The improvement of cephalosporin C production by fed-batch culture ofCephalosporium acremonium M25 using rice oil

The objective of this study is to improve cephalosporin C (CPC) production by optimization of medium and culture conditions. A statistical method was introduced to optimize the main culture medium. The main medium for CPC production was optimized using a statistical method. Glucose and corn steep liquor (CSL) were found to be the most effective factors for CPC production. Glucose and CSL were optimized to 2.84 and 6.68%, respectively. CPC production was improved 50% by feeding of 5% rice oil at day 3rd and 5th day during the shake flask culture ofC. acremonium M25. The effect of agitation speeds on CPC production in a 2.5-L bioreactor was also investigated with fed-batch mode. The maximum cell mass (54.5 g/L) was obtained at 600 rpm. However, the maximum CPC production (0.98 g/L) was obtained at 500 rpm. At this condition, the maximum CPC production was improved about 132% compared to the result with batch flask culture.     

Further studies on recombination in diploid clones from Bacillus subtilis protoplast fusion

Summary Diploid prototrophs were obtained from protoplast fusion of Bacillus subtilis strains. They are unstable but upon further cultivation they stabilize retaining diploidy but are genetically inactive. It has been suggested that recombination between the parental chomosomes is involved in the production of stable prototrophs and recombinants. In this work the occurrence of this recombination was searched for by determining genetic linkages in transformation experiments. In prototrophs two alleles: hisH2 and trpE8 carried originally on each parental chromosome, were shown to be 48% co-transformable in a stable clone whereas they were only cotransformed in 10% of the unstable colonies. For Trp^- recombinants (the most frequent type of a Leu^- Met^- Thr^- x Ade^- Ura^- Trp^- fusion pair) lysed protoplasts were used as donor DNA for the transformations. High values of co-transfer for Ura⁺ Met⁺ were obtained. These results confirm the occurrence of recombination in stable diploid clones, prototrophs or recombinants.     

Effects of nutrients and culture conditions on morphology in the seed culture ofCephalosporium acremonium ATCC 20339

The objective of this study was to investigate the effects of nutrients and culture conditions on morphology during the seed culture ofC. acremonium ATCC 20339. Morphological factors such as hyphal length, number of tips, number of arthrospores were observed to investigate the relationship between seed morphology and CPC production. During the time course of seed culture, hyphal length was shortened and the number of arthrospores increased rapidly. On the other hand, the number of tips decreased rapidly, and this was closely related to the hyphal length. Mixed nitrogen sources of 3% soybean meal and 1% cotton seed flour were determined as the proper organic nitrogen sources, in terms of the morphological factors in the seed culture. This fact was proven in batch culture for the production of Cephalosporin C. It was also found that a proper agitation speed enhanced the morphological differentiation ofC. acremonium ATCC 20339, thus improving the production of Cephalosporin C.     

Genetic recombination by protoplast fusion inNocardia asteroides

Summary Fusion and regeneration of protoplasts ofNocardia asteroides strains ATCC 3318, IMRU W3599 and HIK B971 have been used to study genetic recombination in this species. Protoplasts were produced by treatment with lysozyme, following incubation with glycine. Mutants of ATCC 3318 were grown in peptone yeast extract medium at 32°C prior to protoplast production to maximize protoplast frequency, whereas mutants of IMRU W3599 and HIK B971 were grown in trypticase-soy broth. Glycine concentrations favoring protoplast formation varied from 1.5% to 5% depending on strain. For all strains, protoplast formation was complete 1 h after addition of 5 mg/ml lysozyme. Protoplasts were fused by addition of 50% polyethylene glycol-1000. In general, 25% of the protoplasts could be regenerated. The incidence of recombinant recovery was increased up to 750-fold. The distribution of recombinant phenotypes in matings was similar for protoplast fusion and conventional crosses.     

Brassica carinata resynthesized by protoplast fusion

Brassica carinata (bbcc) was resynthesized by protoplast fusion betweenB. nigra (bb) andB. oleracea (cc). In two fusion experiments 64 hybrid plants were obtained and identified to be true hybrids by isoenzyme analysis, nuclear DNA content, chromosome number, and intermediate morphology. Of these plants 56% were normal amphidiploids with 2n=34 chromosomes and a DNA content equivalent to that of naturalB. carinata. The remaining plants were polyploid, morphologically abnormal, and infertile. The majority of the hybrids contained both chloroplasts and mitochondria fromB. nigra, but some plants combined chloroplast and mitochondria from the different progenitors. Hybrids with a DNA content equivalent to that ofB. carinata had a wide range of male fertility (4–98%), but consistently low female fertility. Only a few selfed seed were produced, but these germinated and grew into vigorous plants.Salaries and research support provided by State and Federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Journal Article No. 296-92     

Cybernetic modeling of the cephalosporin C fermentation process by Cephalosporium acremonium

A cybernetic mathematical model has been developed to describe the production of cephalosporin C. In developing the model, diauxic behavior of substrate consumption, morphological differentiation of cells, and catabolite repression of cephalosporin C production by the preferred substrate, glucose, were considered. The proposed model was tested on the experimental data from the literature and could adequately describe the morphological differentiation of cells, the sequential utilization of carbon sources and the production of cephalosporin C. It could be a useful tool to optimize the production of cephalosporin C by Cephalosporium acremonium in batch, fed-batch or continuous operations.     

Electrical fusion for optimal formation of protoplast heterokaryons in Nicotiana

The electrical fusion of protoplasts has been studied in order to maximize the formation of heterokaryons for culture. Heterokaryons of Nicotiana tabacum L. mesophyll protoplasts and N. plumbaginifolia Viviani supension-cell protoplasts were identified in fixed and stained as well as living material; a quantitative fusion index was thereby developed. With this index the efficiencies of various electric fields and fusion-chamber designs have been determined. Optimal fusion was obtained with an alternating-current (AC) field of 150 V/cm and direct-current (DC) square-wave pulses of 1000 V/cm. A new, simple-to-use, largescale fusion chamber is described in which batches of up to 5·10⁵ protoplasts (0.5 ml of cells at 10⁶/ml) can be fused in 5–7 min with efficiencies approaching 40%. Half of the fusion products are heterokaryons, thus fusion is random. Of the fusion products, 60% are bi- or trinucleate. Using fusion procedures similar to those described here Bates and C. Hasenkampf (1985, Theor. Appl. Genet., in press) have recovered viable somatic hybrids which have been regenerated.Abbreviations AC alternating current - DC direct current - PEG polyethylene glycol     

Plant regeneration from protoplast fusion inPassiflora spp.

Summary Protoplasts were isolated from leaf explants ofPassiflora edulis var.flavicarpa (the yellow passion fruit) and from cell suspensions of fivePassiflora species. Chemical fusion was performed using polyethylene glycol and the microcolonies obtained were transferred to growth medium to produce calli. Electrophoresis of soluble proteins and analysis of isoenzymes from calli produced from the fusion experiments were performed to select somatic hybrids. Specific polypeptide bands allowed the identification of somatic hybrids betweenP. edulis var.flavicarpa (+)P. alata, P. edulis var.flavicarpa (+)P. amethystina, P. edulis var.flavicarpa (+)P. cincinnata, P. edulis var.flavicarpa (+)P. giberti andP. edulis var.flavicarpa (+)P. coccinea. An average of 3 to 5% hybrid calli were obtained. With the exception of theP. edulis var.flavicarpa (+)P. coccinea, whole plants were recovered from all hybrids. These somatic hybrids showed 4n=36 chromosomes, which represents a further evidence of their hybridity.     

Transmission genetics of Nicotiana hybrids produced by protoplast fusion

Somatic cell hybrids were produced by fusing protoplasts isolated from callus cells of a tobacco line transformed by Agrobacterium tumefaciens (octopine synthesizing strain B6S3), and mesophyll protoplasts from haploid plants of Nicotiana plumbaginifolia. Hybrids were selected by using differential medium (hormone-independent growth plus greening capacity), or by mechanical isolation and cloning of individual heterokaryocytes. The analysis of hybrid cell lines included the determination of lysopine dehydrogenase activity (encoded by the T-region of Agrobacterium tumefaciens plasmid), examination of isozymes of esterase, and study of chromosome number and morphology. All eight cell lines selected on the screening medium were identified as nuclear hybrids, while only three of the eight evaluated clones obtained by mechanical isolation and cloning were found to be nuclear hydrids; the rest of them were nuclear segregants of tobacco [1] or N. plumbaginifolia [4] type. These data give independent evidence for the occurrence of non-fusion and segregation of nuclei in fusion products, that can be revealed only by using nonselective methods for hybrid screening. In this paper we demonstrate the value of microisolation for the recovery of cytoplasmic hybrids.     

Regulation of cephalosporin synthesis in Cephalosporium acremonium by phosphate and glucose

The regulation of cephalosporin synthesis in Cephalosporium acremonium was studied in a simple chemically-defined medium with glucose as the carbon source. Antibiotic synthesis depended on the phosphate content of the medium. At phosphate concentrations above 2.75 mM maximum antibiotic titres were not reached while glucose uptake and growth rates were increased. Phosphate exerted its effect indirectly by regulating the rate of glucose consumption. The negative effect of high phosphate concentrations could be overcome completely by controlling the sugar supply in fed-batch and chemostat experiments. High actual concentrations of phosphate or of glucose alone had no direct negative effect on antibiotic synthesis.     

Genetic mapping by means of protoplast fusion in Bacillus subtilis

Summary A new mapping method involving protoplast fusion in Bacillus subtilis is described. Protoplasts from an isogenic standard marker strain containing purA and from a strain containing both purB and the marker, x, to be mapped were fused with polyethylene glycol, and purA ⁺ purB ⁺ fusants were selected. After isolation of single colonies and determination of unselected markers, marker x was mapped between two standard markers. This method was fully applicable to PBS1-resistant strains (e.g., lyt strains). The results obtained by protoplast fusion, conventional transformation and/or lysed protoplast transformation indicated that a lyt strain, Ni15, contained two new autolysin-minus mutations (lyt-151 and lyt-152). The properties of lyt-15 are also discussed.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - SMM 0.5 M sucrose, 0.02 M MgCl₂, 0.02 M maleate buffer, pH 6.5     

Improvement of the cell volume ofCandida blankii through protoplast fusion

Summary The yeastCandida blankii ESP-94, capable of utilizing xylose as substrate, was isolated for the production of single-cell protein (SCP) on bagasse hydrolysates. However, the small cell volume of strain ESP-94 would complicate harvesting of the cells during a continuous fermentation process. Auxotrophic mutants of strain ESP-94 were generated and intraspecific protoplast fusion experiments performed in an attempt to increase the cell volume of strain ESP-94. The fusion products were characterised with respect to cell volume, DNA content and genetic stability. Six genetically stable fusants with bigger cell volumes and higher DNA contents were obtained. One such fusant, fusant F17, had a cell volume 3-times that of strain ESP-94, while exhibiting similar growth rates to strain ESP-94 ond-xylose as carbon source.     

Male-sterile chicory cybrids obtained by intergeneric protoplast fusion

Male-sterile chicory plants were obtained by fusion of chicory mesophyll protoplasts and hypocotyl protoplasts derived from male-sterile sunflower plants. The protoplasts of both species were fused by the PEG method and the products were selected manually and cultivated at very low density in a liquid medium. Three to twenty percent of the heterokaryocytes divided and evolved into microcalli, then into calli where budding could be induced. The mitochondrial genome of ten male-sterile or totally sterile plants was studied. Restriction endonuclease profiles of mitochondrial DNA and molecular hybridization with specific genes of the mitochondrial genome used as probes indicated that mitochondrial DNA rearrangement had occurred between sunflower and chicory and the intensity of the rearrangements correlated with the degree of sterility of the different plants.     

Transfer of organelles of the alga Chlamydomonas reinhardii into carrot cells by protoplast fusion

A mutant of Chlamydomonas reinhardii which lacks a cell wall was fused with Daucus carota protoplasts using polyethylene glycol and the resulting fusion products were cultured. Fusion involved integration of Chlamydomonas and carrot plasma membranes and the release of algal organelles into the carrot cytoplasm. Chlamydomonas basal bodies, nuclei and chloroplasts were frequently observed in the fusion products. Cultured fusion products regenerated cell walls and divided; most Chlamydomonas organelles degenerated during culture but chloroplasts were still recognizable in the carrot cytoplasm after 10.Abbreviations PEG polyethylene glycol - TEM transmission electron microscopy - SEM scanning electron microscopy This study was undertaken during sabbatical leave in The Research School of Biological Sciences. Australian National University     

Enrichment for Nicotiana heterokaryons after protoplast fusion and subsequent growth in agarose microdrops

Protoplasts isolated from Nicotiana tabacum L. leaves and Nicotiana suaveolens Lehm. cell suspensions have been fused with polyethylene glycol (PEG). Enrichment for heterokaryons was based on a Percoll flotation protocol which allowed a preparation with 50% heterokaryons to be obtained. The heterokaryons developed into calli whose hybrid nature was shown by polyacrylamide gel electrophoresis of esterase isoenzymes. Sensitivity of the mesophyll protoplasts to PEG and different buoyant densities of the heterokaryon and cell-suspension protoplasts contribute to the enrichment. The 50%-fusion figure following purification is an improvement on standard PEG procedures.Heterokaryons obtained were embedded in 20l drops of agarose and placed in a liquid nurse culture that allows optimum growth of the heterokaryons and maintains a physical boundary between the heterokaryons and the nurse culture. Once colonies develop, the agarose microdrop is removed from the nurse culture and placed on shoot-induction medium. Agarose microdrops containing the heterokaryons can be readily removed at any stage and processed for electron microscopy to follow the early stages of colony development.The procedures we have utilised provide a robust physical selection method that allows the total variation from a heterokaryon population to be expressed.Abbreviations BAP N⁶-benzylaminopurine - BM basal medium - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthalene acetic acid - PEG polyethylene glycol - PKM modified Kao (1977) medium for protoplast culture     

Somatic hybridization between Brassica juncea and Moricandia arvensis by protoplast fusion

Intergeneric somatic hybrids have been produced between Brassica juncea (2n=36, AABB) cv. RLM-198 and Moricandia arvensis (2n=28, MM) by protoplast fusion. Hypocotyl protoplasts of B. juncea were fused with mesophyll protoplasts of M. arvensis using polyethylene glycol. Fusion frequency, estimated on the basis of differential morphological characterstics of parental protoplasts was about 5%. Of the 156 calli obtained, four calli produced shoots intermediate in morphology between the parents. Hybrid nature of the plants was confirmed using wheat nuclear rDNA probe. Hybridization of total DNA with a mitochondrial DNA probe carrying 5s–18s rRNA genes of maize showed that the mitochondria of the somatic hybrids were derived from the wild species M. arvensis. Meiosis in the only hybrid that produced normal flowers revealed the occurrence of 64 chromosomes, the sum of chromosomes of parental species. Inspite of complete pollen sterility, siliquas were produced in this hybrid by back-crossing with B. juncea. These siliquas on in vitro culture produced 12 seeds.     

Fertility and chromosome stability in Brassica napus resynthesised by protoplast fusion

Summary Fertile somatic hybrids between Brassica campestris and B. oleracea have been produced by protoplast fusion. Fusion products were identified by their intermediate protoplast morphology. Heterokaryons were isolated either with micropipettes using a micromanipulator or by flow sorting. About 2% of the obtained calli differentiated to shoots. Of the shoots obtained from manually selected heterokaryons, 100% were true hybrids as confirmed by isozyme analysis while 87% of the flow sorted ones showed a hybrid pattern. Ploidy level of the hybrid plants was determined by chromosome counting and relative DNA-content analysis. The sum of the chromosome number (38) from the two fusion partners were found in 30% of the hybrids; 9% had fewer and 61% had more chromosomes. Pollen viability and seed set varied with ploidy level. Compared to natural B. napus, a pollen viability of 52%–93% and a fertility of 1%–40% was found for the somatic hybrids with normal chromosome number. Restriction enzyme analysis of chloroplast-DNA showed that either B. campestris or B. oleracea chloroplasts were present in the somatic hybrid plants. Of 11 hybrid plants 5 had the campestris and 6 had the oleracea type (11 ratio).     

Transformation of plant mitochondria with mitochondrial DNA plasmids via protoplast fusion

Summary Brassica napus cybrid plants which contain novel nucleus-mitochondria-chloroplast combinations have been constructed, via protoplast fusion. Such fusions resulted in mitochondrial DNA plasmids being lost (at a frequency of 12.5%) or, more surprisingly, being transferred from mitochondria of one protoplast population to mitochondria of the other population (at a frequency of 6.1%). Mitochondria containing their new DNA complement became the dominant organelle population in regenerated plants and were faithfully maternally inherited through successive sexnal generations. No concomitant alterations in mitochondrial chromosome organization or nuclear chromosome number occurred. Protoplast fusion can, therefore, cure plant mitochondria of extrachromosomal DNA and, more importantly, be used to transform plant mitochondria with naturally occurring mitochondrial plasmids. The potential for mitochondrial transformation with recombinant vectors is discussed.     

Production of Trichoderma strains with pesticide-polyresistance by mutagenesis and protoplast fusion

The sensitivity of two cold-tolerant Trichoderma strains belonging to the species T. harzianum and T.␣atroviride was determined to a series of pesticides widely used in agriculture. From the 16 pesticides tested, seven fungicides: copper sulfate, carbendazim, mancozeb, tebuconazole, imazalil, captan and thiram inhibited colony growth of the test strains significantly with minimal inhibitory concentrations of 300, 0.4, 50, 100, 100, 100 and 50 g/ml, respectively. Mutants resistant to carbendazim and tebuconazole were produced from both wild type strains by means of UV-mutagenesis. The cross-resistance capabilities and in␣vitro antagonistic properties of the mutants were determined. Carbendazim-resistant mutants showed total cross-resistance to benomyl and thiabendazole at a concentration of 20 g/ml. Intraspecific protoplast fusion was carried out between carbendazim- and tebuconazole-resistant mutants of both parental strains, and putative haploid recombinants with stable resistance to both pesticides were produced in the case of T.␣atroviride. These pesticide-polyresistant progenies are potential candidates for application in an integrated pest management system.This work was presented as an oral lecture in section ‘Agriculture, Soil, Forest Microbiology’ at the BioMicroWorld2005 conference.     

Stable inheritance and expression of the CMS traits introduced by asymmetric protoplast fusion

The donor-recipient protoplast fusion method was used to produce cybrid plants and to transfer cytoplasmic male sterility (CMS) from two cytoplasmic male-sterile lines MTC-5A and MTC-9A into a fertile japonica cultivar, Sasanishiki. The CMS was expressed in the cybrid plants and was stably transmitted to their progenies. Only cytoplasmic traits of the male-sterile lines, especially the mitochondrial DNAs, were introduced into the cells of the fertile rice cultivar. More than 80% of the cybrid plants did not set any seeds upon selfing. Sterile cybrid plants set seeds only when they were fertilized with normal pollen by hand and yielded only sterile progenies. This maternally inherited sterility of the cybrid plants showed that they were characterized by CMS. The CMS of cybrid plants could be restored completely by crossing with MTC-10R which had the single dominant gene Rf-1 for restoring fertility. These results indicated that CMS was caused by the mitochondrial genome introduced through protoplast fusion. The introduced CMS was stably transmitted to their progenies during at least eight backcross generations. These results demonstrate that cybrids generated by the donor-recipient protoplast fusion technique can be used in hybrid rice breeding for the creation of new cytoplasmic male-sterile rice lines.