过量表达钝齿棒杆菌柠檬酸合酶编码基因prpC2对L-精氨酸合成的影响

钝齿棒杆菌(Corynebacterium crenatum)SYPA5-5是诱变选育的一株高产精氨酸菌株。柠檬酸合酶作为TCA途径的关键酶,对细胞胞内氨基酸代谢流调节有重要作用。在钝齿棒杆菌SYPA5-5中过量表达同源的柠檬酸合酶(citrate synthase)基因prp C2,研究其对精氨酸及副产物合成的影响。重组菌C.crenatum SYPA5-5/p DXW-10-prp C2胞内柠檬酸合酶比酶活提高了5.37倍,使L-精氨酸产量在5L发酵罐中达到44.7g/L,与对照相比提高了23.1%。同时,有机酸测定分析TCA循环的精氨酸前体柠檬酸及异柠檬酸的量有所提高,且赖氨酸合成前体草酰乙酸量减少,氨基酸测定分析L-精氨酸发酵中最主要的副产物L-赖氨酸浓度由原来的5.96g/L降到1.21g/L,降低了80%。     

钝齿棒杆菌精氨酸琥珀酸酶编码基因argH的克隆表达及其重组菌发酵产精氨酸研究

钝齿棒杆菌（Corynebacterium crenatum）SYPA是本实验室筛选获得的一株高产精氨酸生产菌株。精氨酸琥珀酸酶（AL）是精氨酸合成过程中的最后一个酶,催化底物精氨酸琥珀酸生成产物精氨酸。为进一步提高精氨酸产量,本文以钝齿棒杆菌基因组为模板,扩增得到其编码基因argH,全长为1434 bp,编码476个氨基酸,理论蛋白分子量大小为50.8 kDa,其与C. glutamicum ATCC 13032比对其同源性为99.4%,相差10 bp,3个氨基酸。将其在E.coli BL21(DE3)及C. crenatum SYPA中成功表达。利用载体pET-28a上的6×His?Tag选用Ni柱亲和层析纯化AL,纯化后获得的重组蛋白的比酶活达156.9mU/mg蛋白,总回收率为72.3%,对该酶的部分酶学性质进行了初步研究,并发现产物精氨酸对其具有反馈抑制作用。成功构建钝齿棒杆菌重组穿梭表达质粒pJC1-tac-argH并将其通过电击转化法转入C.crenatum SYPA中,加强其代谢途径中AL蛋白表达量,并对其发酵产精氨酸做了初步分析。结果表明与出发菌株相比,转化子在精氨酸琥珀酸酶酶活增强了66.8%的基础上精氨酸产量达40.9 g/L,比出发菌株产量的35.8 g/L提高了约14.2%。     

以葡萄糖为底物一步法合成γ-氨基丁酸整合型重组钝齿棒杆菌的构建

[目的]为了构建一株直接利用廉价的葡萄糖合成γ-氨基丁酸的重组钝齿棒杆菌,将来自于植物乳杆菌γ-氨基丁酸合成途径的关键酶谷氨酸脱羧酶基因(lpgad)在产谷氨酸菌株钝齿棒杆菌中进行整合表达,实现葡萄糖到GABA的一步法生产.[方法]运用PCR技术扩增得到带有tac启动子的谷氨酸脱羧酶基因tacgad.通过重叠PCR的方法获得钝齿棒杆菌精氨酸合成途径关键酶N-乙酰谷氨酸激酶(NAGK)基因内部缺失型基因△argB.利用自杀载体pK18mobsacB构建同源整合载体pK18-△argB::tacgad,以△argB的上下游序列为同源臂,通过两次同源重组将tacgad基因整合到钝齿棒杆菌基因组,同时将NAGK基因argB灭活,利用蔗糖致死基因sacB反向筛选标记筛选得到谷氨酸脱羧酶的重组钝齿棒杆菌C.crenatum △argB::tacgad.重组钝齿棒杆菌以葡萄糖为底物进行发酵,测定GABA含量.[结果]重组菌C.crenatum △argB::tacgad成功表达谷氨酸脱羧酶,同时阻断了精氨酸合成途径对谷氨酸到GABA代谢途径的竞争,粗酶液基本检测不到NAGK活性,发酵液无精氨酸合成.通过96 h发酵,重组菌可积累约8.28 g/L的GABA.[结论]本研究通过将谷氨酸脱羧酶基因定向整合到钝齿棒杆菌精氨酸合成途径的关键酶基因argB内部,成功表达谷氨酸脱羧酶的同时阻断竞争途径精氨酸的合成.本研究为实现直接利用葡萄糖合成GABA的一步法生产奠定了基础.     

钝齿棒杆菌N-乙酰鸟氨酸转氨酶的克隆表达分析及其重组菌的精氨酸发酵

N-乙酰鸟氨酸转氨酶 (EC 2.6.1.11,ACOAT) 是钝齿棒杆菌Corynebacterium crenatum精氨酸合成途径中的第4个酶,催化底物N-乙酰谷氨酸半醛生成产物N-乙酰鸟氨酸。为研究N-乙酰鸟氨酸转氨酶在钝齿棒杆菌中精氨酸合成中的作用,考察其酶学性质,对培养基成分和发酵过程工艺条件的优化提高精氨酸产量提供依据。从精氨酸高产菌株钝齿棒杆菌SYPA 5-5染色体扩增获得ACOAT编码基因argD,全长1 176 bp,编码390个氨基酸,在Escherichia coli BL21(D     

离子转运蛋白调控钝齿棒杆菌离子和pH稳态促进L-精氨酸合成

L-精氨酸是一种半必需氨基酸,广泛应用于食品、制药、饲料等行业。【目的】当前对L-精氨酸生产菌株的研究,极少涉及离子转运领域。在本研究中,发现在发酵时适量添加外源K~+有利于促进钝齿棒杆菌(Corynebacterium crenatum) SYPA5-5合成L-精氨酸。【方法】在C. crenatum SYPA5-5发酵培养基外源添加0.5 g/L和2.5 g/L的K_3PO_4,取对数期发酵样品进行转录组数据分析,挖掘出K~+转运相关的阳离子转运ATP酶CTAP1以及单价阳离子/H~+逆转运蛋白Mrp1A,研究其在C. crenatum SYPA5-5快速合成L-精氨酸阶段,对菌株生长及L-精氨酸合成的影响。【结果】对基因ctap1和mrp1分别进行敲除和过表达,深入研究突变株对L-精氨酸合成的影响。研究发现同时过表达离子转运蛋白CTAP1和Mrp1A更有利于胞内离子、pH稳态和渗透压调节,最终提高L-精氨酸的产量。在补料分批发酵中分别过表达Mrp1A、CTAP1以及同时过表达Mrp1A和CTAP1的菌株L-精氨酸产量分别达到61.4 g/L、63.9 g/L和65.3 g/L,产率分别为0.383 g/g、0.392 g/g和0.395 g/g,比C. crenatum SYPA5-5分别提高了34.9%、38.0%和39.1%。【结论】CTAP1是特异性的K~+转运ATP酶,可以将培养基中的K~+运输到胞内。同时Mrp1A可将胞内K~+和Na~+等单价阳离子运输到胞外,将胞外H~+运输至胞内,中和胞内L-精氨酸所导致的碱性环境,从而维持胞内pH稳定。CTAP1和Mrp1A的研究为解析离子转运机制和L-精氨酸合成之间的联系奠定了基础。     

钝齿棒杆菌的代谢改造：L-鸟氨酸与L-瓜氨酸合成菌株的构建

【目的】对一株产L-精氨酸的钝齿棒杆菌(Corynebacterium crenatum)SYPA5-5进行代谢工程改造,构建L-鸟氨酸和L-瓜氨酸合成菌株,并考察其发酵生产相应氨基酸的性能。【方法】分别敲除菌株SYPA5-5鸟氨酸氨甲酰转移酶(Ornithine carbamoyltransferase,OTC)的编码基因argF和精胺琥珀酸合成酶(Argininosuccinate synthase,ASS)的编码基因argG,构建能够合成L-鸟氨酸及L-瓜氨酸的重组菌株SYPA5-5△argF和SYPA5-5△argG;考察不同营养条件对上述重组菌株生长和相应氨基酸积累的影响。【结果】添加0.3 g/L L-精氨酸可满足SYPA5-5△argF的生长及L-鸟氨酸积累所需,L-鸟氨酸产量可达21.5 g/L;添加L-精氨酸有利于SYPA5-5△argG的生长,但不利于L-瓜氨酸的积累;不添加L-精氨酸时,L-瓜氨酸产量可达15.2 g/L,同时积累6.8 g/L的L-谷氨酸。【结论】分别敲除L-精氨酸生产菌株SYPA5-5的argF及argG基因,可实现L-精氨酸合成途径的中间代谢物L-瓜氨酸和L-鸟氨酸的积累,拓展了该菌株的工业应用范围。     

结合缺陷型菌株显色法采用离子束诱变筛选高产L-精氨酸突变株

为快速高效筛选L-精氨酸高产突变株,建立一种缺陷菌株平板显色法并采用低能N+离子束对L-精氨酸生产用菌株钝齿棒杆菌SYPA5-5进行诱变处理,通过上述平板显色法筛选获得高产突变株.对突变株进行摇瓶发酵实验,最终选育出一株L-精氨酸产量较高且产酸性能比较稳定的突变菌株钝齿棒杆菌SYPA5-5-36.该菌株摇瓶发酵L-精氨酸产量可达35.85 g/L,比出发菌株提高了19.5％.因此,缺陷型菌株平板显色法可以用于快速、高效筛选高产L-精氨酸突变株.     

proC及putP基因的敲除对钝齿棒杆菌产L-精氨酸生理代谢的影响

为了选育精氨酸高产菌株,基于谷氨酸棒杆菌的基因组尺度代谢网络模型的指导,以钝齿棒杆菌(Corynebacterium crenatum)MT-M4为出发菌株,通过基因敲除技术构建了pro C和put P敲除菌株。摇瓶发酵结果表明,pro C敲除菌株精氨酸产量达到9.94g/L,较出发菌株提高了15.90%,葡萄糖转化率提高了26.02%。由于其生长受到明显抑制,因此在发酵液中外源添加24mmol/L的脯氨酸,结果发现其精氨酸产量达到12.22g/L,且菌株恢复生长。put P敲除菌株精氨酸产量达到12.23g/L,较出发菌株提高了42.70%,葡萄糖转化率提高了49.31%。以上结果显示,put P的敲除比pro C的敲除更有利于精氨酸的合成,put P的敲除对菌株的生理代谢基本无影响且无需外添加脯氨酸。     

重组钝齿棒杆菌全细胞转化生产L-瓜氨酸条件优化

实现了精氨酸脱亚胺酶(ADI)首次在钝齿棒杆菌Corynebacterium crenatum SYPA 5-5中的高效表达。通过Ni-NTA亲和层析纯化得到纯化ADI,经SDS-PAGE测定其分子量约为46.8 k Da,酶学性质研究发现ADI的最适催化温度为37℃,最适pH为6.5,ADI在最佳催化条件下作用于L-精氨酸的米氏常数为12.18 mmol/L,最大反应速率为0.36μmol/(min·mL)。优化了重组菌全细胞转化产L-瓜氨酸的工艺条件,在最优条件下可一次转化300 g/L L-精氨酸,转化速率达8 g/(L·h)。进行重组菌5 L罐发酵并进行罐上全细胞转化300 g/L L-精氨酸,一批菌体可进行多次转化,累计产量达1 900 g以上。     

钝齿棒杆菌中异源表达N-乙酰鸟氨酸脱乙酰基酶合成L-鸟氨酸的研究

目的:对一株产鸟氨酸的钝齿棒杆菌Corynebacterium crenatum SYPA5-5/△proB/△argF(SYPO-1)进行代谢工程改造,筛选不同细菌来源的N-乙酰鸟氨酸脱乙酰基酶在大肠杆菌中克隆与表达,纯化后对其进行酶学性质的比较;将黏质沙雷氏菌Serratia marcescens Y213来源的Smarg E基因编码的N-乙酰鸟氨酸脱乙酰基酶在L-鸟氨酸生产菌株C.crenatum SYPO-1中过量表达,进一步提高L-鸟氨酸的产量。方法:通过利用pDXW10穿梭质粒对不同来源的N-乙酰鸟氨酸脱乙酰化酶进行克隆表达和酶学性质比较,选择性质最优来源的N-乙酰鸟氨酸脱乙酰基酶编码基因Smarg E在产L-鸟氨酸重组钝齿棒杆菌中表达,考察重组菌株发酵过程中参数的变化。结果:来源于S.marcescens Y213的N-乙酰鸟氨酸脱乙酰基酶比酶活最高为798.98U/mg,最适pH为7,最适温度为37℃,0.1mmol/L的Mg~(2+)、Li~+、Mn~(2+)促进酶的比酶活提高了50%;在钝齿棒杆菌中表达N-乙酰鸟氨酸脱乙酰基酶酶活达到128.4U/ml,显著提高了钝齿棒杆菌中胞内乙酰基循环水平;5L发酵罐发酵重组菌株96h,L-鸟氨酸的产量达到38.5g/L,比出发菌株,L-鸟氨酸的产量提高了33.2%,产率达0.401g/(L·h)。结论:筛选出最佳来源的N-乙酰鸟氨酸脱乙酰基酶,在鸟氨酸生产菌株C.crenatum(SYPO-1)中过量表达,可以促进鸟氨酸的前体物质N-乙酰鸟氨酸的快速消耗,实现鸟氨酸的积累。     

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.     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.     

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).     

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.