Efficient protoplast regeneration for some homofermentative lactobacilli and pediococci

Conditions for protoplast regeneration were examined for several strains of homofermentative lactobacilli and pediococci isolated from silage. Attempts to regenerate protoplasts using previously published agar regeneration media for lactobacilli were unsuccessful for most of the strains. Replacing or increasing colloidal substances in a medium containing raffinose and MgCl(2) as osmotic stabilizers enabled efficient regeneration of the protoplasts at a frequency of 10-99%. A medium containing gelatin, polyvinylpyrrolidone (PVP) and no agar was effective for Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus rhamnosus protoplasts. An agar medium containing PVP (PVP medium) was effective for Pediococcus sp. protoplasts, and addition of agarose to the PVP medium enabled regeneration of Lactobacillus casei protoplasts. A medium containing calcium alginate gel and no agar was effective for Lactobacillus curvatus protoplasts. The type of colloidal substance required for protoplast regeneration varied from species to species. This result suggested that several kinds of media may be necessary to regenerate protoplasts for all the genera of lactobacilli and pediococci.     

Protoplast formation, regeneration and plasmid curing in Lactobacillus reuteri

Abstract A method for protoplast formation and regeneration suitable for Lactobacillus reuteri strains was developed.
Lysozyme-treated cells formed protoplasts at a high percentage and regenerative ability varied according to the strains considered.
Moreover, production and regeneration of protoplasts promoted the loss of plasmids harboured by the strains.     

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.     

The methods of protoplast formation and transformation of Streptomyces strains

Factors influencing formation, regeneration and transformation of protoplasts in streptomyces are described. Conditions for formation and regeneration of protoplasts in 4 industrial strains producing the macrolide antibiotic tylosin were studied. It was demonstrated possible to apply the method for transformation of the S. lividans type culture to 3 industrial strains of S. griseus producing grisin, an antibiotic used as a feed additive. Potential increasing of the efficiency of protoplast transformation and transfection in various actinomycetous strains including industrial ones is discussed. The stimulating effect of lyposomes on transformation of protoplasts in S. lividans 66 with DNA of plasmids pVG101 and pIJ350 as well as transfection with DNA of phages SH10 and KS404 was shown. The tylosin resistance genes in S. fradiae strain B45 were cloned which enabled isolating the cluster of the genes participating in tylosin biosynthesis.     

Antibiotic production by variants of Penicillium chrysogenum isolated after protoplast formation and exposure to mutagens at the protoplast stage

A procedure for protoplast formation in the penicillin-producing organism Penicillium chrysogenum was developed. The yield of the protoplasts was high, the protoplasts were stable and capable of regeneration. Two types of the protoplast regeneration were revealed. The spores and protoplasts were treated with UV light and N-nitroso-N'-methyl biuret and their effect on production of the antibiotic by the isolated variants was studied. It was shown that the protoplasts of P. chrysogenum were more liable to the mutagenic effect of UV light and nitroso methyl biuret than the fungus conidia. It is possible to use this specific feature in intensification of selection aimed at isolation of highly productive strains of P. chrysogenum.     

Optimal Cultural and Physiological Conditions for Handling Streptomyces rimosus Protoplasts

A general procedure for manipulating protoplasts of three Streptomyces rimosus strains was developed. More than 50% regeneration efficiency was obtained by optimizing the osmotic stabilizer concentrations and modifying the plating procedure. Preparation and regeneration of protoplasts were studied by both phase-contrast and electron microscopy. After cell wall degradation with lysozyme, protoplasts about 1,000 to 1,500 nm in diameter appeared. The reversion process exhibited normal and aberrant regeneration of protoplasts to hyphae and to spherical cells, respectively. Spherical cells contained no alpha, epsilon-ll-diaminopimelic acid and were colorless or red after Gram staining. They showed consistent stability during at least five subsequent subcultivations. However, the omission of glycine from the precultivation medium reduced the unusual process of regeneration almost completely. After normal protoplast regeneration, the production of oxytetracycline by single isolates was not affected.     

Protoplast fusion in Streptomyces fradiae strains producing neomycin and tylosin]

Interspecies fusion of protoplasts of the Streptomyces fradiae strains producing neomycin (an aminoglycoside antibiotic) and tylosin (a macrolide antibiotic) was performed with a view to isolate strains producing novel antibiotics. Fusion of the protoplasts of the neomycin- and tylosin-producing strains labelled by the resistance to monomycin and lincomycin, respectively, caused no formation of stable strains producing antibiotics differing in chromatographic mobility from the antibiotics produced by the initial strains. In fusion of the protoplasts of the unlabelled strains, heat-inactivated protoplasts of the active line of one strain (donor) and native protoplasts of the inactive line of the other strain (recipient) were used. When the neomycin-producing culture was used as a recipient the fusion led to formation of strain 195-34 producing antibiotics of the benzo(a)anthraquinone group. One of these antibiotics, i.e. antibiotic 34-I, proved to be a novel biologically active substance. After regeneration of the protoplasts of the initial strains, no stable strains producing antibiotics differing from neomycin and tylosin were isolated.     

Formation, Fusion, and Regeneration of Protoplasts from Wild-Type and Auxotrophic Strains of the White Rot Basidiomycete Phanerochaete chrysosporium

A preparation of two commercial enzymes was used to liberate protoplasts from 16-h-old mycelium of Phanerochaete chrysosporium. Regeneration frequencies of up to 5% were attained when the protoplasts were plated in a medium containing 10% sorbose and 3% agar. Fusion of protoplasts from different auxotrophic strains in polyethylene glycol-Ca²⁺ produced heterokaryons. Separation of the heterokaryons into their constituent homokaryotic strains could be effected through protoplast release and formation of colonies on regeneration agar.     

银耳原生质体分离与再生条件优化研究*

应用正交设计,研究不同菌株(Tr01、Tr21)、材料(芽孢、菌丝体、子实体)、溶壁酶浓度和酶解温度对原生质体产量的影响。实验结果表明,实验材料对原生质体产量影响最大,以芽孢为材料原生质体产量可达到2.75×107个/ml,而菌丝体和子实体的原生质体产量仅为2.5×106个/ml 和1.0×106个/ml;在35℃下酶解,原生质体产量高;溶壁酶浓度在1%~3%范围内对原生质体产量影响不大;不同菌株原生质体产量差异不显著。本实验还研究了稳渗剂浓度对原生质体再生率的影响,结果表明,0.5 mol/L~0.7 mol/L的KCl 对原生质体再生没有显著差异,再生率最高可达32.3%。     

金针菇原生质体分离制备、融合、再生及融合子检出

探讨了利用金针菇菌丝体分离、制备原生质体;不同的酶浓度和酶解时间对原生质体得率的影响;以及八种再生培养基的选择性试验.建立了金针菇原生质体的分离、制备、融合及再生的试验体系.试验共获得209株再生菌株,通过核染色检测,共获得37株具有双核和锁状联合的再生株.     

蓝色犁头霉原生质体的制备与再生

研究了氢化可的松生产菌蓝色犁头霉原生质体的形成与再生。通过对溶解酶系统的选择,影响原生质体形成的因素如渗透压稳定剂、酶浓度、菌龄、菌丝培养基和培养方式等因素进行考察,发现以0．4mol/L NH4Cl做为稳定剂、2．5mg/mL溶壁酶和5mg/mL纤维素酶组成的混合酶液溶解菌丝,4h后原生质体量可达10^6cell/mL。通过显微镜观察原生质体的形成过程以及在高渗培养基上的再生情况,再生率为15．6％。     

利用紫外线、利福平、氯化锂诱变原生质体筛选L—亮氨酸高产菌株的研究

本实验是以黄色短杆菌T_(6—13)的诱变株L—亮氨酸产生菌D—R—4为出发菌株,经青霉素、甘氨酸、溶菌酶作用制备原生质体,形成率达91.30％,再生率达53.68％;然后对原生质体进行紫外线、利福平、氯化锂复合诱变处理;在再生培养基平皿上培养,获得再生突变株,从中挑取单独菌落,进行摇瓶发酵筛选,已选育出一株57—4S号高产稳定菌株;经氨基酸分析仪测定其发酵液L—亮氨酸产量由出发菌株的17.35mg/ml提高到23.45mg/ml提高了35％。发酵液中主要副酸——异亮氨酸含量很少。     

Isolation and regeneration of Micromonospora olivoasterospora protoplasts

The conditions for preparation and regeneration of the protoplasts of M. olivoasterospora were developed. It was found that effective formation of the protoplasts required preliminary cultivation of M. olivoasterospora in the medium containing glycine in a concentration inhibiting its growth at least by 60-80 per cent. The strains studied markedly differed in their sensitivity to glycine and were highly sensitive to it. The efficacy of the protoplast formation depended on the culture age and increased with the use of the lytic enzyme 3 of Cytophaga dissolvens. The possibility and advisability of the use of prolonged lysis of the Micromonospora cell walls were shown. A rich organic medium was used for regeneration of the protoplasts.     

Permeability Properties of Aspergillus nidulans Protoplasts

The regeneration of mycelial forms from protoplasts of mutant and wild-type strains of Aspergillus nidulans was followed microscopically under conditions with which the rate of regeneration depends upon a specific transport function. This method has shown that at least four uptake systems-one for divalent anions, two for purines, and one for sugars-remain functional after removal of the cell wall.     

Albofungin formation by a strain isolated as a result of fusion of protoplasts of organisms producing aminoglycoside antibiotics

Strain 344 synthesizing an antibiotic complex was isolated after fusion of the protoplasts of Streptomyces monomycini producing monomycin and Streptomyces kanamyceticus producing kanamycin. The major component of the complex was identified with albofungin and the minor one was suggested to be chloralbofungin. In the cultures of strain 344 variants forming monomycin were detected. After regeneration of the protoplasts of the parent strains there were isolated no stable clones synthesizing antibiotics differing from monomycin and kanamycin.     

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.     

High-frequency fusion of Streptomyces parvulus or Streptomyces antibioticus protoplasts induced by polyethylene glycol.

Conditions were established for the regeneration of protoplasts of Streptomyces parvulus and Streptomyces antibioticus to the mycelial form. Regeneration was accomplished with a hypertonic medium that contained sucrose, CaCl2, MgCl2, and low levels of phosphate. High-frequency fusion of protoplasts derived from auxotrophic strains of S. parvulus or S. antibioticus was induced by polyethylene glycol 4,000 (42%, wt/vol). The frequency of genetic transfer by the fusogenic procedure varied with the auxotrophic strains examined. Fusion with auxotrophic strains of S. parvulus resulted in the formation of true prototrophic recombinants. Similar studies with S. antibioticus revealed that both stable prototrophic recombinants and heterokaryons were formed.     

The formation and regeneration of mycelial protoplasts in hyper lignolytic fungus,strain IZU-154

Over 2 × 10⁷/ml protoplasts were obtained from mycelia of hyper lignolytic fungus (nomenclatured as strain IZU-154) by treatment with the lytic enzyme NovoZym 234 in the presence of 0.05 M maleic acid buffer (pH 5.6) containing 0.6 M MgSO₄. The protoplasts regenerated at more than 10% of frequency on solid 2% agar medium containing 0.6 M sucrose as an osmotic stabilizer overlaid with 0.5% agar containing the stabilizer. In the determination of the lignolytic activities of 50 regenerants from protoplasts, 2 strains which degraded more than 56% of the lignin during incubation for 30 d and showed activity higher than the parent were found. The regeneration from protoplasts of this fungus was suggested to be useful for the breeding of strains having higher lignolytic activity than this fungus.     

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.     

林可链霉菌转化系统的建立

利用基因工程技术改良抗生素生产菌有着广阔的应用前景［１］。值得尝试的是将抗生素生物合成基因片段随机或定向组合导入生产菌中,通过同源重组方式提高生物合成途径中限速步骤相关基因的剂量,以达到提高生产菌发酵单位的目的。此战略被作者称为“基因回转化技术”,并...