代谢工程改造L-半胱氨酸供给模块促地衣芽胞杆菌 高效合成杆菌肽

杆菌肽是一种主要由芽胞杆菌产生的广谱性抗生素,目前作为兽药广泛应用于畜禽养殖领域。前体氨基酸供应不足可能是限制微生物发酵高产杆菌肽的重要因素。文中以杆菌肽工业生产菌株——地衣芽胞杆菌Bacillus licheniformis DW2为出发菌株,研究l-半胱氨酸供给模块强化对杆菌肽合成的影响。首先,构建了l-半胱氨酸合成酶基因cysK强化表达菌株,杆菌肽效价相比于对照菌株提高了9.47%。接着,为提高l-半胱氨酸合成前体供给,对l-丝氨酸乙酰转移酶基因cysE和硫代硫酸盐/硫酸盐胞内转运蛋白基因cysP进行强化,杆菌肽产量分别提高了7.23%和8.52%。随后,结果表明转运蛋白TcyP负责从胞外向胞内转运胱氨酸,强化表达TcyP后胞内l-半胱氨酸浓度和杆菌肽效价分别提高了29.19%和7.79%。通过组合代谢工程育种,在整合表达了基因cysK基础上,利用强启动子PbacA分别替换基因cysP、cysE和tcyP原始启动子,得到工程菌株CYS4 (DW2::cysK-PbacA(cysP)- PbacA(cysE)-PbacA(tcyP)),杆菌肽效价达到910.02 U/mL,相比于出发菌株DW2 (747.71 U/mL) 提高了21.10%。最后,通过3 L发酵罐小试实验,进一步证实了强化l-半胱氨酸有利于杆菌肽合成。研究表明,强化胞内l-半胱氨酸供给水平是提高地衣芽胞杆菌中杆菌肽产量的有效策略,为杆菌肽工业生产提供了一株具有良好应用前景的菌株。     

地衣芽胞杆菌DW2中敲除氨基酸转运蛋白基因yhdG提高杆菌肽产量

杆菌肽是微生物产生的由11种氨基酸残基组成的广谱性抗生素,前体物的供应可能是限制杆菌肽高产的重要因素。文中通过支链氨基酸(异亮氨酸、亮氨酸、缬氨酸)的添加实验考察了前体物质支链氨基酸对杆菌肽高产的影响,证实了异亮氨酸(Ile)和亮氨酸(Leu)的添加可以提高杆菌肽的效价,其中Ile的添加对杆菌肽效价提高的效果较为明显。随后,文中以地衣芽胞杆菌DW2为出发菌株,分别构建了支链氨基酸转运蛋白Yhd G的缺失和强化表达菌株。发酵结果表明,转运蛋白Yhd G缺失工程菌DW2△yhd G的杆菌肽效价达到917.35 U/m L,与原始菌DW2相比提高了11%,而强化Yhd G则会使杆菌肽效价下降25%。最后通过分析胞内胞外支链氨基酸含量,发现缺失转运蛋白Yhd G能够在发酵中后期显著提高胞内支链氨基酸含量,表明氨基酸转运蛋白Yhd G在地衣芽胞杆菌DW2中可能发挥着氨基酸输出的功能。综上,文中通过缺失转运蛋白Yhd G显著提高了地衣芽胞杆菌胞内支链氨基酸的供给水平,从而提高了杆菌肽效价,为杆菌肽高产菌株的构建提供了一种新的策略。     

DegU负调控地衣芽胞杆菌普切明酸的合成及分泌

普切明酸是地衣芽胞杆菌合成并分泌的一种铁离子螯合剂,是细胞维持铁稳态的重要介质。【目的】揭示转录调控因子DegU在调控普切明酸的合成及分泌过程中的作用。【方法】以地衣芽胞杆菌DW2为出发菌株,构建degU缺失菌株DW2ΔdegU和过表达菌株DW2::Pbay-degU,通过产物检测、转录水平检测、凝胶阻滞分析和GFP报告蛋白表达分析等方法分析DegU对普切明酸合成、分泌及调控因子基因的转录调控机制。【结果】DW2ΔdegU的普切明酸产量相比于DW2提高了56.8%,而DW2::Pbay-degU相比于DW2菌株则下降83.7%。同时,degU缺失后,普切明酸合成酶基因yvm C和转运蛋白基因yvm A的转录水平分别上升为DW2的2.85倍和2.71倍,yvmC和yvmA的负调控因子基因yvmB的转录水平则下降为DW2的0.35倍;而在DW2::Pbay-degU菌株中,yvmC和yvmA的转录水平分别下降为DW2的0.47倍和0.24倍,yvmB的转录水平则上升为DW2的1.78倍。凝胶阻滞分析和GFP报告蛋白表达分析表明,DegU可以直接与PyvmC和PyvmB启动子结合,但是与yv...     

谷氨酸棒杆菌S9114中amn基因缺失对生理代谢的影响

摘要:【目的】为了研究腺苷单磷酸核苷酶基因(amn)缺失对谷氨酸棒杆菌S9114生理代谢的影响。【方法】本文构建了amn基因缺失菌株△amn,并对谷氨酸发酵性能以及酸耐受性进行了比对分析。【结果】与野生菌株WT相比,amn基因缺失菌株:(1)细胞干重提高了16.2%,谷氨酸产量降低了58.8%;(2)发酵10 h、25 h和40 h,胞内ATP分别提高了3.0、3.7和2.2倍,异柠檬酸裂合酶活性提高17.1%、4.9%和44.5%,异柠檬酸脱氢酶活性降低76.9%、74.6%和5.0%,谷氨酸脱氢酶活性降低42.4%、50.8%和42.4%;(3)pH4.0条件下存活率降低了64.9%,而胞内ROS和蛋白质羰基化水平提高了31.5%和22.5%。【讨论】amn基因的缺失提高了菌株的胞内ATP水平和生物量,但是对谷氨酸发酵和酸性条件的耐受性却产生不利影响。     

北京棒杆菌芳香族氨基酸转运蛋白基因敲除对L-色氨酸积累的影响

[目的]为了减少北京棒杆菌PD-67(Corynebacterium pekinense PD-67)从细胞外吸收色氨酸,降低细胞内色氨酸库的浓度,从而使色氨酸的反馈控制作用减弱,增加胞外L-色氨酸的积累量,构建北京棒杆菌PD-67的芳香族氨基酸转运蛋白基因aroP敲除的菌株,研究aroP基因敲除对菌株L-色氨酸积累的影响.并进一步研究在aroP敲除菌株中表达邻氨基苯甲酸合成酶(AS)基因对L-色氨酸积累的影响.[方法]运用PCR技术扩增aroP基因,与整合质粒连接后,用限制性内切酶法构建带有内部片段缺失的aroP基因的敲除载体.利用同源重组技术,敲除北京棒杆菌PD-67的aroP基因,构建菌株PD-67 ΔaroP,并用带有aroP基因的表达载体对PD-67ΔaroP进行互补验证.采用PCR技术扩增AS基因,与表达载体连接构建重组质粒.将重组质粒转入菌株PD-67ΔaroP,构建工程菌株PD-67 ΔaroP/pXAS.通过摇瓶发酵研究PD-67 AaroP和PD-67 ΔaroP/pXAS的发酵特性.[结果]经PCR验证获得了aroP基因缺陷的菌株.摇瓶发酵结果表明,与出发菌株相比,PD-67ΔaroP的L-色氨酸的积累量提高了65％.酶活分析结果表明,AS基因在菌株PD-67 △aroP中得到表达.AS基因表达使工程菌单位菌体产酸率提高了25.6％.[结论]北京棒杆菌PD-67中芳香族氨基酸转运蛋白基因arop的敲除能够提高胞外L-色氨酸的积累量.在arop基因敲除菌中表达AS基因,可以进一步提高工程菌的产酸率.     

谷氨酸棒杆菌metX、dapA基因敲除对苏氨酸合成的影响

谷氨酸棒杆菌中metX基因编码蛋氨酸合成途径关键酶高丝氨酸乙酰转移酶,dapA基因编码赖氨酸合成途径关键酶二氢吡啶二羧酸合成酶。为研究这两个基因缺失对苏氨酸积累的影响,以谷氨酸棒杆菌R102(AHVr)为出发菌株,通过重叠延伸PCR及同源重组技术分别构建了metX、dapA单基因缺失突变株R102ΔmetX、R102ΔdapA以及双基因缺失的突变株R102ΔmetXΔdapA。对出发菌以及上述3株重组菌进行初步摇瓶发酵试验,用HPLC法测定发酵液中苏氨酸含量。结果表明,发酵72 h后,3株重组菌的苏氨酸产量分别为2.58、2.38和3.01 g/L,比原始菌株分别提高了42.5%、31.5%和66.3%。     

ccpA基因对蜡样芽胞杆菌氨肽酶生产的影响

【目的】构建蜡样芽胞杆菌(Bacillus cereus)ccp A缺失菌株,并初步探索ccp A基因对其碳代谢及氨肽酶生产的影响。【方法】利用温敏型质粒p KSV7构建蜡样芽胞杆菌CZ ccp A基因缺失突变株CZΔccp A,通过回补菌株对敲除株表型进行验证;不同碳源发酵对比菌株碳代谢的变化,进行氨肽酶发酵优化。【结果】成功构建ccp A缺失菌株CZΔccp A与回补菌株CZ1,三株菌在LB培养基中生长无差异;在柠檬酸钠以及甘露低聚糖为碳源时,菌株的代谢产生明显变化;以D-木糖为单一碳源时,氨肽酶的产量提高48.25%。【结论】CZ ccp A基因对柠檬酸钠、甘露低聚糖、D-木糖为单一碳源时的代谢可能具有调控作用,ccp A基因缺失可以提高蜡样芽胞杆菌CZ的氨肽酶产量。     

芽胞形成相关基因缺失对解淀粉芽胞杆菌生物量及胞外酶表达的影响

解淀粉芽胞杆菌作为公认的安全生产宿主菌(GRAS),在高效表达异源蛋白方面具有巨大的发展潜力和应用前景。本研究以解淀粉芽胞杆菌TCCC111018为出发菌株,通过敲除芽胞形成相关基因(spo0A,sigF和sigE),构建了一系列突变菌株(BAΔspo0A,BAΔsigF,BAΔsigE),并对突变株生物量和胞外酶表达量进行分析比较。结果表明,与出发菌株BAΔupp相比,菌株BAΔspo0A的生物量及胞外酶表达量显著降低,BAΔsigE无明显变化,而菌株BAΔsigF的生产性能有显著提升,菌体生长的稳定期延长约6 h,产酶时间提前,耐酸性α-淀粉酶与碱性蛋白酶酶活分别提高了25.2%和21.3%,该菌株的成功构建为工业酶生产宿主提供了新的选择,也为异源酶的高效生产提出了一种新的策略。     

地衣芽胞杆菌乙醛酸循环对聚谷氨酸合成的影响北大核心

[目的]了解乙醛酸循环在地衣芽胞杆菌WX-02生物合成聚谷氨酸中作用,为聚谷氨酸生产提供新的解决方法。[方法]采用基因工程手段,以地衣芽胞杆菌WX-02为原始菌株,分别增强表达和敲除异柠檬酸裂解酶ace A基因,检测发酵过程中聚谷氨酸产量、生物量、胞内外代谢物和相关基因转录量。[结果]增强表达异柠檬酸裂解酶ace A基因后,胞内谷氨酸浓度显著升高(483.42 ng/m L/Log(CFU)),溢流代谢产物减少(乙酸5.41 g/L、乙偶姻5.82 g/L、2,3-丁二醇7.31 g/L),聚谷氨酸生物合成产量为11.74 g/L,相比原始菌株提高15%。谷氨酸脱氢酶roc G基因、谷氨酸消旋酶glr基因和聚谷氨酸合成酶复合体中pgs B基因转录水平相对原始菌株分别提高1.61倍、1.32倍和1.24倍。[结论]增强乙醛酸循环可以降低地衣芽胞杆菌WX-02乙酸等溢流代谢产物合成,提高胞内谷氨酸合成能力,并上调聚谷氨酸合成酶基因转录水平,最终提高聚谷氨酸生物合成产量。     

枯草芽孢杆菌聚谷氨酸合成途径相关基因功能研究

聚谷氨酸(polyglutamic acid, PGA)作为一种天然多功能的聚合物,近年来成为研究的热点。由于很难通过化学方法合成,微生物发酵是目前生产聚谷氨酸的有效途径。【目的】从基因水平探究枯草芽孢杆菌聚谷氨酸合成途径中degS、degQ、degU、swrA、rocA、putM基因的功能,通过分子改造实现对代谢途径的调控。【方法】以枯草芽孢杆菌为出发菌株,通过对代谢途径中相关基因进行敲除或过表达,分别构建degS、degQ和degU基因缺失的重组菌,swrA、rocA和putM基因过表达的重组菌,借助菌株胞外聚谷氨酸积累的变化分析影响途径的关键节点。【结果】在摇瓶发酵条件下,重组菌Bacillus subtilis 168-swrA、Bacillus subtilis 168-rocA、Bacillus subtilis 168-putM的胞外聚谷氨酸含量分别是原始菌株的1.28倍、1.47倍和1.37倍。重组菌Bacillus subtilis 168-ΔdegS、Bacillus subtilis 168-ΔdegQ、Bacillus subtilis 168-ΔdegU的胞外...     

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.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.