Genetic transformation of xylose-fermenting yeastPichia stipitis

Applied Biochemistry and Biotechnology - A plasmid-mediated transformation system has been developed for the xylose-fermenting yeastPichia stipitis. We found that plasmid vectors containing...     

High-yield shake-flask fermentation of xylose to ethanol

Applied Biochemistry and Biotechnology - Methods of achieving high-yield fermentation ofd-xylose to ethanol in shake flasks were investigated using the microaerophilic yeastPichia stipitis NRRL...     

Construction of a Recombinant <Emphasis Type="Italic">S. cerevisiae</Emphasis> Expressing a Fusion Protein and Study on the Effect of Converting Xylose and Glucose to Ethanol

Gene XYL1 from Candida shehatae and gene XYL2 from Pichia stipitis were amplified by polymerase chain reaction (PCR), and the two genes were both placed under the strong promoter of alcohol dehydrogenase (ADH) of plasmid pAD2 to produce the recombinant expression vector pAD2-P12. Because the amplified XYL1 fragment lacks the stop codon UAA, the polypeptide expressed in yeast cells should be a fusion protein, which is a fusion of xylose reductase and xylitol dehydrogenase. Subsequently, the pAD2-P12 vector was transformed into Saccharomyces cerevisiae YS58 to produce a recombinant S. cerevisiae YS58-12. It was indicated that S. cerevisiae YS58-12 has the ability of metabolizing xylose to produce ethanol by fermentation experiment. The result of cofermentation of glucose and xylose by using this recombinant S. cerevisiae YS58-12 showed a relatively satisfactory result. The highest percentage of xylose consumption rate reached 81.3% and the ethanol yield was equal to 67.14% of the ideal value.     

Isolation of xylose reductase gene ofPichia stipitis and its expression inSaccharomyces cerevisiae

Applied Biochemistry and Biotechnology - A NADPH/NADH-dependent xylose reductase gene was isolated from the xylose-assimilating yeast,Pichia stipitis. DNA sequence analysis showed that the gene...     

Cloning and improving the expression ofPichia stipitis xylose reductase gene inSaccharomyces cerevisiae

Applied Biochemistry and Biotechnology - The intactPichia stipitis xylose reductase gene (XR) has been cloned and expressed inSaccharomyces cerevisiae. The possible further improvement of the...     

Peculiarities of the regulation of fermentation and respiration in the crabtree-negative,xylose-fermenting yeastPichia stipitis

Applied Biochemistry and Biotechnology - The respiration ofPichia stipitis was not repressed by either high concentrations of fermentable sugars or oxygen limitation. Fermentation was not induced...     

The effect of initial cell concentration on xylose fermentation by Pichia stipitis

Xylose was fermented using Pichia stipitis CBS 6054 at different initial cell concentrations. A high initial cell concentration increased the rate of xylose utilization, ethanol formation, and the ethanol yield. The highest ethanol concentration of 41.0 g/L and a yield of 0.38 g/g was obtained using an initial cell concentration of 6.5 g/L. Even though more xylitol was produced when the initial cell concentrations were high, cell density had no effect on the final ethanol yield. A two-parameter mathematical model was used to predict the cell population dynamics at the different initial cell concentrations. The model parameters, a and b correlate with the initial cell concentrations used with an R(2) of 0.99.     

Electrophoretic behavior of plasmid DNA in the presence of various intercalating dyes

In the present study, the electrophoretic behavior of linear, supercoiled and nicked circular plasmid DNA in the presence of various intercalating dyes was characterized using pGL3 plasmid DNA as a model. The enzymatic digestion of pGL3 plasmid DNA with HindIIIwas monitored by capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF). Nicked circular plasmid DNA was found to be relatively sensitive to enzymes, and was almost digested into the linear conformer after 10-min incubation, indicating that nicked circular plasmid DNA has little chance of targeting and entering the cell nucleus. Partly digested plasmid DNA containing only linear and supercoiled conformers can be used as a standard to confirm the migration order of plasmid DNA. In methylcellulose (MC) solution with YO-PRO-1 or YOYO-1, linear plasmid DNA eluted first, followed by supercoiled and nicked plasmid DNA, and nicked plasmid DNA eluted as a broad peak. With SYBR Green 1, nicked plasmid DNA eluted first as three sharp peaks, followed by linear and supercoiled plasmid DNA. The nuclear plasmid DNA from two transfected cell lines was successfully analyzed using the present procedure. Similar results were obtained with an analysis time of seconds using microchip electrophoresis with laser-induced fluorescence detection (mu-CE-LIF). To our knowledge, these results represent the first reported analysis of nuclear plasmid DNA from transfection cells by CE-LIF or mu-CE-LIF without pre-preparation, suggesting that the present procedure is a promising alternative method for evaluating transfection efficiency of DNA delivery systems.     

Supercoiled plasmid quality assessment by analytical arginine-affinity chromatography

Supercoiled plasmids are an important component of gene-based delivery vehicles, applied in new therapeutic strategies such as gene therapy or DNA vaccination. However, aiming at the general distribution of plasmid DNA (pDNA) therapeutics requires a procedure to easily and efficiently assess the purity and recovery yield of the supercoiled (sc) plasmid isoform. Based on affinity interactions between amino acids and nucleic acids, an arginine affinity methodology with UV detection was established to quantify and to control the quality of sc plasmid biopharmaceuticals. The fact that this new technique allows to distinguish between plasmid isoforms represents an advantage, since it allows the selective quantification of the biologically active pDNA topology, and a more accurate analysis of the quality of the isolated plasmid. The analytical experiments were performed in 12 min and the method was found to be accurate, precise, reproducible and linear for a sc plasmid concentration range between 2 and 150 μg/mL. In comparison with other established methods used in the quantification of native pDNA (oc+sc), the main advance introduced by this new method is the possibility to quantify the sc plasmid in a sample containing other plasmid topologies, ensuring the purity of plasmid products to be therapeutically applied.     

Combating drug-resistant bacteria: small molecule mimics of plasmid incompatibility as antiplasmid compounds

A major mechanism for bacterial resistance to antibiotics is through the acquisition of a plasmid coding for resistance-mediating proteins. Described herein is a strategy to eliminate these plasmids from bacteria, thus resensitizing the bacteria to antibiotics. This approach involves mimicking a natural mechanism for plasmid elimination, known as plasmid incompatibility. The compound apramycin was identified as a tight binder to SLI RNA (Kd = 93 nM), the in vivo target of the plasmid incompatibility determinate RNA I, and footprinting/mutagenesis studies indicate apramycin binds SLI in the important regulatory region that dictates plasmid replication control and incompatibility. In vivo studies demonstrate that this compound causes significant plasmid loss and resensitizes bacteria to conventional antibiotics. The demonstration that a small molecule can mimic incompatibility, cause plasmid elimination, and resensitize bacteria to antibiotics opens up new targets for antibacterial research.     

Ru(TAP)3]2+-photosensitized DNA cleavage studied by atomic force microscopy and gel electrophoresis: a comparative study

Topological modifications of plasmid DNA adsorbed on a variety of surfaces were investigated by using atomic force microscopy (AFM). On mica modified with 3-aminopropyltriethoxysilane (APS) or poly-L-lysine, the interaction between the plasmid DNA and the surface "freezes" the plasmid DNA conformation deposited from solution, and the AFM images resemble the projection of the three-dimensional conformation of the plasmid DNA in solution. Modified mica with low concentrations of Mg(2+) leads to a decrease in the interaction strength between plasmid DNA and the substrate, and the AFM images reflect the relaxed or equilibrium conformation of the adsorbed plasmid DNA. Under these optimized deposition conditions, topological modifications of plasmid DNA were produced under irradiation in the presence of [Ru(TAP)(3)](2+) (TAP = 1,4,5,8-tetraazaphenanthrene), which is a non-intercalating complex, and were followed as a function of illumination time. The observed structural changes correlate well with the conversion of the supercoiled covalently closed circular form (ccc form) into the open circular form (oc form), induced by a single-strand photocleavage. The AFM results obtained after fine-tuning of the plasmid DNA-substrate interaction compare well with those observed from gel electrophoresis, indicating that under the appropriate deposition conditions, AFM is a reliable technique to investigate irradiation-induced topological changes in plasmid DNA.     

Plasmid DNA induces dodecyl triethyl ammonium bromide to aggregate into vesicle

Single-chained cationic surfactant dodecyl triethyl ammonium bromide and plasmid DNA together can form vesicles once the concentration of plasmid DNA reaches a critical value(C_CVC).Bigger the size of plasmid DNA,higher the value of C_CVC.     

DNA分子结构的多态性研究 (Ⅱ) 吖啶橙凝聚体变化诱导的质粒DNA大分子构象的研究

利用吸收光谱和荧光光谱方法,研究了吖啶橙（ＡＯ）与质粒ＤＮＡ水溶液、以及含胶束介质的吖啶橙与质粒ＤＮＡ溶液体系的相互结合作用及减色效应。结果表明：吖啶橙对质粒ＤＮＡ的吸收光谱有减色效应;含十二烷基硫酸钠（ＳＤＳ）的ＡＯ水溶液体系中,随着ＳＤＳ浓度的增加,其光谱结果表现为由凝聚态向单体的转化。而在含十二烷基硫酸钠（ＳＤＳ）的ＡＯ与质粒ＤＮＡ溶液体系中,吖啶橙凝聚态随ＳＤＳ浓度的增加,对ＡＯ与质粒ＤＮＡ相互结合产生协同的减色效应,使质粒ＤＮＡ空间结构发生缩拢。进一步采用电泳法研究了ＡＯ凝聚态可能对质粒ＤＮＡ构象的影响,结果表明：在ＡＯ与质粒ＤＮＡ溶液体系中,ＡＯ浓度的增加对质粒ＤＮＡ构象未产生影响;而在含有ＳＤＳ的ＡＯ与质粒ＤＮＡ的溶液体系中,由于ＳＤＳ对ＡＯ凝聚态的解聚作用,以及ＳＤＳ对质粒ＤＮＡ减色效应的协同作用,使得质粒ＤＮＡ的构象发生变化,诱导质粒ＤＮＡ形成超螺旋构象     

Repair of UV-irradiated plasmid DNA in excision repair deficient mutants of Saccharomyces cerevisiae

Abstract— The repair of UV-irradiated DNA of plasmid Yep13 was studied in the incision defective strains by measurement of cell transformation frequency. In Saccharomyces cerevisiae, radl,2,3 and 4 mutants could repair UV-damaged plasmid DNA. In Escherichia coli, uvrA mutant was unable to repair UV-damaged plasmid DNA; however, pretreatment of the plasmid with Micrococcus luteus endo-nuclease increased repair. We concluded that all the mutations of yeast were probably limited only to the nuclear DNA.     

Adsorption behavior of large plasmids on the anion-exchange methacrylate monolithic columns

The objective of this study was to investigate the behavior of large plasmids on the monolithic columns under binding and nonbinding conditions. The pressure drop measurements under nonbinding conditions demonstrated that the flow velocities under which plasmid passing monolith became hindered by the monolithic pore structure depended on the plasmid size as well as on the average monolith pore size; however, they were all very high exceeding the values encountered when applying CIM monolithic columns at their maximal flow rate. The impact of the ligand density and the salt concentration in loading buffer on binding capacity of the monolith for different sized plasmids was examined. For all plasmids the increase of dynamic binding capacity with the increase of salt concentration in the loading solution was observed reaching maximum of 7.1 mg/mL at 0.4M NaCl for 21 kbp, 12.0 mg/mL at 0.4 M NaCl for 39.4 kbp and 8.4 mg/mL at 0.5M NaCl for 62.1 kbp. Analysis of the pressure drop data measured on the monolithic column during plasmid loading revealed different patterns of plasmid binding to the surface, showing "car-parking problem" phenomena under certain conditions. In addition, layer thickness of adsorbed plasmid was estimated and at maximal dynamic binding capacity it matched calculated plasmid radius of gyration. Finally, it was found that the adsorbed plasmid layer acts similarly as the grafted layer responding to changes in solution's ionic strength as well as mobile phase flow rate and that the density of plasmid layer depends on the plasmid size and also loading conditions.     

Purification of supercoiled plasmid DNA from clarified bacterial lysate by arginine‐affinity chromatography: Effects of spacer arms and ligand density

Efficient loading on a chromatographic column is the dilemma of the process development faced by engineers in plasmid DNA purification. In this research, novel arginine‐affinity chromatographic beads were prepared to investigate the effect of spacer arm and ligand density to their chromatographic performance for the purification of plasmid. The result indicated that dynamic binding capacity for plasmid increased with an increasing ligand density and carbon number of spacer arm, and the highest binding capacity for plasmid of 6.32 mg/mL bead was observed in the column of arginine bead with a ligand density of 47 mmol/L and 10‐atom carbon spacer. Furthermore, this arginine bead exhibited better selectivity to supercoiled (sc) plasmid. The evidence of a linear gradient elution suggested further that the binding of plasmid on arginine beads was driven by electrostatic interaction and hydrogen bonding. Hence, sc plasmid could successfully be purified from clarified lysate by two‐stepwise elution of salt concentration. By the refinement of the elution scheme and loading volume of clarified lysate, the column of arginine bead with a ligand density of 47 mmol/L exhibited the highest recovery yield and a much higher productivity among arginine‐affinity columns. Therefore, reshaped arginine beads provided more feasible and practical application in the preparation of sc plasmid from clarified lysate.     

巯基吡啶配基液相色谱固定相的制备及其对质粒的快速纯化

在基因治疗中,质粒是一种常用的非病毒型载体。由于目前市场上对质粒的需求量很大,因此有必要开发大规模的质粒制备技术。该文以双孔型流通色谱介质为基质,以巯基吡啶为配基,制得了液相色谱固定相,并考察了其对质粒DNA的纯化效果,分析了分离机理。结果表明:该分离介质对质粒的纯化是基于疏水作用;当上样量为10 mL (质粒质量浓度为0.30 mg/mL)、流速高达4 mL/min时,质粒仍然能以100%的收率实现纯化,其纯度为100%。该色谱介质对RNA的柱容量高达2.2 mg/mL。     

Size exclusion chromatography of plasmid DNA isoforms

There is considerable interest in using size exclusion chromatography (SEC) to analyze and purify specific plasmid isoforms, but there is currently no fundamental understanding of the effects of plasmid size and morphology on plasmid behavior in SEC. Experiments were performed for plasmids from 3.0 to 17.0 kbp in size. The linear and open-circular isoforms were generated from the supercoiled plasmid by appropriate enzymatic digestion. SEC retention data were obtained using a Sephacryl S-1000 SF resin packed column and an Agilent HPLC system over a range of flow rates using buffers of different ionic strength and composition. The plasmid partition coefficients, K_P, were evaluated from the first statistical moment of the chromatographic peak. The partition coefficient decreased with increasing plasmid size as expected; K_P varied from 0.299 to 0.045 for supercoiled plasmids of 3.0 to 17.0 kbp. The partition coefficient also increased with increasing ionic strength due to the compaction of the DNA associated with the shielding of the intramolecular electrostatic interactions. For any plasmid size, the supercoiled isoform had the highest K_P followed by the open-circular and then the linear isoform, consistent with independent estimates of the plasmid radius of gyration as determined by static light scattering. The experimental data were analyzed using available theoretical models for the partitioning of linear and cyclic polymer chains in well-defined pore geometries. These results provide important insights into the behavior of different plasmid isoforms in size exclusion chromatography.     

Effects of pulse strength and pulse duration on in vitro DNA electromobility

Interstitial transport of DNA is a rate-limiting step in electric field-mediated gene delivery in vivo. Interstitial transport of macromolecules, such as plasmid DNA, over a distance of several cell layers, is inefficient due to small diffusion coefficient and inadequate convection. Therefore, we explored electric field as a novel driving force for interstitial transport of plasmid DNA. In this study, agarose gels were used to mimic the interstitium in tissues as they had been well characterized and could be prepared reproducibly. We measured the electrophoretic movements of fluorescently labeled plasmid DNA in agarose gels with three different concentrations (1.0%, 2.0% and 3.0%) subjected to electric pulses at three different field strengths (100, 200 and 400 V/cm) and four different pulse durations (10, 50, 75, 99 ms). We observed that: (1) shorter pulses (10 ms) were not as efficient as longer pulses in facilitating plasmid transport through agarose gels; (2) plasmid electromobility reached a plateau at longer pulse durations; and (3) plasmid electromobility increased with applied electric energy, up to a threshold, in all three gels. These data suggested that both pulse strength and duration needed to be adequately high for efficient plasmid transport through extracellular matrix. We also found that electric field was better than concentration gradient of DNA as a driving force for interstitial transport of plasmid DNA.     

Extraction of plasmid DNA from Escherichia coli cell lysate in a thermoseparating aqueous two-phase system

The primary purification of a 6.1 kilo base pair (kbp) plasmid from a desalted alkaline lysate has been accomplished by a thermoseparating aqueous two-phase system [(50% ethylene oxide-50% propylene oxide)-Dextran T 500]. The partitioning of the different nucleic acids (plasmid DNA, RNA, genomic DNA) in the thermoseparating aqueous two-phase system was followed both qualitatively by agarose gel electrophoresis and quantitatively by analytical chromatography (size exclusion- and anion-exchange mode) and PicoGreen fluorescence analysis. The experimental results showed a complete recovery of the plasmid DNA to the top phase, while 80% of total RNA and 58% of total protein was discarded to the bottom phase. Moreover, a 3.8-fold volume reduction of the plasmid DNA solution was achieved. By using a final thermoseparating step, the EO50PO50 polymer could be efficiently recycled, resulting in plasmid solution containing less than 1% polymer. The developed thermoseparating aqueous two-phase system shows great potential for the large-scale processing of plasmid DNA.