红霉素基因工程研究进展

红霉素是一类广谱大环内酯类抗生素 ,在临床上具有广泛的应用。近 10年来用基因工程方法对红霉素结构改造已经取得了很大的进展。通过基因工程不仅可以改造红霉素内酯环环的大小、环的骨架和环的侧链 ,而且可以对后修饰的羟基、糖基和甲基进行改造。迄今用基因工程方法合成的新的大环内酯环结构已超过 10 0种 ,所合成的各种红霉素类似物也有数十种 ,且经过基因改造的红霉素类似物都具有生物活性。但基因工程产物产量都普遍降低 ,抑菌活性也不理想 ,因此未来红霉素基因工程研究的重点应加强产量和高活性结构筛选的研究。     

红霉素生物合成的生物化学和基因学

从基因重组技术问世以来,对红霉素(大环内酯类抗生素)生物合成的生物化学和基因学的研究到目前已经累积了大量的研究结果。就这2个领域中对红霉素生物合成的研究历史、研究现状和研究前景(组合生物合成)作以简要的介绍。     

前体代谢工程提高红霉素产量的研究进展

红霉素是十四元大环内酯类抗生素,具有广泛的医药价值和巨大的新药开发潜力。红霉素的主要成分红霉素A由丙酰辅酶A和甲基丙二酰辅酶A作为前体通过聚酮合酶合成大环内酯骨架,再经羟基化、糖基化、甲基化等一系列修饰合成。根据红霉素A的生物合成路线,我们从前体喂养途径、糖基化和甲基化优化等方面,简要综述近年来利用前体代谢工程手段提高红霉素产量的研究进展。     

白介素6与肿瘤相关性的研究进展

白介紊-6(interleukin 6,IL-6)是一种由多种细胞产生的多功能细胞因子,在免疫应答、急性期反应、造血调节中起重要作用.最近研究发现IL-6与肿瘤的发生发展、预后及治疗密切相关.本文主要介绍IL-6的结构、生物学功能、及其与肿瘤的关系.探讨其在肿瘤发生发展中的作用机制及在肿瘤的早期诊断,预后和治疗方面的应用前景.     

C6胶质瘤侵袭机制的研究进展

恶性肿瘤的侵袭转移机制是当今肿瘤学研究的热点之一,多形性胶质母细胞瘤(glioblastoma multiforme,GBM)死亡率居高不下的主要原因在于其在脑内发生广泛的浸润,GBM的侵袭是一个受多因素调控、多种基因参与的多步骤、多阶段、连续复杂的主动过程,很多机制还不十分清楚.但是,近年来许多学者通过C6胶质瘤模型对GBM的研究表明.一些黏附分子、蛋白酶及细胞因子等参与C6胶质瘤的侵袭.本文就目前C6胶质瘤侵袭机制的研究进展进行综述.     

内消旋-二氨基庚二酸脱氢酶关键氨基酸位点的改造提高对烷基取代2-酮酸的催化活力

【背景】高效实现D-氨基酸的生物合成一直是人们关注的热点。内消旋-二氨基庚二酸脱氢酶(meso-diaminopimelate dehydrogenase,DAPDH)能够直接催化2-酮酸和氨合成D-氨基酸。【目的】提高DAPDH对烷基取代2-酮酸的催化活力,并解释其催化机制。【方法】以来源于嗜热共生杆菌(Symbiobacteriumthermophilum)的内消旋-二氨基庚二酸脱氢酶(StDAPDH)为模板,在前期结构分析结合被选择位点突变结果的基础上,确定对H227位进行定点饱和突变,并以D-丙氨酸、D-2-氨基丁酸、D-正缬氨酸、D-谷氨酸为底物进行筛选。【结果】获得突变体H227Q和H227N。突变体H227Q对丙酮酸、2-氧代丁酸、2-氧代戊酸、2-酮戊二酸的比活力比野生型分别提高了10.9、11.5、8.6和7.6倍。动力学参数表明,突变体H227Q同时提高了酶对底物的亲和力及催化常数,使其对丙酮酸的催化效率(k_(cat)/K_m)相较于野生型提高了9.4倍。利用分子模拟技术分析突变体H227Q与产物氨基酸之间的相互作用表明,227位的谷氨酰胺通过与氨基酸的羧酸形成氢键,使得氨基酸产物Cα上的氢和辅酶烟酰胺环C4原子之间的距离缩短。【结论】利用定向进化技术提高DAPDH对烷基取代2-酮酸的催化活力,有助于开发新型的高效生物催化剂,这些工作也为下一步继续进行更具挑战性的D-氨基酸研究提供了基础。     

RNA中6-甲基腺嘌呤的研究进展

RNA酶促共价修饰研究, 尤其是m⁶A(6-甲基腺嘌呤), 是RNA生物学研究的一个新兴领域。m⁶A是真核生物mRNA内部序列中最常见的一种转录后修饰形式, 由包含3个独立组分的复合物mRNA: m⁶A甲基转移酶催化生成。最新研究发现肥胖相关蛋白FTO可以脱掉m⁶A上的甲基, 表明该甲基化过程是可逆的。抑制或敲除m⁶A甲基转移酶会引起重要的表型变化, 但是由于过去的检测方法受限, m⁶A确切的作用机制目前为止还不甚清楚。二代测序技术结合免疫沉淀方法为大规模检测m⁶A修饰并研究其作用机制提供了可能。文章主要综述了m⁶A的发现史、生成机制、组织和基因组分布、检测方法、生物学功能等及其最新研究进展, 并通过比较3种IP-seq技术和数据分析的异同及优缺点, 对m⁶A这种RNA表观修饰研究中尚未解决的问题进行了讨论。     

△6-脂肪酸脱氢酶的分子生物学研究进展

包括γ-亚麻酸在内的多不饱和脂肪酸由于在人类健康中的重要作用而成为有价值的产品,目前市场上对γ-亚麻酸的需求持续增长,然而当前来源难以满足市场的需求,寻找合适的替代来源将有助于解决这一问题。△^6-脂肪酸脱氢酶是多不饱和脂肪酸合成途径中的限速酶,这里从△^6-脂肪酸脱氢酶的基因克隆、结构和功能的研究、系统进化和基因工程应用等方面探讨了△^6-脂肪酸脱氢酶的研究进展。     

IL-6与2型糖尿病关系的研究进展

2型糖尿病(T2DM)已成为全世界危害人类健康的主要疾病,且患病率逐年增加.虽然T2DM确切的发病机制尚未完全阐明,但越来越多的证据提示T2DM的发生同炎症密切相关.IL-6是参与炎症反应的重要的细胞因子,研究发现IL-6在T2DM及其并发症的发生发展中起重要的作用.通过阻断炎症因子及其受体的抗炎治疗明显降低糖尿病的发生率或延迟其发展.这将有望给T2DM的防治提供新途径,为临床早发现及治疗提供更多的科学依据.     

跨膜蛋白AgrC_(TM6-7C)分离纯化方法的优化北大核心CSCD

旨在通过优化AgrC_(TM6-7C)蛋白的纯化过程,获得高纯度的目的蛋白,为后续研究提供材料。详细探讨了金属离子螯合层析缓冲液中咪唑浓度、离子交换层析缓冲液的pH值对AgrC_(TM6-7C)蛋白纯化效果的影响,并研究了β-巯基乙醇对AgrC_(TM6-7C)蛋白聚集及其激酶活性的影响。经过优化后,在不影响AgrC_(TM6-7C)蛋白激酶活性的前提下,AgrC_(TM6-7C)蛋白在浓度和纯度上均有明显提高,纯度可达98%以上,产量可达15 mg/L。     

红霉素生物合成的分子生物学

近年来,国外对大环内酯类抗生素生物合成和基因工程的研究非常迅速,不仅认识了许多抗生素生物合成的过程,而且利用基因工程技术改造抗生素生物合成基因,合成了100多种非天然的“天然”抗生素。抗生素生物合成的分子生物学是抗生素基因工程的基础。本全面介绍了五厌内酯类抗生素的代表－红霉素生物合成分子生物学的历史、现状及发展趋势。     

Knocking out of tailoring genes eryK and eryG in an industrial erythromycin-producing strain of Saccharopolyspora erythraea leading to overproduction of erythromycin B, C and D at different conversion ratios

Aims: To overproduce erythromycin C, B or D and evaluate the effect of disruption of tailoring genes eryK and eryG in an industrial erythromycin producer. Methods and Results: The tailoring genes eryG and eryK were inactivated individually or simultaneously by targeted gene disruption in an industrial strain Saccharopolyspora erythraea HL3168 E3, resulting in the overproduction of erythromycin C (2·48 g l^?1), B (1·70 g l^?1) or D (2·15 g l^?1) in the mutant strain QL‐G, QL‐K or QL‐KG, respectively. Analysis of the erythromycin congeners throughout the fermentation indicated that, at the end of fermentation, comparatively large amount of erythromycin D (0·67 g l^?1) was accumulated in QL‐G, whereas only small amount of erythromycin D (0·10 g l^?1) was produced in QL‐K. Conclusions: Inactivation of tailoring genes eryG and eryK in the high producer did not affect the biosynthesis of erythromycin. However, erythromycin D could be more efficiently methylated by EryG than be hydroxylated by EryK. Significance and Impact of the Study: Development of the mutant strains provides a method for the economical large‐scale production of potent lead compounds. The information about the accumulation and conversion of erythromycins in the industrial strains may contribute to further improving erythromycin production.     

Synthesis of A 2′- O -(Carbomoylmethyl)Ribonucleoside H-Phosphonate Building Block and A Model Dinucleotide

In order to obtain higher quality 2 ′-O-carbamoylmethyl oligoribonucleotides we are conducting studies of this modification. Here we present synthesis of 2 ′-O-carbamoylmethyl containing H-phosphonate building blocks as well as synthesis of model dinucleotides needed for these studies.     

A conjugative macrolide resistance gene, mef(A), in environmental Clostridium perfringens carrying multiple macrolide and/or tetracycline resistance genes

Aims:  To determine if environmental Clostridium perfringens carry antibiotic resistance genes and if the genes are mobile.
Methods and Results:  Clostridium perfringens from water, soil and sewage (2003–2006) were screened for the tetracycline and macrolide resistance genes previously described in animal and human C. perfringens [ erm (B), erm (Q), tetA (P), tetB (P) and tet (M) genes] and the macrolide resistance mef (A) gene. Of the 160 isolates, 108 (67·5%) carried ≥1 of the six antibiotic resistance gene(s). The tetA (P), tetB (P) and tet (M) genes were in 53%, 22% and 8%, and the erm (B), erm (Q) and mef (A) genes in 26%, 1% and 18% of the isolates, respectively. The mef (A) gene and flanking regions were sequenced. The tet (M), erm (B), erm (Q) and mef (A) genes transfer independently from C. perfringens donors to the Enterococcus faecalis recipient.
Conclusions:  Six resistance genes were found in the environmental C. perfringens with the most common being the tetA (P) gene and the erm (Q) gene the least common.
Significance and Impact of the Study:  This is the first time conjugal transfer of macrolide resistance genes and/or the tet (M) gene from C. perfringens has been demonstrated. The data presented supports the hypothesis that antibiotic-resistant environmental C. perfringens are capable of acting as reservoirs for these antibiotic resistance genes.     

Macrolide resistance genotypes and phenotypes among erythromycin-resistant clinical isolates of Staphylococcus aureus and coagulase-negative staphylococci, Italy

One hundred macrolide-resistant staphylococcal isolates from clinically relevant infections in Italy during a 19-month period were studied. Four distinct resistance phenotypes were observed using the triple-disk induction test (erythromycin, clindamycin, telithromycin): the cMLS_B phenotype (24 isolates); the iMLS_B phenotype (41 isolates); the MS phenotype (three isolates); and the iMTS phenotype (erythromycin-induced telithromycin resistance) (32 isolates). ermC and ermA genes predominated within erythromycin-resistant Staphylococcus aureus isolates with iMLS_B phenotype and cMLS_B phenotype, respectively. Among erythromycin-resistant CoNS isolates, half of the strains showed the iMTS or MS/ msrA association, and ermC gene predominated among isolates with MLS_B phenotype. By pulsed-field gel electrophoresis, high genetic heterogeneity was observed among the isolates studied. Both independent acquisition of macrolide resistance genes and spread of specific resistant clones were observed. Association between certain clonal types and specific types of infection could be detected. To our knowledge, this is the first report on characterization of erythromycin-resistant staphylococci in Italy.     

大环内酯类9 位羰基结构修饰衍生物的研究进展

大环内酯类抗生素自上市以来一直用于治疗呼吸系统等疾病,但随着该类药物的广泛应用,耐药菌日益增多。为此,研究者们一直致力于半合成大环内酯类抗生素的研究,希望开发出对耐药菌有效、抗菌谱广的半合成衍生物。综述近年来对大环内酯9 位羰基结构修饰的研究进展,并着重介绍一些活性较好的化合物。     

Versatility of Enzymes Catalyzing Late Steps in Polyene 67-121C Biosynthesis

Ribonuclease-A (RNase-A) has been a model for studying protein folding and unfolding. However, we show here that its unfolding at neutral pH is complicated by aggregation. Circular dichroism thermal scans showed that reversibility of RNase-A after heating is only about 63%. In accordance with this observation, native-polyacrylamide gel electrophoresis of the sample heated at 75°C showed formation of soluble oligomers. Ammonium sulfate at 0.4 M caused about a 3°C higher melting temperature and nearly complete reversibility, while glycine and NaCl at 0.4 M significantly increased reversibility and decreased aggregation without affecting melting temperature. These results demonstrate that aggregation makes thermal unfolding of RNase-A at least partially irreversible and salts and glycine increase reversibility and decrease aggregation.