Microalgae protoplasts isolation and fusion for biotechnology research

Protoplasts are microbial or vegetable cells lacking a cell wall. These can be obtained from microalgae by an enzymatic hydrolysis process in the presence of an osmotic stabilizer. In general, protoplasts are experimentally useful in physiological, geneticand bio-chemical studies, so their acquisition and fusion will continue to be an active research area in modern biotechnology. The fusion of protoplasts in microalgae constitutes a tool for strain improvement because it allows both intra and interspecific genetic recombina-tion, resulting in organisms with new or improved characteristics of industrial interest. In this review we briefly describe themethod-ology for obtaining protoplasts, as well as fusion methods and the main applications of microalgal platforms.

Preparation and Regeneration of Protoplasts from the Ethyl Vincamine Producing Fungus CH1 (Geomyces sp.)

Vinpocetine, a semi-synthetic compound derived from the alkaloid vincamine, exhibits effective pharmacological activities for the treatment and prevention of cerebrovascular circulation and vascular cognitive disorders. Vinpocetine can be produced through a one-step chemical reaction beginning with ethyl vincamine, and a two-step chemical reaction beginning with vincamine. In our previous study, the endophytic fungus CH1, Geomyces sp., was isolated and identified as a producer of ethyl vincamine, which was first obtained by endophytic fungal fermentation. However, the production was largely limited. Fungal protoplasts are a valuable experimental tool for physiological and genetic research such as protoplast fusion, gene transfer and metabolite production. In this paper, we optimized some key factors for the preparation and regeneration of protoplasts from strain CH1. Using an enzymes mixture consisting of cellulase (2.0%, w/v), glusulase (3.0%, w/v) and driselase (1.0%, w/v) in osmotic stabilizer (0.7 mol/L NaCl), the highest yield of protoplasts (6.78×107/mL) was obtained with mycelia after 72 h at pH 5.0-6.0 by digesting for 1.5 h at 30°C. After purification of the prepared protoplasts, they were regenerated in the regeneration medium using a bilayer plate culture method.

Protein Kinase C and the stress response pathways are required for kinetochore homeostasis

Kinetochores serve both a structural role linking chromosomes to the mitotic spindle and a regulatory role, controlling the timing of mitosis via the spindle assembly checkpoint. To identify proteins that regulate the kinetochore we used a genome-wide fluorescence microscopy approach. We combined an array of mutants of either non-essential gene deletions or essential temperature-sensitive alleles with fluorescently tagged spindle pole bodies (centrosome) and outer kinetochores. Quantitative and qualitative analysis revealed mutants that affect the levels and distribution of kinetochores respectively. These mutants are enriched for those involved in mRNA processing, chromatin organization, DNA replication/repair and mitosis. Our data show that the Pkc1 kinase maintains the kinetochore focus via its ability to prevent cell stress and this phenotype is rescued by an osmotic stabilizer. These data support the notion that kinetochore and microtubule homeostasis are perturbed by the stress response pathways. Hence this observation provides a candidate mechanism for extracellular stress leading to chromosome segregation defects.

Basidiospore and Protoplast Regeneration from Raised Fruiting Bodies of Pathogenic Ganoderma boninense

Ganoderma boninense, a phytopathogenic white rot fungus had sought minimal genetic characterizations despite huge biotechnological potentials. Thus, efficient collection of fruiting body, basidiospore and protoplast of G. boninense is described. Matured basidiocarp raised under the glasshouse conditions yielded a total of 8.3 × 104 basidiospores/ml using the low speed centrifugation technique. Mycelium aged 3-day-old treated under an incubation period of 3 h in lysing enzyme from Trichoderma harzianum (10 mg/ml) suspended in osmotic stabilizer (0.6 M potassium chloride and 20 mM dipotassium phosphate buffer) yielded the highest number of viable protoplasts (8.9 × 106 single colonies) among all possible combinations tested (regeneration media, age of mycelium, osmotic stabilizer, digestive enzyme and incubation period).

Efficient production of Aschersonia placenta protoplasts for transformation using optimization algorithms

The insect pathogenic fungus Aschersonia placenta is a highly effective pathogen of whiteflies and scale insects. However, few genetic tools are currently available for studying this organism. Here we report on the conditions for the production of transformable A. placenta protoplasts using an optimized protocol based on the response surface method (RSM). Critical parameters for protoplast production were modelled by using a Box–Behnken design (BBD) involving 3 levels of 3 variables that was subsequently tested to verify its ability to predict protoplast production (R2 = 0.9465). The optimized conditions resulted in the highest yield of protoplasts ((4.41 ± 0.02) × 107 cells/mL of culture, mean ± SE) when fungal cells were treated with 26.1 mg/mL of lywallzyme for 4 h of digestion, and subsequently allowed to recover for 64.6 h in 0.7 mol/L NaCl–Tris buffer. The latter was used as an osmotic stabilizer. The yield of protoplasts was approximately 10-fold higher than that of the nonoptimized conditions. Generated protoplasts were transformed with vector PbarGPE containing the bar gene as the selection marker. Transformation efficiency was 300 colonies/(μg DNA·107 protoplasts), and integration of the vector DNA was confirmed by PCR. The results show that rational design strategies (RSM and BBD methods) are useful to increase the production of fungal protoplasts for a variety of downstream applications.

Production and regeneration of protoplasts from orchid Mycorrhizal Fungi Epulorhiza repens and Ceratorhiza sp.

The aim of this work was to study the standardization of conditions to obtain and regenerate Epulorhiza repens and Ceratorhiza sp. protoplasts. For E. repens, the largest number of protoplasts (8.0 × 10(6) protoplasts/mL) was obtained in 0.6 M KCl, using 15 mg/mL of Lysing Enzymes, and 2-day-old fungal mycelium. When 0.5 M sucrose was used as osmotic stabilizer, the highest frequency of regeneration was achieved (8.5 %); 80.0 % of protoplasts were nucleated, and 20.0 % anucleated. For Ceratorhiza sp., the largest number of protoplasts (4.0 × 10(7) protoplasts/mL) was achieved in 0.6 M NaCl, when 15 mg/mL of Lysing Enzymes and 15mg/mL of Glucanex, with 2-day-old fungal mycelium were used. The highest frequency of regeneration was 6.7 % using 0.5 M sucrose as osmotic stabilizer; 88.8 % of protoplasts were nucleated, and 11.2 % anucleated.

Factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum

The present work reports factors affecting the production and regeneration of protoplasts from Colletotrichum lindemuthianum. The usefulness of protoplast isolation is relevant for many different applications and has been principally used in procedures involving genetic manipulation. Osmotic stabilizers, lytic enzymes, incubation time and mycelial age were evaluated in terms of their effects on protoplast yield. The optimal condition for protoplast production included the incubation of young mycelia (48 h) in 0.6 mol l-1 NaCl as the osmotic stabilizer, with 30 mg ml-1 Lysing Enzymes from Trichoderma harzianum for 3 h of incubation. In these conditions protoplasts production was higher than 10(6) protoplatos ml-1 in the digestion mixture, number suitable enough for experiments of transformation in fungi. Sucrose concentrations of 1.2 mol l-1 and 1 mol l-1 were the most suitable osmotic stabilizers for the regeneration after 48 h, with rates of 16.35% and 14.54%, respectively. This study produced an efficient method for protoplast production and reverted them into a typical mycelial morphology using a Colletotrichum lindemuthianum LV115 isolate.

Protein O-Mannosyltransferases B and C Support Hyphal Development and Differentiation in Aspergillus nidulans

ABSTRACT Aspergillus nidulans possesses three pmt genes encoding protein O-d-mannosyltransferases (Pmt). Previously, we reported that PmtA, a member of the PMT2 subfamily, is involved in the proper maintenance of fungal morphology and formation of conidia (T. Oka, T. Hamaguchi, Y. Sameshima, M. Goto, and K. Furukawa, Microbiology 150:1973-1982, 2004). In the present paper, we describe the characterization of the pmtA paralogues pmtB and pmtC. PmtB and PmtC were classified as members of the PMT1 and PMT4 subfamilies, respectively. A pmtB disruptant showed wild-type (wt) colony formation at 30°C but slightly repressed growth at 42°C. Conidiation of the pmtB disruptant was reduced to approximately 50% of that of the wt strain; in addition, hyperbranching of hyphae indicated that PmtB is involved in polarity maintenance. A pmtA and pmtB double disruptant was viable but very slow growing, with morphological characteristics that were cumulative with respect to either single disruptant. Of the three single pmt mutants, the pmtC disruptant showed the highest growth repression; the hyphae were swollen and frequently branched, and the ability to form conidia under normal growth conditions was lost. Recovery from the aberrant hyphal structures occurred in the presence of osmotic stabilizer, implying that PmtC is responsible for the maintenance of cell wall integrity. Osmotic stabilization at 42°C further enabled the pmtC disruptant to form conidiophores and conidia, but they were abnormal and much fewer than those of the wt strain. Apart from the different, abnormal phenotypes, the three pmt disruptants exhibited differences in their sensitivities to antifungal reagents, mannosylation activities, and glycoprotein profiles, indicating that PmtA, PmtB, and PmtC perform unique functions during cell growth.

Genetic variability in regenerated Metarhizium flavoviride protoplasts

Protoplast isolation and regeneration were evaluated in two wild-type and two colour mutant strains of Metarhizium flavoviride. Cultivation in liquid medium, followed by mycelium treatment with Novozym 234 in the presence of KCl 0.7M as osmotic stabilizer, produced 5.05 x 10(6) to 1.15 x 10(7)x mL-1 protoplasts. The percentage of regeneration ranged from 6.65 to 27.92%. Following protoplast regeneration, one strain produced spontaneously stable morphological variant colonies. Although colonies with altered morphology have been reported in bacteria following protoplast regeneration, this is the first time that the same is described in a filamentous fungus. The original strain and one derived variant were tested for sensitivity to the fungicides benomyl and captan.