【作者】 蹇晓红；

【导师】 邓子新；

【作者基本信息】 上海交通大学 ， 微生物学， 2008， 博士

【摘要】 有效霉素是一种氨基糖苷类抗生素,在防治水稻纹枯病中被广泛使用。在已调出吸水链霉菌5008中有效霉素部分生物合成基因簇的研究基础上,通过基因置换试验失活可能的2-表-5-表-有效醇酮合成酶基因valA,对基因置换菌株的生物活性测定及HPLC分析均表明此菌株失去合成有效霉素的能力。有效霉素生物合成基因簇中有一个可能的转运蛋白基因valH,生物信息学分析表明它具有12个跨膜螺旋,属于主要异化超家族(The Major Facilitator Superfamily),推测ValH可能的功能是负责将合成好的有效霉素转运到细胞外,通过基因置换试验中断valH后,突变株(JXH4)仍然能够产生有效霉素,但是产量下降约17%,将valH基因构建在强启动子下回补valH突变株JXH3后,有效霉素的产量与野生型相比,提高20%。将吸水链霉菌10-22的一系列突变株染色体缺失范围与野生型染色体物理图谱比较发现,染色体上某些片段的缺失与5102-Ⅰ号素的合成密切相关,5102-Ⅰ号素是吸水链霉菌10-22产生的一种与有效霉素类似的一种抗生素,分析28个失去抑制水稻纹枯生长能力的菌株发现,这些菌株染色体缺失大小是不同的,但有一个共同的特点就是都没有一个300 kb的AseI-F片段,在S. lividans ZX1异源表达实验显示300 kb AseI-F片段中的一个文库质粒的发酵产物具有抑菌活性。并且以2-表-5-表-有效醇酮合成酶基因valA为探针杂交实验也证实了5102-Ⅰ号基因合成簇的位置, valA的同源基因(orf1)缺失后的基因改造菌株(JXH4)失去抑菌活性,回补后恢复产素能力。同时,在鉴定5102-Ⅰ抗生素结构时发现它与有效霉素相同,为方便描述将其命名为井岗霉素。10-22产生的井岗霉素与5008产生的有效霉素相比,液体发酵产量相差悬殊,分析两者的基因簇发现,大部分的合成基因都高度同源,但两个基因簇各有自己可能的调节基因,敲除井岗霉素基因簇中的可能的调节基因jin2后,10-22产量无变化,把有效霉素基因簇中已经证明的调节基因valP和valQ导入10-22后,井岗霉素产量上升约40%。为探明井岗霉素基因簇和有效霉素基因簇在产量上差别悬殊的可能原因,分别将两个基因簇整合到异源宿主Streptomyces lividans染色体上,有效霉素异源表达菌株JXH21产量与原始产生菌5008接近,井岗霉素异源表达菌株JXH10产量约为JXH21的1/4,但是远高于原始产生菌10-22。由此推测井岗霉素基因簇在菌株10-22产素能力低下的主要原因为菌株本身,但是有效霉素基因簇与井岗霉素基因簇基因簇相比,其基因簇中内部存在导致高产的原因。井岗霉素基因簇在异源宿主Streptomyces lividans中整合表达的菌株JXH10可以产生与有效霉素相同的几个组分,敲除JXH10中井岗霉素基因簇中的可能与B组分形成相关的氧化还原酶jinJ基因后得到的突变菌株JXH12仍然可以产生B组分,进一步分析有效霉素基因簇中异源表达菌株XH-9和XH-6暗示可能形成B组分并不需要特异的羟化酶,同时探讨了有效霉素可能的合成机制。更多还原

【Abstract】 Validamycin, an animoglyciside antibiotics, is used widely against sheath blight disease of rice plants. Based on the gene cluster responsible for the biosynthesis of validamycin identified from Streptomyces hygroscopicus 5008, valA gene encoding a putative 2-epi-5-epi-valiolone synthase is esential for validamycin biosynthesis as confirmed by both bioassay and HPLC analysis.The gene valH in the validamycin biosynthetic gene cluster harbours 12 transmembrane regions, and pertains to a member of the Major Facilitator Superfamily (MFS). The ValH may be a validamycin exporter. An engineered mutant (JXH3) with disrupted valH was obtained, to our surprise; the mutant JXH3 also has the capability of validamycin production, although the yield decreased about 17%. The valH gene placed under the control of ermE* promoter was introduced into the mutant for complementation, and the yield of validamycin increased about 20% compared with the wild type strain.A series of large chromosomal deletions in Streptomyces hygroscopicus 10-22 were aligned on the physical map of the wild-type strain and the mutants were assessed for their ability to produce the aminocyclitol antibiotic 5102-I. Twenty-eight mutants were blocked for jinggangmycin production and all of them were found to lack a 300 kb AseI-F fragment of the wild-type chromosome. One of the cosmids of 300 kb AseI-F fragment conferred 5102-I productivity to Streptomyces lividans ZX1. Three of the overlapping cosmids also hybridized to the valA gene of the validamycin pathway from S. hygroscopicus 5008 as a probe. The valA-homolog (orf1) of S. hygroscopicus 10-22 was shown to be essential for 5102-I. biosynthesis as an engineered mutant with a deletion of orf1 completely abolished 5102-I production. The corresponding knock-out mutant (JXH4) could be complemented by the introduction of an orf1-containing construct. Concurrently, the identities of the genes common to S. hygroscopicus strains 10-22 and 5008 prompted a comparison of the chemical structures of antibiotic 5102-I and validamycin, which led to a clear demonstration that they are identical. And the former is named jinggangmycin.The yield of jinggangmycin produced by S. hygroscopicus 10-22 is very low comparing with the validamycin produced by S. hygroscopicus 5008 in liquid-state fermentation. The most genes in the two gene cluster have high homology, but the regulatory gene between them seem to be different. After disruption of a putative regulatory gene, jin2, the mutant(JXH6) has not obvious effect on yield of jinggangmycin as detected by HPLC analysis, while the yield of jinggangmycin increased about 40% after the regulatory genes valP and valQ were introduced into the S. hygroscopicus 1022.To explore the possible reason that led to different production of jingangmycin between the val cluater and jin cluster. The jin cluster and val gene cluster were integrated into the chromosome of Streptomyces lividans, generating the engineered strain JXH10 and JXH21 respectively. The yield of validamycin produced by the JXH21 is similar to S. hygroscopicus 5008, while the yield of jinggangmycin produced by JXH10 is about 25% comparing with JXH21 and is much higher than the S. hygroscopicus 10-22. For S. hygroscopicus 10-22, the poor ability of jinggangmycin production is predominantly related with the 10-22 strain, instead of the jin cluster. It could be deduced that there are some reason in validamycin gene cluster caused higher yield comparing with the JXH21 and JXH10.With comparison of the three gene cluster responsible for the same antibitic produced by S. hygroscopicus 10-22, S. hygroscopicus 5008 and S. hygroscopicus var. limoneus KCCM 11405, the jinJ gene encoding a putative oxidoreductase may be responsible for hydroxylation of jinggangmycin B/validamyicn B But the engineered strain JXH12 in which the jinJ gene was disruption using JXH10 as start strain also produced the jinggangmycin B. With analysis of the engineered strain XH-9 and XH-6 ( the jinJ gene is absent in both strains), it is seemed that biosynthesis of validamycin B should not need the special oxidoreductase.更多还原