A genetic transformation system based on trp1 complementation in Candida glycerinogenes

Candida glycerinogenes WL2002-5 is an osmotolerant yeast used for the commercial production of glycerol. The TRP1 gene of Candida glycerinogenes (CgTRP1), encoding phosphoribosylanthranilate isomerase (PRAI) was cloned by complementation of the trp1 mutation of Saccharomyces cerevisiae W303-1A. DNA sequence analysis revealed a 735 bp open reading frame (ORF) encoding a polypeptide of 244 amino acids, which shared 32.9 ~ 49.2% amino acid sequence similarity to PRAI proteins from other species of Saccharomycetales. A trp1 auxotrophic mutant of C. glycerinogenes was selected in medium containing 5-fluoroanthranilic acid, and confirmed by functional and sequence analysis. An integrative vector was constructed with the 18S rDNA gene as integration target and CgTRP1 gene as selectable marker. The trp1 mutant of C. glycerinogenes was transformed with integrative vector, transformants were screened by trp1 complementation. Diagnostic PCR show that the plasmid could be integrated in the site of the 18S rDNA gene of C. glycerinogenes.     

Improved glutathione production by gene expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

To utilize Pichia pastoris to produce glutathione, an intracellular expression vector harboring two genes (gsh1 and gsh2) from Saccharomyces cerevisiae encoding enzymes involved in glutathione synthesis and regulated by the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter was transformed into P. pastoris GS115. Through Zeocin resistance and expression screening, a transformant that had higher glutathione yield (217 mg/L) in flask culture than the host strain was obtained. In fed-batch culture process, this recombinant strain displayed high activity for converting precursor amino acids into glutathione. The glutathione yield and biomass achieved 4.15 g/L and 98.15 g (dry cell weight, DCW)/L, respectively, after 50 h fermentation combined with addition of three amino acids (15 mmol/L glutamic acid, 15 mmol/L cysteine, and 15 mmol/L glycine).     

Developing phosphinothricin-resistant transgenic sunflower (Helianthus?annuus L.) plants

Most investigations on genetic transformations of sunflower have used the neomycin transferase (nptII) gene as the selectable marker. We previously reported a PPT-based selection system for sunflower transformation that uses the bialaphos resistance (bar) gene as the selectable marker and 20 mg/l of phosphinothricin (PPT) as the selective agent. Sunflower (Helianthus annuus L.) variety Skorospeliy 87 was genetically transformed via Agrobacterium tumefaciens strain EHA 105 harbouring the binary plasmid vector pBAR. Two-day-old explants from mature embryos competent for direct shooting were used. Southern blot and ELISA experiments confirmed the stability of expression in two generations of transgenic plants. Transformed plants transferred to soil in the greenhouse exhibited resistance to the herbicide Basta^? at 3 l/ha.     

A genomic island provides <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> ATCC 53993 additional copper resistance: a possible competitive advantage

There is great interest in understanding how extremophilic biomining bacteria adapt to exceptionally high copper concentrations in their environment. Acidithiobacillus ferrooxidans ATCC 53993 genome possesses the same copper resistance determinants as strain ATCC 23270. However, the former strain contains in its genome a 160-kb genomic island (GI), which is absent in ATCC 23270. This GI contains, amongst other genes, several genes coding for an additional putative copper ATPase and a Cus system. A. ferrooxidans ATCC 53993 showed a much higher resistance to CuSO₄ (>100 mM) than that of strain ATCC 23270 (<25 mM). When a similar number of bacteria from each strain were mixed and allowed to grow in the absence of copper, their respective final numbers remained approximately equal. However, in the presence of copper, there was a clear overgrowth of strain ATCC 53993 compared to ATCC 23270. This behavior is most likely explained by the presence of the additional copper-resistance genes in the GI of strain ATCC 53993. As determined by qRT-PCR, it was demonstrated that these genes are upregulated when A. ferrooxidans ATCC 53993 is grown in the presence of copper and were shown to be functional when expressed in copper-sensitive Escherichia coli mutants. Thus, the reason for resistance to copper of two strains of the same acidophilic microorganism could be determined by slight differences in their genomes, which may not only lead to changes in their capacities to adapt to their environment, but may also help to select the more fit microorganisms for industrial biomining operations.     

Construction of a flocculent brewer's yeast strain secreting Aspergillus nigerβ-galactosidase

One way of improving heterologous protein production is to use high cell density systems, one of the most attractive being the flocculating yeast production system. Also, lactose is available in large amounts as a waste product from cheese production processes. The construction of flocculent and non-flocculent brewer's yeast strains secreting β-galactosidase and growing on lactose is presented. A plasmid was constructed coding for an extracellular β-galactosidase of Aspergillus niger and having, as selective marker, the yeast CUP1 gene conferring resistance to copper. This selective marker allows for the transformation of wild-type yeasts. This work represents an important step towards the study of heterologous protein secretion by flocculent cells. Received: 13 January 2000 / Accepted: 23 January 2000     

Improved <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of GNA transgenic sugarcane

Six plasmids carrying a snowdrop lectin (Galanthus nivalis agglutinin, GNA) and one of three selection markers were successfully transferred into two sugarcane cultivars (FN81–745 and Badila) via Agrobacterium-mediated transformation. Agrobacterium strains LBA4404, EHA105 and A281 that harboured a super-binary vector were used for sugarcane transformation. The use of the hygromycin (Hyg) resistance gene (hpt II), phosphinothrincin (PPT) resistance gene (bar) or G418 resistance gene (npt II) as a screenable marker facilitated the initial selection of GNA transgenic sugarcane callus with different efficiencies and helped the rapid segregation of individual transformation events. All the three selective marker genes were controlled by CaMV 35S promoter, while GNA gene was controlled by promoter of RSs-1 (rice sucrose synthase-1) or Ubi (maize ubiquitin). Factors important to successful transformation mediated by Agrobacterium tumefaciens were optimized, which included concentration of A. tumefaciens, medium composition, co-cultivated methods with plant tissue, strain virulence and different selective marker genes. An efficient protocol for sugarcane transformation mediated by A. tumefaciens was established. The GNA gene has been integrated into sugarcane genome as demonstrated by PCR and Southern dot blotting detections. The preliminary results from bioassay demonstrated a significant resistance of the transgenic sugarcane plants to woolly aphid (Ceratovacuna lanigera Zehnther) indicating thus the possibility for obtaining a transgenic sugarcane cultivar with resistance to woolly aphid.     

Overexpression of a sterol C-24(28) reductase increases ergosterol production in Saccharomyces cerevisiae

Three plasmids, pHX4, pHXA4 and pHXC4, containing sterol C-24(28) reductase gene (ERG4) under the control of ERG4, ADH1 or CUP1 promoters, respectively, and the copper resistance gene as the selection marker were constructed, and they were then introduced into Saccharomyces cerevisiae. Ergosterol production in recombinant strains was enhanced. Under the optimal culture condition, ergosterol content in recombinant strains YEH56(pHX4), YEH56(pHXA4) and YEH56(pHXC4) was 1.2, 1.4 and 1.5-fold (47 mg g^–1) of that in the original strain.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of the plant pathogenic fungus <Emphasis Type="Italic">Rosellinia necatrix</Emphasis>

Rosellinia necatrix is a soil-borne root pathogen affecting a wide range of commercially important plant species. The mycelium of R. necatrix was transformed to hygromycin B resistance by an Agrobacterium tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) gene controlled by the heterologous fungal Aspergillus nidulans P-gpd (glyceraldehyde 3-phosphate dehydrogenase) promoter and the trpC terminator. Co-cultivation of R. necatrix strain W1015 and A. tumefaciens strain AGL-1 at 25°C using the binary vector pAN26-CB1300, which contained the hygromycin B resistance cassette based on pAN26 and pCAMBIA1300, resulted in high frequencies of transformation. The presence of the hph gene in the transformants was detected by PCR, and single-copy integration of the marker gene was demonstrated by Southern b lot analy s is. This report of an Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system for insertional mutagenesis in R. necatrix and provide a simple and reliable method for genetic manipulation.     

<Emphasis Type="Italic">Rol</Emphasis> (root loci) gene as a positive selection marker to produce marker-free <Emphasis Type="Italic">Petunia hybrida</Emphasis>

The presence of marker genes conferring antibiotic or herbicide resistance in transgenic plants has been a serious problem for their public acceptance and commercialization. MAT (multi-auto-transformation) vector system has been one of the strategies to excise the selection marker gene from transgenic plants. Agrobacterium tumefaciens strain EHA105 harboring a rol-type MAT vector, pMAT101, was used to produce morphologically normal transgenic Petunia hybrida ‘Dainty Lady’ employing rol gene as the selection marker gene. LacZ gene was used as a model gene of interest. Infected explants were cultured on plant growth regulator (PGR)- and antibiotic-free half-strength MS medium. Sixty-five percent of the infected explants produced hairy roots. The hairy roots were separated and proliferated on 1/2 MS hormone-free medium. Shoots produced from the hairy roots on 1/2 MS medium supplemented with benzylaminopurine (BA) and naphthalene acetic acid (NAA) exhibited hairy root syndrome (Ri syndrome) such as dwarfed, reduced apical dominance, short internodes and increased rooting, but subsequently produced normal-looking marker-free shoots. Molecular analysis of DNA from the hairy roots, shoots with Ri syndrome and morphologically normal shoots revealed that the normal shoots had only LacZ gene, and the removable cassette consisting of rol, R (recombinase) and GUS genes was excised. From this study it can be concluded that the chimeric rol genes can be used as a selection marker for Agrobacterinum-mediated transformation of Petunia hybrida and that the production of marker-free normal transgenic plants is possible without using selective chemical agents employing rol-type MAT vector.     

在无天然游离质粒产甘油假丝酵母中非整合表达合成咖啡酸

【背景】咖啡酸（3,4-二羟基肉桂酸）是一种有多种生物活性和药用价值的天然酚类化合物,产甘油假丝酵母（Candida glycerinogenes）具有咖啡酸前体代谢途径,高耐酸且生长与发酵速率快,是潜在高产咖啡酸的底盘细胞,但无游离载体将影响咖啡酸合成的深入研究。【目的】探索在无天然游离质粒的C. glycerinogenes中构建操作更简便、表达能力更强的游离载体合成咖啡酸的可行性。【方法】筛选自主复制序列（autonomously replicating sequence,ARS）,构建适用于C. glycerinogenes合成咖啡酸的游离载体,并通过改造其ARS位置、标记基因URA5启动子长度、基因表达元件和利用Kozak序列优化表达并合成咖啡酸。【结果】构建的5个分别含不同ARS的载体中,pTGAPU-CA-AOX1t-KLARS在C. glycerinogenes中能自我复制并表达合成咖啡酸的基因,而且当ARS位于目的基因表达元件上游、URA5启动子截短250 bp,或分别采用Kozak序列与终止子URA5t后,咖啡酸产量较改造前均有明显提升,最高产量为初始产量的3.73倍,达29.1 mg/L,高于前期整合表达产量。【结论】在C. glycerinogenes中非整合表达合成咖啡酸且优于整合表达,为今后利用游离载体改造咖啡酸合成代谢途径提供了新工具,同时为其他无游离质粒菌株构建非整合表达体系提供参考。     

Isolation and Characterization of Cytotoxic Compounds from Corn

A host-vector system was constructed in Bacillus megaterium strain NK84–0128, an oxetanocin A producer. The replication origin of an endogeneous plasmid, P–4, was used to construct a potential plasmid vector, pSM5, which had a chloramphenicol resistance gene as a selective marker. Plasmid transformation by a protoplast method was used in B. megaterium strain NK84–0128. The maximum transformation frequency attained with the pSM5 plasmid was 2.0 x 10⁴cfu/µg DNA.     

Adaptation dynamics and evolutionary rescue under sulfide selection in cyanobacteria: a comparative study between Microcystis aeruginosa and Oscillatoria sp. (cyanobacteria)

Experimental evolution studies using cyanobacteria as model organisms are scarce despite the role of cyanobacteria in the evolution of photosynthesis. Three different experimental evolution approaches have been applied to shed light on the sulfide adaptation process, which played a key role in the evolution of this group. We used a Microcystis aeruginosa sulfide‐sensitive strain, unable to grow above ~0.1 mM, and an Oscillatoria sp. strain, isolated from a sulfureous spa (~0.2 mM total sulfide). First, performing a fluctuation analysis design using the spa waters as selective agent, we proved that M. aeruginosa was able to adapt to this sulfide level by rare spontaneous mutations. Second, applying a ratchet protocol, we tested if the limit of adaptation to sulfide of the two taxa was dependent on their initial sulfide tolerance, finding that M. aeruginosa adapted to 0.4 mM sulfide, and Oscillatoria sp. to ~2 mM sulfide, twice it highest tolerance level. Third, using an evolutionary rescue approach, we observed that both speed of exposure to increasing sulfide concentrations (deterioration rate) and populations’ genetic variation determined the survival of M. aeruginosa at lethal sulfide levels, with a higher dependence on genetic diversity. In conclusion, sulfide adaptation of sensitive cyanobacterial strains is possible by rare spontaneous mutations and the adaptation limits depend on the sulfide level present in strain’s original habitat. The high genetic diversity of a sulfide‐sensitive strain, even at fast environmental deterioration rates, could increase its possibility of survival even to a severe sulfide stress.     

Genetic transformation and overexpression of a rice <Emphasis Type="Italic">Hd3a</Emphasis> induces early flowering in <Emphasis Type="Italic">Saussurea involucrata</Emphasis> Kar. et Kir. ex Maxim

Saussurea involucrata is a valuable traditional Chinese medicinal herb. This is the first report of a successful genetic transformation protocol for S. involucrata using Agrobacterium tumefaciens. Leaf explants were incubated with A. tumefaciens strain EHA105 harboring the binary vector pCAMBIA 1301, which contains the hpt gene as a selectable marker for hygromycin resistance and an intron-containing β-glucuronidase gene as a reporter gene. Following co-cultivation, about 23.7% of the explants produced hygromycin-resistant calli on MS basal medium (Murashige and Skoog in Physiol Plant 15: 473–497, 1962) supplemented with 1 mg l⁻¹ benzyladenine (BA), 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 0.1 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), 20 mg l⁻¹ hygromycin, and 500 mg l⁻¹ cefotaxime. Shoots were regenerated following transfer of the resistant calli to shoot induction medium containing 1.5 mg l⁻¹ BA, 0.1 mg l⁻¹ NAA, 0.25 mg l⁻¹ gibberellic acid (GA3), 20 mg l⁻¹ hygromycin, and 250 mg l⁻¹ cefotaxime, and about 67.5% of the resistant calli differentiated into shoots. Finally, 80% of the hygromycin-resistant shoots rooted on MS media supplemented with 0.2 mg l⁻¹ NAA, 20 mg l⁻¹ hygromycin, and 250 mg l⁻¹ cefotaxime. The transgenic nature of the transformants was demonstrated by detection of β-glucuronidase activity in the primary transformants and by Southern blot hybridization analysis. About 16% of the total inoculated leaf explants produced transgenic plants after approximately 5 months. Using this optimized transformation system, a rice ortholog of the Arabidopsis FLOWERING LOCUS T gene, Hd3a, was transferred into S. involucrata. Introduction of this gene caused an early-flowering phenotype in S. involucrata.     

Establishment of a transgene expression system for the marine microalga Schizochytrium by 18S rDNA-targeted homologous recombination

Schizochytrium is an established candidate for commercial production of long chain polyunsaturated fatty acids (PUFAs) that are important in human health and aquaculture. Genetic engineering technology has been applied successfully to increase the metabolites in many organisms. In order to use genetic engineering technology to enhance lipid accumulation in Schizochytrium for economically feasible PUFAs production, the transgene expression system should be established first. In the present study, we investigated a novel transgene expression system in Schizochytrium by 18S rDNA-targeted homologous recombination. The targeting vector pBS-18S-ZeoR contains a portion of 18S rDNA from Schizochytrium and the Zeocin resistance gene (Streptoalloteichus hindustanus bleomycin gene, Sh ble gene) expression cassette. This targeting vector was transformed into Schizochytrium by electroporation and the transformants were then selected on Zeocin-containing plates. The exogenous Sh ble gene has been incorporated into the genome of Schizochytrium according to PCR amplification. More importantly, the majority of the transformants showed similar biomass and total lipid to the wild type strain. Our results suggest that the 18S rDNA is a suitable recombination site and this system could be used to introduce new functional genes into Schizochytrium.     

RecET driven chromosomal gene targeting to generate a RecA deficient Escherichia coli strain for Cre mediated production of minicircle DNA

Background  

Minicircle DNA is the non-replicating product of intramolecular site-specific recombination within a bacterial minicircle producer plasmid. Minicircle DNA can be engineered to contain predominantly human sequences which have a low content of CpG dinucleotides and thus reduced immunotoxicity for humans, whilst the immunogenic bacterial origin and antibiotic resistance marker gene sequences are entirely removed by site-specific recombination. This property makes minicircle DNA an excellent vector for non-viral gene therapy. Large-scale production of minicircle DNA requires a bacterial strain expressing tightly controlled site-specific recombinase, such as Cre recombinase. As recombinant plasmids tend to be more stable in RecA-deficient strains, we aimed to construct a recA ^- bacterial strain for generation of minicircle vector DNA with less chance of unwanted deletions.     

Accumulation of copper in <Emphasis Type="Italic">Trichoderma reesei</Emphasis> transformants,constructed with the modified <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation technique

A modified Agrobacterium tumefaciens-mediated transformation method was established for the construction of mutants with improved copper tolerance and accumulation capability in Trichoderma reesei. One transformant, AT01, exhibited the highest copper accumulation capability. With copper at 0.7 mM, AT01 removed 13 mg copper/g biomass (removal rate of 96%), whereas the wild-type strain removed only 6 mg copper/g biomass (removal rate of 50%). Optimal conditions were a pH of 5.0 at 28°C. The pigment change of Trichoderma mycelia was a potential indicator of copper accumulation. Electron microscopy revealed that copper was mainly accumulated in cell vacuoles.