柔红霉素产生菌SIPI-1482中dnmV基因功能的阻断及恢复

dnmV基因产物为柔红霉素生物合成途径中TDP-6-脱氧己糖C4酮基还原酶,破坏该基因能阻断柔红糖胺的合成,进而阻断柔红霉素的产生。从天蓝淡红链霉菌(S. coeruleorubidus)SIPI-1482基因组DNA中经PCR扩增出dnmV及其上游dnmU基因片段,并由此构建了用于阻断dnmV基因的同源重组质粒pYG817,转化SIPI-1482菌株后成功地破坏了dnmV基因,发酵结果显示阻断突变株不再代谢产生柔红霉素,为引入新的基因来改变代谢产物的糖基结构打下了基础。通过导入dnmV基因表达质粒可重建该突变株的生物合成途径,恢复产生柔红霉素,但产量比出发菌株要低。     

黑暗链霉菌DNA同源重组系统的构建

以黑暗链霉菌Tt-49基因组为模板,利用PCR方法,扩增安普霉素生物合成关键基因aprF-G的上、下游序列,作为同源交换臂,并将红霉素抗性基因筛选标记及其启动子插入两交换臂之间,以温敏型质粒pKC1139为基础,构建用于阻断黑暗链霉菌Tt-49安普霉素生物合成的重组质粒pFD8.该质粒通过E.coil ET12567/pUZ8002去甲基化修饰后,经接舍转移进入黑暗链霉菌Tt-49,利用红霉素抗性筛选得到3株阳性转化子,分别命名为Tt-49 AG1、Tt-49 AG2和Tt-49 AG3.通过PCR鉴定,证明pFD8已插入黑暗链霉菌Tt-49基因组的目标位点.以亲株作对照,对3株工程菌进行红霉素抗性能力考察,发现3株工程菌的抗红霉素能力均高迭1 000 μg/mL以上.     

通过一种新方法使T7基因Ⅰ置换araBAD基因簇

目的：用T7RNA聚合酶基因T7基因Ⅰ置换大肠杆菌基因组中的araBAD基因簇。方法：通过分析大肠杆菌基因组中araBAD基因序列,设计两侧同源臂,构建高拷贝数的打靶质粒,并且在这个质粒上的打靶片段两侧各加上一个归巢内切酶位点;将辅助质粒和打靶质粒同时转化到宿主体内,在L-阿拉伯糖的诱导下表达归巢内切酶和Red重组酶,实现体内重组,使T7基因I置换araBAD基因簇。结果：在4株不同的大肠杆菌中用T7基因I置换了araBAD基因簇。结论：成功地将T7基因Ⅰ整合到araBAD基因位置,为构建PAra-PT7表达系统奠定了基础。     

通过一种新方法使T7基因I置换araBAD基因簇

目的:用T7 RNA聚合酶基因T17基因I置换大肠杆菌基因组中的araBAD基因簇.方法:通过分析大肠杆菌基因组中araBAD基因序列,设计两侧同源臂,构建高拷贝数的打靶质粒,并且在这个质粒上的打靶片段两侧各加上一个归巢内切酶位点;将辅助质粒和打靶质粒同时转化到宿主体内,在L-阿拉伯糖的诱导下表达归巢内切酶和Red重组酶,实现体内重组,使T7基因I置换araBAD基因簇.结果:在4株不同的大肠杆菌中用T7基因I置换了araBAD基因簇.结论:成功地将T7基因,整合到araBAD基因位置,为构建Para-PT7表达系统奠定了基础.     

天蓝淡红链霉菌SIPI-1482中酮还原酶基因dnrU的阻断突变研究

目的：研究柔红霉素产生菌天蓝淡红链霉菌SIPI-1482中酮还原酶基因dnrU阻断后的产物（13s）-13-二氢柔红霉素及其他发酵产物的变化。方法：利用同源重组的原理,以大肠杆菌质粒pUC18为基础构建了dnrU基因交换质粒,通过在SIPI-1482染色体上的dnrU基因中插入安普霉素抗性基因来筛选dnrU的阻断突变株。结果和结论：PCR验证表明成功地阻断了dnrU基因。dnrU基因敲除后,重组菌发酵产物中（13s）-13-二氢柔红霉素消失,而其他发酵中间产物也有一定变化。     

产诺加霉素链霉菌中失活snogA基因的研究

诺加霉素是重要的蒽环类抗肿瘤抗生素,由黑胡桃链霉菌ATCC27451发酵产生。本研究从诺加霉素产生茵中克隆得到560bp的氨基甲基化酶（snogA）编码基因片段,并将其插入基因整合型质粒pKCll39的多克隆位点,构建得到基因中断质粒pLMX-3-58。通过接合转移和同源重组,构建得到氨基甲基化酶编码基因被中断的重组菌株删-3-59。基因重组突变株基因型验证结果表明,中断质粒以正确方式整合入基因组,将氨基甲基化酶编码基因中断。发酵验证结果表明,重组茵株发酵产物中不含有诺加霉素。本研究表明snogA基因在诺加霉素生物合成途径中是必需的。这为进一步阐明诺加霉素生物合成途径和组合生物合成改造诺加霉素提供了参考。     

阿维链霉菌孢子色素生物合成对阿维菌素产量的影响

以野生型阿维链霉茵NRRL8165为出发菌株,用PCR方法克隆孢子色素基因簇直系同源基因(whiEa)侧翼片段,并构建基因置换载体pHL643.将pilL643跨属接合转移进入阿维链霉菌NRRL8165,通过置换载体和染色体之间的同源双交换,对染色体上的whiEa基因簇进行置换,得到3株阿泊拉霉素抗性、硫链丝菌素敏感的重组菌株,均表现为孢子色素合成缺陷.通过Southern杂交分析,证明whiEa基因簇被置换.通过摇瓶发酵和HPLC检测,发现whiEa基因簇置换菌株所产阿维菌素产量明显提高,表明孢子色素与阿维菌素生物合成之间可能有竞争底物的现象.     

同源重组置换衣藻叶绿体chlL基因及其同质化鉴定

设计以 aad A基因置换衣藻 chl L基因并作为转基因衣藻筛选标记 ,将 chl L基因两侧的基因片段 clp P- trn L- pet B- chl L5′- 3′作为同源片段 ,构建衣藻叶绿体同源重组质粒 p64D.通过改进的基因枪法转化程序 ,将此质粒轰击入衣藻叶绿体 .经抗性筛选、暗培养及 PCR、Southern- blotting等鉴定 ,证实衣藻叶绿体基因组中大部分 chl L结构基因已被外源基因 aad A所置换     

人CCL20基因打靶载体的构建与鉴定

目的:构建针对人CC亚族趋化因子配体20(CC chemokine ligand 20,CCL20)基因外显子2的置换型打靶栽体.方法:设计和合成引物,利用长距离聚合酶链反应(polymerase chain reaction,PCR)从永生化人角质形成细胞系(HaCaT)基因组DNA中克隆出CCL20基因组DNA片段,再以CCL20基因为模板扩增出长度为1969 bp的同源短臂和2356 bp的同源长臂.分别插入pioxP栽体的Neo基因上游和下游,构建针对人CCL20基因外显子2的置换型打靶载体(ploxP-hCCL20).将打靶载体线性化并以电穿孔方法转移入HaCaT,观察克隆的形成.结果:经过PCR、限制性内切酶鉴定及DNA序列测定.证实该载体的两条同源臂包含人CCL20基因的外显子1、3、4及其邻近的部分内含子.结论:ploxP-hCCL20载体构建成功,增加Neo基因下游同源臂长度的策略有可能提高细胞基因敲除的同源重组率.     

基于同源双交换的聚球藻PCC 7942基因组大片段无标记删除

在蓝藻合成生物学中,对大片段进行无标记删除可以加快基因组简化的进程.研究以聚球藻PCC 7942为材料,基于同源重组技术对基因组中大于10 kb的3个非必需区域进行无标记删除.构建带有两侧同源片段的不可在聚球藻复制的质粒,利用接合转移将其导入藻细胞获得同源单交换株,再借助于条件致死基因sacB筛选第二步交换的克隆,获得...     

Cloning and characterization of the Streptomyces peucetius dnmZUV genes encoding three enzymes required for biosynthesis of the daunorubicin precursor thymidine diphospho-L-daunosamine.

Characterization of the dnmZ, dnmU, and dnmV genes from the daunorubicin-producer Streptomyces peucetius by DNA sequence analysis indicated that these genes encode a protein of unknown function plus a putative thymidine diphospho-4-keto-6-deoxyglucose-3(5)-epimerase and thymidine diphospho-4-ketodeoxyhexulose reductase, respectively. Inactivation of each of the three genes by gene disruption and replacement in the wild-type strain demonstrated that all of them are required for daunosamine biosynthesis.     

Construction of an expression system for site-directed mutagenesis of the lantibiotic mersacidin

The lantibiotic (i.e., lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide of 20 amino acids which is produced by Bacillus sp. strain HIL Y-85,54728. Mersacidin inhibits bacterial cell wall biosynthesis by binding to the precursor molecule lipid II. The structural gene of mersacidin (mrsA) and the genes for the enzymes of the biosynthesis pathway, dedicated transporters, producer self-protection proteins, and regulatory factors are organized in a biosynthetic gene cluster. For site-directed mutagenesis of lantibiotics, the engineered genes must be expressed in an expression system that contains all of the factors necessary for biosynthesis, export, and producer self-protection. In order to express engineered mersacidin peptides, a system in which the engineered gene replaces the wild-type gene on the chromosome was constructed. To test the expression system, three mutants were constructed. In S16I mersacidin, the didehydroalanine residue (Dha) at position 16 was replaced with the Ile residue found in the closely related lantibiotic actagardine. S16I mersacidin was produced only in small amounts. The purified peptide had markedly reduced antimicrobial activity, indicating an essential role for Dha16 in biosynthesis and biological activity of mersacidin. Similarly, Glu17, which is thought to be an essential structure in mersacidin, was exchanged for alanine. E17A mersacidin was obtained in good yields but also showed markedly reduced activity, thus confirming the importance of the carboxylic acid function at position 17 in the biological activity of mersacidin. Finally, the exchange of an aromatic for an aliphatic hydrophobic residue at position 3 resulted in the mutant peptide F3L mersacidin; this peptide showed only moderately reduced activity.     

橙色红曲菌As3.4384 orf7基因缺失株的构建及其功能分析

红曲菌能产生多种有益的次级代谢产物,但红曲菌也产生一种对人和哺乳动物肝和肾有毒害的毒素,即桔霉素。因此控制毒素的产生是保障红曲产品安全性所必须的。故对桔霉素的合成途径及相关的基因做深入了解。6个桔霉素合成相关的基因成簇位于21 kb的DNA片段上。克隆了一个新基因(orf7基因),其位于该基因簇的外侧。采用基因敲除技术,构建红曲菌orf7基因缺失菌株。并采用紫外分光光度法检测orf7基因缺失菌株的红曲色素产量,HPLC法检测其桔霉素产量。orf7缺失菌株产红曲色素能力与出发菌株As3.4384相比没有变化;产桔霉素培养13~19 d,桔霉素的产量与出发菌株As3.4384相比增加了 142.4%。从而证实orf7基因与桔霉素代谢相关。     

Construction of an Expression System for Site-Directed Mutagenesis of the Lantibiotic Mersacidin

The lantibiotic (i.e., lanthionine-containing antibiotic) mersacidin is an antimicrobial peptide of 20 amino acids which is produced by Bacillus sp. strain HIL Y-85,54728. Mersacidin inhibits bacterial cell wall biosynthesis by binding to the precursor molecule lipid II. The structural gene of mersacidin (mrsA) and the genes for the enzymes of the biosynthesis pathway, dedicated transporters, producer self-protection proteins, and regulatory factors are organized in a biosynthetic gene cluster. For site-directed mutagenesis of lantibiotics, the engineered genes must be expressed in an expression system that contains all of the factors necessary for biosynthesis, export, and producer self-protection. In order to express engineered mersacidin peptides, a system in which the engineered gene replaces the wild-type gene on the chromosome was constructed. To test the expression system, three mutants were constructed. In S16I mersacidin, the didehydroalanine residue (Dha) at position 16 was replaced with the Ile residue found in the closely related lantibiotic actagardine. S16I mersacidin was produced only in small amounts. The purified peptide had markedly reduced antimicrobial activity, indicating an essential role for Dha16 in biosynthesis and biological activity of mersacidin. Similarly, Glu17, which is thought to be an essential structure in mersacidin, was exchanged for alanine. E17A mersacidin was obtained in good yields but also showed markedly reduced activity, thus confirming the importance of the carboxylic acid function at position 17 in the biological activity of mersacidin. Finally, the exchange of an aromatic for an aliphatic hydrophobic residue at position 3 resulted in the mutant peptide F3L mersacidin; this peptide showed only moderately reduced activity.     

大肠杆菌莽草酸途径限速酶多基因盒的构建及基因替换

优化大肠杆菌芳香族氨基酸生物合成代谢途径 ,构建莽草酸代谢途径限速酶的多基因盒PtacaroAaroCaroBkan .利用Red重组系统 ,在破坏整体调控基因csrA时 ,替换多基因盒 .Southern印迹证实 ,基因破坏和基因替换是成功的 .摇瓶发酵表明 ,构建的基因工程菌株比原始菌株基础产酸率提高了 4 5 3倍     

Cloning and characterization of the Streptomyces peucetius dnrQS genes encoding a daunosamine biosynthesis enzyme and a glycosyl transferase involved in daunorubicin biosynthesis.

The dnrQS genes from the daunorubicin producer Streptomyces peucetius were characterized by DNA sequencing, complementation analysis, and gene disruption. The dnrQ gene is required for daunosamine biosynthesis, and dnrS appears to encode a glycosyltransferase for the addition of the 2,3,6-trideoxy-3-aminohexose, daunosamine, to epsilon-rhodomycinone.     

Streptomyces clavuligerus HlmI is an intramolecular disulfide-forming dithiol oxidase in holomycin biosynthesis

Holomycin and related dithiolopyrrolone antibiotics display broad-spectrum antimicrobial activities and contain a unique 5,5-bicyclic ring structure with an N-acylated aminopyrrolone fused to a cyclic ene-disulfide. Here we show that the intramolecular disulfide bridge is constructed from the acyclic ene-dithiol at a late stage in the pathway by a thioredoxin oxidoreductase-like enzyme HlmI from the holomycin producer Streptomyces clavuligerus. Recombinant HlmI was purified from E. coli with bound flavin adenine dinucleotide (FAD) and converts reduced holomycin to holomycin utilizing O(2) as cosubstrate. As a dithiol oxidase, HlmI is functionally homologous to GliT and DepH, which perform a similar dithiol to disulfide oxidation in the biosynthesis of fungal natural product gliotoxin and epigenetic regulator compound FK228, respectively. Deletion of the hlmI gene in the wild type S. clavuligerus and in a holomycin-overproducing mutant resulted in decreased level of holomycin production and increased sensitivity toward holomycin, suggesting a self-protection role of HlmI in the holomycin biosynthetic pathway. HlmI belongs to a new clade of uncharacterized thioredoxin oxidoreductase-like enzymes, distinctive from the GliT-like enzymes and the DepH-like enzymes, and represents a third example of oxidoreductases that catalyzes disulfide formation in the biosynthesis of small molecules.