短刺小克银汉霉与卷枝毛霉对昂丹司琼的转化

采用微生物转化法考察11株放线菌及11株小型丝状真菌对昂丹司琼的转化能力,通过高效液相色谱-多级质谱(HPLC-MSn)检测转化产物。7株真菌可将昂丹司琼转化为7-羟基昂丹司琼和N-去甲基昂丹司琼,与文献中报道的人体内主要代谢产物相同,其中短刺小克银汉霉AS 3.153转化能力最强,在优化的转化系统中7-羟基昂丹司琼和N-去甲基昂丹司琼的产率分别达到57.80%和15.60%。此外,3株真菌和7株放线菌将昂丹司琼转化为1-羟基昂丹司琼,其中卷枝毛霉AS 3.3421转化能力最强,在优化的转化系统中,1-羟基异构体的总产率达43.10%。表明筛选出的2模型菌株对形成昂丹司琼的类哺乳动物代谢产物具有互补能力,在确定药物代谢产物种类及制备相应的对照品中具有应用价值,可作为药物代谢体外研究的有效辅助工具。     

Electroporation of Tobacco Protoplasts with Homologous and Nonhomologous Transformation Vectors

A modified protoplast selection/plating technique was used to quantitate and compare the transformation efficiencies of tobacco NTl protoplasts electroporated with plasmid molecules harboring a chimeric nos-neo gene (pMON213) or both this chimeric gene and a region of homology with the host chromosome (pCPI). The latter plasmid was constructed by cloning the pMON213 EcoR I cassette carrying the nos-neo gene into pNtSS233, a pBR322 derivative harboring a 1.2-kbp piece of Nicotiana tabacum DNA comprising the 5'-end of a gene coding for 1,5-ribulose bisphosphate carboxylase-oxygenase (Rubisco). These plasmids were linearized by restriction endonuclease digestion and electroporated into tobacco protoplasts which were subsequently selected on kanamycin-containing medium. Both plasmids displayed the same transformation efficiency, indicating that the presence of a homologous region in the selectable vector had no influence on the rates of transformation.     

Production of gamma-linolenic acid by Mucor circinelloides and Mucor rouxii with acetic acid as carbon substrate

Summary The production of gamma-linolenic acid (GLA) by Mucor circinelloides CBS 203.28 and M. rouxii CBS 416.77 in fed-batch cultures operated in pH-stat mode with acetic acid as carbon substrate and titrant compared favourably with the performance of M. circinelloides in batch culture on glucose. On acetic acid M. circinelloides accumulated up to 39.8 mg GLA/g biomass, with a crude oil content of 28% containing 91% neutral lipids. The GLA content of the neutral lipid fraction was 15.6%.     

Enhancement of Heterologous Gene Expression in Flammulina velutipes Using Polycistronic Vectors Containing a Viral 2A Cleavage Sequence

Agrobacterium tumefaciens-mediated transformation for edible mushrooms has been previously established. However, the enhancement of heterologous protein production and the expression of multi-target genes remains a challenge. In this study, heterologous protein expression in the enoki mushroom Flammulina velutipes was notably enhanced using 2A peptide-mediated cleavage to co-express multiple copies of single gene. The polycistronic expression vectors were constructed by connecting multi copies of the enhanced green fluorescent protein (egfp) gene using 2A peptides derived from porcine teschovirus-1. The P2A peptides properly self-cleaved as shown by the formation of the transformants with antibiotic resistant capacity and exciting green fluorescence levels after introducing the vectors into F. velutipes mycelia. The results of western blot analysis, epifluorescent microscopy and EGFP production showed that heterologous protein expression in F. velutipes using the polycistronic strategy increased proportionally as the gene copy number increased from one to three copies. In contrast, much lower EGFP levels were detected in the F. velutipes transformants harboring four copies of the egfp gene due to mRNA instability. The polycistronic strategy using 2A peptide-mediated cleavage developed in this study can not only be used to express single gene in multiple copies, but also to express multiple genes in a single reading frame. It is a promising strategy for the application of mushroom molecular pharming.     

Enhanced sunflower oil utilization and gamma-linolenic acid production by Mucor circinelloides f.circinelloides CBS 108.16 in the presence of acetate

World Journal of Microbiology and Biotechnology -     

Integrative Transformation of Aspergillus oryzae with a Plasmid Containing the Aspergillus nidulans argB Gene

The transformation of Aspergillus oryzae has been achieved with a plasmid carrying the Aspergillus nidulans argB gene coding for ornithine carbamoyltransferase (OCTase). The frequency of transformation was relatively low (0.7 transformants/μg DNA) but the transformed phenotype was extremely stable for many generations without selective pressure.

Southern blot analysis revealed that transformation had occurred by integration of multiple tandem copies of plasmid DNA into the host genome through non-homologous recombination. There was no evidence of the existence of free plasmid in the transformants. The number of integrated copies of the plasmid ranged from 15 to 60. The specific activity of OCTase in the cell- free extract was proportional to the copy number of the plasmid, indicating that most of the integrated argB gene was expressed.     

油菜叶绿体基因组同源重组片段的克隆及phb基因定点整合载体的构建

利用叶绿体基因组在进化过程中高度保守的特点,根据烟草、水稻和玉米叶绿体基因组全序列资料,设计合成引物,PCR扩增并克隆了油菜叶绿体两个重要的功能基因rbcL和atpB（GenBank登录号分别为AF267640和AF267641）,并以此作为定点整合外源基因的同源重组片段。以来自叶绿体的强启动子PpsbA和Prrn等驱动PHB合成途径中3个关键酶基因phbA、phbB和phbC,分别构建表达盒,并将它们按照其在原始菌株中的自然转录顺序phbC-phbA-phbB相串联,最后连同选择标记基因aadA表达盒一起,克隆到油菜叶绿体同源片段中,构建成phb基因定点整合载体pRCABZ和pRCABF。酶切及Southern杂交结果证明所构建的转化载体符合预期设计。叶绿体转化及后续工作目前正在进行之中。     

Expression of three isoprenoid biosynthesis genes and their effects on the carotenoid production of the zygomycete Mucor circinelloides

The zygomycete Mucor circinelloides accumulates β-carotene as the main carotenoid compound. In this study, the applicability of some early genes of the general isoprenoid pathway to improve the carotenoid production in this fungus was examined. The isopentenyl pyrophosphate isomerase gene (ipi) was cloned and used together with the genes encoding farnesyl pyrophosphate synthase (isoA) and geranylgeranyl pyrophosphate synthase (carG) in overexpression studies. Transformation experiments showed that the first bottleneck in the pathway, from the aspect of carotenoid production, is the step controlled by the carG gene, but overexpression of the ipi and isoA genes also contributes to the availability of the precursors. Transformations with these isoprenoid genes in combination with a bacterial β-carotene ketolase gene yielded Mucor strains producing canthaxanthin and echinenone.     

Vip3a基因植物表达载体的构建及其对烟草的遗传转化

为了研究Vip3A基因在转基因抗虫植物中的应用,利用PCR技术克隆了苏云金芽孢杆菌的Vip3A基因和烟草的EF1α启动子,以pB1121质粒为基本载体,构建了分别由组成型CaMV35S启动子和花特异表达的EF1α启动子驱动Vip3A基因的植物表达载体pBIVip3A和pBIEFVip3A,并通过农杆菌介导的方法对烟草进行了遗传转化。经PCR检测,外源基因已整合到烟草基因组中。