Analysis of factors responsible for the regeneration to intact cells from sphaeroplasts of Saccharomyces cerevisiae

Abstract: An attempt to produce ethanol from a cellulosic substrate by a single-stage process using intergeneric hybrids obtained from Trichoderma reesei QM 9414 and Saccharomyces cerevisiae NCIM 3288 fusants is described. Two hundred one fused strains were isolated using two different combinations of hybridization, followed by polyethylene glycol treatment of protoplasts derived from parent and mutant strains. Some fusants were either diploids or heterokaryons, as verified by a protoplast fused system obtained by auxotrophic mutants derived from T. reesei QM 9414 and S. cerevisiae NCIM 3288. Of the 201 fusants, 170 were found to be relatively stable. Two fusants (M14 and M62) showed the highest synthesis of ethanol from filter paper cellulose, producing 0.0149 and 0.0191 g l −1 , respectively.

Abstract: Rhizopus oryzae and Rhizopus microsporus strains were screened for their ability to produce fumaric acid on glycerol as the sole carbon source in the medium. After seven days of stationary culture, fumaric acid was assayed by HPLC analysis, and maximum concentrations of 0.3% (w/v) and 0.33% (w/v) were recorded. Protoplast fusion was used to improve fumaric acid production. A selective medium for the fusant culture was composed on the basis of biochemical differences between parental strains, as examined using the Biolog FF MicroPlate TM Fungi Identification Test. Double fusion rounds led to a 1.46-fold increase in fumaric acid productivity relative to the parental strains. Individual Rhizopus fusants demonstrated a various ability to produce fumaric acid from 2.0% (w/v) of glycerol, with the most effective ones producing from 0.2 to 0.27 g@ g !1 of this acid. To date, no studies have been carried out to improve fumaric acid production by Rhizopus with the use of glycerol as the only carbon source in the medium.

Abstract: Rhizopus oryzae and Rhizopus microsporus strains were screened for their ability to produce fumaric acid on glycerol as the sole carbon source in the medium. After seven days of stationary culture, fumaric acid was assayed by HPLC analysis, and maximum concentrations of 0.3% (w/v) and 0.33% (w/v) were recorded. Protoplast fusion was used to improve fumaric acid production. A selective medium for the fusant culture was composed on the basis of biochemical differences between parental strains, as examined using the Biolog FF MicroPlate TM Fungi Identification Test. Double fusion rounds led to a 1.46-fold increase in fumaric acid productivity relative to the parental strains. Individual Rhizopus fusants demonstrated a various ability to produce fumaric acid from 2.0% (w/v) of glycerol, with the most effective ones producing from 0.2 to 0.27 g@ g !1 of this acid. To date, no studies have been carried out to improve fumaric acid production by Rhizopus with the use of glycerol as the only carbon source in the medium.

Abstract: To clarify the involvement of actin in the formation of the yeast cell wall, reverting protoplasts of Schizosaccharomyces pombe were used as a simple model system. Actin of reverting protoplasts was labeled with rhodamine-conjugated phalloidin and observed by conventional fluorescence microscopy and laser scanning confocal microscopy. A close spatial as well as temporal relationship between actin and cell wall formation was observed in protoplast reversion. That is, the site of actin 'dots' in the reverting protoplasts coincided with the site of new wall formation and the timing of rearrangement of actin coincided with the initiation of cell wall formation and with the timing of cell wall expansion. Treatment of reverting protoplasts with cytochalasin D (CD) further clarified the close relationship between actin and cell wall organization. The effect of CD was dose dependent. A high dose of CD caused the absence of actin as well as the complete inhibition of cell wall formation. A low dose of CD caused weakly stained unlocalized actin, which induced grossly aberrant cell wall deposition as well as substantial changes in the morphology of the reverting protoplasts. These results demonstrated that actin is associated with initiation of cell wall formation, the proper deposition of cell wall materials, and maintaining the normal morphology of reverting protoplasts. Scanning electron microscopy revealed the presence of a fibrillar net structure on the surface of non-treated control reverting protoplasts. However, the absence of a fibrillar network on the surface of reverting protoplasts was observed in the presence of a high concentration of CD. Lack of localization of microfibrils as well as poor development of the fibrillar network were also observed in the presence of a low concentration of CD. Recovery experiments confirmed the close relationship between actin and cell wall formation.

Abstract: The ultrastructure of regenerating cell wall in Schizosaccharomyces pombe protoplasts was studied with a high resolution, low voltage scanning electron microscope (LVSEM). In contrast to the transmission electron microscopy, the LVSEM images give three-dimensional information on the cell wall regeneration in yeast protoplasts. We found that, after only a few minutes of incubation, the protoplasts began to show protuberances in a unipolar manner, and a fibrilar network was formed asymmetrically which covered the whole surface of the protoplasts after 5 hr. The network consisted of microfibrils about 8 to 10 nm wide, forming flat and wavy bundles of various widths and lengths, up to about 200 nm wide and 1 micron long, mainly made of yeast glucan. Free ends of microfibrils were seldom found. Interfibrillar spaces were progressively filled with granular particles and finally the complete cell wall was formed after 12 hr. The fibrillar network was destroyed by the digestion with beta (1----3)-glucanase. When protoplasts were regenerating in the presence of aculeacin A, the fibrillar networks were not formed, resulting in incomplete cell wall formation. These observations suggest that beta-glucan is the main component of the microfibrils and that it plays an important role in the formation of the cell wall in S. pombe.

Abstract: SUMMARY: The process of regeneration of Fusarium culmorum protoplasts, obtained by means of strepzyme RA, has been followed. Protoplast regeneration started with the formation of cellular aggregates originating a mass of globular forms which later gave rise to the formation of a pseudomycelial form. Regeneration of protoplasts was formed in three different ways: (a) by means of a chain of yeast-like forms and later originating a germ tube; (b) by direct formation of the germ tube from the protoplast; (c) through a complicated process with the formation of various globular forms and giving rise to the formation of the germ tube at the end. Maximum regeneration, about 80 % of the protoplasts, was found in the presence of 2% (w/v) sucrose (or 1% (w/v) glucose + 1% (w/v) fructose) + 10% (w/v) sorbose + 0·2% agar + the mineral salts of the Czapek medium. No other sugars were able to substitute for sucrose and sorbose. Agar was the best substance for regeneration; gelatin produced an inhibition. Regeneration was greater under acid conditions, alkaline pH values interfering with the phenomenon. The number of nuclei/protoplast varied from 1 to 4, the lack of them in some spherical forms perhaps being the cause of failure to regenerate. No differences were found in the regenerative process as between protoplasts obtained by the use of snail or by microbial lytic enzymes.