The xylose isomerase gene from Thermoanaerobacterium thermosulfurogenes allows effective selection of transgenic plant cells using D-xylose as the selection agent

The xylose isomerase gene (xylA) from Thermoanaerobacterium thermosulfurogenes (formerly Clostridium thermosulfurogenes) has been expressed in three plant species (potato, tobacco, and tomato) and transgenic plants have been selected on xylose-containing medium. The xylose isomerase gene was transferred to the target plant by Agrobacterium-mediated transformation. The xylose isomerase gene was expressed using the enhanced cauliflower mosaic virus (CaMV) 35S promoter and the translation enhancer sequence from tobacco mosaic virus. Unoptimized selection studies showed that, in potato and tomato, the xylose isomerase selection was more efficient than the established kanamycin resistance selection, whereas in tobacco the opposite was observed. Efficiency may be increased by optimization. The xylose isomerase system enables the transgenic cells to utilize xylose as a carbohydrate source. It is an example of a positive selection system because transgenic cells proliferate while non-transgenic cells are starved but still survive. This contrasts to antibiotic or herbicide resistance where transgenic cells survive on a selective medium but non-transgenic cells are killed. The results give access to a new selection method which is devoid of the disadvantages of antibiotic or herbicide selection.     

Positive selection: a plant selection principle based on xylose isomerase, an enzyme used in the food industry

A new method for the selection of transgenic plants has been developed. It is based upon selection of transgenic plant cells expressing the xylA gene from Streptomyces rubiginosus, which encodes xylose isomerase, on medium containing xylose. The xylose isomerase selection system was tested in potato and the transformation frequency was found to be approximately ten fold higher than with kanamycin selection. The level of enzyme activity in the transgenic plants selected on xylose was 5- to 25-fold higher than the enzyme activity in control plants. Potato transformants were stable over two generations in Southern blotting analysis. This novel selection system is more efficient than the traditionally used kanamycin-based selection systems. In addition, the xylose isomerase system is independent of antibiotic or herbicide resistance genes, but depends on an enzyme that is generally recognized as safe for use in the starch industry and which is already being widely utilized in specific food processes. Received: 13 August 1997 / Revision received: 26 November 1997 / Accepted: 15 December 1997     

Crystal structure of Streptomyces diastaticus No. 7 strain M1033 xylose isomerase

The crystal structures of Streptomyces diastaticus No. 7 strain M1033 xylose isomerase (SDXyI) have been analysed and refined at 0.19nm. The crystal space group is I222, with unit cell dimensions of a=9.884 ran, b=9.393nm and c=8.798nm. Based on the coordinates of the Streptomyces rubiginosus xylose isomerase (SRXyI), the initial model of SDXyl was built up by the dose packing analysing and R-factor searching and refined by PROLSQ to a final R-factor of 0.177 with the rms deviations of bond lengths and bond angles of 0.001 9nm and 2.1°, respectively. No significant global conformation change existed between SRXyI and SDXyI except the local conformation in the active site.     

木糖异构酶在酿酒酵母细胞表面的展示

将来源于嗜热细菌Thermus thermophilus的木糖异构酶基因xylA,与酿酒酵母(Sac-charomyces cerevisiae)a-凝集素表面展示载体pYD1的Aga2p亚基C端序列融合。编码融合蛋白的基因序列前接上半乳糖诱导型启动子。用LiAc完整细胞法转化酿酒酵母EBY100。含重组质粒的菌株EBY100/pYD-xylA经半乳糖诱导表达外源融合蛋白,免疫荧光显微镜结果显示外源蛋白被锚定在细胞壁上,木糖异构酶活性测定结果表明,细胞壁上酶活测定值为1.52U,木糖异构酶在酿酒酵母细胞壁上得到活性表达。     

Plant selectable markers and reporter genes

In recent years, considerable progress has been made in genetic engineering of various plant species, both agronomically important crops as well as model plants. The bases of this progress were, in addition to efficient transformation methods, the design of appropriate signals regulating transgene expression and the use of selection marker or reporter genes. In most cases, a gene of interest is introduced into plants in association with a selectable marker gene (nptII, hpt, acc3, aadA, bar, pat). Recovery of a transgenic plant is, therefore, facilitated by selection of putative transformants on a medium containing a selection agent, such as antibiotic (nptII, hpt, acc3, aadA), antimetabolite (dhfr), herbicide (bar, pat), etc. On the other hand, use of reporter genes (cat, lacZ, uidA, luc, gfp) allows not only to distinguish transformed and non-transformed plants, but first of all to study regulation of different cellular processes. In particular, by employing vital markers (Luc, GFP) gene expression, protein localization and intracellular protein traffic can be now observed in situ, without the need of destroying plant.     

Agrobacterium and biolistic transformation of onion using non-antibiotic selection marker phosphomannose isomerase

A new selection system for onion transformation that does not require the use of antibiotics or herbicides was developed. The selection system used the Escherichia coli gene that encodes phosphomannose isomerase (pmi). Transgenic plants carrying the manA gene that codes for pmi can detoxify mannose-6-phosphate by conversion to fructose-6-phosphate, an intermediate of glycolysis, via the pmi activity. Six-week-old embryogenic callus initiated from seedling radicle was used for transformation. Transgenic plants were produced efficiently with transformation rates of 27 and 23% using Agrobacterium and biolistic system, respectively. Untransformed shoots were eliminated by a stepwise increase from 10 g l⁻¹ sucrose with 10 g l⁻¹ mannose in the first selection to only10 g l⁻¹ mannose in the second selection. Integrative transformation was confirmed by PCR, RT-PCR and Southern hybridization. An erratum to this article can be found at     

Purification and characterization of thermostable xylose(glucose) isomerase from Bacillus thermoantarcticus

Xylose isomerase produced by Bacillus thermoantarcticus was purified 73-fold to homogeneity and its biochemical properties were determined. It was a homotetramer with a native molecular mass of 200 kDa and a subunit molecular mass of 47 kDa, with an isoelectric point at 4.8. The enzyme had a K _m of 33 mM for xylose and also accepted D-glucose as substrate. Arrhenius plots of the enzyme activity of xylose isomerase were linear up to a temperature of 85°C. Its optimum pH was around 7.0, and it had 80% of its maximum activity at pH 6.0. This enzyme required divalent cations for its activity and thermal stability. Mn²⁺, Co²⁺ or Mg²⁺ were of comparable efficiency for xylose isomerase reaction, while Mg²⁺ was necessary for glucose isomerase reaction. Journal of Industrial Microbiology & Biotechnology (2001) 27, 234–240. Received 18 March 2001/ Accepted in revised form 03 July 2001     

Display of Clostridium cellulovorans xylose isomerase on the cell surface of Saccharomyces cerevisiae and its direct application to xylose fermentation

Xylose isomerase (XI) is a key enzyme in the conversion of d ‐xylose, which is a major component of lignocellulosic biomass, to d ‐xylulose. Genomic analysis of the bacterium Clostridium cellulovorans revealed the presence of XI‐related genes. In this study, XI derived from C. cellulovorans was produced and displayed using the yeast cell‐surface display system, and the xylose assimilation and fermentation properties of this XI‐displaying yeast were examined. XI‐displaying yeast grew well in medium containing xylose as the sole carbon source and directly produced ethanol from xylose under anaerobic conditions. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 346–351, 2013     

An efficient and novel plant selectable marker based on organomercurial resistance

A selectable marker gene facilitates the detection of genetically modified plant cells during transformation experiments. So far, these marker genes are almost exclusively of two types, conferring either antibiotic resistance or herbicide tolerance. However, more selectable markers must be developed as additional transgenic traits continue to be incorporated into transgenic plants. Here, we used mercury resistance, conferred by the organomercurial lyase gene, as a selectable marker for transformation. The merB gene fromStreptococcus aureus was modified for plant expression and transferred to a hybrid poplar(Populus alba xPopulus glandulosa), using the stem segment-agrobacteria co-cultivation method. The transformed cells were selected on a callus-inducing medium containing as little as 1 μM methylmercury. Subsequent plant regeneration was done in the presence of methylmercury. Resistance to Hg was stably maintained in mature plants after two years of growth in the nursery. We suggest that this gene could serve as an excellent selectable marker for plant transformation.     

Selectable Markers and Reporter Genes: A Well Furnished Toolbox for Plant Science and Genetic Engineering

Marker genes (MGs) are essential tools for plant research and biotechnology. Positive selectable marker genes (SMGs) are used in genetic transformation to allow only transgenic cells to grow and develop and are necessary for efficient transformation. Negative SMGs confer a selective disadvantage to the cells that express them, and have several uses in both basic and applied research. Reporter genes (RGs) make it possible to easily screen cells or tissues for their expression. Several tens of different genes from bacteria, fungi, plants, and animals have been demonstrated to function as SMGs. Here, SMGs are classified based on the mechanism of action of the gene products. To provide the readers with practically useful information, details on transformation and selection efficiency are given. RGs are the object of intense research. Refinement of existing RGs and development of new ones is constant, and has provided powerful aids for fine studies on cell biology and more efficient genetic engineering. They are classified as vital and non vital, depending on the possibility to screen their expression in living cells. The effect of MG expression on the phenotype and their safety in crops is briefly discussed. The picture emerging from this literature review is that a plentiful array of powerful and versatile tools for basic and applied research is available.     

Analysis of mannose selection used for transformation of sugar beet

Various factors affecting mannose selection for the production of transgenic plants were studied using Agrobacterium tumefaciens-mediated transformation of sugar beet (Beta vulgaris L.) cotyledonary explants. The selection system is based on the Escherichia coli phosphomannose isomerase (PMI) gene as selectable gene and mannose as selective agent. Transformation frequencies were about 10-fold higher than for kanamycin selection but were only obtained at low selection pressures (1.0–1.5 g/l mannose) where 20–30% of the explants produced shoots. The non-transgenic shoots were eliminated during the selection procedure by a stepwise increase in the mannose concentration up to 10 g/l. Analysis of the transformed shoots showed that the PMI activity varied from 2.4 mU/mg to 350 mU/mg but the expression level was independent of the selection pressure. Complete resistance to mannose of transformed shoots was observed already at low PMI activities (7.5 mU/mg). Genomic DNA blot analysis confirmed the presence of the PMI gene in all transformants analysed. The possible mode of action of mannose selection compared to other selection methods is discussed.     

An efficient method for organic acetylation and use of dl-phosphinothricin as a negative selection agent in argE transgenic rice

We present an efficient method for the production of N-acetyl-l-phosphinothricin (N-AcPt) from commercial dl-phosphinothricin (DL-PPT) by organic acetylation for use as a negative selection agent (NSA) that induces cell death in argE transgenic rice. DL-PPT was efficiently converted into N-AcPt with tetrahydrofuran (THF) and acetic anhydride (Ac₂O). Chemical changes were confirmed using NMR and ATR-FTIR analyses. DL-PPT was toxic but N-AcPt did not show cytotoxic effects on leaf discs or seed germination of wild-type rice. Conversely, in argE–hpt transgenic rice, non-toxic N-AcPt showed the negative selection (NS) effect by inducing cell destruction in leaf discs and restricting seed germination. For inducing NS, ?0.1 mg ml⁻¹ and ?0.5 mg ml⁻¹ of N-AcPt were effective in leaf and seed assays, respectively. Further, the NS effect occurred faster in the leaf assay compared with the seed germination assay, again indicating the leaf assay was a more sensitive indicator of N-AcPt as an NSA to argE transgenic rice than the seed germination assay. This negative selection approach could be useful for the development of selectable marker free transgenic plants in the economically important monocot species and its commercialization for multiple gene transformation.     

Application of a compatible xylose isomerase in simultaneous bioconversion of glucose and xylose to ethanol

Simultaneous isomerisation and fermentation (SIF) of xylose and simultaneous isomerisation and cofermentation (SICF) of glucose-xylose mixture was carried out by the yeastSaccharomyces cerevisiae in the presence of a compatible xylose isomerase. The enzyme converted xylose to xylulose andS. cerevisiae fermented xylulose, along with glucose, to ethanol at pH 5.0 and 30°C. This compatible xylose isomerase fromCandida boidinii, having an optimum pH and temperature range of 4.5–5.0 and 30–50°C respectively, was partially purified and immobilized on an inexpensive, inert and easily available support, hen egg shell. An immobilized xylose isomerase loading of 4.5 IU/(g initial xylose) was optimum for SIF of xylose as well as SICF of glucose-xylose mixture to ethanol byS. cerevisiae. The SICF of 30 g/L glucose and 70 g xylose/L gave an ethanol concentration of 22.3 g/L with yield of 0.36 g/(g sugar consumed) and xylose conversion efficiency of 42.8%.     

Overexpression of bacterial xylose isomerase and yeast host xylulokinase improves xylose alcoholic fermentation in the thermotolerant yeast Hansenula polymorpha

The thermotolerant methylotrophic yeast Hansenula polymorpha is able to ferment xylose to ethanol. To improve characteristics of xylose fermentation, the recombinant strain Delta xyl1 Delta xyl2-ADelta xyl2-B, with deletions of genes encoding first enzymes of xylose utilization (NAD(P)H-dependent xylose reductase and NAD-dependent xylitol dehydrogenases, respectively), was constructed and used as a recipient for co-overexpression of the Escherichia coli xylA gene coding for xylose isomerase and endogenous XYL3 gene coding for xylulokinase. The expression of both genes was driven by the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase promoter. Xylose isomerase activities of obtained transformants amounted to approximately 80% of that of the bacterial host strain. Xylulokinase activities of the transformants increased twofold when compared with the parental strain. The recombinant strains displayed improved ethanol production during the fermentation of xylose.     

Analysis of substrate protection of an immobilized glucose isomerase reactor

The effect of substrate protection on enzyme deactivation was studied in a differential bed and a packed bed reactor using a commercial immobilized glucose isomerase (Swetase, Nagase Co.). Experimental data obtained from differential bed reactor were analyzed based on Briggs-Haldane kinetics in which enzyme deactivation accompanying the protection of substrate was considered. The deactivation constant of the enzyme-substrate complex was found to be about half of that of the free enzyme. The mathematical analysis describing the performance of a packed bed reactor under the considerations of the effects of substrate protection, diffusion resistance, and enzyme deactivation was studied. The system equations for the packed bed reactor were solved using an orthogonal collocation method. The presence of substrate protection and the diffusion effect within the enzyme particles resulted in an axial variation of effectiveness factor, eta(D), along the length of the packed bed. The axial distribution profile of eta(D) was found to be dependent on the operation temperature, Based on the effect of substrate protection, a better substrate feed policy could be theoretically found for promoting productivity in long-term operation. (c) 1993 John Wiley & Sons, Inc.     

Cloning,expression and characterization of xylose isomerase from the marine bacterium Fulvimarina pelagi in Escherichia coli

Production of a xylose isomerase (XI) with high tolerance to the inhibitors xylitol and calcium, and high activity at the low pH and temperature conditions characteristic of yeast fermentations, is desirable for a simultaneous isomerization/fermentation process for cellulosic ethanol production. A putative XI gene (xylA) from the marine bacterium Fulvimarina pelagi was identified by sequence analysis of the F. pelagi genome, and was PCR amplified, cloned, and expressed in Escherichia coli. The rXI was produced in shake flask and fed‐batch fermentations using glucose as the growth substrate. The optimum pH for rXI was approximately 7, although activity was evident at pH as low as 5.5. The purified rXI had a molecular weight in 160 kDA, a V_max of 0.142 U/mg purified rXI, and a K_M for xylose in the range of 1.75–4.17 mM/L at pH 6.5 and a temperature of 35°C. The estimated calcium and xylitol K_I values for rXI in cell‐free extracts were 2,500 mg/L and >50 mM, respectively. The low K_M of the F. pelagi xylose isomerase is consistent with the low nutrient conditions of the pelagic environment. These results indicate that Ca²⁺ and xylitol are not likely to be inhibitory in applications employing the rXI from F. pelagi to convert xylose to xylulose in fermentations of complex biomass hydrolysates. A higher V_max at low pH (<6) and temperature (30°C) would be preferable for use in biofuels production. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1230–1237, 2016     

Purification and characterization of a highly thermostable glucose isomerase produced by the extremely thermophilic eubacterium, Thermotoga maritima

Thermotoga maritima, among the most thermophilic eubacteria currently known, produces glucose isomerase when grow in the presence of xylose. The purified enzyme is a homotetramer with submit molecular Wight of about 45,000. It has a number of features in common with previously described glucose isomerases-pH optimum of 6.5 to 7.5, presence of activesite histidine, requirement for metal cations such as Co(2+) and Mg(2+), and preference for xylose as substrate. In addition, it has significant sequence/structural homology with other glucose isomerases, as shown by both N-terminal sequencing and immunological crossreactivity. The T. maritima enzyme is distinguished by its extreme thermostability-a temperature optimum of 105 to 110 degrees C, and an estimated half-life of 10 minutes at 120 degrees C, pH 7.0. The high degree of thermostability, coupled with a neutral to slightly acid pH optimum, reveal this enzyme to be a promising candidate for improvement of the industrial glucose isomerization process (c) 1993 Wiley & Sons, Inc.     

An efficient mannose selection protocol for tomato that has no adverse effect on the ploidy level of transgenic plants

A protocol for Agrobacterium-mediated transformation with mannose selection was developed for cotyledon petiole, hypocotyl and leaf explants of tomato (Lycopersicon esculentum L. Mill). More than 400 transgenic plants from three tomato varieties were selected with 1% mannose in combination with 0.1–0.5% glucose. Average transformation frequencies ranged from 2.0 to 15.5% depending on the construct, genotype and type of tissue used for transformation. The highest transformation rate was obtained for hypocotyl explants from tomato variety SG048. The ploidy levels of 264 independent transgenic events and 233 non-transgenic plants regenerated from tissue culture were assessed by flow cytometry. The incidence of polyploids within the total population of transgenic plants varied from 10 to 78% and was not significantly different from the non-transgenic population. The greatest variation in the proportion of polyploids was observed in plants derived from different explant types, both in transgenic and non-transgenic regenerants, across three studied genotypes. Transgenic and non-transgenic plants regenerated from leaves included the highest number of normal diploid plants (82–100%), followed by cotyledon petiole-derived plants (63–78%). Transgenic plants produced from hypocotyls contained 22–58% diploids depending on the genotype used in transformation. Results described in this study demonstrate that, although transformation frequencies for leaf tissue are still lower under current protocols, the high percentage of diploids obtained make leaf tissue an attractive transformation target.Abbreviations BAP Benzylaminopurine - MS Murashige-Skoog - MsCHI Medicago sativa chalcone isomerase - PMI Phosphomannose isomerase     

Green fluorescent protein as a visual selection marker for papaya (<Emphasis Type="Italic">Carica papaya</Emphasis> L.) transformation

Chemical-based selection for plant transformation is associated with a number of real and perceived problems that might be avoided through visual selection. We have used green fluorescent protein (GFP), as a visual selectable marker to produce transformed papaya (Carica papaya) plants following microprojectile bombardment of embryogenic callus. GFP selection reduced the selection time from 3 months on a geneticin (G418) antibiotic-containing medium to 3–4 weeks. Moreover, GFP selection increased the number of transformed papaya plants by five-to eightfold compared to selection in the presence of antibiotics. Overall, the use of GFP for selecting transgenic papaya lines improved our throughput for transformation by 15- to 24-fold while avoiding the drawbacks associated with the use of antibiotic resistance-based selection markers.Abbreviations BA: Benzyladenine - 2, 4-D: 2,4-Dichlorophenoxyacetic acid - GFP: Green fluorescent protein - IBA: Indole-3-butyric acid - NAA: -Naphthaleneacetic acid - MS: Murashige and Skoog plant culture mediumCommunicated by R.J. Rose