不同来源的谷氨酸棒杆菌氨基脱氧分支酸合成酶的活性分析

分别以高产L-丝氨酸的谷氨酸棒杆菌(Corynebacterium glutamicum)SYPS-062与模式菌株谷氨酸棒杆菌(Corynebacterium glutamicum) ATCC 13032的基因组DNA为模板,运用PCR技术扩增出氨基脱氧分支酸合成酶(ADC synthase)的编码基因pabAB。实验结果表明：来源于SYPS-062和ATCC 13032的pabAB片段全长均为1863bp,编码620个氨基酸。两片段存在16个碱基的差异,引起了7个氨基酸的突变。将pabAB连接表达载体pET-28a(+),构建表达质粒pET-28a-pabAB,并转化E.coli BL21(DE3),在IPTG诱导下,E.coli BL21(DE3)(pET-28a-pabAB)高效表达分子量约为67kDa的可溶性蛋白。表达产物带有His-tag标记,选用Ni柱对表达产物进行纯化,纯化后酶活测定结果表明,来源于SYPS-062氨基脱氧分支酸合成酶的比酶活低于ATCC 13032达46.6%。     

产丁二酸谷氨酸棒状杆菌基因缺失代谢工程菌株的构建

【目的】提高谷氨酸棒状杆菌(Corynebacterium glutamicum)ATCC13032厌氧条件下的丁二酸产量,并降低发酵产物中副产物的含量。【方法】以谷氨酸棒状杆菌(Corynebacterium glutamicum)ATCC13032为出发菌,首先敲除乳酸形成的关键酶乳酸脱氢酶基因(ldh),构建ldh缺失株谷氨酸棒状杆菌ATCC13032Δldh;然后以缺失株谷氨酸棒状杆菌ATCC13032Δldh为出发菌,敲除该菌的丙酮酸脱氢酶系的E1p酶基因(aceE),构建一株双缺失突变菌株谷氨酸棒状杆菌ATCC13032ΔldhΔaceE。【结果】与供试菌比较,谷氨酸棒状杆菌ATCC13032Δldh的丁二酸产量和转化率分别提高了94.9%和32%,并且主要的副产物乳酸产量由出发菌产量的63.5 g/L降低到很微量的程度。丙酮酸脱氢酶的失活并不能完全消除副产物乙酸的形成,但乙酸的产量较ATCC13032Δldh降低了37.9%,丁二酸的产量略有提高。【结论】该重组菌具有较强的丁二酸生产工业化潜力,并且该研究方法为微生物代谢育种提供参考。     

谷氨酸棒杆菌高丝氨酸脱氢酶编码基因hom的敲除

本研究以谷氨酸棒杆菌（Corynebacterium glutamicum）标准菌株ATCC 13032染色体为模板,设计引物PCR扩增高丝氨酸脱氢酶编码基因（hom）,在hom基因内部插入一段来源于质粒pET28a的卡那霉素抗性基因（Km）,得到基因元件hom::Km;通过电击转化法将hom::Km转入出发菌株替换原菌株的hom,在含卡那霉素的平板上挑取阳性转化子,通过PCR验证得到高丝氨酸脱氢酶缺陷的重组菌。发酵结果表明重组菌C.g- hom::Km -8发酵60小时赖氨酸产量达到4.7 g／L,是出发菌株谷氨酸棒杆菌ATCC 13032（0.7 g／L）的6.7倍。     

谷氨酸棒杆菌1dh基因的敲除

谷氨酸棒杆菌中ldh基因编码乳酸脱氢酶,可催化丙酮酸转化生成乳酸.利用重叠延伸PCR的方法,获得中间缺失部分序列的dldh基因片段,将其与载体pk 18mobsacB连接,转化大肠杆菌感受态,筛选出阳性转化子后,转化谷氨酸棒杆菌ATCC 13032感受态细胞.分别在卡那霉素抗性平板及10％蔗糖平板上进行两次筛选,利用PCR方法鉴定,成功获得ldh基因缺失的谷氨酸棒杆菌突变株ATCC 13032-(4)ldh.应用荧光定量PCR检测,ATCC 13032-(z)ldh中的ldh基因在转录水平与野生型菌株ATCC 13032相比,相对表达量为O.ldh基因的敲除对菌株的生长造成了一定的影响.     

直接利用糖质原料产L-丝氨酸谷氨酸棒杆菌glyA基因序列分析

以能够利用糖质原料产L-丝氨酸的谷氨酸棒杆菌（Corynebacterium glutamicum）SYPS-062 glyA基因为研究对象,比较cglutamicum SYPS-062与C．glutamicum模式菌株ATCC13032的glyA基因的异同。分别以SYPS-062及ATCC13032基因组为模板,利用PCR技术获得丝氨酸羟甲基转移酶编码基因glyA。核苷酸序列分析结果表明,来源于SYPS-062和ATCC13032的glyA基因片段全长均为1305bp,编码434个氨基酸,分子量为46．5kD,基因的同源性为99．54％,存在6个核苷酸的差异,引起一个氨基酸残基的突变。将获得的基因分别在大肠杆菌（Escherichia coli）BL21（DE3）中诱导表达。酶活测定结果显示,2种不同菌株来源的重组SHMT的比活力稍有差异,说明SYPS-062 glyA基因的差异对其表达产物SHMT蛋白构型及功能影响不大。提示对于C.glutamicum SYPS-062能够利用糖质原料产L-丝氨酸的机制解析应进一步从glyA基因的转录水平、翻译水平及胞内SHMT辅酶的供给情况等方面进行深入研究探讨。     

Corynebacterium glutamicum组成型启动子及核糖体结合位点的研究

谷氨酸棒杆菌(Corynebacterium glutamicum,C.glutamicum)是一种广泛用于工业生产的生物安全性菌株。本研究旨在丰富组成型启动子表达元件库,从C.glutamicum ATCC 13032中克隆了5种基因启动子,分别为锰超氧化物歧化酶基因启动子(psod)、延伸因子基因启动子(ptuf)、三磷酸甘油醛脱氢酶基因启动子(pgap)、苹果酸合成酶基因启动子(pms)和二氢吡啶二羧酸合酶基因启动子(pa16)。通过构建工具质粒,以绿色荧光蛋白基因(gfp)为报告基因,研究了这5种基因启动子的启动活性。结果表明5种基因启动子的启动活性由高到低依次为pa16、psod、pms、ptuf和pgap,荧光强度分别为465 RFU/OD_(600)、420 RFU/OD_(600)、305 RFU/OD_(600)、200 RFU/OD_(600)和175 RFU/OD_(600)。此外,基于已构建的载体pa16gfp-p XMJ19,以卡那霉素为报告基因,构建了包含两个核糖体结合位点序列(CGAAAGGATTTTTTACCC及CAGGAGGACATACA)的psod和ptuf的验证质粒,为构建不同C.glutamicum工程菌提供了可选择性调控元件。     

适用于异源DNA高效整合转化的谷氨酸棒杆菌电转化法

根据最近文献报道的方法,以基因组测序用典型菌株Corynebacterium glutamicum ATCC13032为宿主,采用E.coli-C.glutamicum穿梭质粒pC2以及可以整合到谷氨酸棒杆菌基因组DNA上的质粒pAK进行电转化条件优化的实验,建立了一种简便的,适用于异源DNA高效整合转化的谷氨酸棒杆菌电转化方法。建立的新方法与原方法相比,细胞预培养时间缩短4h,细胞培养时间缩短1d左右。细胞培养基被简化,不再需要添加进口生化试剂。转化率可达5.5×106cfu／μgDNA,是一种适用于异源DNA高效整合转化的谷氨酸棒杆菌电转化法。     

谷氨酸棒杆菌SYPS-062 L-丝氨酸脱水酶活性分析及其基因敲除对L-丝氨酸积累的影响

以谷氨酸棒杆菌(Corynebacterium glutamicum) SYPS-062基因组DNA为模板,扩增得到L-丝氨酸脱水酶(L-SerDH)的编码基因sdaA。将其克隆到表达载体pET-28a(+),并在E.coli BL21(DE3)中诱导表达,对纯化的L-SerDH进行了酶活测定,并与来自C.glutamicum ATCC13032的重组L-SerDH进行了比较,结果显示,两种不同菌株来源的重组L-SerDH降解L-丝氨酸的酶比活力差异并不显著。在此基础上敲除菌株SYPS-062 的sdaA基因,探讨该基因对C.glutamicum SYPS-062生长及产酸的影响。通过构建自杀型重组质粒pK18mobsacB-△sdaA,电击转入C.glutamicum SYPS-062中,以同源重组的方式获得了sdaA基因缺失突变株,并用PCR方法对突变株C.glutamicum SYPS-062△sdaA进行了验证。与出发菌株相比,突变菌株生长缓慢,单位菌体L-丝氨酸的产量(Y_P/X)提高了15.13%。     

代谢木糖重组谷氨酸棒杆菌的构建

为了使谷氨酸棒杆菌较好地利用木糖生产有机酸,将来自Escherichia coli K-12的木糖异构酶基因xylA构建到表达载体pXMJ19中,导入Corynebacterium glutamicum ATCC13032Δldh中,成功表达了该酶基因。结果表明:重组菌株在以木糖为唯一C源进行发酵时,木糖的消耗速率为0.54 g/(L·h),木糖异构酶比酶活约为0.54 U/mL;在以木糖和葡萄糖的混合糖为C源进行发酵时,菌株优先利用葡萄糖,在葡萄糖完全消耗后,菌株开始有效利用木糖;以木糖为唯一C源进行两阶段发酵时,琥珀酸的收率可达(0.62±0.003)g/g。     

磷酸烯醇式丙酮酸羧化酶基因的敲除对于谷氨酸棒杆菌V1生理代谢的影响

【目的】L-缬氨酸生物合成的前体物质是丙酮酸。为了增加磷酸烯醇式丙酮酸向丙酮酸的代谢流向,优化L-缬氨酸前体物质的供应,以一株积累L-缬氨酸的谷氨酸棒杆菌V1(Corynebacterium glutamicum V1)为对象,构建磷酸烯醇式丙酮酸羧化酶(PEPC)基因敲除的重组菌株C.glutamicum V1-Δpepc,并研究pepc敲除后菌株生理特性的改变。【方法】运用交叉PCR方法得到pepc基因内部缺失的同源片段Δpepc,并构建敲除质粒pK18mobsacB-Δpepc。利用同源重组技术获得pepc基因缺陷突变株C.glutamicum V1-Δpepc。采用摇瓶发酵对C.glutamicum V1-Δpepc进行发酵特性的研究。对谷氨酸棒杆菌模式菌株C.glutamicum ATCC 13032、出发菌株C.glutamicum V1和敲除菌株C.glu-tamicum V1-Δpepc的丙酮酸激酶(Pyruvate kinase,PK)、丙酮酸脱氢酶(Pyruvate dehydro-genase,PDH)、丙酮酸羧化酶(Pyruvate carboxylase,PC)分别进行测定和分析。【结果】PCR验证以及PEPC酶活测定都表明筛选到pepc缺陷的突变菌株C.glutamicum V1-Δpepc,摇瓶发酵结果表明,突变菌株C.glutamicum V1-Δpepc不再积累L-缬氨酸而是积累L-精氨酸达到7.48 g/L。酶活测定结果表明出发菌株的PDH和PC酶活均低于模式菌株C.glu-tamicum ATCC13032和重组菌株C.glutamicum V1-Δpepc,出发菌株的PK与PEPC酶活与模式菌株没有较大的差异。【结论】研究表明,通过切断PEPC参与的三羧酸循环的回补途径,增加磷酸烯醇式丙酮酸向丙酮酸的流向使丙酮酸向TCA循环的流量增加,精氨酸的累积量提高。同时,以丙酮酸为前体的L-缬氨酸和丙氨酸的积累量降低。     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

鸡传染性法氏囊病病毒研究进展

传染性法氏囊病(Infection bursal disease, IBD)是由鸡传染性法氏囊病毒(Infectious bursal disease virus, IBDV)引起的鸡和火鸡的一种高度接触性传染病,给世界各国的禽养殖业带来了巨大损失.自IBDV发现至今新的变异株不断出现,分子结构的改变导致病毒致病力的改变及宿主对疫苗应答的改变,使得传统的疫苗已不能控制其流行,因此各国学者对其基因组结构和功能进行了广泛深入的研究,并积极研制新型有效的疫苗以达到防治的目的.     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).