and phosphate in the basal medium exceeded 20 g/L, 10 mmol/L, and 10 mmol/L, respectively, avilamycin biosynthesis greatly decreased. When 20 g/L glucose was added at 32 h, avilamycin yield decreased by 70.2%. Avilamycin biosynthesis hardly continued when 2-deoxy-glucose was added into the basal medium at 32 h. There was little influence on avilamycin biosynthesis with the addition of the 3-methyl-glucose (20 g/L) at 32 h. In the presence of excess (NH₄)₂SO₄ (20 mmol/L), the activities of valine dehydrogenase and glucose-6-phosphate dehydrogenase were depressed 47.7 and 58.3%, respectively, of that of the control at 48 h. The activity of succinate dehydrogenase increased 49.5% compared to the control at 48 h. The intracellular adenosine triphosphate level and 6-phosphate glucose content of S. viridochromogenes were 128 and 129%, respectively, of that of the control at 48 h, with the addition of the 40 mmol/L of KH₂PO₄. As a result, high concentrations of glucose, ammonium ions, and inorganic phosphate all led to the absence of the precursors for avilamycin biosynthesis and affected antibiotic synthesis.