氮源对阿维菌素合成的影响及基于二氧化碳释放速率的阿维菌素发酵调控

中国是世界上最大的也是唯一的阿维菌素原料生产国,但在工业规模生产中与同类型大环内酯类抗生素相比其产量相对偏低。文中通过研究不同氮源对阿维链霉菌生长、代谢的影响,发现氮源在发酵中后期对菌丝活性、菌丝浓度以及阿维菌素B1a的合成都有较为显著的影响。在100 L生物反应器中,于发酵中后期基于二氧化碳释放速率(CER)控制补入酵母粉,效价达到8697mg/L,与原工艺相比,提高了26.9%。这一结论若在实际工业生产中应用,有望带来实际的经济效益。     

从阿维菌素获得诺贝尔奖到中国创造

纵观世界历史,大国崛起无不伴随着科技的兴起和机制体制的突破。来自大自然的天然产物对于人类的健康起到非常重要的作用,从抗肿瘤明星分子紫杉醇到挽救了无数人生命的抗感染药物青霉素,从治疗代谢疾病到营养保健,都离不开天然产物。此外,还有大量天然产物资源没有被开发过。而随着阿维菌素的发现者Satoshiōmura教授和William C.Campbell博士,及青蒿素的发现者屠呦呦研究员因为这两种天然产物在治疗寄生虫感染病和疟疾上的应用而获得2015年诺贝尔生理与医学奖后,天然产物有望迎来其发展的第二个黄金时代。我国是世界工厂也是天然药物的资源大国,为了实现产业升级,弯道超车,实现大国崛起的中国梦,我国科学家在"十二五"期间围绕着天然产物的高产和创新两大主题开展了富有成效的合成生物学研究。阿维菌素是由阿维链霉菌产生的高效低毒生物杀虫剂,其原料产能占国际市场的100%。但我国原有生产菌株的单位发酵产量较低,高消耗、高污染、片面追求生产规模的粗放型发展模式已经成了阻碍低碳经济持续高速发展的瓶颈。如何从根本上解决问题,提高生产菌株的单位发酵产量和原料利用率,降低能耗和生产成本,减少环境污染,是促进我国从"发酵大国"向"发酵强国"转变的关键。本文以阿维菌素为例,综述其基础研究的技术发展,特别是中国科学院微生物研究所引入合成生物学技术,将阿维菌素的单位产量提高了1000倍,至9 g/L,在内蒙古新威远与阿维菌素产业联盟的公司应用,迫使默克公司全面退出阿维菌素历史舞台,从而引领产业迅速发展的过程,为我国其它天然产物生物制造品种的改良提供思路和方法。     

Insecticides with novel modes of action: Mechanism, selectivity and cross-resistance

Efforts have been made during the past two decades to develop insecticides with selective properties that act specifically on biochemical sites present in particular insect groups, but whose properties differ from other insecticides. This approach has led to the discovery of compounds that affect the hormonal regulation of molting and developmental processes in insects; for example, ecdysone agonists, juvenile hormone mimics and chitin synthesis inhibitors. In addition, compounds that selectively interact with the insect nicotinic acetylcholine receptor, such as imidacloprid, acetamiprid and thiamethoxam, have been introduced for the control of aphids, whiteflies and other insect species. Natural products acting selectively on insect pests, such as avermectins, spinosad and azadirachtin, have been introduced for controlling selected groups of insect pests. Compounds acting on the nervous site that controls the sucking pump of aphids and whiteflies, such as pymetrozine, or respiration, such as diafenthiuron, have been introduced for controlling sucking pests. All the above compounds are important components in pest and resistance management programs.     

Stimulation of avermectin B1a biosynthesis in Streptomyces avermilitis by feeding glucose and propionate

The biosynthetic ability of avermectin B1a in Streptomyces avermilitis was improved with propionate addition and glucose feeding. The results showed that B1a production was increased by 12.8–13.8% through supplement of 0.8% propionate at 24 h of cultivation. A stronger stimulation on B1a biosynthesis in S. avermilitis was observed by 3.0% glucose feeding at 5 d of cultivation. The B1a biosynthesis could be further enhanced by repeated glucose fed-batch process in a 10-l bench-top fermentor. A maximal B1a concentration of 780.0 mg/l was obtained by repeated 1.0% glucose addition on 4 d, 5 d and 6 d, which was 2.1-fold higher than that in a control fermentation. Corresponding with this, an additional 6.8% increase of B1a proportion was observed in comparison with the control process. This stimulation on avermectin B1a production was obvious even in a 2000-l fermentor under suitable control of aeration.     

紫外吸收剂抑制阿维菌素光解及对小菜蛾防治的增效作用

利用高效液相色谱法测定了紫外吸收剂在一定紫外光强度（420 lx,254 nm）下对阿维菌素光解速率的影响,同时测定了其与阿维菌素混用对小菜蛾的毒力和田间防效.结果表明,加入紫外吸收剂UV-73和UV-75后,可以显著地提高阿维菌素的光稳定性,光解4 h,阿维菌素的分解率分别降低76.74%和77.46%;光解8h,阿维菌素的分解率分别降低37.92%和47.09%;光解16 h,阿维菌素的分解率分别降低33.55%和48.55%;光解24 h,阿维菌素的分解率分别降低23.57%和42.09%.室内生物测定结果表明,紫外光下加入紫外吸收剂UV-73和UV-75后,阿维菌素对小菜蛾的增效比值分别为1.59和1.74.田间药效结果也表明,与紫外吸收剂混用的阿维菌素对小菜蛾的防效明显高于单用.     

甲氰菊酯和阿维菌素对柑橘全爪螨的亚致死效应

通过叶碟饲养的方法, 利用生命表技术,研究了甲氰菊酯和阿维菌素亚致死剂量LC₂₀处理柑橘全爪螨若螨后,对试验种群当代(F₀)和后代(F₁、F₂代)生长发育及繁殖的影响.结果表明: 甲氰菊酯LC₂₀处理若螨后,当代雌成螨产卵量显著增加;F₁、F₂代的产卵前期缩短,后代雌性比例增大,且均与对照差异显著;同时,F₁和F₂代种群内禀增长率(r_m)和周限增长率(λ)增大,世代历期(T)和种群加倍时间(Dt)缩短,且F₂代与对照相比差异显著.用阿维菌素LC₂₀处理若螨后,当代种群雌成螨产卵量显著下降; F₁和F₂代的产卵量也显著下降,但后代雌性比例增大,产卵前期显著缩短;F₁和F₂代的种群r_m和λ增大,T和Dt缩短,且F₂代比F₁代更为明显.总体来看,甲氰菊酯和阿维菌素亚致死浓度LC₂₀对柑橘全爪螨的影响并不完全相同,甲氰菊酯能够促进当代种群的发展,而阿维菌素对当代种群有一定的抑制作用;但两种杀螨剂亚致死浓度处理柑橘全爪螨对后代种群都有一定的促进作用.研究结果对柑橘全爪螨综合防治策略的制定有一定的指导意义.     

Actions of avermectin B1a on the γ-aminobutyric AcidA receptor and chloride channels in rat brain

The interaction of avermectin B_1a (AVM) with the γ-aminobutyric acid (GABA) receptor of rat brain was studied using radioactive ligand binding and tracer ion flux assays. Avermectin potentiated the binding of [³H]flunitrazepam and inhibited the binding of both [³H]muscimol and [³⁵S]t-butylbicyclo-phosphorothionate to the GABA_A receptor. Inhibition of muscimol binding by AVM suggested competitive displacement. Two kinds of ³⁶chloride (Cl) flux were studied. The ³⁶Cl efflux from preloaded microsacs was potentiated by AVM and was highly inhibited by the Cl-channel blocker 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS). However, it was not potentiated by GABA nor was it sensitive to the convulsants picrotoxin or bicuculline. On the other hand, ³⁶Cl-influx measurement in a different microsac preparation of rat brain was very sensitive to GABA and other GABA-ergic drugs. Avermectin induced ³⁶Cl influx into these microsacs in a dose–dependent manner, but to only 35% of the maximal influx induced by GABA. The AVM-induced ³⁶Cl influx was totally blocked by bicuculline. It is suggested that AVM opens the GABA_A-receptor Cl channel by binding to the GABA recognition site and acting as a partial receptor agonist, and also opens a voltage–dependent Cl channel which is totally insensitive to GABA but is very sensitive to DIDS.     

Efficacy of Four Nematicides Against the Reproduction and Development of Pinewood Nematode,Bursaphelenchus xylophilus

To understand the efficacy of emamectin benzoate, avermectin, milbemectin, and thiacloprid on the reproduction and development of Bursaphelenchus xylophilus, seven parameters, namely population growth, fecundity, egg hatchability, larval lethality, percent larval development, body size, and sexual ratio, were investigated using sublethal (LC₂₀) doses of these compounds in the laboratory. Emamectin benzoate treatment led to a significant suppression in population size, brood size, and percent larval development with 411, 3.50, and 49.63%, respectively, compared to 20850, 24.33, and 61.43% for the negative control. The embryonic and larval lethality increased obviously from 12.47% and 13.70% to 51.37% and 75.30%, respectively. In addition, the body length was also significantly reduced for both males and females in the emamectin benzoate treatment. Avermectin and milbemectin were also effective in suppressing population growth by increasing larval lethality and reducing larval development, although they did not affect either brood size or embryonic lethality. Body length for both male and female worms was increased by avermectin. Thiacloprid caused no adverse reproductive effects, although it suppressed larval development. Sexual ratio was not affected by any of these four nematicides. Our results indicate that emamectin benzoate, milbemectin, and avermectin are effective against the reproduction of B. xylophilus. We think these three nematicides can be useful for the control of pine wilt disease.     

Dynamics of serine/threonine protein kinase activity during the growth of the wild-typeStreptomyces avermitilis strain and its chloramphenicol-resistant mutant

The dynamics of serine/threonine protein kinase activity during the growth of the wild-typeStreptomyces avermitilis strain 964 and its chloramphenicol-resistant (Cml^r) pleiotropic mutant with an enhanced production of avermectins was studied by measuring the transfer of radiolabeled phosphate from [y-³²P]ATP to the serine and threonine residues of proteins in cell-free extracts. In both of the strains studied, radiolabeled phosphate was found to incorporate into polypeptides with molecular masses of 32, 35, 41, 68, 75, 79, 83, and 137 kDa; however, the degree and the dynamics of phosphorylation of particular peptides were different in these strains. The differences revealed could not be accounted for by the interference of ATPases or phosphoprotein phosphatases. The data obtained may be interpreted as evidence that Cml^r mutation activates the protein kinase signalling system ofS.avermitilis cells in the early stationary growth phase and thus enhances the production of avermectins and leads to some other physiological changes in the mutant strain.     

The effect of avermectin B1 on developmental stability in the bush fly, Musca vetustissima, as measured by fluctuating asymmetry

Fluctuating asymmetry is used as an indicator of developmental stability to assess the effect of exposure to Avermectin B₁ on normal developmental processes in the bush fly, Musca vetustissima Walker (Diptera: Muscidae). Flies breeding in dung from cattle treated with Avermectin B₁ were found to be significantly more asymmetric for two morphometric characters (wing vein lengths), than those breeding in dung from either untreated cattle or cattle treated with Levamisole-HCl at times when fly survival was not affected by the treatments. Fluctuating asymmetry is shown to be a valuable means of defining developmental stress in situations where conventional estimates such as survival show no response.

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Résumé La fluctuation de l'asymétrie a été utilisée comme indice de la stabilité du développement pour mettre en évidence l'effet de l'exposition à l'avermectine B₁ au cours du développement de M. vetustissima. Les mouches élevées sur bouses de vaches traitées à l'avermectine B₁ sont devenues significativement plus asymétriques pour 2 caractères morphométriques (longueurs des veines de l'aile) que celles élevées sur bouses de vaches témoins ou de vaches traitées au Levamisole-HCl, même quand la survie des mouches n'était pas affectée par le traitement. La fluctuation de l'asymétrie s'est avérée un bon critère pour définir des stress pendant le développement, alors que des critères conventionnels comme la survie ne permettent pas de déceler quoi que ce soit.