Expression of vde Gene Integrated Into Tobacco Genome in Antisense and Overexpressed Ways

Violaxanthin de-epoxidase (VDE) is localised in the thylakoid lumen of chloroplasts and catalyses de-epoxidation of violaxanthin into antheraxanthin and zeaxanthin. Tobacco vde gene was inserted into a binary vector pCAMBIA1301 with the hygromycin resistant gene for selection in antisense and overexpressed ways. Two constructs with antisense and overexpressed vde gene were introduced in tobacco (Nicotiana tabacum L.) using Agrobacterium tumefaciens strain LBA4404, PCR and Southern blot analyses demonstrated that the exogenous gene was integrated into genome of tobacco plants. VDE activity assay and HPLC analysis of pigments showed that the vde gene was expressed in the overexpressed transformants, whereas suppressed in the antisense ones. The chlorophyll fluorescence measurements proved that the contents of VDE in transgenic plants have a significant function in non-photochemical quenching.     

Comparison of the xylose reductase-xylitol dehydrogenase and the xylose isomerase pathways for xylose fermentation by recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

Two heterologous pathways have been used to construct recombinant xylose-fermenting Saccharomyces cerevisiae strains: i) the xylose reductase (XR) and xylitol dehydrogenase (XDH) pathway and ii) the xylose isomerase (XI) pathway. In the present study, the Pichia stipitis XR-XDH pathway and the Piromyces XI pathway were compared in an isogenic strain background, using a laboratory host strain with genetic modifications known to improve xylose fermentation (overexpressed xylulokinase, overexpressed non-oxidative pentose phosphate pathway and deletion of the aldose reductase gene GRE3). The two isogenic strains and the industrial xylose-fermenting strain TMB 3400 were studied regarding their xylose fermentation capacity in defined mineral medium and in undetoxified lignocellulosic hydrolysate.     

A new approach to the production of the recombinant SOD protein by methylotrophic <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The gene for the copper, zinc–superoxide dismutase (SOD) from the yeast Saccharomyces cerevisiae was cloned, characterized, and overexpressed in the methylotrophic Pichia pastoris. The sod gene sequence obtained is 465 bp and encodes 154 amino acid residues. The sod gene sequence was cloned into the pPIC9K vector, yielding pAB22. The linearized pAB22 DNA, digested with restriction enzyme SacI, was transformed into the genome of the GS115 strain of yeast P. pastoris. The overexpressed SOD protein was shown to have immunologically biological activity and to be enzymatically active. The SOD protein was purified from the cultured yeast by ammonium sulfate precipitation and diethylaminoethyl–cellulose column chromatography. This relatively simple purification method produced a single band on analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which indicated that the SOD protein obtained attained to higher purity and specific activity.     

A Proteomic Approach Towards the Analysis of Salt Tolerance in Rhizobium etli and Sinorhizobium meliloti Strains

Soluble proteins from the salt-tolerant Rhizobium etli strain EBRI 26 were separated by two-dimensional (2D) gel electrophoresis and visualised by Commassie staining. Six proteins are highly expressed after induction by 4% NaCl compared to the non-salt-stressed cells. These proteins have pI between 5 and 5.5 and masses of approximately 22, 25, 40, 65, 70, and 95 kDa. These proteins were analysed by Matrix-assisted laser adsorption ionization time of flight (MALDI-TOF) after digestion with trypsin. Despite having very good peptide mass fingerprint data, these proteins could not be identified, because the genome sequence of R. etli is not yet published. In a second approach, soluble proteins from salt-induced or non-salt-induced cultures from R. etli strain EBRI 26 were separately labelled with different fluorescent cyano-dyes prior to 2D difference in gel electrophoresis. Results revealed that 49 proteins are differentially expressed after the addition of sodium chloride. Fourteen proteins are overexpressed and 35 were downregulated. The genome of Sinorhizobium meliloti, a closely related species to R. etli, has been published. Similar experiments using Sinorhizobium meliloti strain 2011 identified four overexpressed and six downregulated proteins. Among the overexpressed protein is a carboxynospermidin decarboxylase, which plays an important role in the biosynthesis of spermidin (polyamine). The enzyme catalase is among the downregulated proteins. These proteins may play a role in salt tolerance.     

Overexpression of the gene encoding HMG-CoA reductase in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for production of prenyl alcohols

To develop microbial production method for prenyl alcohols (e.g., (E,E)-farnesol (FOH), (E)-nerolidol (NOH), and (E,E,E)-geranylgeraniol (GGOH)), the genes encoding enzymes in the mevalonate and prenyl diphosphate pathways were overexpressed in Saccharomyces cerevisiae, and the resultant transformants were evaluated as to the production of these alcohols. Overexpression of the gene encoding hydroxymethylglutaryl (HMG)-CoA reductase was most effective among the genes tested. A derivative of S. cerevisiae ATCC 200589, which was selected through screening, was found to be the most suitable host for the production. On cultivation of the resultant transformant, in which the HMG-CoA reductase gene was overexpressed, in a 5-liter bench-scale jar fermenter for 7 d, the production of FOH, NOH, and GGOH reached 145.7, 98.8, and 2.46 mg/l, respectively.     

A genetic overhaul of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> 424A(LNH-ST) to improve xylose fermentation

Robust microorganisms are necessary for economical bioethanol production. However, such organisms must be able to effectively ferment both hexose and pentose sugars present in lignocellulosic hydrolysate to ethanol. Wild type Saccharomyces cerevisiae can rapidly ferment hexose, but cannot ferment pentose sugars. Considerable efforts were made to genetically engineer S. cerevisiae to ferment xylose. Our genetically engineered S cerevisiae yeast, 424A(LNH-ST), expresses NADPH/NADH xylose reductase (XR) that prefer NADPH and NAD⁺-dependent xylitol dehydrogenase (XD) from Pichia stipitis, and overexpresses endogenous xylulokinase (XK). This strain is able to ferment glucose and xylose, as well as other hexose sugars, to ethanol. However, the preference for different cofactors by XR and XD might lead to redox imbalance, xylitol excretion, and thus might reduce ethanol yield and productivity. In the present study, genes responsible for the conversion of xylose to xylulose with different cofactor specificity (1) XR from N. crassa (NADPH-dependent) and C. parapsilosis (NADH-dependent), and (2) mutant XD from P. stipitis (containing three mutations D207A/I208R/F209S) were overexpressed in wild type yeast. To increase the NADPH pool, the fungal GAPDH enzyme from Kluyveromyces lactis was overexpressed in the 424A(LNH-ST) strain. Four pentose phosphate pathway (PPP) genes, TKL1, TAL1, RKI1 and RPE1 from S. cerevisiae, were also overexpressed in 424A(LNH-ST). Overexpression of GAPDH lowered xylitol production by more than 40%. However, other strains carrying different combinations of XR and XD, as well as new strains containing the overexpressed PPP genes, did not yield any significant improvement in xylose fermentation.     

Overexpression and purification of the galactose operon enzymes from Escherichia coli

A convenient new procedure for the purification of galactokinase, galactose-1-phosphate uridyltransferase, and UDP-galactose 4-epimerase overexpressed in Escherichia coli is presented. The procedure is shorter than any other described in the literature and facilitates the purification of the three recombinant enzymes in considerable amounts and at high purity and specific activity. The purified gal operon enzymes were biochemically cheracterized by gel-filtration column chromatography and isoelectric focusing, and the K_m values for their substrates were determined.     

Immunohistochemical detection of transgene expression in the brain using small epitope tags

Background  

In vivo overexpression of proteins is a powerful approach to study their biological function, generate disease models or evaluate gene therapy approaches. In order to investigate an exogenously expressed protein, specific and sensitive detection is essential. Unfortunately, antibodies that allow histological detection of the protein of interest are not always readily available. The use of an epitope tag fused to the protein can circumvent this problem as well as provide the possibility to discriminate endogenous from overexpressed proteins. In order to minimize impact on the bioactivity and biodistribution of the overexpressed protein, preference is given to small tags.     

鸡zp1基因对成骨细胞矿化的影响

本研究旨在克隆编码鸡透明带1（zona pellucida 1,Zp1）蛋白的zp1基因,并研究其组织表达谱及在成骨细胞矿化中的作用。利用实时荧光定量聚合酶链式反应（real-time fluorescence quantitative polymerase chain reaction,RT-qPCR）检测蛋鸡不同组织和性成熟启动前后胫骨中的zp1表达水平;将Zp1过表达载体转染至诱导分化8 d的鸡颅骨成骨细胞内,检测细胞外矿化基质和矿化相关基因表达。结果表明,鸡zp1基因全长3 045 bp,编码958个氨基酸残基,具有2个N-糖基化位点。zp1基因在产蛋期鸡肝脏中高水平表达,其次为胫骨和卵黄膜,在蛋壳腺和心脏中不表达。鸡性成熟启动后血浆雌激素（estrogen,E2）、胫骨糖胺聚糖（glycosaminoglycan,GAG）含量和zp1表达量均极显著高于性成熟启动前。在过表达Zp1的鸡成骨细胞中添加雌激素后,细胞外矿化基质和矿化相关基因表达水平均显著升高。因此,zp1基因表达具有组织特异性,在雌激素作用下正向调控鸡成骨细胞矿化,为阐明Zp1在鸡产蛋期骨骼中的功能特性奠定了理论基础。     

Flux control-based design of furfural-resistance strains of Saccharomyces cerevisiae for lignocellulosic biorefinery

We have previously developed a dynamic flux balance analysis of Saccharomyces cerevisiae for elucidation of genome-wide flux response to furfural perturbation (Unrean and Franzen, Biotechnol J 10(8):1248–1258, 2015). Herein, the dynamic flux distributions were analyzed by flux control analysis to identify target overexpressed genes for improved yeast robustness against furfural. The flux control coefficient (FCC) identified overexpressing isocitrate dehydrogenase (IDH1), a rate-controlling flux for ethanol fermentation, and dicarboxylate carrier (DIC1), a limiting flux for cell growth, as keys of furfural-resistance phenotype. Consistent with the model prediction, strain characterization showed 1.2- and 2.0-fold improvement in ethanol synthesis and furfural detoxification rates, respectively, by IDH1 overexpressed mutant compared to the control. DIC1 overexpressed mutant grew at 1.3-fold faster and reduced furfural at 1.4-fold faster than the control under the furfural challenge. This study hence demonstrated the FCC-based approach as an effective tool for guiding the design of robust yeast strains.

     

Rad3 protein of Saccharomyces cerevisiae: overexpression and preliminary characterization using specific antibodies

Summary The cloned RAD3 gene of Saccharomyces cerevisiae was tailored into expression vectors for overexpression of Rad3 protein in Escherichia coli and in yeast. In both organisms the overexpressed protein is detected as a species of molecular weight ca. 90 kDa, the size expected from the sequence of the cloned gene. The protein overexpressed in E. coli is largely insoluble; however the insoluble fraction was used to generate affinity-purified polyclonal antisera which proved to be powerful reagents for the initial characterization of Rad3 protein expressed in yeast. These studies showed that: (1) when overexpressed in yeast most of the Rad3 protein is detected in the soluble fraction of cell extracts; (2) endogenous Rad3 protein is untransformed cells is also ca. 90 kDa in size and is located in the cell nucleus; (3) Rad3/-galactosidase fusion protein partially purified on an affinity matrix is associated with DNA-dependent ATPase activity that is inhibited in the presence of anti-Rad3 antibodies, suggesting that Rad3 protein is an ATPase; and (4) Rad3 antibodies cross-react with two electrophoretically distinguishable polypeptides present in the nuclear fraction of human cells, and with a single polypeptide in extracts of Drosophila cell.     

Use of the Recombinant 38-kDa Antigen of Mycobacterium tuberculosis as an Immunogen for Specific Antisera Production

The Mycobacterium tuberculosis 38-kDa protein antigen is one of the secreted immunodominant antigens showing high immunogenicity at B-cell and T-cell levels. Although monoclonal antibodies to this antigen have been produced, specific polyclonal antisera is required for standardization of specific immunodiagnostic assays. This protein has been overexpressed and purified from recombinant Escherichia coli using an inducible vector system. During each stage of expression and purification, the recombinant protein was used to immunize mice and rabbits by several methods: 1) as overexpressed protein present as inclusion bodies in recombinant E. coli; 2) embedded in a polyacrylamide gel; 3) fixed to a solid-phase nitrocellulose membrane and 4) emulsified with an adjuvant. All strategies yielded specific antisera as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analyses. The results obtained, both quantitative (ELISA) and qualitative (immunoblot) demonstrate that the purified recombinant antigen retains its antigenicity and immunogenicity throughout the various steps in the process of expression and purification and serves as a potent antigen for production of specific antisera to be used in immunoassays.     

The cold shock response inLactococcus lactis subsp.lactis

The lactic acid bacterium,Lactococcus lactis subsp.lactis IL1403 was subjected to defferent cold temperatures for various times. Physiological experiments showed that this strain had an improved survival capacity in stationary phase as the temperature decreased. Two-dimensional electrophoresis of proteins extracted from cold-temperature exposed cultures showed that a dozen proteins are overexpressed up to threefold compared with exposure at 30°C. Most of these proteins are overexpressed first, temporarily and second, in the first 10 h after the transfer to 8°C. These observations indicate that response to cold stress inL. lactis subsp.lactis is an active phenomenon.     

Heterologous and homologous expression of the arginine biosynthetic argC~H cluster from Corynebacterium crenatum for improvement of (L) -arginine production

The genes involved in l-arginine biosynthesis in Corynebacterium crenatum are organized as the argCJBDFRGH cluster like in Corynebacterium glutamicum. However, the argC~H cluster of the C. crenatum SYPA 5-5, which is an industrialized l-arginine producer, had a lethal mutation occurring in the ArgR repressor encoding gene. The argC~H cluster with an inactive argR was overexpressed in E. coli and C. crenatum. In the recombinant E. coli JM109 enzyme activities were increased, and more l-arginine was found in the supernatants from l-glutamine. When the argC~H cluster was overexpressed in C. crenatum under its native promoter Parg, l-arginine production was increased by 24.9%, but the presence of the recombinant plasmid pJC-9039 had a negative effect on cell growth. Surprisingly, the DO value of the recombinant strain dropped gently and stayed at a lower level from 24 h to the end of fermentation. The results demonstrated an increasing utilization of oxygen and the distinct enhancement of unit cell l-arginine yields with the cluster argC~H-bearing in C. crenatum SYPA-9039. This study provides a kind of Corynebacteria with improved l-arginine-producing ability and an efficient elevation for producing amino acid. Moreover, the promoter Parg would be used as a valid promoter to express objective genes for metabolic engineering in Corynebacteria.     

Metabolic engineering for the production of prenylated polyphenols in transgenic legume plants using bacterial and plant prenyltransferases

Prenylated polyphenols are secondary metabolites beneficial for human health because of their various biological activities. Metabolic engineering was performed using Streptomyces and Sophora flavescens prenyltransferase genes to produce prenylated polyphenols in transgenic legume plants. Three Streptomyces genes, NphB, SCO7190, and NovQ, whose gene products have broad substrate specificity, were overexpressed in a model legume, Lotus japonicus, in the cytosol, plastids or mitochondria with modification to induce the protein localization. Two plant genes, N8DT and G6DT, from Sophora flavescens whose gene products show narrow substrate specificity were also overexpressed in Lotus japonicus. Prenylated polyphenols were undetectable in these plants; however, supplementation of a flavonoid substrate resulted in the production of prenylated polyphenols such as 7-O-geranylgenistein, 6-dimethylallylnaringenin, 6-dimethylallylgenistein, 8-dimethylallynaringenin, and 6-dimethylallylgenistein in transgenic plants. Although transformants with the native NovQ did not produce prenylated polyphenols, modification of its codon usage led to the production of 6-dimethylallylnaringenin and 6-dimethylallylgenistein in transformants following naringenin supplementation. Prenylated polyphenols were not produced in mitochondrial-targeted transformants even under substrate feeding. SCO7190 was also expressed in soybean, and dimethylallylapigenin and dimethylallyldaidzein were produced by supplementing naringenin. This study demonstrated the potential for the production of novel prenylated polyphenols in transgenic plants. In particular, the enzymatic properties of prenyltransferases seemed to be altered in transgenic plants in a host species-dependent manner.