代谢工程改造酿酒酵母合成葡萄糖二酸

葡萄糖二酸是一种高附加值的有机酸,广泛用于食品、医药和化工领域。为获得生产葡萄糖二酸的微生物细胞工厂,通过共表达小鼠来源的肌醇加氧酶(MIOX)及恶臭假单胞菌来源的醛酸脱氢酶(Udh),在酿酒酵母Saccharomyces cerevisiae CEN.PK2-1C中构建了葡萄糖二酸合成途径,产量为(28.28±3.15)mg/L。在此基础上,通过调控前体肌醇的合成途径,发现肌醇-1-磷酸合成酶(INO1)是葡萄糖二酸合成途径的限速酶,过量表达INO1,葡萄糖二酸产量达到(107.51±10.87)mg/L,提高了2.8倍。进一步弱化竞争支路中磷酸果糖激酶(PFK1)的表达,最终葡萄糖二酸的产量达到(230.22±10.75)mg/L,为进一步获得高产葡萄糖二酸细胞工厂提供基础。     

定向进化改造酪氨酸解氨酶提高大肠杆菌合成对香豆酸产量

对香豆酸是一种具有多种药理活性的天然酚类化合物,也是多种天然药用产物生物合成的前体物质,广泛应用于食品、化妆品、医药等领域。通过微生物合成对香豆酸相对于化学合成和植物提取工艺具有节能减排等优势。但是,目前微生物合成对香豆酸产量较低,难以满足大规模工业发酵生产的要求。为了进一步提高对香豆酸产量,对粘红酵母酪氨酸解氨酶 (Tyrosine ammonia-lyase,TAL) 进行定向进化改造,利用高通量筛选方法从随机突变体文库中筛选TAL催化活性提高的突变体。通过初筛和复筛两轮筛选,从大约10 000个突变体中获得1个TAL催化活性提高1倍的突变体。该突变体包含3个氨基酸突变位点,分别为S9Y、A11N、E518A。进一步通过单点氨基酸饱和突变验证,当S9位点突变为Y、I、N和A11位点突变为N、T、Y时,TAL的催化活性提高1倍以上。通过对S9和A11位点3种类型突变进行组合突变验证,S9Y/A11N和S9N/A11Y突变体的TAL催化活力显著高于其他组合。将S9N/A11Y突变体质粒转入酪氨酸高产菌株CP032。通过摇瓶发酵,该菌株在48 h时的对香豆酸产量达到394.2 mg/L,比对照菌提高2.2倍。本研究工作对促进微生物合成对香豆酸的代谢工程研究具有一定的参考价值。     

代谢工程改造黑曲霉生产葡萄糖二酸

葡萄糖二酸是葡萄糖的一种二元羧酸衍生物,是重要的平台化合物,被应用于医药、化工等领域。本研究以黑曲霉为底盘细胞,通过表达来自恶臭假单胞菌Pseudomonas putida KT2440的糖醛酸脱氢酶基因ppudh,成功在黑曲霉中实现了葡萄糖二酸的合成,产量为18.74 mg/L;在此基础上通过共过表达黑曲霉自身来源的肌醇加氧酶(anmioxA)和肌醇-1-磷酸合酶(aninoA)、酿酒酵母Saccharomyces cerevisiae S288C来源的羧酸转运蛋白(scJEN1),强化了合成通路和外泌途径,将产量提高至102.10 mg/L;通过表达来自乳酸乳球菌Lactococcus lactis subsp.cremoris MG1363的NADH氧化酶(llnox),建立NAD⁺辅因子循环系统,使产量进一步提高至115.65 mg/L;利用RNA干扰技术对竞争支路中的关键酶磷酸果糖激酶(pfkA)和葡萄糖-6-磷酸脱氢酶(zwf)进行弱化表达,葡萄糖二酸最终的产量达到313.65 mg/L。本研究为微生物高效生产葡萄糖二酸和下游相关产品生产奠定基础。     

嗜盐古菌Haloferax volcanii WFD11中龙胆酸1,2-双加氧酶HagA的研究

【目的】研究嗜盐古菌Haloferax volcanii WFD11菌株以不同芳香酸作为碳源的生长情况;鉴定其通过龙胆酸途径代谢芳香酸过程中的开环酶龙胆酸1,2-双加氧酶的基因,并对其进行生化水平的研究;初步揭示古菌和细菌代谢芳香酸的可能差异。【方法】分别以4 mmol/L的6种不同芳香酸为唯一碳源培养菌株WFD11,利用全自动生长曲线分析仪测定菌株生长情况并绘制生长曲线;利用高效液相色谱检测菌株WFD11代谢3-羟基苯甲酸的中间产物;对菌株WFD11的基因组进行生物信息学分析,寻找潜在的龙胆酸1,2-双加氧酶编码基因,并在Haloferax volcanii H1424中异源表达;通过快速纯化系统(采用Ni2+-NTA亲和层析柱)纯化异源表达的蛋白,以龙胆酸为底物通过紫外分光光度计检测粗酶液和纯化后的龙胆酸1,2-双加氧酶和相关酶学特性;通过实时定量PCR观察hag A的表达类型。【结果】菌株WFD11能以4 mmol/L的3-羟基苯甲酸和3-羟基苯丙酸为唯一碳源和能源生长;高效液相色谱检测证明菌株WFD11通过龙胆酸代谢3-羟基苯甲酸(3HBA);克隆和异源表达了龙胆酸1,2-双加氧酶基因hag A;Hag A粗酶液和纯化蛋白均具龙胆酸1,2-双加氧酶的活性,催化龙胆酸开环生成顺丁二酸单酰丙酮酸;Hag A的龙胆酸1,2-双加氧酶比活力为0.024 8 U/mg,且其活性不依赖于Fe2+;荧光定量PCR实验结果证明hag A是组成型表达。【结论】嗜盐古菌H.volcanii WFD11可能是通过龙胆酸途径代谢芳香酸类物质,为进一步研究古菌和细菌代谢芳香酸的可能差异打下了基础。     

核黄素工业菌株高通量筛选方法的建立和应用

枯草芽孢杆菌是主要的核黄素工业生产菌之一,高通量筛选技术是选育获得高产核黄素菌株的关键环节。为实现工业菌种选育与高通量筛选技术相结合,对流式细胞分选、液滴微流控分选和96孔板筛选在核黄素工业菌株筛选中的应用进行了研究,并对96孔板筛选方法进行了优化。在流式细胞分析中,来源于同一株工业菌的低产菌株P1与高产菌株R1的细胞荧光与核黄素产量不成正相关。在液滴微流控分析中,P1和R1的发酵液上清荧光与核黄素产量成正相关,然而液滴分选后活细胞的数量很少。在96孔板筛选实验中,振荡培养后P1和R1的发酵液荧光分别为22 264 a.u.、28 647 a.u.;静置培养后荧光分别为7 095 a.u.、10 189 a.u.,核黄素产量与荧光成正相关,且二者荧光差异显著。利用96孔板静置培养的方法对工业菌株S1的突变体库进行筛选,得到的优选菌株核黄素产量为2.53 g/L,相比S1提高了15%。这些结果表明96孔板静置培养-荧光检测筛选可以应用于核黄素工业生产菌产量的提高。     

高效合成葡萄糖二酸酿酒酵母工程菌的构建

葡萄糖二酸是天然存在的一种重要二元酸,其在医疗保健和化工工业等领域具有很高的实际应用价值,因此被称为"最具价值的生物炼制产品之一".以酿酒酵母(Saccharomyces cerevisiae)为底盘微生物,文中考察了过量表达肌醇转运蛋白Itr1、融合表达肌醇加氧酶和葡萄糖醛酸脱氢酶以及弱化表达葡萄糖6-磷酸脱氢酶基因...     

利用基因组改组技术选育阿维拉霉素高产菌

为选育阿维拉霉素高产菌株,对野生型绿色产色链霉菌(Streptomyces viridochromogenes)Tü57进行传统物理和化学诱变,并获得一系列以阿维拉霉素A的含量为指标的正向突变体,再采用基因组改组技术将不同的正向突变体进行融合杂交,获得融合子。在筛选融合子的过程中,采用传统薄层色谱(TLC)和管碟法(二剂量法)结合进行初筛、高效液相色谱法进行复筛的筛选策略。经过基因组改组,筛选得到一株高产菌株R708,其在摇瓶实验中阿维拉霉素A产量达到0.208 g/L,比野生菌株提高了20倍。基因组改组选育阿维拉霉素高产菌,能提高子代菌株的遗传多样性,比传统诱变选育更快速有效。     

不同碳源对生防荧光假单胞菌2P24产抗生素2,4-二乙酰基间苯三酚的影响

【目的】包括碳源代谢等不同环境因子可调控生防菌株生防相关因子表达,进而影响其防病效果。荧光假单胞菌2P24可防治多种植物病原真菌、细菌引起的土传病害,抗生素2,4-二乙酰基间苯三酚(2,4-diacetylphoroglucinol,2,4-DAPG)是其主要生防因子之一。本文利用平板对峙法及遗传学方法研究不同碳源对菌株2P24产生2,4-DAPG的影响及相关的调控途径。【方法】利用平板对峙法检测了菌株2P24在添加葡萄糖、果糖和蔗糖等碳源的土豆浸液培养基中对棉花立枯丝核菌(Rhizoctonia solani)的拮抗能力及菌株2P24中影响2,4-DAPG产生的相关基因的表达。另外,利用Tn5转座子对含有2,4-DAPG合成基因phl A报告质粒p970Gm-phl Ap的野生型菌株2P24进行随机突变,在果糖土豆浸液培养基中筛选提高phl A基因表达的突变菌株。【结果】平板对峙实验表明,菌株2P24以葡萄糖为碳源时其抑菌活性最强,蔗糖次之,而以果糖等为碳源时菌株2P24无抑菌活性;转录融合实验进一步表明葡萄糖可促进phl A基因的表达,果糖则不影响phl A基因的表达。在果糖土豆浸液培养基中,转座子随机突变实验获得了5株可明显提高phl A基因表达的突变菌株。Tn5插入位点和序列分析显示其中一个突变体是Tn5破坏了che B基因。转录检测表明与野生菌株相比,che B突变体中phl A基因的表达和2,4-DAPG的前体物质间苯三酚(phloroglucinol,PG)产量都显著提高。游动性实验发现突变che B基因可显著降低该菌株的游动性。【结论】上述结果表明菌株2P24中不同碳源在转录水平上可影响phl A基因的表达,进而影响2,4-DAPG产生。遗传学结果也显示,che B基因参与调控2,4-DAPG生物合成过程。     

高效菌株S对TNT的降解条件和机理初探

确立了筛选菌株S对TNT的降解条件并初步探讨了TNT的降解机理.研究表明,葡萄糖的加入显著促进了TNT的生物降解,培养16h后100mg/L的TNT去除率达100%;但不加入葡萄糖时TNT仍有12%的去除,S菌也有微量生长.硝酸钾的加入同样促进了TNT的完全降解,而不加入硝酸钾时TNT有80%的去除.TNT浓度的增加并没有影响其去除效果,高达200 mg/L的TNT仍可完全降解.TNT的降解过程包括TNT生物转化为缩合产物A和A物质的开环分解.生物催化体系中硝基还原酶、甲苯双加氧酶和邻苯二酚双加氧酶的检出为这一降解途径的存在提供了有力的酶学支持.     

代谢工程改造大肠杆菌提高乙醇酸产率

乙醇酸(Glycolate)是一种在工业上有多种用途的重要化合物。本研究首先在大肠杆菌MG1655(DE3)中敲除了ldh A(乳酸脱氢酶),获得菌株Mgly1,作为出发菌株。然后通过调节乙醇酸合成途径的关键酶——异柠檬酸裂解酶(ace A)、乙醛酸还原酶(ycd W)、异柠檬酸脱氢酶激酶/磷酸化酶(ace K)的表达水平,得到乙醇酸产率为0.24 g/g葡萄糖(占理论产率的28.2%)。过量表达柠檬酸合成酶(glt A),乙醇酸产率提高到0.326 g/g葡萄糖(占理论产率的38.3%)。然后在Mgly1中敲除了glc B和ace B(苹果酸合成酶),减少了乙醇酸合成的前体乙醛酸的消耗。最终获得的工程菌株Mgly335乙醇酸产率达到0.522 g/g葡萄糖(占理论产率的61.4%)。     

Phylogenetic assignment and mechanism of action of a crop growth promoting <Emphasis Type="Italic">Rhizobium radiobacter</Emphasis> strain used as a biofertiliser on graminaceous crops in Russia

The taxonomic position of “Agrobacterium radiobacter strain 204,” used in Russia as a cereal crop growth promoting inoculant, was derived by a polyphasic approach. The phenotypic analyses gave very similar biochemical profiles for strain 204, Rhizobium radiobacter NCIMB 9042 (formerly the A. radiobacter type strain) and R. radiobacter NCIMB 13307 (formerly the Agrobacterium␣tumefaciens type strain). High percentage similarities, above the species separation level, were observed between the 16S rRNA, fusA and rpoB housekeeping gene sequences of these three strains, and the genomic DNA–DNA hybridisation of strain 204 against the type strain of R. radiobacter NCIMB 9042 was over 70%. Strain 204 is not phytopathogenic and it does not fix atmospheric N₂ or form a physical association with the roots of barley. Strain 204 culture and culture supernatant stimulated the rate of mobilisation of seed reserves of barley in darkness and promoted its shoot growth in the light. Gibberellic acid (GA) concentration was 1.3 μM but indole acetic acid was undetectable (<50 nM) in cultures of strain 204. It is concluded that strain 204 is phenotypically and genotypically very similar to the current R. radiobacter type strain and that the mechanism of its effect on growth of cereals is via the production of plant growth promoting substances. GA is likely to play an important role in the strain 204 stimulation of early growth of barley.     

定向进化-易错PCR方法提高华根霉Rhizopus chinensis CCTCC M201021脂肪酶的活力

运用定向进化-易错PCR的方法,提高了华根霉Rhizopus chinensis CCTCC M201021脂肪酶的活力。经过两轮易错PCR和pNPP顶层琼脂法筛选,从第一轮和第二轮突变库中分别筛选获得最佳突变株1-11和2-28,脂肪酶酶活与野生菌株相比分别提高2倍和4倍。基因比对结果表明,突变脂肪酶2-28有4个氨基酸发生了突变:A129S、K161R、A230T、K322R。蛋白质分子空间结构模拟显示,突变A129S、K161R、A230T位于脂肪酶分子表面。突变A230T增强了α-螺旋盖结构的稳定性。突变K322R处在loop上,靠近脂肪酶底物结合区域,与邻近的Asp(带负电)形成盐桥。静电引力将该loop向底物进入酶活性中心的通道口反方向牵引,使底物分子更易进入酶活性中心。酶学性质研究表明,突变株2-28脂肪酶的Km值比出发菌株下降了10%,Kcat值提高为原来的2.75倍。     

猪消化道中产苯乳酸乳酸菌的益生特性研究

【目的】建立一种简单有效的产苯乳酸乳酸菌的筛选方法,并筛选到高产苯乳酸的乳酸菌。【方法】菌株经过添加了苯丙氨酸的培养基厌氧培养后,利用高效液相色谱法检测发酵液中苯乳酸的含量。【结果】从猪的消化道中共分离得到31株产苯乳酸的乳酸菌,其中菌株R53的苯乳酸产量最大,为321.7 mg/L。菌株R53对.OH、O2和DPPH都有清除能力,清除率分别达到11.2%、52.7%和63.2%。同时R53也能降低培养基中胆固醇的含量,清除率为32.5%。【结论】乳酸菌R53经菌落形态、细胞形态、生化反应实验、16S rDNA测序,最终确定为植物乳杆菌。植物乳杆菌R53能产生苯乳酸,具有清除胆固醇和抗氧化能力。     

Screening of microorganisms producing esterase for the production of (R)-β-Acetylmercaptoisobutyric acid from methyl (R,S)-β-acetylmercaptoisobutyrate

(R)-β-acetylmercaptoisobutyric acid (RAM), a chiral compound, is an important intermediate for the chemical synthesis of various antihypertensive and congestive heart failure drugs. Microorganisms capable of converting (R,S)-β-acetylmercaptoisobutyric acid ((R,S)-ester) to RAM were screened from soil microorganisms. A strain ofPseudomonas sp. 1001 screened from a soil sample was selected to be the best. Cells showed an activity of 540 U/mL from culture broth and the enzyme was thermostable up to 70°C. This strain could produce RAM asymmetrically from (R,S)-ester.     

Purification and characterization of a 14-kilodalton protein that is bound to the surface of polyhydroxyalkanoic acid granules in Rhodococcus ruber.

The N-terminal amino acid sequence of the polyhydroxyalkanoic acid (PHA) granule-associated M(r)-15,500 protein of Rhodococcus ruber (the GA14 protein) was analyzed. The sequence revealed that the corresponding structural gene is represented by open reading frame 3, encoding a protein with a calculated M(r) of 14,175 which was recently localized downstream of the PHA synthase gene (U. Pieper and A. Steinbüchel, FEMS Microbiol. Lett. 96:73-80, 1992). A recombinant strain of Escherichia coli XL1-Blue carrying the hybrid plasmid (pSKXA10*) with open reading frame 3 overexpressed the GA14 protein. The GA14 protein was subsequently purified in a three-step procedure including chromatography on DEAE-Sephacel, phenyl-Sepharose CL-4B, and Superose 12. Determination of the molecular weight by gel filtration as well as electron microscopic studies indicates that a tetrameric structure of the recombinant, native GA14 protein is most likely. Immunoelectron microscopy demonstrated a localization of the GA14 protein at the periphery of PHA granules as well as close to the cell membrane in R. ruber. Investigations of PHA-leaky and PHA-negative mutants of R. ruber indicated that expression of the GA14 protein depended strongly on PHA synthesis.     

Proteinase K-resistant material in ARR/VRQ sheep brain affected with classical scrapie is composed mainly of VRQ prion protein

Classical scrapie is a prion disease in sheep and goats. In sheep, susceptibility to disease is genetically influenced by single amino acid substitutions. Genetic breeding programs aimed at enrichment of arginine-171 (171R) prion protein (PrP), the so-called ARR allele, in the sheep population have been demonstrated to be effective in reducing the occurrence of classical scrapie in the field. Understanding the molecular basis for this reduced prevalence would serve the assessment of ARR adaptation. The prion formation mechanism and conversion of PrP from the normal form (PrP(C)) to the scrapie-associated form (PrP(Sc)) could play a key role in this process. Therefore, we investigated whether the ARR allele substantially contributes to scrapie prion formation in naturally infected heterozygous 171Q/R animals. Two methods were applied to brain tissue of 171Q/R heterozygous sheep with natural scrapie to determine the relative amount of the 171R PrP fraction in PrP(res), the proteinase K-resistant PrP(Sc) core. An antibody test differentiating between 171Q and 171R PrP fragments showed that PrP(res) was mostly composed of the 171Q allelotype. Furthermore, using a novel tool for prion research, endoproteinase Lys-C-digested PrP(res) yielded substantial amounts of a nonglycosylated and a monoglycosylated PrP fragment comprising codons 114 to 188. Following two-dimensional gel electrophoresis, only marginal amounts (<9%) of 171R PrP(res) were detected. Enhanced 171R(res) proteolytic susceptibility could be excluded. Thus, these data support a nearly zero contribution of 171R PrP in PrP(res) of 171R/Q field scrapie-infected animals. This is suggestive of a poor adaptation of classical scrapie to this resistance allele under these natural conditions.     

微小杆菌(Exiguobacterium sp.)对肉桂酸降解行为

【目的】为有效缓解自毒物质肉桂酸对西瓜等作物生长的危害,从宁夏中卫硒砂瓜连作土壤中分离筛选得到一株高效降解肉桂酸的菌株,研究其基本降解特性。【方法】分离筛选得到一株能有效利用肉桂酸生长的菌株,采用16S r RNA基因序列分析进行菌株鉴定,运用高效液相色谱法和西瓜幼苗生长毒性实验检测降解特性。【结果】从多年西瓜连作土壤中筛选得到一株高效降解肉桂酸的细菌R30,鉴定为Exiguobacterium sp.,其96 h内对肉桂酸的降解率可达99%以上,最适降解温度和p H分别为30°C、p H 7.0。除肉桂酸外,该菌也能够高效降解香豆酸、阿魏酸、苯甲酸等其他酚酸类物质,表现出一定的底物广谱性;检测96 h降解液对西瓜种子萌发直至幼苗生长阶段的影响表明,该菌株可有效缓解肉桂酸对西瓜幼苗的生长抑制作用。【结论】菌株R30在肉桂酸、香豆酸、阿魏酸、苯甲酸等酚酸类物质导致的农作物连作障碍治理领域具有潜在的开发应用价值。