谷氨酸棒状杆菌厌氧产丁二酸的发酵条件

考察谷氨酸棒状杆菌ATCC13032Δldh厌氧产丁二酸的发酵条件。结果发现:补加NaHCO3的效果最好,并且考察了NaHCO3浓度对葡萄糖转化速率及丁二酸生成速率的影响。运用代谢流分析方法分析了乳酸脱氢酶基因敲除对谷氨酸棒状杆菌厌氧代谢的影响,发现乳酸脱氢酶基因敲除导致磷酸烯醇式丙酮酸生成丁二酸的流量提高了214.3%,流向乳酸的流量变为0;分批厌氧转化36 h生成41.2 g/L丁二酸,产率45.0%。     

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.     

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。     

敲除aceE基因对猪链球菌丙酮酸代谢的影响

【目的】探究缺失编码丙酮酸脱氢酶蛋白的aceE基因对猪链球菌生长特性、三羧酸循环和丙酮酸代谢的影响。【方法】通过测量菌液的OD600值,绘制野生型菌株与aceE基因缺失突变株的生长曲线;利用试剂盒测定三羧酸循环和丙酮酸代谢旁路中乙酰CoA、琥珀酸CoA、延胡索酸、草酰乙酸、丙酮酸、乳酸和ATP的含量,通过荧光定量qRT-PCR确定柠檬酸合酶基因、苹果酸脱氢酶基因、琥珀酸脱氢酶基因、异柠檬酸脱氢酶基因、丙酮酸脱羧酶基因、乳酸脱氢酶基因、乙醇脱氢酶基因和乙醛脱氢酶基因的表达水平。【结果】与野生株相比,菌株ΔaceE在平台期OD600值下降;添加1g/L乙酸盐能够显著提升菌株ΔaceE平台期OD600值。菌株ΔaceE的丙酮酸含量上升,ATP含量下降;三羧酸循环代谢中乙酰CoA、琥珀酸CoA、延胡索酸含量降低;柠檬酸合酶基因和苹果酸脱氢酶基因表达水平上升,琥珀酸脱氢酶基因和异柠檬酸脱氢酶基因表达水平下调;在丙酮酸代谢旁路中丙酮酸脱羧酶基因、乳酸脱氢酶基因、乙醇脱氢酶基因和乙醛脱氢酶基因表达水平上升。【结论】结果显示,菌株ΔaceE三羧酸循环活性降低,虽然能够通过PDH旁路将部分丙酮酸分解为乙...     

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。     

嗜乙酰乙酸棒杆菌Corynebacterium acetoacidophilum-Δldh缺氧条件下代谢葡萄糖途径的变化

缺氧条件下嗜乙酰乙酸棒杆菌Corynebacterium acetoacidophilum ATCC13870生长停滞,却能够代谢葡萄糖产生以乳酸和琥珀酸为主的有机酸。采用以sacB基因为反向筛选标记的同源重组染色体基因敲除系统,敲除嗜乙酰乙酸棒杆菌的乳酸脱氢酶基因,得到的Δldh菌株CCTCC NO.M20122041在缺氧条件下不产乳酸,葡萄糖消耗速率降低了29.3%,产琥珀酸和乙酸浓度分别提高45.6%和182%;NADH/NAD+值小于1(约0.7);磷酸烯醇式丙酮酸羧化酶和乙酸激酶的比酶活分别提高84%和12倍。说明嗜乙酰乙酸棒杆菌中乳酸合成途径的阻断驱使了琥珀酸和乙酸代谢途径加强,推测加强NADH供给和阻断乙酸产生支路可能是提高C.acetoacidophilum菌株产琥珀酸产量的有效途径。     

代谢组学分析提高谷氨酸棒杆菌L-瓜氨酸产量

利用基于气相色谱-质谱联用(GC-MS)的代谢组学分析方法,研究Corynebacterium glutamicum ATCC 13032和C.glutamicum ATCC 13032Δarg GΔargR-p XMJ19-arg B(ec)(CIT4)重组菌胞内代谢物差异性,挖掘L-瓜氨酸合成关键代谢物及代谢途径作用机制,并通过添加关键代谢物进行理性强化,提高L-瓜氨酸的产量。利用GC-MS共检测124种化合物,结合主成分分析(PCA)和偏最小二乘判别分析(PLS)统计发现10种为区别这两种菌的生物标志物,理性强化添加α-酮戊二酸、丙酮酸、谷氨酸、鸟氨酸、氨甲酰磷酸和谷氨酰胺,CIT4中L-瓜氨酸产量从18.50 g/L增加到20.86 g/L。     

谷氨酸棒杆菌的乙醛酸循环与谷氨酸合成

为阐明谷氨酸棒杆菌的乙醛酸循环与菌体的生长以及谷氨酸合成之间的关系 ,以谷氨酸棒杆菌基因组测序用典型菌株Corynebacteriumglutamicum ATCC 130 32为出发菌株 ,构建了乙醛酸循环途径缺失的谷氨酸棒杆菌突变株Corynebacteriumglutamicum WTΔA。该菌株没有异柠檬酸裂解酶活性 ,不能在以乙酸盐为唯一碳源的基本培养基上生长。与出发菌株ATCC13032相比 ,WTΔA在以葡萄糖为唯一碳源的培养基上生长时不受影响 ,说明谷氨酸棒杆菌并不需要乙醛酸循环途径提供菌体生长所需的能量和生物合成反应所需的中间产物。但是 ,与出发菌株ATCC13032相比 ,WTΔA的谷氨酸合成能力大幅下降。     

丙酮酸和乙酰-CoA协同促进L-亮氨酸的合成

【目的】通过理性改造柠檬酸合酶(citrate synthase,CS)、丙酮酸脱氢酶系E1p (pyruvate dehydrogenase complex,PDHC,编码基因aceE)和ATP-柠檬酸裂解酶(ATP-Citrate lyase,ACL),有效供应胞内丙酮酸和乙酰-CoA,以提高L-亮氨酸产量。【方法】以谷氨酸棒杆菌(Corynebacterium glutamicum)为底盘细胞,分析不同CS和PDHC酶活水平对L-亮氨酸合成的影响。随后,考查协同改造CS和PDHC或引入绿硫菌(Chlorobium tepidum)中ACL对L-亮氨酸合成的影响。【结果】低强度的CS酶活(即重组菌XL-3 P_(dapA-R2)gltA)有利于L-亮氨酸的合成,L-亮氨酸产量达到17.5±0.6 g/L。而改变PDHC酶活水平不利于L-亮氨酸的合成。此外,以启动子P_(dapA-R2)控制CS表达,而以启动子P_(gapA)控制PDHC表达时(即重组菌XL-4),可实现胞内丙酮酸和乙酰-CoA的有效供给,L-亮氨酸产量达到20.2±1.7 g/L,且显著降低副产物产量。若在重组菌XL-4中引入C.tepidum,ACL会显著抑制菌体生长而不利于L-亮氨酸合成,而引入到出发菌XL-3中因胞内丙酮酸和乙酰-CoA得到有效供给,目标重组菌XL-5L-亮氨酸产量达到18.5±1.2 g/L,比出发菌株XL-3增加了14.2%。【结论】重组菌XL-4中因协同控制CS和PDHC酶活,从而实现胞内丙酮酸和乙酰-CoA有效供给,促进L-亮氨酸的合成。该研究结果对后续利用代谢工程技术强化微生物合成L-亮氨酸等支链氨基酸具有重要的参考价值。     

光滑球拟酵母中AMP代谢对其生理功能的影响北大核心CSCD

【目的】研究光滑球拟酵母(Candida glabrata)中AMP代谢影响其碳流代谢和耐酸胁迫的生理机制。【方法】同源重组法敲除基因cgade12(ORF CAGL0K05027g)和cgade13(ORF CAGL0B02794g)构建双缺菌株cgade12Δade13Δ,与出发菌株(ATCC55)比较ATP水平、碳流代谢酶活性和中间代谢物含量变化分析和AMP代谢对其碳流代谢的影响,对比菌株有机酸胁迫下生长情况及胞内环境变化,研究AMP代谢变化对光滑球拟酵母酸胁迫耐受性的影响。【结果】与出发菌株ATCC55相比,cgade12Δade13Δ的ATP水平下降了12.50%。与ATCC55相比,cgade12Δade13Δ中柠檬酸合成酶、苹果酸脱氢酶、异柠檬酸脱氢酶、α-酮戊二酸脱氢酶的活性分别上升了31.26%、19.45%、28.96%、18.36%,柠檬酸、α-酮戊二酸、苹果酸、琥珀酸含量分别提高了44.11%、73.60%、50.00%、65.68%。胞内丙酮酸浓度下降20.00%,丙酮酸产量下降73.11%。与ATCC55相比,cgade12Δade13Δ在0.4%丙酮酸、0.6%苹果酸和0.2%乙酸胁迫下的菌体浓度分别提高了8.71%、11.21%和12.71%。在0.2%乙酸胁迫下,菌株cgade12Δade13Δ的H^+-ATPase活性、细胞膜完整度、细胞膜电势分别比出发菌株ATCC55上升了7.04%、8.71%、25.14%,ROS水平下降了19.51%。【结论】基因cgade12、cgade13的缺失导致菌株的ATP水平下降,TCA循环活性和有机酸耐受性上升。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

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     

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.     

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.     

Synthesis and antimicrobial studies of hydrazone derivatives of 4-[3-(2,4-difluorophenyl)-4-formyl-1H-pyrazol-1-yl]benzoic acid and 4-[3-(3,4-difluorophenyl)-4-formyl-1H-pyrazol-1-yl]benzoic acid

Microbial resistance to antibiotics is an unresolved global concern, which needs urgent and coordinated action. One of the guidelines of the Centers for Disease Control and Preventions (CDC) to combat antibiotic resistance is the development of new antibiotics to treat drug-resistant bacteria. In our effort to find new antibiotics, we report the synthesis and antimicrobial studies of 30 new pyrazole derivatives. These novel molecules have been synthesized by using readily available starting materials and benign reaction conditions. Some of these molecules have shown activity with MIC values as low as 0.78?µg/mL against four bacterial strains; Staphylococcus aureus, methicillin-resistant S. aureus, Bacillus subtilis, and Acinetobacter baumannii. Furthermore, active molecules are non-toxic to mammalian cell line.

     

Novel compounds with potent CDK9 inhibitory activity for the treatment of myeloma

Cyclin-dependent kinases (CDKs) and Polo-like kinases (PLKs) play key role in the regulation of the cell cycle. The aim of our study was originally the further development of our recently discovered polo-like kinase 1 (PLK1) inhibitors. A series of new 2,4-disubstituted pyrimidine derivatives were synthesized around the original hit, but their PLK1 inhibitory activity was very poor. However the novel compounds showed nanomolar CDK9 inhibitory activity and very good antiproliferative effect on multiple myeloma cell lines (RPMI-8226).