Dehydrogenase Promoter Research Articles

The transport of phenylacetic acid across the peroxisomal membrane is mediated by the PaaT protein in Penicillium chrysogenum

Penicillium chrysogenum, an industrial microorganism used worldwide for penicillin production, is an excellent model to study the biochemistry and the cell biology of enzymes involved in the synthesis of secondary metabolites. The well-known peroxisomal location of the last two steps of penicillin biosynthesis (phenylacetyl-CoA ligase and isopenicillin N acyltransferase) requires the import into the peroxisomes of the intermediate isopenicillin N and the precursors phenylacetic acid and coenzyme A. The mechanisms for the molecular transport of these precursors are still poorly understood. In this work, a search was made, in the genome of P. chrysogenum, in order to find a Major Facilitator Superfamily (MFS) membrane protein homologous to CefT of Acremonium chrysogenum, which is known to confer resistance to phenylacetic acid. The paaT gene was found to encode a MFS membrane protein containing 12 transmembrane spanners and one Pex19p-binding domain for Pex19-mediated targeting to peroxisomal membranes. RNA interference-mediated silencing of the paaT gene caused a clear reduction of benzylpenicillin secretion and increased the sensitivity of P. chrysogenum to the penicillin precursor phenylacetic acid. The opposite behavior was found when paaT was overexpressed from the glutamate dehydrogenase promoter that increases phenylacetic acid resistance and penicillin production. Localization studies by fluorescent laser scanning microscopy using PaaT-DsRed and EGFP-SKL fluorescent fusion proteins clearly showed that the protein was located in the peroxisomal membrane. The results suggested that PaaT is involved in penicillin production, most likely through the translocation of side-chain precursors (phenylacetic acid and phenoxyacetic acid) from the cytosol to the peroxisomal lumen across the peroxisomal membrane of P. chrysogenum.

Constitutive high-level expression of a codon-optimized β-fructosidase gene from the hyperthermophile Thermotoga maritima in Pichia pastoris

Enzymes for use in the sugar industry are preferred to be thermotolerant. In this study, a synthetic codon-optimized gene encoding a highly thermostable β-fructosidase (BfrA, EC 3.2.1.26) from the bacterium Thermotoga maritima was expressed in the yeast Pichia pastoris. The gradual increase of the transgene dosage from one to four copies under the control of the constitutive glyceraldehyde 3-phosphate dehydrogenase promoter had an additive effect on BfrA yield without causing cell toxicity. Maximal values of cell biomass (115g/l, dry weight) and overall invertase activity (241U/ml) were reached at 72h in fed-batch fermentations using cane sugar as the main carbon source for growth. Secretion driven by the Saccharomyces cerevisiae α-factor signal peptide resulted in periplasmic retention (44%) and extracellular release (56%) of BfrA. The presence of N-linked oligosaccharides did not influence the optimal activity, thermal stability, kinetic properties, substrate specificity, and exo-type action mode of the yeast-secreted BfrA in comparison to the native unglycosylated enzyme. Complete inversion of cane sugar at initial concentration of 60% (w/v) was achieved by periplasmic BfrA in undisrupted cells reacting at pH 5.5 and 70°C, with average productivity of 4.4g of substrate hydrolyzed per grams of biomass (wet weight) per hour. The high yield of fully active glycosylated BfrA here attained by recombinant P. pastoris in a low-cost fermentation process appears to be attractive for the large-scale production of this thermostable enzyme useful for the manufacture of inverted sugar syrup.

Heterologous protein expression in Pichia thermomethanolica BCC16875, a thermotolerant methylotrophic yeast and characterization of N-linked glycosylation in secreted protein

This study describes Pichia thermomethanolica BCC16875, a new methylotrophic yeast host for heterologous expression. Both methanol-inducible alcohol oxidase (AOX1) and constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoters from Pichia pastoris were shown to drive efficient gene expression in this host. Recombinant phytase and xylanase were expressed from both promoters as secreted proteins, with the former showing different patterns of N-glycosylation dependent on the promoter used and culture medium. In addition, growth temperature also had an effect on N-glycan modification of cell wall mannoproteins. The major glycoprotein oligosaccharide species produced from P.thermomethanolica BCC16875 is Man(8-12) GlcNAc(2) , which is similar to that from other methylotrophs. Moreover, mannosylphosphate and α-1,6- and α-1,2-linked mannose modifications of heterologous secreted protein were also detected. The attainably high level of protein production in complement to distinctive thermotolerance rarely found in other industrial yeasts makes this microorganism an attractive host for large-scale fermentation.

Constitutive expression of Botrytis aclada laccase in Pichia pastoris

The heterologous expression of laccases is important for their large-scale production and genetic engineering—a prerequisite for industrial application. Pichia pastoris is the preferred expression host for fungal laccases. The recently cloned laccase from the ascomycete Botrytis aclada (BaLac) has been efficiently expressed in P. pastoris under the control of the inducible alcohol oxidase (AOX1) promoter. In this study, we compare these results to the constitutive expression in the same organism using the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. The results show that the amounts of BaLac produced with the GAP system (517 mgL-1) and the AOX1 system (495 mgL-1) are comparable. The constitutive expression is, however, faster, and the specific activity of BaLac in the culture supernatant is higher (41.3 Umg-1 GAP, 14.2 Umg-1 AOX1). In microtiter plates, the constitutive expression provides a clear advantage due to easy manipulation (simple medium, no methanol feeding) and fast enzyme production (high-throughput screening assays can already be performed after 48 h).

Codon optimization, constitutive expression and antimicrobial characterization of hen egg white lysozyme (HEWL) in Pichia pastoris

Fusarium oxysporum (F. oxysporum) and Verticillium dahlia (V. dahlia) causes severe cotton disease in China and other cotton-producing countries. Hen egg white lysozyme (HEWL) has antimicrobial properties. In this study, a codon-optimized HEWL gene was synthesized and cloned into the yeast expression vector, pPIC9K, under the control of the Pichia pastoris (P. pastoris) glyceraldehyde-3phosphate dehydrogenase promoter (pGAP). Results showed that codon-optimized HEWL (oHEWL) was constitutively expressed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the molecular weight of recombinant HEWL (roHEWL) was 14 kDa which corresponds to the standard HEWL. The expression of the roHEWL reached to 54 mg/L. Activity of the roHEWL was 1680 U/mL. The optimum pH for roHEWL was from 6.0 to 7.0, and the optimum temperature was 55°C. In vitro antimicrobial activity assay revealed that roHEWL can lyse cell walls of the gram positive bacteria, Micrococcus lysodeikticus (M. lysodeikticus). In vivo studies showed that it inhibits plant fungi, F. oxysporum and V. dahlia. roHEWL anti-fungal properties might be useful for future genetically engineered cotton plant resistance against pathogenic fungal disease.

The secreted lipase FGL1 is sufficient to restore the initial infection step to the apathogenic Fusarium graminearum MAP kinase disruption mutant Δgpmk1

Mitogen activated protein (MAP) kinases are key components in signalling networks. In Fusarium graminearum, one of the most devastating fungal plant pathogens, two MAP kinases are known to be involved in pathogenicity, Mgv1 and the Gibberella pathogenicity MAP kinase Gpmk1. Δgpmk1 mutants with a disrupted GPMK1 gene are unable to infect wheat spikelets. They exhibit altered secretion of several extracellular enzymes. Among those, the lipase FGL1 is known to be a major virulence factor of F. graminearum. FGL1 gene expression was decreased in Δgpmk1 strains during wheat head infection. To uncouple FGL1 expression from Gpmk1 activity, we generated Δgpmk1 strains that constitutively express FGL1 under control of the Cochliobolus heterostrophus glyceraldehyde 3-phosphate dehydrogenase (GPD) promoter. The level of extracellular lipolytic activity of these strains in culture was comparable to the wild-type. These mutants showed fully restored conidiation and partial complementation of defects in development that were reported from the Δgpmk1 mutant. They also partially complemented the apathogenic disease phenotype of the Δgpmk1 mutants causing lesions in directly inoculated wheat spikelets. But in contrast to wild-type, their growth was restricted to directly inoculated spikelets. A barrier-like formation was observed at the rachis node. Based on these results, we could show that the lipase FGL1 is necessary but not sufficient to restore complete pathogenicity to the apathogenic Δgpmk1 mutant. Hence, we hypothesize that the MAP kinase Gpmk1 is involved in the regulation of additional factors required for complete virulence of F. graminearum.

Molecular genetic analysis reveals that a nonribosomal peptide synthetase-like (NRPS-like) gene in Aspergillus nidulans is responsible for microperfuranone biosynthesis

Genome sequencing of Aspergillus species including Aspergillus nidulans has revealed that there are far more secondary metabolite biosynthetic gene clusters than secondary metabolites isolated from these organisms. This implies that these organisms can produce additional secondary metabolites, which have not yet been elucidated. The A. nidulans genome contains 12 nonribosomal peptide synthetase (NRPS), one hybrid polyketide synthase/NRPS, and 14 NRPS-like genes. The only NRPS-like gene in A. nidulans with a known product is tdiA, which is involved in terrequinone A biosynthesis. To attempt to identify the products of these NRPS-like genes, we replaced the native promoters of the NRPS-like genes with the inducible alcohol dehydrogenase (alcA) promoter. Our results demonstrated that induction of the single NRPS-like gene AN3396.4 led to the enhanced production of microperfuranone. Furthermore, heterologous expression of AN3396.4 in Aspergillus niger confirmed that only one NRPS-like gene, AN3396.4, is necessary for the production of microperfuranone.

Application of adaptive DO-stat feeding control to Pichia pastoris X33 cultures expressing a single chain antibody fragment (scFv)

In this study, fed-batch cultures of a Pichia pastoris strain constitutively expressing a single chain antibody fragment (scFv) under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter were performed in a pilot 50 L bioreactor. Due to the very high cell density achieved within the first 75 h, typically between 140 and 160 g-DCW/L of dry cell weight (DCW), most of the scFv is produced under hard oxygen transfer limitation. To improve scFv productivity, a direct adaptive dissolved oxygen (DO)-stat feeding controller that maximizes glycerol feeding under the constraint of available oxygen transfer capacity was developed and applied to this process. The developed adaptive controller enabled to maximize glycerol feeding through the regulation of DO concentration between 3 and 5 % of saturation, thereby improving process productivity. Set-point convergence dynamics are characterized by a fast response upon large perturbations to DO, followed by a slower but very robust convergence in the vicinity of the boundary with almost imperceptible overshoot. Such control performance enabled operating closer to the 0 % boundary for longer periods of time when compared to a traditional proportional-integral-derivative algorithm, which tends to destabilize with increasing cell density.

Production and characterisation of recombinant α-l-arabinofuranosidase for production of xylan hydrogels

A recombinant strain of the protease-deficient, non-acidifying pH mutant Aspergillus niger D15 (A. niger D15 [abfB]) strain was developed to secrete α-L-arabinofuranosidase (AbfB) free of endo-1,4-β-xylanases for selective hydrolysis of xylan into hydrogels. The A. niger D15 [abfB] strain expressed the α-L-arabinofuranosidase abfB gene under the transcriptional control of the glyceraldehyde-3-phosphate dehydrogenase promoter (gpd(P)) and glucoamylase terminator (glaA(T)) in fermentation cultures containing 10 % glucose. The yield, activity, purity, kinetics and ability of the recombinant AbfB to selectively hydrolyse xylans into hydrogels were assessed. The recombinant AbfB secreted in 125-mL shake flasks and 10-L bioreactor fermentation cultures had specific activities against ρ-nitrophenyl-α-arabinofuranoside of up to 4.4 and 2.7 U g⁻¹ (dry weight), respectively. In addition, the recombinant AbfB was present as a single protein species on silver-stained 10 % sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The recombinant AbfB had optimal activity at 40-55 °C and pH 3.0 to pH 5.0 and was stable at temperature and pH of up to 60 °C and pH 6.0, respectively. About 20 % of the available arabinose in the xylan was released by the recombinant AbfB from the hydrolysis of low viscosity wheat and oat spelt arabinoxylans and about 9 and 5 % from bagasse and bamboo arabinoglucuronoxylans, respectively, that led to the formation of the hydrogels. Therefore, the constructed A. niger D15 [abfB] strain presented a microbial system for the production of recombinant AbfB with the required purity for the modification of xylans into hydrogels.

Improved biomass saccharification by Trichoderma reesei through heterologous expression of lacA gene from Trametes sp. AH28-2

The Trametes sp. AH28-2 laccase gene lacA fused to cellobiohydrolase I signal peptide coding sequence was heterologously expressed in T. reesei. The lacA cDNA was under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase promoter. Native PAGE analysis indicated that two transformants, L8 and L38, were able to secrete recombinant laccase A, and their laccase activities corresponding to ABTS oxidation reached 3.62 IUml(-1) and 1.50 IUml(-1) respectively. Most of the characteristics of the recombinant laccase were similar to those of the native enzyme. Reducing sugar yields of L8 and L38 obtained from saccharification of corn residue by crude enzyme increased by 31.3% and 71.6% respectively compared to the host strain. These results indicated that the engineering strains developed in this work could be potentially used for laccase production and tailoring cellulase properties with laccase proteins through genetic manipulation would be a feasible strategy to improve saccharification efficiency of biomass by cellulase preparation.

Glucose-induced production of a Penicillium purpurogenum xylanase by Aspergillus nidulans

The heterologous secretion of xylanase B from Penicillium purpurogenum using glucose as inducer was performed in Aspergillus nidulans. For this purpose, plasmid pEVXB, containing the xylanase B cDNA (including its own signal peptide) under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter, was constructed and used to transform A. nidulans. Analysis of transformed clones showed that several of them secreted extracellular xylanase activity when grown in a medium containing glucose. The clone showing the highest xylanase activity was chosen for further work. When this clone was grown on glucose, xylanase activity (0.72U/ml), was detected in the culture supernatant. This was confirmed by a zymogram analysis and by the amplification of xynB cDNA from this clone. To our knowledge, this is the first example of the production of a xylanase from Penicillium in heterologous fungal hosts using glucose as inducer.

A Novel Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Promoter for Expressing Transgenes in the Halotolerant Alga Dunaliella salina

A major challenge for efficient transgene expression in Dunaliella salina is to find strong endogenous promoters to drive the transgene expression. In the present study, a novel glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter was cloned and used to drive expressions of the bialaphos resistance (bar) gene and of the N-terminal fragment of human canstatin (Can-N). The results showed that the bar gene was transcribed by the GAPDH promoter and integrated into the genome of the transformants of D. salina. Furthermore, the PCR identification, Southern and western blots indicated that Can-N was expressed in transgenic D. salina, demonstrating that the promoter of the D. salina GAPDH gene is suitable for driving expression of heterologous genes in transgenic D. salina.

Expression of recombinant green fluorescent protein in Bacillus methanolicus

Microbial biocatalysts are used in a wide range of industries to produce large scale quantities of proteins, amino acids, and commodity chemicals. While the majority of these processes use glucose or other low-cost sugars as the substrate, Bacillus methanolicus is one example of a biocatalyst that has shown sustained growth on methanol as a carbon source at elevated temperature (50-53°C optimum) resulting in reduced feed and utility costs. Specifically, the complete chemical process enabled by this approach takes methane from natural gas, and following a low-cost conversion to methanol, can be used for the production of high value products. In this study, production of recombinant green fluorescent protein (GFPuv) by B. methanolicus is explored. A plasmid was constructed that incorporates the methanol dehydrogenase (mdh) promoter of B. methanolicus MGA3 together with the GFPuv gene. The plasmid, pNW33N, was shown to be effective for expression in other Bacillus strains, although not previously in B. methanolicus. A published electroporation protocol for transformation of B. methanolicus was modified to result in expression of GFP using plasmid pNW33N-mdh-GFPuv (pNmG). Transformation was confirmed by both agarose gel electrophoresis and by observation of green fluorescence under UV light exposure. The mass yield of cells and protein were measured in shake flask experiments. The optimum concentration of methanol for protein production was found to be at 200 mM. Higher concentrations than 200 mM resulted in slightly higher biomass production but lower amounts of recombinant protein.

Constitutive expression of hydrophobin HFB1 from Trichoderma reesei in Pichia pastoris and its pre‐purification by foam separation during cultivation

AbstractHydrophobins are small surface‐active proteins that have considerable potential for use in applications ranging from medical and technical coatings, separation technologies, biosensors, and personal care. Their wider use would be facilitated by the availability of recombinant tailor‐made hydrophobins. We successfully expressed the class II hydrophobin HFB1 from Trichoderma reesei in Pichia pastoris under the control of the constitutive GAP (glyceraldehyde 3‐phosphate dehydrogenase) promoter. Avoiding the use of the AOX1 (alcohol oxidase 1) promoter prevents the costs and risks associated with the storage and delivery of methanol used as an inducer. Efficient secretion of hydrophobin was achieved using either the alpha‐factor prepro‐peptide or the native secretion signal of HFB1. The secreted hydrophobins have been isolated with a purity of up to 70% using in situ foam separation during the cultivation process. Coating experiments and surface pressure measurements demonstrated the activity of the hydrophobins. An immunodot assay showed the accessibility of carboxyterminally fused tags of the hydrophobin, which is necessary for potential applications using functionalized hydrophobins. The presented data show that Pichia pastoris is a suitable system for production of constitutively expressed and secreted active hydrophobin, allowing for in situ pre‐purification using foam separation.

Glucose-induced production of a <i>Penicillium purpurogenum</i> xylanase by <i>Aspergillus nidulans</i>

The heterologous secretion of xylanase B from Penicillium purpurogenum using glucose as inducer was performed in Aspergillus nidulans. For this purpose, plasmid pEVXB, containing the xylanase B cDNA (including its own signal peptide) under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter, was constructed and used to transform A. nidulans. Analysis of transformed clones showed that several of them secreted extracellular xylanase activity when grown in a medium containing glucose. The clone showing the highest xylanase activity was chosen for further work. When this clone was grown on glucose, xylanase activity (0.72 U/ml), was detected in the culture supernatant. This was confirmed by a zymogram analysis and by the amplification of xynB cDNA from this clone. To our knowledge, this is the first example of the production of a xylanase from Penicillium in heterologous fungal hosts using glucose as inducer.

Enhancement of xylitol production in Candida tropicalis by co-expression of two genes involved in pentose phosphate pathway

The yeast Candida tropicalis produces xylitol, a natural, low-calorie sweetener whose metabolism does not require insulin, by catalytic activity of NADPH-dependent xylose reductase. The oxidative pentose phosphate pathway (PPP) is a major basis for NADPH biosynthesis in C. tropicalis. In order to increase xylitol production rate, xylitol dehydrogenase gene (XYL2)disrupted C. tropicalis strain BSXDH-3 was engineered to co-express zwf and gnd genes which, respectively encodes glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6-PGDH), under the control of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter. NADPH-dependent xylitol production was higher in the engineered strain, termed "PP", than in BSXDH-3. In fermentation experiments using glycerol as a co-substrate with xylose, strain PP showed volumetric xylitol productivity of 1.25 g l(-1) h(-1), 21% higher than the rate (1.04 g l(-1) h(-1)) in BSXDH-3. This is the first report of increased metabolic flux toward PPP in C. tropicalis for NADPH regeneration and enhanced xylitol production.

Xylitol production is increased by expression of codon-optimized Neurospora crassa xylose reductase gene in Candida tropicalis

Xylose reductase (XR) is the first enzyme in d-xylose metabolism, catalyzing the reduction of d-xylose to xylitol. Formation of XR in the yeast Candida tropicalis is significantly repressed in cells grown on medium that contains glucose as carbon and energy source, because of the repressive effect of glucose. This is one reason why glucose is not a suitable co-substrate for cell growth in industrial xylitol production. XR from the ascomycete Neurospora crassa (NcXR) has high catalytic efficiency; however, NcXR is not expressed in C. tropicalis because of difference in codon usage between the two species. In this study, NcXR codons were changed to those preferred in C. tropicalis. This codon-optimized NcXR gene (termed NXRG) was placed under control of a constitutive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter derived from C. tropicalis, and integrated into the genome of xylitol dehydrogenase gene (XYL2)-disrupted C. tropicalis. High expression level of NXRG was confirmed by determining XR activity in cells grown on glucose medium. The resulting recombinant strain, LNG2, showed high XR activity (2.86 U (mg of protein)−1), whereas parent strain BSXDH-3 showed no activity. In xylitol fermentation using glucose as a co-substrate with xylose, LNG2 showed xylitol production rate 1.44 g L−1 h−1 and xylitol yield of 96% at 44 h, which were 73 and 62%, respectively, higher than corresponding values for BSXDH-3 (rate 0.83 g L−1 h−1; yield 59%).

An In Vitro Test to Screen Skin Sensitizers Using a Stable THP-1–Derived IL-8 Reporter Cell Line, THP-G8

Several studies have suggested that interleukin (IL)-8 can serve as a biomarker for discrimination of skin sensitizers from nonsensitizers. We established a stable THP-1-derived IL-8 reporter cell line, THP-G8, which harbors SLO and SLR luciferase genes under the control of IL-8 and glyceraldehyde 3-phosphate dehydrogenase promoters, respectively. After 6 h treatment with chemicals, normalized SLO luciferase activity (nSLO-LA) was calculated by dividing SLO-LA by SLR-LA, and the fold induction of nSLO-LA (FInSLO-LA) was calculated by dividing nSLO-LA of chemically treated cells by that of nontreated cells. The nSLO-LA of THP-G8 cells increased in response to lipopolysaccharide (LPS) and several sensitizers. The FInSLO-LA in THP-G8 cells induced by LPS or sensitizers positively correlated with their induction of IL-8 messenger RNA in THP-1 cells. The nSLO-LA value of THP-G8 cells was significantly increased (FInSLO-LA ≥ 1.4) by 13 of the 15 sensitizers as well as by 5 of the 7 nonsensitizers. Interestingly, pretreatment with N-acetylcysteine suppressed the increase in FInSLO-LA induced by all sensitizers (inhibition index (II) ≤ 0.8) but did not suppress that induced by most of the nonsensitizers. We then evaluated the performance of this assay using values of FInSLO-LA ≥ 1.4 and II ≤ 0.8 in at least two of three independent experiments as the criteria of a sensitizer, which resulted in test accuracies of 82% for the 22 chemicals used and of 88% for the chemicals proposed by European Center for the Validation of Alternative Methods. This newly developed assay is a candidate replacement for animal tests of skin sensitization because of its accuracy, convenience, and high throughput performance.

Transcription Factor Rational Design Improves Directed Differentiation of Human Mesenchymal Stem Cells Into Skeletal Myocytes

There is great interest in transdifferentiating cells from one lineage into those of another and in dedifferentiating mature cells back into a stem/progenitor cell state by deploying naturally occurring transcription factors (TFs). Often, however, steering cellular differentiation pathways in a predictable and efficient manner remains challenging. Here, we investigated the principle of combining domains from different lineage-specific TFs to improve directed cellular differentiation. As proof-of-concept, we engineered the whole-human TF MyoDCD, which has the NH(2)-terminal transcription activation domain (TAD) and adjacent DNA-binding motif of MyoD COOH-terminally fused to the TAD of myocardin (MyoCD). We found via reporter gene and marker protein assays as well as by a cell fusion readout system that, targeting the TAD of MyoCD to genes normally responsive to the skeletal muscle-specific TF MyoD enforces more robust myogenic reprogramming of nonmuscle cells than that achieved by the parental, prototypic master TF, MyoD. Human mesenchymal stem cells (hMSCs) transduced with a codon-optimized microdystrophin gene linked to a synthetic striated muscle-specific promoter and/or with MyoD or MyoDCD were evaluated for complementing the genetic defect in Duchenne muscular dystrophy (DMD) myocytes through heterotypic cell fusion. Cotransduction of hMSCs with MyoDCD and microdystrophin led to chimeric myotubes containing the highest dystrophin levels.

Development of a simple and efficient transformation system for the basidiomycetous medicinal fungus Ganoderma lucidum

In this study, we report the development of a simple and efficient system for genetic transformation of the medicinal fungus Ganoderma lucidum. Various parameters were optimized to obtain successful Agrobacterium tumefaciens-mediated transformation. Co-cultivation of bacteria and protoplast at a ratio of 1,000:1 at 25°C in medium containing 0.2mM acetosyringone was found to be the optimum condition for high efficiency transformation. Four plasmids, each carrying a different promoter driving the expression of an antibiotic resistance marker, were tested. The construct carrying the Ganoderma lucidum glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter showed good transformation efficiency, whereas constructs with the GPD promoter from ascomycetes were ineffective. Our analysis showed that over 70% of the transformants tested remained mitotically stable even after five successive rounds of subculturing. We were able to detect the expression of EGFP and GUS reporter genes in the Ganoderma lucidum transformants by fluorescence imaging and histochemical staining assays respectively. Our results demonstrate a new transgenic approach that will facilitate Ganoderma lucidum research.