Protoplast fusion — a tool for genetic manipulation and breeding in industrial microorganisms

Protoplasts can be isolated from microbial cells by enzymatic digestion of the cell wall, in the presence of an osmotic stabilizer. Such protoplasts can be induced to fuse in the presence of agents such as the poly (ethylene glycols). When suitably selected auxotrophic strains are used, the fusion products can be recovered by selection on the basis of nutritional complementation. Cultivation of the protoplasts on a hypertonic growth medium induces regeneration of new cell wall material and their subsequent reversion to the normal cell form of the organism. The protoplast fusion technique has been applied sucessfully to both bacterial and fungal systems leading to the recovery of recombinant progeny. In the fungi, the recovery of non-parental segregants from inter-species crosses has also been demonstrated. In assessing the value of the fusion technique, caution may be necessary at this stage in its application to genetic mapping in bacteria. The behaviour of protoplasts, especially with respect to reversion, could be an additional factor that operates during selection, distorting recombination frequencies. However, the fusion technique, in providing a mechanism by which genetic recombination can be readily achieved, should be of great potential in empirical breeding and strain improvement. These aspects are reviewed.     

Visualization of protoplast fusion and quantitation of recombination in fused protoplasts of auxotrophic strains of Escherichia coli

Protoplast fusion has been used to combine genes from different organisms to create strains with desired properties. A recently developed variant on this approach, genome shuffling, involves generation of a genetically heterogeneous population of a single organism, followed by recursive protoplast fusion to allow recombination of mutations within the fused protoplasts. These are powerful techniques for engineering of microbial strains for desirable industrial properties. However, there is a prevailing opinion that it will be difficult to use these methods for engineering of Gram-negative bacteria because the outer membrane makes protoplast fusion more difficult. Here we describe the successful use of protoplast fusion in Escherichia coli. Using two auxotrophic strains of E. coli, we obtained prototrophic strains by recombination in fused protoplasts at frequencies of 0.05-0.7% based on the number of protoplasts subjected to fusion. This frequency is three-four orders of magnitude better than those previously reported for recombination in fused protoplasts of Gram-negative bacteria such as E. coli and Providencia alcalifaciens.     

An interspecific aggregate cell capable of cell wall regeneration

Intraspecifically fused protoplasts of soybean were found tobe capable of cell wall regeneration and limited mitotic activityover a period of several weeks. When interspecific fusion wasaccomplished between soybean and crabgrass protoplasts a cellwall was regenerated around the aggregate, but no mitotic activitywas observed. (Received October 26, 1971; )     

Electron microscopic study of Bacillus subtilis protoplast fusion.

When protoplasts derived from sporulating cells of Bacillus subtilis were fused by exposure to polyethylene glycol (PEG) and fixed immediately thereafter, protoplasts with two enclosed prespores could be seen by electron microscope. The number of fusion events was greatly increased, and multiply fused protoplasts appeared, when the PEG-treated suspension was diluted in hypertonic broth and reincubated before fixation. This post-PEG incubation effect is taken to indicate a fusion mechanism of two steps: a short, PEG-dependent step of membrane activation, followed by a slow, metabolism-requiring step completing fusion. When prespore-bearing protoplasts from two genetically different strains were mixed and fused, the extent of fusion could also be followed by counting clones of recombinant bacteria. Maximal from the start, their number (1% of each parent type protoplast present) was unaffected by post-PEG incubation. Fusion in this case is apparently completed after plating on the wall-regeneration medium. After optimal post-PEG incubation, the majority of the protoplasts were seen to participate in fusion, and the cytological fusion observed, corrected for wall-regeneration frequency, accounted quantitatively for the prototrophic bacteria eventually recovered. These results are in good agreement with those obtained independently by Sanchez-Rivas and Garro (J. Bacteriol. 137:1340--1345, 1979).     

甘蓝型油菜与芝麻菜体的细胞杂交

为拓宽油菜育种的基因资源库, 改良油菜品种, 以甘蓝型油菜(Brassica napus)花油3号下胚轴和芝麻菜(Eruca sativa)下胚轴为材料分离制备原生质体; 然后采用PEG-高Ca2+-高pH法进行原生质体融合, 当PEG浓度为35%, 原生质体融合密度为5×105个/mL时, 融合25 min时, 融合率可达18.2%。融合后在培养密度为1×105个/mL时, 以附加1.0 mg/L 2,4-D +0.5 mg/L 6-BA+0.5 mg/L NAA+ 200 mg/L肌醇+300 mg/L水解酪蛋白的改良的KM8p为融合体培养基, 以0.1 mol/L 蔗糖+0.2 mol/L葡萄糖+0.2 mol/L甘露醇作渗透稳定剂进行液体浅层培养, 效果较好, 愈伤组织再生率最高为6.8%。将融合体再生的小愈伤组织转移至培养基(B5无机盐+0.087 mol/L蔗糖+0.2 mg/L 2, 4-D+0.5 mg/L NAA+0.2 mg/L 6-BA+ 0.5% Agar, pH 5.8)上增殖培养, 待愈伤组织长至直径为3~5 mm时, 及时将其转至分化培养基(MS无机盐+0.087 mol/L 蔗糖+0.1 mg/L IAA+0.8 mg/L 6-BA+0.8% Agar, pH 5.8)中诱导不定芽再生, 芽分化率为35.7%。当不定芽长为2~3 cm时, 将其切下转入附加0.5 mg/L IBA+0.2 mg/L 6-BA的1/2MS生根培养基中诱导生根, 14 d左右即可形成再生植株, 生根率可达88%。同时, 以紫外线(60 μW/cm2)照射芝麻菜原生质体, 进行不对称融合, 照射2 min的获得了愈伤组织和再生植株, 照射4 min的只获得愈伤组织, 而照射5 min以上的没有获得愈伤组织, 但其愈伤组织再生、增殖及植株再生均不如对称融合。从细胞学鉴定的21块杂种愈伤组织上再生出16株杂种植株。     

甘蓝型油菜与芝麻菜体的细胞杂交

为拓宽油菜育种的基因资源库, 改良油菜品种, 以甘蓝型油菜(Brassica napus)花油3号下胚轴和芝麻菜(Eruca sativa)下胚轴为材料分离制备原生质体; 然后采用PEG-高Ca2+-高pH法进行原生质体融合, 当PEG浓度为35%, 原生质体融合密度为5×105个/mL时, 融合25 min时, 融合率可达18.2%。融合后在培养密度为1×105个/mL时, 以附加1.0 mg/L 2,4-D +0.5 mg/L 6-BA+0.5 mg/L NAA+ 200 mg/L肌醇+300 mg/L水解酪蛋白的改良的KM8p为融合体培养基, 以0.1 mol/L 蔗糖+0.2 mol/L葡萄糖+0.2 mol/L甘露醇作渗透稳定剂进行液体浅层培养, 效果较好, 愈伤组织再生率最高为6.8%。将融合体再生的小愈伤组织转移至培养基(B5无机盐+0.087 mol/L蔗糖+0.2 mg/L 2, 4-D+0.5 mg/L NAA+0.2 mg/L 6-BA+ 0.5% Agar, pH 5.8)上增殖培养, 待愈伤组织长至直径为3~5 mm时, 及时将其转至分化培养基(MS无机盐+0.087 mol/L 蔗糖+0.1 mg/L IAA+0.8 mg/L 6-BA+0.8% Agar, pH 5.8)中诱导不定芽再生, 芽分化率为35.7%。当不定芽长为2~3 cm时, 将其切下转入附加0.5 mg/L IBA+0.2 mg/L 6-BA的1/2MS生根培养基中诱导生根, 14 d左右即可形成再生植株, 生根率可达88%。同时, 以紫外线(60 μW/cm2)照射芝麻菜原生质体, 进行不对称融合, 照射2 min的获得了愈伤组织和再生植株, 照射4 min的只获得愈伤组织, 而照射5 min以上的没有获得愈伤组织, 但其愈伤组织再生、增殖及植株再生均不如对称融合。从细胞学鉴定的21块杂种愈伤组织上再生出16株杂种植株。     

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium.

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.     

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.     

Mating reaction in yeast protoplasts

Protoplasts prepared from complementary haploid strains ofSaccharomyces cerevisiae were studied with regard to their ability of conjugating. Neither fresh protoplasts nor the growing protoplasts possessing fibrillar walls exhibited sex specific agglutination or fusion. However, they were capable of inducing sexual activation in normal cells of opposite mating type. After completing the regeneration of cell walls the protoplasts could conjugate either with each other or with cells of opposite sex. The frequency of conjugations was low, about 1%, and was largely dependent on the degree of completition of the wall during regeneration. From the results the following conclusions may be drawn: 1. The initiation of mating is dependent on the integrity of the cell wall. 2. The sex specific morphogenetic changes do not occur in wall-less protoplasts but may happen after the protoplasts have regenerated their cell walls. 3. The lysis of cell walls does not occur until the walls come into close contact. 4. The fusion of plasma membranes in sex-activated protoplasts cannot be induced by artefucial agglutination.     

Protoplast fusion: a tool for intergeneric gene transfer in bacteria

Protoplasts can be isolated from bacterial cells by digestion of the cell wall with the help of lysozyme in presence of osmotic stabilizers. Fusion of protoplasts can be induced by chemical fusogens like polyethylene glycol. The electrofusion technique has been reported in bacteria in which the fusion frequency is much higher than that obtained by PEG induced protoplast fusion. This technology allows recombination to take place not only between related species but also between unrelated genera and is of great potential in the breeding and improvement of industrial strains. This review includes the information and developments on the protoplast fusion in bacteria with special reference to genetic recombination by protoplast fusion between phylogenetically unrelated bacteria.     

The localization of SPO1 phage resistance in the genome of Bacillus subtilis as revealed by fusion of protoplasts

We localized the gene for resistance to phage SPO1 relatively to the markers pur B 34 and ura by means of the polyethylene-glycol induced fusion of bacterial protoplasts of three-fold auxotrophic Bacillus subtilis strains S3 and S13. By this same method, the site of some auxotrophic markers was tentatively determined. The application of the protoplast fusion technique to exact genetic analysis will not be possible until the exo- and endogenous factors influencing cell wall regeneration are standardized. Fluctuations of this kind are very significant for the determination of genetic segregation.     

Studies of protoplast fusion in Streptomyces chrysomallus

Conditions for the regeneration of cells from protoplasts of Streptomyces chrysomallus, a producer of the peptide antibiotic actinomycin, are described. Regeneration of fusion products was most efficient at 27-30 degrees C on regeneration R2 medium (Okanishi et al., 1974) containing 0.25 M-sucrose. The addition of phosphate (150-300 mg 1(-1) to the medium and incubation at 23 degrees C proved to be optimal for the regeneration of individual strains. Highest recombination frequencies after protoplast fusion were obtained by fusing protoplasts in the presence of 45% (w/v) polyethylene glycol 6000. With strains that produce no, or little antibiotic, protoplasts must be present in excess in fusion mixtures in order to overcome inhibition of regeneration by the antibiotic-producing partner.     

Regeneration and molecular characterization of an intergeneric hybrid between Graphium putredinis and Trichoderma harzianum by protoplasmic fusion

The fungal strains Graphium putredinis and Trichoderma harzianum were selected as parents for fusant development. Protoplasts were isolated using the combination of lysing enzymes Novozym 234 and cellulase with 0.6 M KCl as osmotic stabilizer. The optimum conditions for release of viable protoplasts from the fungal mycelium viz. age of the mycelium, lytic enzymes, osmotic stabilizers, pH, incubation period and regeneration medium were determined. Intergeneric protoplast fusion was carried out using 50% polyethylene glycol with calcium chloride (CaCl₂) and glycine buffer and the conditions for effective protoplast fusion, viz. fusogen, osmotic stabilizer, pH, incubation period and regeneration medium were optimized. At optimum conditions, the regeneration frequency of the fused protoplasts on potato dextrose agar (PDA) medium and fusion frequency were calculated. The regeneration frequency on non-selective (PDA) and selective media (PDA amended with starch) was determined for the parental and fusant strains in which, fusant showed a higher rate of regeneration. Fusant formation was confirmed by morphological markers (colony morphology and spore size and shape) and genetical markers like, mycelial protein pattern, restriction digestion pattern and random amplified polymorphic DNA (RAPD) analysis. The efficiency of these parental strains and their intergeneric fusant in the production of hydrolytic enzymes – amylases (treatment plant for sago factory effluent), cellulases (bioethanol), xylanases (bleaching agents for waste paper pulp) and proteases (additives in commercial detergents) – have probable applications in various industrial processes.     

Protoplast transformation of glutamate-producing bacteria with plasmid DNA

A method for polyethylene glycol-induced protoplast transformation of glutamate-producing bacteria with plasmid DNA was established. Protoplasts were prepared from cells grown in the presence of penicillin by treatment with lysozyme in a hypertonic medium. The concentration of penicillin during growth affected the efficiency of formation, regeneration, and polyethylene glycol-induced DNA uptake of protoplasts. Regeneration of protoplasts was accomplished on a hypertonic agar medium containing sodium succinate and yeast extract. The spectinomycin and streptomycin resistance plasmid pCG4, originally from Corynebacterium glutamicum T250, could transform various glutamate-producing bacteria such as C. glutamicum, Corynebacterium herculis, Brevibacterium flavum, and Microbacterium ammoniaphilum. The plasmid was structurally unchanged and stably maintained in new hosts. The transformation frequency of most competent protoplasts with pCG4 DNA isolated from primary transformants was high (ca. 10(6) transformants per microgram of covalently closed circular DNA) but was still two orders of magnitude below the frequency of transfection with modified DNA of the bacteriophage phi CGI. The difference was ascribed to the involvement of regeneration in transformation.     

Characteristic features of protoplast formation and regeneration in Fusidium coccineum

The methods for preparation and regeneration of protoplasts were tested with respect to the strains of F. coccineum markedly differing in their capacity for antibiotic production, sporulation and the growth rate. It was found that the substrate used for the culture growth had a significant effect on the cell wall and sensitivity of the mycelium to lytic enzymes. An enzyme from Hellix pomatia and its combination with lysozyme were used for lysing the culture. The cytological investigation of the time course of the culture lysis revealed a stage-by-stage pattern of protoplast formation by means of fragmentation of the hyphal contents till a ball was formed. Two to 4 protoplasts differing in their size and structure were formed within a cell. The pH value and osmotically stabilizing component had some effect on the rate of protoplast formation. Highly productive strains were characterized by formation of protoplasts heterogenous in their size and by decreased frequency of regenerations. The enzyme-free protoplasts preserved their viability and capacity for germination in osmotically stabilizing media for 72-96 hours of storage at 4 degrees C. On solid media the regeneration frequency reached 38 per cent. The regenerated cells formed colonies morphologically similar to those of the intact culture.     

Efficient plant regeneration from protoplasts isolated from embryogenic suspension cultures of Cyclamen persicum Mill.

A protocol for plant regeneration from protoplasts has been developed, and then successfully applied to different genotypes of Cyclamen persicum Mill. Protoplasts were isolated from embryogenic suspension cultures by enzymatic digestion in 2% cellulase R10 and 0.5% macerozyme R10. Yields obtained varied between 1 and 5 × 10⁵ protoplasts per gram fresh mass depending on the genotype. Protoplasts were immobilized in alginate films, which promoted proper cell wall regeneration. The highest cell division frequencies were found in modified Kao and Michayluk (1975, Planta 126:105–110) medium containing the same types and concentrations of plant growth regulators that were applied for suspension culture (2.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid and 0.8 mg l⁻¹ 6-(γ,γ-dimethylallylamino)purine). Cell division was recorded for all 11 tested genotypes in frequencies of up to 12% and 18% after 7 and 14 days, respectively. However, cell division frequency varied strongly between different genotypes. After 4–6 weeks calluses were released from the alginate films and further cultured on hormone-containing medium for continued growth or transferred to hormone-free medium for regeneration of somatic embryos. Plant regeneration via somatic embryogenesis succeeded in 9 out of the 11 genotypes under investigation. Up to now protoplast-derived plants from four genotypes have been successfully transferred to soil.     

Preparation and regeneration of protoplasts from Bacillus megaterium cells dried by sublimation]

Bacterial protoplasts are widely used in genetical research, for instance, in protoplasts fusion experiments and the transfer of heterologous DNA into bacterial cells. The usage of a new fresh grown culture of bacteria in every experiment restricts the reproducibility of the results preventing the technique becoming widespread. The use of antioxidants as components of stabilizing medium for sublimation drying of Bacillus megaterium cells supported cellular viability in bacterial culture. It also made possible preservation of such cellular fundamental properties as the ability to form protoplasts and regenerate the cell wall. Efficiencies of protoplasts formation and generation are similar for lyophilized and fresh grown cells. Cellular properties are conserved for 6 months of storage at least. Experiments with a lot of lyophilized biomass samples are highly reproducible. The potential of the technique was demonstrated in obtaining the hybrid Bacillus megaterium colonies by fusion of protoplasts derived from lyophilized genetically marked strains stored for up to 6 months.     

Preparation and regeneration of protoplasts and spheroplasts for fusion and transformation ofSchizosaccharomyces pombe

Preparation and regeneration of protoplasts is essential for somatic hybridization and transformation of yeasts. We present conditions that were found to be optimal for preparing and regeneratingSchizosaccharomyces pombe protoplasts for cell fusion. In contrast to these conditions, genetic transformation ofS. pombe requires spheroplasts that are osmotically sensitive, but still have some wall material attached to the cell. The main finding were as follows: (a) For protoplast formation with Novozym SP234, 0.9M sorbitol was found to be the optimal osmotic milieu and -mercaptoethanol is not necessary. (b) Embedding in soft agar yields considerably better regeneration frequencies than direct plating. (c) Cell fusion is optimal when both fusion partners are fully protoplasted, although considerable fusion occurs between spheroplasted cells as well. (d)Schizosaccharomyces pombe transformation frequencies are much higher with spheroplasts than with protoplasts. Inclusion of -mercaptoethanol did not enhance transformation frequency.     

Transmission, segregation, and recombination of mitochondrial genomes in zygote clones and protoplast fusion clones of yeast

Summary The average transmission-and recombination frequencies of mitochondrial markers are similar in diploid clones derived from zygotes or from fusion of haploid protoplasts of identical mating type. The transmissional patterns of mitochondrial markers in individual fusion-or zygote-clones, however, are very different. The time needed for regeneration of cell walls of fused protoplasts is found to be mainly responsible for this difference, since delay of first cell division in zygotes leads to similar results.     

Quantitative studies on the viability of yeast protoplasts following dielectrophoresis

Abstract Protoplasts from Saccharomyces cerevisiae and Saccharomyces diastaticus were collected in a non-homogeneous alternating electric field. The dependence of the viability of the protoplasts on different conditions of collection was tested by determining the regeneration rates in each case. The parameters varied in collection were the field strength (0.33 kV/cm–6.67 kV/cm), the frequency of the alternating field (1–2 MHz) and the collection time (2–10 min). The introduction of a new type of fusion chamber (meander chamber) permitted, for the first time, quantitative exposure of protoplasts to the electric field as well as their complete transference into the regeneration medium. The regeneration rates of yeast protoplasts collected under those conditions employed for electrofusion did not differ from those of protoplasts which had been maintained under the same experimental conditions but were not subject to the influence of an alternating electric field. The two yeast strains were fused together (collection 1 kV/cm; pulse 15 kV/cm; duration of pulse 40 μs) and the fusion products were introduced into a selection medium for regeneration. The fusion rate was about 4.8 × 10⁻⁴; on average 272 colonies grew on the selection medium for each chamber filling.