链霉菌抗生素生物合成的基因调控

链霉菌中抗生素生物合成受到许多调控基因的调控,它们通过途径特异性调控方式、全局性调控以及双组分调控方式对基因表达进行调控.概述这些调控基因将有利于改良抗生素生产菌株及新型抗生素的研究,并为改造链霉菌抗生素生物合成相关基因、提高产量提供理论依据.本文归纳了国内外链霉菌抗生素生物合成基因簇各调控基因及其研究进展.     

抗生素产生菌核糖体相关基因改变与次级代谢产物的生物合成

利用抗生素抗性筛选方法及定点突变方法可以得到引起抗生素产生菌核糖体相关基因改变的突变株,这些突变对次级代谢产物的生物合成产生深刻影响,如能够显著地提高抗生素的产量也能够产生一些新的次级代谢产物。这种方法不仅对抗生素产生菌的菌种选育具有重要的意义,且为获得新的抗生素提供了新的途径。本文就抗生素产生菌核糖体蛋白基因、核糖体RNA基因、核糖体修饰相关基因及核糖体相关蛋白基因的改变引起次级代谢改变研究进展做简要综述。     

基因克隆产生杂合蒽环类抗生素研究进展

随着人们对芳香聚酮体的生物合成机理的深入了解，将有越来越多的Ⅱ型PKS基因和DOS基因被克隆。人们有可能应用基因克隆或组合生物合成方法对现有抗生素进行改造或创造出结构新颖的杂合化合物。主要介绍了应用基因克隆的方法产生的杂合蒽环类抗生素的研究进展，同时还介绍了蒽环类抗生素的生物合成，链霉菌基因克隆策略与组合生物合成，以及杂合蒽环类抗生素及其前体等研究进展。     

只产阿维菌素B组分的基因工程菌的构建

目的通过对阿维菌素生物合成基因C5-O-甲基转移酶基因(aveD)内部进行缺失,使aveD基因失活,从而获得只产生阿维菌素B组分的基因工程菌。方法构建大肠杆菌-链霉菌重组质粒pZHJ06,通过接合转移将缺失部分片段的aveD基因以双交换的方式整合到阿维链霉菌的染色体上。采用摇瓶进行发酵,采用高效液相色谱(HPLC)检测发酵液中阿维菌素的组分。结果重组菌株不再产生阿维菌素A组分,只产生阿维菌素B组分,并且B组分的总产量也有所提高。结论将阿维链霉菌的aveD基因失活,并不影响下游酮基还原酶基因(aveF)基因的表达,重组菌株只产生阿维菌素B组分。     

脱氧糖组合生物合成的研究进展

天然活性物质的糖基对其发挥生物活性十分重要。目前已从许多抗生素产生菌中分离出各种糖生物合成基因簇以及糖苷转移酶基因，对其生物合成途径的认识也不断深入。近年的研究结果表明抗生素的糖合成酶和糖苷转移酶具有一定的底物宽泛性。本文在总结脱氧糖生物合成基因及糖苷转移酶基因的发展概况基础上，重点综述了运用组合生物合成技术改造脱氧糖分子结构的研究进展。     

微生物次级代谢产物生物合成基因簇与药物创新

微生物产生众多结构和生物活性多样的次级代谢产物，其生物合成基因簇的克隆是药物创新和产量提高的必要前提。迄今为止已有超过150种生物合成基因簇通过各种方式被克隆，并被用于组合生物合成、体外糖类随机化、代谢工程的定向改造。我们研究室已经克隆并测定了氨基糖苷类井冈霉索／有效霉索、多烯类抗生素FR-008／克念菌索、聚醚类南昌霉索、聚酮类梅岭霉索、杂合聚酮一多肽类略唑霉索等生物合成基因簇。深入的基因功能分析揭示了他们独特的生物合成途径和调节机理，为正在进行的组合生物合成结构改造和代谢工程产量提高奠定了基础。     

四环素生产菌与柔红霉素产生菌的种间原生质体融合

采用原生质体融合法,以提高重组频率,从而提高抗生素高产菌株的筛选机率,或获得产生新抗生素的菌株,已得到广泛应用~[1～3]。在提高产率方面,国内外报道的仅限于同类抗生素产生菌的理论性探索且未见实效~[6]。为寻找菌种选育新途径,我们选取了具有共同生物合成主路——聚酮体(polyke-tide)途径的二株不同大类的抗生素生产菌株,通过种间原生质体融合,以探索提高抗生素产率的可能性。     

双拷贝aveC基因对阿维菌素产量的影响

目的通过在阿维链霉菌染色体上增加aveC基因的拷贝数,提高阿维菌素"1"组分的产量。方法采用基因工程技术,在阿维链霉菌染色体DNA的bkdAB基因中插入一套aveC基因,通过同源双交换得到重组菌株,采用HPLC考察各组分的含量。结果第二套aveC基因正确地插入到基因组DNA的预定部位。然而,对重组菌株发酵产物的HPLC分析显示,阿维菌素"1"组分比例并没有明显的变化,但B1a效价却有显著提高。结论aveC基因产物的活性可能不是决定阿维菌素"1"组分比例的主要因素,但aveC基因产物的活性可能是阿维菌素生物合成的限制因素。     

利用基因工程技术选育氨甲酰妥布霉素高含量菌种

目的以产生安普霉素、氨甲酰妥布霉素和氨甲酰卡那霉素3个组分的黑暗链霉菌为出发菌株,利用基因工程技术选育氨甲酰妥布霉素含量高的菌种。方法克隆安普霉素生物合成基因,通过基因阻断技术破坏安普霉素生物合成基因,阻断安普霉素生物合成。结果获得了氨甲酰妥布霉素含量高的菌种。结论利用基因工程技术可以有效地改良抗生素组分。     

氨基糖苷类抗生素生物合成基因研究进展

目的对氨基糖苷类抗生素生物合成基因的研究进展作一综述。方法按照化学结构类型 ,分别介绍了有糖苷取代的氨基环己醇类抗生素和 2 脱氧链霉胺类抗生素的生物合成基因的研究状况 ,以及氨基糖苷类抗生素生物合成基因的特点。结果与结论氨基糖苷类抗生素生物合成基因具有很多共同的特点 ,为进一步对氨基糖苷类抗生素生物合成基因进行克隆、研究及基因改造提供了参考     

Branched-chain fatty acid requirement for avermectin production by a mutant of Streptomyces avermitilis lacking branched-chain 2-oxo acid dehydrogenase activity

The eight natural avermectins produced by Streptomyces avermitilis have the carbon skeleton of either isobutyric or S-2-methylbutyric acid incorporated into their structures. A mutant of S. avermitilis has been isolated that contains no functional branched-chain 2-oxo acid dehydrogenase activity. The mutant, in contrast to its parent, is unable to grow with isoleucine, valine and leucine as carbon sources. In medium lacking both S(+)-2-methylbutyric and isobutyric acid, the mutant is also incapable of making the natural avermectins, while supplementation with either one of these compounds restores production of the corresponding four natural avermectins. These facts indicate that in S. avermitilis the branched-chain 2-oxo acid dehydrogenase enzyme functions not only to catabolize the cellular branched-chain amino acids in order to meet energy and growth requirements but also to provide the small branched-chain organic acid precursor molecules necessary for avermectin biosynthesis. Supplementation of the mutant strain with R(-)-2-methylbutyric acid yields novel isomeric avermectins unseen in the (unsupplemented) wild-type strain. It was also concluded that acetate and propionate production by branched-chain 2-oxo acid degradation is not absolutely essential for avermectin production.     

Novel avermectins produced by mutational biosynthesis

Avermectins with a wide range of novel C-25 substituents have been prepared by feeding carboxylic acids or their biosynthetic precursors to a Streptomyces avermitilis mutant strain ATCC 53568. This organism lacks the ability to form isobutyric and S-2-methylbutyric acids from their 2-oxo acid precursors and thus is unable to produce natural avermectins unless supplied with these acids. The novel avermectins produced by mutational biosynthesis possess broad-spectrum antiparasitic activity.     

几种调控基因对除虫链霉菌工业生产株的抗生素效价的影响

在模式链霉菌（如天蓝色链霉菌和变铅青链霉菌）中导入许多抗生索生物合成的调控基因可以大幅度提高抗生索的含量。本文报道利用链霉菌的整合质粒克隆几种已知的调控基因。并通过接合转移从大肠埃希菌中导入产生avermectin和多拉菌素的除虫链霉菌工业生产菌株中。发现3种调控基因afiR、aveR和orfX对菌株MMR630中avermectin的含量均可以提高约1倍。但是，以上的3种，加上另外3种调控基因分别导入菌株G11后，发现除aft8提高约13％外，其余调控基因使菌株产生多拉菌素的含量反而有不同程度的降低。将调控基因币B置于链霉菌强启动子PerrnE^＊下表达降低了菌株G11中多拉菌素的含量。上述结果表明，调控基因对不同链霉菌的抗生素生物合成具有不同的影响，反映了抗生素生物合成确实受到了复杂网络的调控。     

Demethylavermectins. Biosynthesis, isolation and characterization

Streptomyces avermitilis normally produces eight avermectins. Avermectin A components contain three methoxyl groups; two on the oleandrose disaccharide and one on the aglycone moiety at C5. Avermectin B components contain methoxyl groups only on the oleandrose disaccharide. Sinefungin inhibits methylation at all three sites. Addition of sinefungin to S. avermitilis Agly-1, a mutant which produces virtually only avermectin aglycone A components, alters the fermentation and causes an accumulation of avermectin aglycone B components. Addition of sinefungin to S. avermitilis 08, a high producing strain, results in accumulation of 8 new avermectins which lack methoxyl groups on the oleandrose moieties as well as the aglycone. These new avermectins were isolated and shown to possess anthelmintic and insecticidal activity.     

Biosynthesis of the avermectins by Streptomyces avermitilis. Incorporation of labeled precursors

The biosynthesis of the avermectins, a group of 16 membered macrolides with potent anthelmintic and insecticidal activity produced by Streptomyces avermitilis, was studied by supplying cultures with 14C and 13C precursors. [1-14C] and [2-14C]acetate and propionate were poor precursors of the avermectins and were instead rapidly oxidized to 14CO2. The S-methyl of methionine in contrast was incorporated extensively and equally into the three methoxyl groups of the avermectins. The carbon backbone of methionine was not a precursor of the avermectins. Feeding of [1-13C]glucose yielded avermectins labeled specifically in the C1' and C1" of the oleandrose moiety and in the aglycone moiety in carbons known to be derived from the methyl of acetate. Feeding [U-13C]glucose showed that the entire avermectin molecule is derived from glucose carbons.     

The leptomycin gene cluster and its heterologous expression in Streptomyces lividans

Leptomycin exerts its antifungal and anti-tumoral activity via inhibiting nucleo-cytoplasmic translocations in eukaryotic cells. To learn more about the biosynthesis of leptomycin and in an effort to generate leptomycin analogues through genetic engineering, 90 kb segment of DNA containing the putative leptomycin (lep) biosynthesis cluster from Streptomyces sp. ATCC 39366 was cloned and sequenced. The lep cluster consist of 12 polyketide synthase (PKS) modules distributed in four genes (lepA, B, C and D) and a P450 encoding gene. The lep gene cluster was confirmed by its successful expression in Streptomyces lividans, where it directed the production of the two natural congeners-leptomycins A and B. The production of leptomycin B showed that the host has the capability to synthesize ethylmalonyl-CoA.     

The metabolism of avermectins B1a, H2B1a, and H2B1b by liver microsomes

The avermectins area a new class of structurally related antiparasitic agents isolated from Streptomyces avermitilis. The major polar metabolites isolated from in vitro incubations of [3H]avermectins B1a, H2B1a, and H2B1b with either rat or steer liver microsomes have been isolated and identified as the C24-methyl alcohols of the parent compounds. A smaller quantity of a more polar metabolite has also been identified as the monosaccharide of the C24-methyl alcohols of avermectin H2H1b from rat liver microsomal incubation and avermectin H2B1a from steer liver microsomal incubation. The mass spectra and 300-MHz 1H-NMR spectra permitted assignment of structures to these metabolites. Together these two metabolites represent 50-80% of the total radioactivity more polar than the parent compounds. The metabolite profiles on reverse-phase HPLC demonstrate that the rat and steer are qualitatively similar in the production of these two polar metabolites.     

Streptomyces griseochromogenes菌株milbemycin生物合成基因簇的筛选

经BamHI酶切的柯氏质粒pku4 0 3与MboⅠ部分消化并经碱性磷酸酶处理的Streptomycesgriseochromogenes染色体DNA片段相连接 ,体外包装后 ,转化大肠杆菌E .coliJM1 0 8,挑取转化子1 1 5 2个 ,构建Streptomycesgriseochromogenes的基因文库。分别以avermectin聚酮体合成酶 (PKS)的酰基转移酶基因 (AT)、酮基合成酶基因 (KS)及参与avermectin生物合成的内酯化酶基因(aveE)、C5 O 甲基转移酶基因 (aveD)为探针 ,利用菌落原位杂交、核酸杂交和以烯酰基还原酶基因(ER)引物的PCR扩增等手段 ,从文库中筛选出 2 4个阳性克隆。根据克隆DNA之间的重叠关系 ,将阳性克隆分为 7组 ,这 7组阳性克隆群可能覆盖控制milbemycin生物合成的基因簇     

The complete biosynthetic gene cluster of the 28-membered polyketide macrolactones, halstoctacosanolides, from Streptomyces halstedii HC34

Halstoctacoanolides A and B are 28-membered polyketide macrolactones and were isolated from Streptomyces halstedii HC34. The biosynthetic gene cluster (hls cluster) of halstoctacosanolides was completely identified from the genome library of Streptomyces halstedii HC34. DNA sequence analysis of ca. 100 kb region revealed that there were seven type I polyketide synthases (PKSs) and two cytochrome P450 monooxygenases in this cluster. Involvement of the gene cluster in the halstoctacosanolide biosynthesis was demonstrated by the gene disruption of P450 monooxygenase genes. The mutants produced a new deoxygenated halstoctacosanolide derivative, halstoctacosanolide C, which confirmed that the hls gene cluster was essential for the biosynthesis of halstoctacosanolides.