黑曲霉htmA基因的分离鉴定及其功能分析

本研究通过分析比较黑曲霉基因组与人、哺乳动物和酿酒酵母基因组序列同源性,首次分离鉴定了黑曲霉htmA基因,该基因长3459bp,编码1083个氨基酸。已知真核生物的htmA基因编码一种类α-甘露糖苷酶I的非必须蛋白HTMA,在内质网中参与降解非正确折叠的糖蛋白,htmA基因的破坏会延迟非正确折叠糖蛋白的降解。为分析htmA基因在黑曲霉中的功能,运用同源重组技术敲除黑曲霉基因组中的htmA基因,获得htmA基因缺失突变菌株,并进行了缺失株外源漆酶分泌能力的检测。结果表明黑曲霉htmA基因的破坏延缓了外源漆酶的降解,由此推测黑曲霉htmA基因编码蛋白HTMA具有与酵母、哺乳动物HTM1P/EDEM类似的功能作用。     

黑曲霉葡萄糖淀粉酶Ⅰ基因的克隆及在酵母中的表达

采用RT—PCR技术,从黑曲霉的菌丝体RNA中扩增出葡萄糖淀粉酶GAI的结构基因,将其连接到pPIC9载体中,转入巴斯德毕赤酵母GSll5中,获得12株Mut＇重组酵母;甲醇诱导目的蛋白分泌表达,葡萄糖淀粉酶酶活最高达180U／ml,占上清液蛋白的38％。通过GAI在巴斯德毕赤酵母中的表达,重点讨论了目的基因拷贝数,基因的偏爱性等对外源基因分泌表达的影响。     

黑曲霉果胶裂解酶基因毕赤酵母在Pichia pastoris GS115中的表达

利用RT-PCR技术从黑曲霉(EIM-6)中扩增得到去除信号肽的果胶裂解酶基因A,将其插入到毕赤酵母表达载体pPIC9k上,构建重组表达质粒pPIC9K-pelA,电击转化毕赤酵母GS115,得到了表达成功的工程菌株。用终浓度为1.5%的甲醇对其进行诱导,将发酵上清液浓缩后,用盐酸法测定其酶活可以达到2.3U/mL。通过对重组毕赤酵母诱导表达产物进行SDS-PAGE鉴定,发现重组毕赤酵母分泌了1个约38kD的蛋白,与该酶基因产物的理论值相符,并通过水解圈法测定验证,均说明果胶裂解酶得到正确的分泌表达.     

MHF组蛋白折叠复合体组分编码基因MHF1过表达对重组酿酒酵母纤维素酶生产的影响

【背景】重组酿酒酵母可用于生产多种药用蛋白和工业酶等外源蛋白,但蛋白分泌水平低是限制其异源蛋白高效生产的重要因素。异源蛋白表达和分泌过程可能会对宿主细胞产生多种胁迫,因此,研究胁迫响应相关基因对重组酵母异源蛋白生产的影响具有重要意义。Mhf1p是MHF组蛋白折叠复合体的组分之一,与DNA损伤修复及维持基因组稳定性有关,但其对异源蛋白生产的作用尚不清楚。【目的】研究MHF1过表达对重组酿酒酵母蛋白生产的影响。【方法】在分泌表达纤维素酶的重组酿酒酵母菌株中利用基于CRISPR-Cas9的基因组编辑技术整合过表达MHF1,分析其对产酶的影响,并探讨影响产酶的分子机理。【结果】与出发菌株相比,过表达MHF1菌株的外切纤维素酶CBH酶活性提高了38%。对过表达MHF1的CBH生产菌株中蛋白合成和分泌途径相关基因转录水平进行检测,发现与对照菌株相比,CBH1基因和与分泌相关的SEC22、ERV29等基因在不同时间点呈现不同程度显著上调。【结论】MHF1过表达可促进酿酒酵母异源外切纤维素酶的生产,并影响外源酶基因和分泌途径基因的表达,可能通过对多基因的协同表达影响促进产酶。     

黑曲霉pepD基因阻断突变菌株的构建及功能分析

运用同源重组技术破坏了黑曲霉基因组中的pepD基因,该基因编码一种类subtilisin的胞外蛋白酶PEPD。实验以黑曲霉GICC2773基因组DNA为模板,PCR扩增pepD基因,并在此基因中间插入潮霉素抗性基因(hph)表达单元,由此产生了3.7kb的pepD阻断基因片段。将此阻断基因片段与载体pBS连接,构建成pepD基因阻断质粒pBSDH。采用原生质体-CaCl2/PEG法将酶切阻断质粒得到的含pepD基因和hph表达单元的3.7kb线性片段转化AspergillusnigerGICC2773菌株,在含潮霉素的平板上筛选潮霉素抗性转化子,从这些抗性转化子中经PCR检测分离到到1个pepD基因阻断突变菌株?pepD66。外源漆酶分泌活性分析显示,黑曲霉pepD基因的破坏使其外源漆酶的分泌表达有所提高。     

黑曲霉pepD基因阻断突变菌株的构建及功能分析

运用同源重组技术破坏了黑曲霉基因组中的pepD基因,该基因编码一种类subtilisin的胞外蛋白酶PEPD。实验以黑曲霉GICC2773基因组DNA为模板,PCR扩增pepD基因,并在此基因中间插入潮霉素抗性基因(hph)表达单元,由此产生了3.7kb的pepD阻断基因片段。将此阻断基因片段与载体pBS连接,构建成pepD基因阻断质粒pBSDH。采用原生质体-CaCl₂/PEG法将酶切阻断质粒得到的含pepD基因和hph表达单元的3.7kb线性片段转化AspergillusnigerGICC2773菌株,在含潮霉素的平板上筛选潮霉素抗性转化子,从这些抗性转化子中经PCR检测分离到到1个pepD基因阻断突变菌株?pepD66。外源漆酶分泌活性分析显示,黑曲霉pepD基因的破坏使其外源漆酶的分泌表达有所提高。     

黑曲霉木聚糖酶在工业酒精酵母中的分泌表达

用重叠延伸PCR方法从黑曲霉 (Aspergillusniger)UV 11的基因组DNA中克隆出木聚糖酶的cDNA基因 ,构建了由酵母乙醇脱氢酶 (ADH1)启动子和终止子引导表达、木聚糖酶自身信号肽引导分泌、rDNA序列介导的酵母整合型分泌表达质粒pAX2。用pAX2与酵母YEp型G4 18抗性质粒共转化野生型工业酒精酵母S .cerevisiae 2 346 ,获得了整合型分泌表达木聚糖酶的酵母重组菌株XY2。发酵分析表明该工程菌能够明显提高酒精生产率     

透明颤菌血红蛋白基因vgb和腈水解酶基因bxn在毕赤酵母中的表达研究

为获得高效表达外源蛋白的Pichia pastoris菌株而设计了重组质粒pPIC9K—vgbbxn,其中透明颤菌血红蛋白基因vgb胞内表达以提高菌体的发酵密度,腈水解酶基因bxn分泌表达。转化GS115菌株后,通过PCR、SDS-PAGE检测证实两基因已经整合进酵母基因组且能高效表达,以及用准确的蛋白活性测定方法成功地检测到二所表达的产物均具有正常的活性。摇瓶发酵实验证明,血红蛋白在贫氧条件下可明显促进酵母菌体生长和bxn基因分泌表达。     

人分泌粒蛋白Ⅲ的克隆和表达

研究了一个新的人分泌蛋白基因－分泌粒蛋白Ⅲ（secretograninⅢ,SgⅢ),SgⅢ蛋白序列共有468个氨基酸残基,N端有一段疏水信号肽,序列中含有DSTK重复序列和7对二元碱性氨基酸（dibasic sites),这些结构特点同其他分泌蛋白家族成员相类似,人源SgⅢ蛋白在小鼠,大鼠和爪蟾中各有一个同源蛋白,基因组分析表明,SgⅢ基因位于15号染色体上,含有12个外显子, 分布在39kb长的基因组DNA上,Western印迹和免疫细胞化学实验证实,SgⅢ蛋白同其他分泌粒蛋白家族成员一样,通过分泌途径被分泌到胞外,SgⅢ在多种组织中都表达,Northern印迹显示SgⅢ的mRNA主要有2．2kb和1．9kb两种形式,但在脑中还有4．5kb和3．3kb大小的两种特异转录本。     

嗜热脂肪芽孢杆菌羧酸酯酶的异源表达及酶学性质研究

运用生物信息学技术从嗜热脂肪芽孢杆菌(Geobacillus stearothermophilus) CICC 20156中克隆获得羧酸酯酶基因,构建黑曲霉和毕氏酵母表达质粒,将重组质粒分别转化毕氏酵母GS115和黑曲霉pyrG基因缺陷株M54．SDS-PAGE和Western blot检测显示：携带His标记的外源蛋白在转化真菌宿主中均获得了高效分泌性表达,毕氏酵母和黑曲霉表达的外源蛋白分子质量均约为29 ku,蛋白质浓度分别为30.7 mg/L和15.3 mg/L．生物学活性测定表明,毕氏酵母与黑曲霉表达的羧酸酯酶单位蛋白酶活分别为22 671 U/mg和21 438 U/mg．酶学性质研究显示,两种表达系统表达的重组羧酸酯酶的酶学特性基本一致,它们在40～70℃范围内均显示较好的酶活性,最适反应温度为60℃．70℃处理30 min,毕氏酵母和黑曲霉表达重组羧酸酯酶残余酶活分别为 76.7%和67.6%,显示出良好的热稳定性．在pH 6.5～8.5的范围内显示较高酶活性,最适pH为8.0．上述研究首次实现了具有良好热稳定性的嗜热脂肪芽孢杆菌羧酸酯酶在黑曲霉和毕氏酵母中高效异源分泌性表达,其中毕氏酵母羧酸酯酶的产量要高于黑曲霉的酶产量,但考虑到重组黑曲霉表达外源性蛋白无需使用任何诱导剂,黑曲霉菌表达热稳定性羧酸酯酶可能具有更好的应用前景．     

Characterisation of the gptA gene,encoding UDP N-acetylglucosamine: dolichol phosphate N-acetylglucosaminylphosphoryl transferase,from the filamentous fungus,Aspergillus niger

The production of asparagine (N)-linked oligosaccharides is of vital importance in the formation of glycosylated proteins in eukaryotes and is mediated by the dolichol pathway. As part of studies to allow manipulation of this pathway, the gene coding for the production of the enzyme UDP N-acetylglucosamine: dolichol phosphate N-acetylglucosaminylphosphoryl transferase (GPT), catalysing the first step in the assembly of dolichol-linked oligosaccharides, was cloned from the filamentous fungus Aspergillus niger. Degenerate-PCR was used to amplify a 470-bp fragment of the gene, which was labelled as a probe to obtain a full-length clone from a genomic library of A. niger. This contained a 1557-bp open reading frame encoding a highly hydrophobic protein of 468 amino acids with a predicted molecular weight of 51.4 kDa. The gene contained two intron sequences and putative dolichol recognition sites (PDRSs) were present in the deduced amino acid sequence. Comparison with other eukaryotic GPTs revealed the A. niger GPT to share 45-47% identity with yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe) and 41-42% identity with mammals (mouse, hamster, human). Nested-PCR of a cDNA library was used to confirm the position of an intron. A complete cDNA clone of A. niger gpt was obtained by employing a recombinant PCR approach. This was used to rescue a conditional lethal mutant of S. cerevisiae carrying a dysfunctional gpt gene by heterologous expression, confirming that the gpt genes from A. niger and S. cerevisiae are functionally equivalent.     

Cloning and characterization of glucokinase from a methylotrophic yeast Hansenula polymorpha: different effects on glucose repression in H. polymorpha and Saccharomyces cerevisiae

Glucokinase gene (HPGLK1) was cloned from a methylotrophic yeast Hansenula polymorpha by complementation of glucose-phosphorylation deficiency in a H. polymorpha double kinase-negative mutant A31-10 by a genomic library. An open reading frame of 1416 nt encoding a 471-amino-acid protein with calculated molecular weight 51.6 kDa was characterized in the genomic insert of the plasmid pH3. The protein sequence deduced from HPGLK1 exhibited 55 and 46% identity with glucokinases from Saccharomyces cerevisiae and Aspergillus niger, respectively. The enzyme phosphorylated glucose, mannose and 2-deoxyglucose, but not fructose. Transformation of HPGLK1 into A31-10 restored glucose repression of alcohol oxidase and catalase in the mutant. Transformation of HPGLK1 into S. cerevisiae triple kinase-negative mutant DFY632 showed that H. polymorpha glucokinase cannot transmit the glucose repression signal in S. CEREVSIAE: synthesis of invertase and maltase in respective transformants was insensitive to glucose repression similarly to S. cerevisiae DFY568 possessing only glucokinase.     

Identification and characterization of a gene and protein required for glycosylation in the yeast Golgi

The MNN2 gene of Saccharomyces cerevisiae has been cloned by complementation of the mnn2 mutant phenotype scored by a change in cell surface carbohydrate structure resulting from a lack of alpha 1----2-mannose branching in the outer chain. The gene was subcloned as a 3 kb DNA fragment that integrated at the MNN2 locus, and a gene disruption yielded the mnn2 phenotype. A lacZ-MNN2 gene fusion protein, produced in Escherichia coli, was used to raise a specific antiserum that recognized a 65 kD wild-type yeast protein. This MNN2 gene product lacks N-linked carbohydrate but appears to be an integral membrane protein. Overproduction of MNN2p does not enhance the alpha 1----2-mannosyltransferase activity of yeast cells. The results suggest that MNN2p is a Golgi-associated protein that is involved in mannoprotein sorting rather than glycosylation.     

Molecular cloning and characterization of the fructooligosaccharide-producing beta-fructofuranosidase gene from Aspergillus niger ATCC 20611

The fopA gene encoding a fructooligosaccharide-producing beta-fructofuranosidase was isolated from Aspergillus niger ATCC 20611. The primary structure deduced from the nucleotide sequence showed considerable similarity to those of two other beta-fructofuranosidases from A. niger, but the fopA gene product had several amino acid insertions and an extra C-terminal polypeptide consisting of 38 amino acids that could not be found in the two others. We could successfully express the fopA gene in S. cerevisiae and the fopA gene product obtained from the culture supernatant of the S. cerevisiae transformant had similar characteristics to the beta-fructofuranosidase purified from A. niger ATCC 20611. However, we could not detect any beta-fructofuranosidase activity in either the culture supernatant or cell lysate when the C-terminal truncated fopA gene product by 38 amino acids was used to transform S. cerevisiae. In western analysis of those samples, there was no protein product that is cross-reacted with anti-beta-fructofuranosidase antibody. These results suggested that the C-terminal region of the fopA gene product consisting of 38 amino acids was essential for the enzyme production.     

Yeast expression of the VP8* fragment of the rotavirus spike protein and its use as immunogen in mice

The VP8* fragment from the rotavirus spike protein was expressed as a fusion protein with two different cell wall proteins of Saccharomyces cerevisiae, Icwp (Ssr1p) and Pir4, to achieve cell wall targeting or secretion to the growth medium of the fusion proteins. Two different host strains were used for the expression of the fusion proteins, a standard S. cerevisiae strain and a mnn9 glycosylation deficient strain, the later to reduce hyper-glycosylation. The Icwp-VP8* fusion could only be detected in the growth medium, indicating that the presence of the VP8* moiety interferes with the anchorage of Icwp to the cell wall. In the case of the Pir4-VP8* fusion proteins, we achieved cell wall targeting or secretion depending on how the gene fusion had been performed. In all cases, the fusion proteins expressed in the mnn9 strain showed a reduced level of glycosylation. Mice were inoculated intraperitoneally either with Pir4-VP8* or Icwp-VP8* fusion proteins purified from the growth medium of mnn9 strains expressing them or with whole cells of an mnn9 strain expressing a Pir4-VP8 fusion protein on its cell walls. Hundred percent of mice inoculated with the Pir4-VP8* fusion protein and 25% of those inoculated with the Icwp-VP8* fusion protein showed high titers of anti-VP8* antibodies. No specific immune response was detected in those mice inoculated with whole cells. Finally, susceptibility to rotavirus infection of the offspring of immunized dams was determined and protection was found in a percentage of approximately 60% with respect to the control group.     

Haptoglobin Proved a Prognostic Biomarker in Peripheral Blood of Patients with Personalized Peptide Vaccinations for Advanced Castration-Resistant Prostate Cancer

The fopA gene encoding a fructooligosaccharide-producing β-fructofuranosidase was isolated from Aspergillus niger ATCC 20611. The primary structure deduced from the nucleotide sequence showed considerable similarity to those of two other β-fructofuranosidases from A. niger, but the fopA gene product had several amino acid insertions and an extra C-terminal polypeptide consisting of 38 amino acids that could not be found in the two others. We could successfully express the fopA gene in S. cerevisiae and the fopA gene product obtained from the culture supernatant of the S. cerevisiae transformant had similar characteristics to the β-fructofuranosidase purified from A. niger ATCC 20611. However, we could not detect any β-fructofuranosidase activity in either the culture supernatant or cell lysate when the C-terminal truncated fopA gene product by 38 amino acids was used to transform S. cerevisiae. In western analysis of those samples, there was no protein product that is cross-reacted with anti-β-fructofuranosidase antibody. These results suggested that the C-terminal region of the fopA gene product consisting of 38 amino acids was essential for the enzyme production.     

Secretory expression and purification of Aspergillus niger glucose oxidase in Saccharomyces cerevisiae mutant deficient in PMR1 gene

The gene encoding glucose oxidase (GOD) from Aspergillus niger was expressed as a secretory product in the yeast Saccharomyces cerevisiae. Six consecutive histidine residues were fused to the C-terminus of GOD to facilitate purification. The recombinant GOD-His(6) secreted by S. cerevisiae migrated as a broad diffuse band on SDS-PAGE, with an apparent molecular weight higher than that in natural A. niger GOD. To investigate the effects of hyperglycosylation on the secretion efficiency and enzyme properties, GOD-His(6) was expressed and secreted in a S. cerevisiae mutant in which the PMR1 gene encoding Ca(++)-ATPase was disrupted. The pmr1 null mutant strain secreted an amount of GOD-His(6) per unit cell mass higher than that in the wild-type strain. In contrast to the hyperglycosylated GOD-His(6) secreted in the wild-type strain, the pmr1 mutant strain secreted GOD-His(6) in a homogeneous form with a protein band pattern similar to that in natural A. niger GOD, based on SDS-PAGE. The hyperglycosylated and pmr1Delta mutant-derived GOD-His(6) enzymes were purified to homogeneity by immobilized metal ion-affinity chromatography and their specific activities and stabilities were compared. The specific activity of the pmr1Delta mutant-derived GOD-His(6) on a protein basis was very similar to that of the hyperglycosylated GOD-His(6), although its pH and thermal stabilities were lower than those of the hyperglycosylated GOD-His(6).     

Yeast mannan structure necessary for co-aggregation with Lactobacillus plantarum ML11-11

Lactic acid bacteria (LAB) Lactobacillus plantarum ML11-11, an isolate from Fukuyama pot vinegar, and yeast Saccharomyces cerevisiae form significant mixed-species biofilm with direct cell-cell contact. Co-aggregation of L. plantarum ML11-11 and S. cerevisiae cells, mediated by the interaction between surface protein(s) on L. plantarum ML11-11 cells and surface mannan of S. cerevisiae cells, contributes significantly to mixed-species biofilm formation. In this study, co-aggregation activities of yeast mutants that were deleted of genes related to mannan biosynthesis were investigated to clarify the mannan structures essential for interaction with L. plantarum ML11-11. Among the 12 deletion mutants which had various incomplete mannan structures, only the mnn2 mutant lost the co-aggregation activity. In the mnn2 mutant, the gene coding the activity of attaching first branching mannose residue to mannan main chain is deleted and therefore the mnn2 mutant has unbranched mannan. From this result, it is clarified that the specific structure, consisted of mannan main chain to which are attached side chains containing one or more mannose residues, is critical for co-aggregation with L. plantarum ML11-11.