Protoplast fusion

The use of protoplasts in studies of stress physiology and biochemistry expands the advantages of cell culture systems discussed in the preceding sections. Additional applications are related to the fusion of protoplasts. Intraspecifie and interspecific protoplast fusion greatly enhance genetic variability of the fused protoplasts (Kumar Cocking, 1987). The resulting somatic hybrids provide cells which can be used for selection of specific traits (e.g. environmental stress tolerance) provided by one or both donor cells and for basic studies on cytoplasmic and nuclear inheritance of desired characteristics. 

Kumar, A. Cocking, E.C. (1987). Protoplast fusion A novel approach to organelle genetics in higher plants. American Journal of Botany, 74, 1289-303. 

Endo-pectinase production by intraspecific hybrids of Aspergillus sp. CH-Y-1043 obtained by protoplast fusion... 

Protoplast fusion induced by polyethyleneglycol and Ca was carried out between two auxotrophic mutants of Aspergillus sp. CH-Y-1043. The hybrids obtained showed significant differences in endopectinase activity and morphology compared to the prototrophic strain. Strains grown on lemon peel showed production improvement with respect to the parental strain. Since H15 hybrid showed up to 90% higher endopectinase production than the wild type CH-Y-1043, kinetics of enzyme production in Fernbach flasks and Fermentor (14L) by H15 were determined. 

Nishihara, and M. Kito. Phospholipids of membranes of cultured cells and the products of protoplast fusion. Phytochemistry 1979 18 423-426. 

Protoplast fusion has also been successfiilly used to produce novel mold isoprenoids, such as citreoanthrasteroids and citreohybridones (Nakada 2000). Long chain aldehydes, valuable as a bio-flavor, have also been secured along similar procedures fiom tMli cultures of the green seaweed Ulva pertusa, regenerated fiom protoplasts (Fujimura 1990). 

Two major advances describe the construction of mutants with an apparently significantly higher probability of improved phenotype. The method involves whole-genome shuffling - with protoplast fusion - of a small number of parental strains... 

S. avermitilis and the biosynthesis of avermectins constitute an interesting example where traditional techniques such as chemical mutagenesis and protoplast fusion combined with recombinant DNA technology have been successfully applied in mutant isolation and strain improvement. In addition, this system offered the first opportunity to apply mutasynthesis to the production of better analogs, an application that had never before been exploited commercially. An intense doramectin development effort was therefore initiated with the Mrf-deficient mutants of S. avermitilis. The first step in this process involved the isolation of mutants deficient in 5-Omethyltransferase activity to maximize levels of the more bioactive class B avermectins [13], Thereafter, a combination of strain im-... 

If the current S. cerevisiae strains do not exhibit pronounced strain-specific effects on wine flavor, is it possible to genetically improve them to produce desired aroma components Such improvement could be accomplished by utilizing classical hybridization, protoplast fusion, mutation/selection or genetic engineering techniques (70), presupposing that the characteristic(s) to be selected for is under the control of a single gene (59). 

Similar techniques were used by Shinohara et al (71) to develop hybrids with increased production of fusel alcohols and esters. Protoplast fusion techniques have been used to confer amylolytic activity to brewery yeasts (22) and ethanol tolerance to wine yeasts (70) Farris et al (72) used protoplast fusion to produce hybrids with killer factor that is, the ability to secrete proteinic toxins. Kunkee and coworkers (25) utilized a leucine auxotrophic mutant strain of S. cerevisiae (UCD Montrachet 522) to produce base wine for brandy production the mutant strain produces less isoamyl alcohol, reducing the quantity of fusel alcohols in the subsequent brandy. And Thornton (48) discussed the progress in utilizing plasmid vectors to introduce new genes into wine yeasts he cautioned, however, that until the yeast genome is better understood that direct gene manipulation techniques will be of limited value. 

Antibiotic 34-1 (207) is the most recent of several known antibiotics (34-2 X-14881 E=8-0-methyl-3, 34-3 6-deoxy-8-0-methylrabelomycin 155, 34-4 8-0-methylrabelomycin 156). All are active against gram-positive bacteria and were isolated from Streptomyces fradiae strain 34, which is a construct obtained by intraspecific protoplast fusion of two S. fradiae strains. The parent strains were known as producers of the aminoglycoside antibiotic neomycin and the macrolide antibiotic tylosin, respectively [146]. Because of its reduced C-1 carbonyl, 207 resembles emycin A and hatomarubigin C (203), its closest relative. 

Cellulases and other carbohydrases are being used to isolate higher plant cells and to produce protoplasts (67, 68), A new era in plant breeding is envisioned as cell lines are manipulated genetically by mutagens, protoplast fusion, or possibly transformed by DNA transfer. 

Haloferax volcanii is particularly attractive as a halobacterial experimental system because it is a relatively fast grower and is capable of growing on a simple minimal medium [53], so that there is the possibility of isolating auxotrophic strains and thus identifying genes for many biosynthetic functions. Auxotrophic strains and methods for mating [53] and protoplast fusion [28] of Haloferax volcanii have been developed. 

Kavanagh, K. Whittaker, P. A. (1996) Application of protoplast fusion to the non-conventional yeasts. Enz Microbiol Tecbnol, 18,45-51. 

One of the drawbacks in the current commercial fermentation process is that the predominant form of the product is the deprotonated lactate rather than lactic acid, requiring more expensive and wasteful product purification steps. This is because the Lactobacillus fermentation operates at a minimum pH of 5.0-5.5 which is above the pA a of lactic acid (3.87). To overcome this limitation, a powerful strain improvement method, genome shuffling, was used to improve the acid tolerance of a poorly characterized industrial strain of LactobacillusA population of strains with subtle improvement in pH tolerance was isolated using classical strain improvement methods such as chemostats, and were then shuffled by recursive pool-wise protoplast fusion to create mutant strains that grow at substantially lower pH than does the wild-type strain. 

Various techniques are employed to obtain the improved biocatalysts for potential improvement. These include the following mutation and selection, hybridization, protoplast fusion, and recombinant DNA methods. One example of strain improvement is the development of an ethanol-tolerant yeast strain, Saccharomyces 1400, through protoplast fusion of S. distaticus and S. uvarum as reported by D Amore et al. [4]. This yeast strain was used as the biocatalyst by Krishnan et al. [5] for the rapid fermentation of high concentrations of glucose... 

With the objective of producing itaconic acid directly from starchy materials, Kirimura et al. [117] studied the interspecific hydridization between A. terreus and A. usamii through protoplast fusion. Although A. terreus has amylase activity, the activity is not strong enough to produce itaconic acid from starch di-... 

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