产胞外多糖嗜热链球菌eps基因簇的表达及生物信息学分析

目的：胞外多糖（EPS）作为乳酸菌（LAB）重要的次级代谢产物,在发酵乳制品的生产中发挥着重要作用。采用生物信息学方法分析乳酸菌产EPS的分子机制,为开发利用EPS提供理论依据。方法：采用二代测序技术（Illumina Hiseq 4000）及实时荧光定量PCR技术（RT-qPCR）对分离自蒙古国酸牛奶中一株高产EPS的嗜热链球菌IMAU20756进行基因组测序,并对不同时间点eps基因表达量进行定量分析。结果：该菌株基因组全长为1 838 440 bp,GC含量为38.8%,共编码2 120个基因,包含1 962个功能基因,36个tRNA、515个假基因、1个tmRNA和2个重复单位。其中和EPS生产相关的基因有12个,epsA、epsB是调控基因,epsC、epsD决定多糖链长,eps1E、eps2E、epsG、epsF、epsH、epsI、epsJ是编码糖基转移酶基因,epsK调控多糖聚合及输出。结论：所有和EPS生物合成相关的基因在发酵过程中均能表达,特别是糖基转移酶的基因表达量在发酵6 h时达最高。     

大肠杆菌cdd和thrA基因的敲除及其对胞苷积累量的影响

以大肠杆菌AU39作为出发菌株,通过基因工程手段对其进行改造,旨在提高胞苷产量。首先,通过Red重组系统敲除了大肠杆菌AU39基因组上的胞苷脱氨酶基因cdd,阻断了胞苷的分解代谢;敲除了E．coliAU39（Acrid）基因组上的高丝氨酸脱氢酶基因thrA,阻断天冬氨酸向高丝氨酸的代谢途径。然后,分别对不同基因缺失菌株进行培养发酵,与出发菌株E．coliAU39相比,两株突变株都有不同程度的胞苷积累,E．coliAU39（Acdd）与E．coliAU39（AcddAthrA）的胞苷产量提高了1．25倍与1．6倍,而尿苷产量均相对有所降低。     

植物乳植杆菌YZH81胞外多糖基因簇的功能鉴定

为了研究植物乳植杆菌胞外多糖（EPS）的合成机制，本文选取一株具有较高EPS产量的植物乳植杆菌YZH81开展其EPS合成基因簇（cps）的研究。经全基因组测序、比对和分析，结果确定了YZH81菌株基因组含有两个cps基因簇，其中一个cps基因簇（cps1）的结构与功能尚未鉴定，而另一个cps基因簇（cps2）与其他已报道的植物乳植杆菌同源性很高。本研究实验敲除了cps1基因簇，与野生型YZH81菌株相比，YZH81△cps1菌株的EPS产量降低了52.28%，自聚集能力加速，粘附性降低，且清除1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2-picryl-hydrazyl radical,DPPH·）能力下降了32.42%。该结果说明YZH81菌株的cps1基因簇与EPS合成相关，也为进一步研究该菌株EPS生物合成机制建立有利条件。     

产L-丝氨酸谷氨酸棒杆菌诱变后突变基因对生长和产酸的影响

在前期研究中,通过比较基因组学分析发现产L-丝氨酸野生型谷氨酸棒杆菌SYPS-062及其突变株SYPS-062-33a之间有12个基因突变。对其中5个差异基因进行回复突变或敲除的研究,发现丙酮酸脱氢酶亚基ace E点突变与突变株SYPS-062-33a副产物积累量直接相关。在此基础上其余7个突变基因对菌株产酸和生长的影响将进一步研究,在高产L-丝氨酸谷氨酸棒杆菌ΔSSAAI中分别敲除这7个基因,发现敲除基因SD36_RS01255后,菌株OD562下降25.8%,L-丝氨酸产量下降26.4%,仅积累19.25 g/L。通过比对NCBI蛋白数据库,对基因SD36_RS01255编码的蛋白进行功能预测分析,发现其编码的蛋白部分区域与质粒pRi A4b ORF-3编码的蛋白类似,参与DNA修复。敲除基因SD36_RS01255影响DNA的修复,从而造成菌株生长和L-丝氨酸产量的下降。而过表达基因SD36_RS01255对谷氨酸棒杆菌ΔSSAAI的生长、L-丝氨酸的产量却未造成显著影响。     

Streptococcus thermophilus IMAU20551胞外多糖基因簇及其表达分析

以1 株产胞外多糖（exopolysaccharide，EPS）的嗜热链球菌（Streptococcus thermophilus）IMAU20551为研究对象，提取其DNA后采用Illumina HiSeq技术对DNA进行二代基因组重测序，并利用SOAPdenovo软件对其进行组装，通过与RAST、直系同源集、基因本体论、京都基因与基因组百科全书等数据库进行比对，注释基因组中功能基因。发现S. thermophilus IMAU20551基因组全长1 725 107 bp，共注释出1 884 个功能基因，其中包含一个长度为19 376 bp的完整eps基因簇，该基因簇中与EPS合成相关的基因共有18 个，主要包括epsA、epsB、epsC、epsD、epsE、eps1F、eps2F、epsJ、wzx、epsP和epsX等，这些基因分别对EPS的合成、组装以及转运输出进行调控。另一方面，利用实时聚合酶链式反应对S. thermophilus IMAU20551 eps基因簇表达进行验证，发现所有基因均可表达，且除个别糖基转移酶基因外，其他被测基因均在6 h达到最大表达量。本研究介绍了S. thermophilus IMAU20551的基因组特征、eps基因簇及其表达能力，为今后深入研究S. thermophilus EPS基因簇及其表征结构的互作关系奠定基础。     

基因改组选育高产杆菌肽地衣芽孢杆菌及改组菌株差异分析

为了提高地衣芽孢杆菌肽产量,采用全基因组改组技术进行菌株选育。以地衣芽孢杆菌TJ12为原始菌株,通过紫外诱变、亚硝基胍诱变、常压室温等离子体诱变构建了TJ12菌的突变体库。在优化其原生质体制备和再生条件的基础上,以其中4 株诱变菌株（U11、U23、H1、L71）作为亲本,采用聚乙二醇介导的方法进行3 轮多亲本的全基因组改组,同时,结合双亲灭活的筛选方法,最终选出1 株杆菌肽产量提高并能稳定遗传的优良菌株F3,对其摇瓶发酵36 h,杆菌肽A产量达760 mg/L,为野生菌株TJ12的1.7 倍。与野生菌株TJ12相比,改组菌株提前进入生长稳定期,两者发酵过程pH值几乎无差异;最终改组菌生长量虽低于野生菌,但菌株单位细胞还原糖产量及杆菌肽产量都高于野生菌株;其合成基因和调控基因表达量相对野生菌株都上调,其中合成基因上调幅度较大。推测改组菌株自身有了更强大的杆菌肽耐受机制,且合成基因相关部位可能发生了改变。     

1 株产γ-氨基丁酸植物乳杆菌谷氨酸脱羧酶基因的克隆与表达

以1 株产γ-氨基丁酸（γ-aminobutyric acid,GABA）植物乳杆菌（Lactobacillus plantarum）BC114谷氨酸脱羧酶为研究对象,通过聚合酶链式反应（polymerase chain reaction,PCR）技术获得该酶基因,对其进行生物信息学分析,并转入大肠杆菌BL21（DE3）中实现异源表达。采用实时荧光定量PCR、聚丙烯酰胺凝胶电泳和高效液相色谱分别测定重组菌不同发酵时间点谷氨酸脱羧酶基因的表达量、蛋白表达量以及产GABA能力。结果表明：植物乳杆菌中谷氨酸脱羧酶基因大小为1?410?bp,编码469?个氨基酸,蛋白二级结构由α-螺旋（32.2%）、β-折叠（11.5%）和无规则卷曲（56.3%）构成,完成了该酶的三维结构同源建模;成功构建了重组谷氨酸脱羧酶大肠杆菌,谷氨酸脱羧酶基因在诱导6?h相对表达量最大,而蛋白表达量较基因在转录水平表达量有一定滞后,在8?h达最大值,此时GABA产量也达最高,为2?387?mg/L。     

猪肉源大肠杆菌分离鉴定及其生物被膜形成

为研究猪肉来源大肠杆菌分离菌株的生物被膜形成及相关基因表达变化，首先从生猪屠宰线、猪胴体表面及生鲜猪肉中采用选择平板和特异性聚合酶链式反应（polymerase chain reaction，PCR）分离鉴定大肠杆菌，采用微孔板结晶紫染色法评价分离菌株15 ℃培养72 h时生物被膜形成能力，进而选取代表菌株研究生物被膜形成过程中被膜量、胞外聚合物（extracellular polymeric substances，EPS）组成，以及基于反转录荧光定量PCR的成膜基因表达的变化。结果表明：共分离到猪肉源大肠杆菌31 株，包括生猪屠宰线11 株、猪胴体表面4 株、生鲜猪肉16 株；微孔板结晶紫染色结果显示，被膜形成能力存在明显菌株差异，64.52%菌株成膜能力较弱，选取其中1 株（D4-18）进行成膜过程研究；微孔板中菌株D4-18 15 ℃培养168 h过程中被膜量持续增加，培养72 h和168 h时，菌株D4-18分泌EPS，培养72 h时，papC、fimH、csgA基因表达量分别为0.095、0.933、0.435 copies/cm2，随着培养时间延长，松散型EPS中蛋白质及多糖含量、紧密型EPS中蛋白质含量极显著增加（P＜0.01），培养168 h时，papC和fimH基因表达量增加，csgA基因表达量无显著变化，表明上述基因在大肠杆菌生物被膜形成过程中发挥了不同程度的调控作用。     

马奶酒样乳杆菌乳糖酶LacZ基因的克隆,原核表达及活性分析

从开菲尔粒(Kefir)中分离出1株马奶酒样乳杆菌(Lactobacillus kefiranofaciens ZW3),具有较高的乳糖酶活性,以此菌株为材料,从其基因组中克隆得到LacZ型乳糖酶基因,该基因全长2007 bp,编码669个氨基酸。随后将该基因插入原核表达载体pET-32a中转入大肠杆菌BL21(DE3)进行过量表达,获得了其重组蛋白,纯化后分析了该重组蛋白的乳糖水解活性特点。结果显示,此蛋白在50℃,pH 7.0时乳糖水解活力最高,并在30~55℃,pH 5.0~9.0的范围仍能保持50%以上的酶活力,具有良好的工业应用潜力。     

芽孢杆菌活性多糖的分离纯化及合成基因研究

选用自行选育的1 株活性多糖高产菌株（Bacillus amyloliquefaciens LPL061）,经醇沉、除蛋白、透析、冷冻干燥从其发酵上清中分离得到活性多糖粗品,并采用色谱纯化技术进一步纯化,得到1 个中性糖组分（EPS1）和1 个酸性糖组分（EPS2）。根据GeneBank中已报道芽孢杆菌产糖相关基因设计引物,经PCR扩增获得解淀粉芽孢杆菌LPL061的5 个EPS生物合成相关基因。对其功能预测分析表明这些基因主要参与EPS合成过程多糖聚合、糖基转移、糖链长度控制以及多糖转运和输出。     

基于氨基酸热点突变对腈水合酶底物亲和力的改造

目的:以Rhodococcus erythropolis CCM2595来源的腈水合酶为研究对象,利用半理性设计以及定点突变以获取其对烟腈底物亲和力更高的突变体.方法:通过序列比对找到同源性很高的1AHJ蛋白并采用Swiss-Model和iTASSER等软件进行评估.利用Discovery Studio 2016(DS...     

带有突变穿膜肽重组凋亡素可溶性表达的研究

目的构建apoptin（凋亡素）-HBDCK-pET28a突变体重组表达载体,在大肠杆菌BL21（DE3）中实现高效可溶性表达,并观察突变后穿膜肽（CPP）在HeLa细胞中的转导活性。方法采用PCR介导突变方法将重组蛋白apoptin-HBD及EGFP-HBD的HBD结构域中的Cys（C）突变为Lys（K）。突变的重组质粒在大肠杆菌BL21（DE3）中表达,Ni2＋-NTA亲和层析纯化目的蛋白,进一步观察HeLa细胞突变后HBD的转导活性。结果经双酶切鉴定和序列分析,突变后的重组质粒apoptin-HBDCK-pET28a及EGFP-HBDCK-pET28a构建正确。转化E.coli BL21（DE3）后,融合蛋白均获得可溶性表达。EGFP-HBDCK作用于HeLa细胞13 h后,可观察到细胞内强绿色荧光。结论 HBD结构域中C突变为K,可达到融合蛋白可溶性表达的目的,且仍能保持很强的转导活性,可携带EGFP蛋白进入HeLa细胞。     

北京棒杆菌天冬氨酸激酶五突变体的构建及酶学性质表征

目的:天冬氨酸激酶（aspartate kinase,AK）是催化天冬氨酸族氨基酸合成的第一个关键别构酶,为了提高其活力,并削弱或解除末端产物赖氨酸（Lys）与苏氨酸（Thr）对AK的协同反馈抑制。方法:在获得的北京棒杆菌四突变体T379N/A380C/G171I/Y198N AK（NCIN AK）的基础上,发现G295位点与抑制剂Thr通过氢键相连且高度保守, 对G295位点进行定点饱和突变,提取质粒,转入大肠杆菌BL21感受态细胞中诱导表达,采用高通量筛选获得酶活力显著提高的突变株,对野生型（Wild type,WT）、五突变体T379N/A380C/G171I/Y198N/G295L AK（NCINL AK）进行酶动力学研究和酶学性质表征。结果:成功构建五突变体NCINL AK,最大反应速率V_max为259 U/（mg·min）。与野生型相比,米氏常数K_m值由3.44 mmol/L减小到0.93 mmol/L,酶与底物结合更加紧密;希尔系数n值由2.73降为1.21,正协同性降低;最适反应温度由25 ℃提高到28 ℃,最适pH由8.0升至8.5,半衰期由4.7 h延长至5.2 h;五突变体NCINL AK较野生型WT AK,不同底物抑制剂浓度在0.2、1.0、5.0、10.0 mmol/L时,抑制作用被不同程度的减弱,甚至Lys抑制作用被完全解除,且在10 mmol/L Thr条件下有激活作用。结论:本实验获得酶活力提高87.20倍的五突变体NCINL AK,酶学性质得到明显改善,在一定抑制剂浓度范围内,基本解除Lys对AK的反馈抑制作用,Thr的反馈抑制得到一定缓解,提高了积累大量天冬氨酸族氨基酸的可能性,为构建高产天冬氨酸族氨基酸菌株提供参考,使甲硫氨酸（Met）等氨基酸的无污染、高效生产成为可能。     

ZMVHA-B1, the gene for subunit B of vacuolar H+-ATPase from the eelgrass Zostera marina L. Is able to replace vma2 in a yeast null mutant

A vacuolar H(+)-ATPase (VHA) gene (ZMVHA-B1) was isolated from an eelgrass (Zostera marina) leaf cDNA library and was characterized to be approximately 1.4 kbp in length and to encode the B subunit protein of VHA comprising 488 amino acids. ZMVHA-B1 was highly expressed in all organs of eelgrass; the expression level was highest in the leaves. On transformation of a yeast vma2 null mutant with ZMVHA-B1, yeast cells became able to grow at pH 7.5, accompanied by the vesicular accumulation of LysoSensor green DND-189. Thus, ZMVHA-B1 expressed in yeast cells produced a functional B subunit that was efficiently incorporated into the VHA complex and eventually restored vacuolar morphology and activity. This success expedites the application of heterologous expression in yeast mutant cells to the screening of eelgrass genes involved in salt-resistance mechanisms, which are to be utilized in improving important crops.     

Tryptophan accumulation in Saccharomyces cerevisiae under the influence of an artificial yeast TRP gene cluster

Plasmid pME559, carrying all five yeast TRP genes, was constructed. This plasmid is a yeast/Escherichia coli shuttle vector based on pBR322 and 2 μm-DNA sequences derived from plasmid pJDB207. We studied in yeast (i) the stability of the plasmid under selective and non-selective conditions, (ii) expression of all five TRP genes and (iii) tryptophan accumulation in yeast transformants. These studies were conducted in comparison with an earlier construction, pME554, which differs from plasmid pME559 in the expression of the TRP1 gene and which carries the TRP2 wild type instead of the TRP2^fbr mutant allele. For stable maintenance of the plasmids in yeast a selection was necessary. Plasmid pME559 displayed normal expression of all TRP genes, and enzyme levels on average 23-fold higher than in the wild type strain were found. In comparison, the maximal tryptophan flux observed in such a plasmid-carrying strain was about ten-fold higher than the maximal flux capacity in the wild type strain.     

嗜热杜邦菌α-淀粉酶的定向进化及高效表达

目的:对嗜热杜邦菌来源α-淀粉酶进行分子改造,以提高其耐热性和产酶水平。方法:基于易错PCR技术构建嗜热杜邦菌来源α-淀粉酶（Td-amy）的随机突变文库,高通量筛选耐热性和比酶活提高的突变体,通过定点突变及同源结构模拟对突变体进行分析,并将其在毕赤酵母中表达。结果:筛选得到一个正向突变体（mTd-amy）。该突变体最适温度（60 ℃）较野生型（55 ℃）提高了5 ℃,比酶活（466.3 U/mg）较野生型（227.9 U/mg）提高至2.0倍。经序列对比,mTd-amy有四个氨基酸发生了变化,分别为Ala4Val、Ala122Val、Lys194Arg和Ala468Asp,定点突变结果表明Ala122Val和Ala468Asp位点为影响其比酶活和最适反应温度的关键。进一步将突变体mTd-amy在毕赤酵母中高效表达,经高密度发酵其酶活达64696 U/mL。结论:定向进化获得了嗜热杜邦菌来源α-淀粉酶的正向突变体,该突变体的最适温度和比酶活力均明显提高,为α-淀粉酶的分子改造以及工业化应用等提供了理论参考。     

Cloning and heterologous expression of the NADPH cytochrome P450 oxidoreductase genes from an industrial dicarboxylic acid-producing Candida tropicalis

NADPH cytochrome P450 oxidoreductase (CPR) catalyses the transfer of electrons during P450-mediated oxidation, which plays an important role in the omega-oxidation pathway of Candida tropicalis. Two putative allelic genes, CPR-a and CPR-b, were cloned from the long chain dicarboxylic acid-producing Candida tropicalis 1230, using cassette PCR methods. Both the identified open reading frames predict the gene products of 679 amino acid residues. The deduced amino acid sequences of CPR-a and CPR-b are highly homologous to CPR genes from C. tropicalis ATCC 750 and Candida maltosa. Both genes were individually expressed in a cpr mutant of Saccharomyces cerevisiae with high CPR activities, in which only a small distinction was observed between recombinant CPR-a and CPR-b. Both CPR-a and CPR-b contain one CTG codon, which codes for serine (amino acid 50) in C. tropicalis rather than universal leucine. A mutated cDNA of CPR-a with a TCG codon instead of CTG codon was constructed and expressed, resulting in little increase in CPR activity. This indicates that the alteration of Ser-50 has little effect on functional expression of CPR. Furthermore, high ketoconazole sensitivity for the cpr mutant was complemented by heterologous expression of the cloned CPR-a or CPR-b.     

Performance of <Emphasis Type="Italic">Lactobacillus sanfranciscensis</Emphasis> TMW 1.392 and its levansucrase deletion mutant in wheat dough and comparison of their impact on bread quality

The performance of Lactobacillus sanfranciscensis TMW 1.392 and its levansucrase deletion mutant TMW 1.392 Δlev in wheat dough was compared. The effects of both strains on dough and bread characteristics were determined in order to find benchmarks for in situ production of exopolysaccharides (EPS). Growth and acidification were lower in doughs prepared with the Δlev mutant than in those employing the wild type. Extensogram resistance of the dough was reduced and extensibility increased with the addition of L. sanfranciscensis levan. Added EPS positively influenced water absorption, bread volume and firming of the crumb. In situ production of EPS was not sufficient to achieve the same positive effects of EPS, as they partially overlapped with effects resulting from enhanced acidification. Control doughs were made to separate effects of predough, EPS and different metabolism/acidification. High acetic acid levels decreased extensibility and volume. High lactic acid levels negatively influenced crumb hardness and firming kinetics. The use of knock out mutants proved helpful to judge overall performance of a strain, although the interpretation of specific effects must consider all changes in its metabolism.     

Environmental influences on exopolysaccharide formation in Lactobacillus reuteri ATCC 55730

Lactobacillus reuteri is known to produce exopolysaccharides (EPS), which have the potential to be used as an alternative biothickener in the food industry. In this study, the effect of several environmental conditions on the growth and EPS production in the L. reuteri strain ATCC 55730 was determined. The expression of the corresponding reuteransucrase gene, gtfO, was investigated over time and the results indicated that the expression increased with growth during the exponential phase and subsequently decreased in the stationary phase. Fermentation with glucose and/or sucrose as carbon and energy source revealed that gtfO was constitutively expressed and that the activity profile was independent of the sugar source. In the applied ranges of parameter values, temperature and pH were the most important factors for EPS formation and only temperature for growth. The best EPS yield, 1.4 g g(-1) CDW, was obtained at the conditions 37 degrees C, pH 4.5 and 100 g l(-1) sucrose, which were close to the estimated optimal conditions: pH 4.56 and 100 g l(-1) sucrose. No EPS formation could be detected with glucose. In addition, no direct connection between the expression and the activity of reuteransucrase could be established. Finally, the strain ATCC 55730 was benchmarked against 14 other L. reuteri strains with respect to EPS production from sucrose and abilities to metabolise sucrose, glucose and fructose. Eight strains were able to produce glucan and a corresponding glucansucrase gene was confirmed for each of them.