Protoplast Fusion Technique Research Articles

Application of the Protoplast Fusion Technique to Engineer a Recombinant Microorganism to More Efficiently Degrade Chlorophenols

Recombinant strain Xz6-1 was constructed by the protoplast fusion technique with the goal of endowing it with the ability to efficiently degrade pentachlorophenol (PCP). This compound was considered as a representative of possible compounds that can be obtained during the bleaching of pulp for papermaking. The potential of Xz6-1 and Pseudomonas putida to treat PCP synthetic wastewater was explored. The majority of PCP was removed within the first 20 h; two degradation curves were obtained that followed first-order reaction kinetics. The kinetics data revealed that the rate constant for degradation of PCP for Xz6-1 was 0.063 h-1, a value that was over 50% greater than that of Pseudomonas putida (0.040 h-1). Aerobic granular sludge was highly fortified with Xz6-1 and Pseudomonas putida to provide PCP degradability improvements of 180.9% and 98.3%, respectively, relative to the original sludge. All results demonstrate that the protoplast fusion technique is an effective approach to construct a high-activity chlorophenol-degrading strain.

Intergenus Protoplast Fusion between Pichia manshurica and Rhodosporidium paludigenum to Increase the Production of Inulinase

The purpose s of this study was to identify the optimum concentration of the lytic enzyme Glucanex for protoplast isolation and to conduct fusion for the purpose of increasing inulinase production . The study performs the protoplast fusion technique using Pichia manshurica and Rh odosporidium paludigenum . Protoplast fusion consists of a series of stages: protoplast isolation , protoplast fusion , protoplast regeneration , and analysis of hybrid fusion results . Protoplast isolation and fusion success rate are determined by various factors, including age of the culture , media type, and type of lytic enzymes used . H ybrid results were analyzed using a fungicide as a marker and measuring specific growth rate ( μ) of the hybrid compared with parental growth rates. Results demonstrated that a concentration of 4 mg/ mL of Glucanex produces the greatest number of protoplasts, 7.2 x 10 10 ( c ell/mL) for P. manshurica and 8.8 x 10 10 ( c ell/mL) for Rh . paludigenum . The results of analysis of hybrid fusions indicate that the study has identified a new fusant , called fusant F4 . Fusant F4 is capable of producing the highest inulinase, 0.6892 IU, compared with parentals P. manshurica, 0557 IU, and Rh . paludigenum, 0.3263 IU. Fusant F4 has specific growth rate ( μ) of 0.3360/h and generation time ( g) of 2.0629 h .

Development and Characterization of Somatic Hybrids of Ulva reticulata Forsskål (×) Monostroma oxyspermum (Kutz.)Doty.

Ulvophycean species with diverse trait characteristics provide an opportunity to create novel allelic recombinant variants. The present study reports the development of seaweed variants with improved agronomic traits through protoplast fusion between Monostroma oxyspermum (Kutz.) Doty and Ulva reticulata Forsskål. A total of 12 putative hybrids were screened based on the variations in morphology and total DNA content over the fusion partners. DNA-fingerprinting by inter simple sequence repeat (ISSR) and amplified fragment length polymorphism (AFLP) analysis confirmed genomic introgression in the hybrids. The DNA fingerprint revealed sharing of parental alleles in regenerated hybrids and a few alleles that were unique to hybrids. The epigenetic variations in hybrids estimated in terms of DNA methylation polymorphism also revealed sharing of methylation loci with both the fusion partners. The functional trait analysis for growth showed a hybrid with heterotic trait (DGR% = 36.7 ± 1.55%) over the fusion partners U. reticulata (33.2 ± 2.6%) and M. oxyspermum (17.8 ± 1.77%), while others were superior to the mid-parental value (25.2 ± 2.2%) (p < 0.05). The fatty acid (FA) analysis of hybrids showed notable variations over fusion partners. Most hybrids showed increased polyunsaturated FAs (PUFAs) compared to saturated FAs (SFAs) and mainly includes the nutritionally important linoleic acid, α-linolenic acid, oleic acid, stearidonic acid, and docosahexaenoic acid. The other differences observed include superior cellulose content and antioxidative potential in hybrids over fusion partners. The hybrid varieties with superior traits developed in this study unequivocally demonstrate the significance of protoplast fusion technique in developing improved varients of macroalgae.

Production and evaluation of antimycotic and antihepatitis C virus potential of fusant MERV6270 derived from mangrove endophytic fungi using novel substrates of agroindustrial wastes.

Among forty endophytic fungal isolates derived from the mangrove plant Avicennia marina, thirty-seven isolates (92.5 %) shown vary antimycotic activity against clinical Trichophyton, Microsporum, and Epidermophyton isolates. The hyperactive wild antagonistic strains Acremonium sp. MERV1 and Chaetomium sp. MERV7 were subjected to intergeneric protoplast fusion technique, and out of 20 fusants obtained, the fusant MERV6270 showed the highest antimycotic activity with the broadest spectrum against all dermatophytes under study. Solid-state fermentation (SSF) showed its superiority for antimycotic/antiviral metabolite production using cost-effective agroindustrial residues. Low-cost novel fermentation medium containing inexpensive substrate mixture of molokhia stalk, lemon peel, pomegranate peel, peanut peel (2:1:1:1) moistened with potato, and meat processing wastewaters (2:1, at moisture content of 60 %) provided a high antimycotic metabolite yield, 33.25 mg/gds, by the fusant MERV6270. The optimal parameters for antimycotic productivity under SSF were incubation period (4 days), incubation temperature (27.5-30 °C), initial pH (6), initial moisture level (60 %), substrate particle size (1.0 mm), and inoculum size (2 × 10(6) spores/gds), which elucidated antimycotic activity to 44.19 mg/gds. Interestingly, wild mangrove Acremonium sp. MERV1 and Chaetomium sp. MERV7 strains and their fusant MERV6270 showed significant inhibition of hepatitis C virus with viral knockdown percent of -82.48, -82.44, and -97.37 %, respectively, compared to the control (100 %), which open a new era in combat epidemic viral diseases.

A xylose-fermenting yeast hybridized by intergeneric fusion between Saccharomyces cerevisiae and Candida intermediamutants for ethanol production

Bioethanol production from lignocellulosic biomass, in particular xylose, is currently of great concern, given the abundance of this sugar in the world, because Saccharomyces cerevisiae, which is widely used for bioethanol production, is unable to naturally ferment xylose. The aim of this study was to obtain a novel yeast capable of stably producing ethanol from biomass containing xylose by protoplast fusion between S. cerevisiae and xylose-utilizing yeast. We describe a novel xylose-fermenting yeast strain, FSC1, developed for ethanol production by intergeneric hybridization between S. cerevisiae and Candida intermedia mutants by using a protoplast fusion technique. The characteristics of the FSC1 strain are reported with respect to xylose fermentation, morphology, gene, and protein expression. Mutation of the parental strains prior to protoplast fusion endowed the FSC1 strain with the ability to convert xylose to ethanol. Microscopic analysis confirmed that the parental and FSC1 strains produced spores in the potassium acetate medium. The FSC1 strain is uninucleate diploid, has a stable metabolism, and expresses proteins at a higher level than the parental strains. We found that FSC1 strain could stably achieve an ethanol yield of 0.38 g/g-substrate in fermentation of a mixture of glucose and xylose. In addition, the fermentation ability of FSC1was improved by successive chemical mutation, resulting in a higher ethanol yield of 0.42 g/g-substrate, corresponding to 82% theoretical yield. The mutation-fusion technique we have described here is very useful for the development of intergeneric hybrids capable of xylose fermentation, and the FSC strains generated using this technique have the potential for industrial use in ethanol production from lignocellulosic biomass.

Phylogeny and antagonistic activity of some protoplast fusants in Trichoderma and Hypocrea

The present work aimed to apply mutagenesis and inter-specific protoplast fusion techniques of two locally isolated Hypocrea and Trichoderma to enhancement their biocontrol abilities against some important plant fungal pathogens which cause damping-off diseases that attacking different crops. The mutants were selected after EMS/UV treatment of Trichoderma isolates. The obtained protoplasts were fused by polyethylene glycol, and six fusants were selected for further studies. The phylogeny of the parental strains was carried out using sequence of ITS region. The BLAST of the obtained sequence was identified these isolates as H. koningii and T. asperellum. The fused protoplasts of the two mutant strains have been regenerated on PDA medium supplemented with the two fungicides. Most of the fusants exhibited fast mycelial growth on PDA as compared to parent strains. The obtained results indicated that partial or incomplete genetic recombination may be possible during nuclear and cytoplasmic protoplast fusion. Most of the fusants have shown powerful antagonistic activity against the grapevine pathogens as Pythium ultimum and Fusarium roseum as shown in dual culture and observed by SEM technique. Results of the present study demonstrated the scope and significance of the protoplast fusion technique, which can be used to develop superior hybrid strains of filamentous fungi that absent sexual stages in Trichoderma enhance biological control activity.DOI: http://dx.doi.org/10.3126/ijasbt.v2i2.10113 Int J Appl Sci Biotechnol, Vol. 2(2): 146-151

Aplikasi Pakan Kaya Karotenoid Hasil Fusi ProtoplasmIntergenera Dunaliella salina dan Chlorella vulgaris pada Udang Windu (Penaeus monodon F.) Stadia PL-20 Di Desa Asempapan, Pati, Jawa Tengah

Dunaliella salina and Chlorella vulgaris is a natural feed microalgae with high carotenoid content that can be increased using protoplast fusion technique. Protoplast fusion as one of the application fields of genetic engineering is a method for obtaining recombinant with the desired properties and profitable in a short time. This study aimed to see the effect of the addition of carotenoid-rich feed results from protoplast fusion recombinant D. salina and C. vulgaris on the survival rate and weight of shrimp post larvae. Mixed fusion results feed and artificial feed needed for the growth of post-larval shrimp, moulting and skin pigmentation. The results showed that the recombinant from protoplast fusion intergenera D. salina and C. vulgaris contains carotenoid pigments higher, reaching 124.6 mg / g bks from the second parent, namely D. salina reached 101.83 mg / g bks, while C. vulgaris 97.18 ug / g bks. Feed manufacturing is done by mixing pellets and 80-100x103 cells per 0.0225 g of feed. Application of feed carried on Penaeus monodon F. (tiger prawn) stadia PL-20 for a month. The results of weight measurements showed the highest prawn post larvae reached at artificial feeding plus recombinant protoplast fusion results intergenera D. salina and C. vulgaris and was able to raise the level of post-larval shrimp survival rate reached 88%. Keywords: D. salina, C. vulgaris, Protoplast Fusion, Carotenoid, Penaeus monodon F.

Construction of engineering microorganism degrading chlorophenol efficiently by protoplast fusion technique

Protoplast fusion is an important technique for engineering microbial strains. It has been used to combine genes from different organisms to create new strains with desired properties. This investigation was aimed to construct a new strain with an ability of degrading chlorophenols efficiently by protoplast fusion between Pseudomonas putida and Psathyrella candolleana. Twenty recombinant strains were screened and used to degrade pentachlorophenol (PCP) synthetic wastewater in order to determine their capabilities of chlorophenol degradation. To rapidly and accurately measure PCP content in synthetic wastewater after treatment by different strains, a new method quantifying PCP was proposed based on derivative spectroscopy. Chlorophenol biodegradability of different strains could be measured rapidly according to the new method. The results showed that PCP degradation % of four recombinant strains viz., Xz 6‐1, Xz 6‐3, Xz 6‐5, Xz 8‐2 were 78.98%, 69.12%, 31.40%, 46.26%, respectively, and that of parent strain (Pseudomonas putida.) was 57.27%, which indicated that the degradation % of Xz 6‐1 is the highest and improved by 21.71% compared with Pseudomonas putida. Furthermore, the colony morphologies and microscopic feature of Xz 6‐1 and Pseudomonas putida were compared in this article. © 2012 American Institute of Chemical Engineers Environ Prog, 32: 443–448, 2013

English

&nbsp; In an attempt to construct superior&nbsp;Trichoderma harzianum&nbsp;isolates for improving &beta;-glucosidase productivity, protoplast fusion technique was applied. After application of different mutagenic treatments, twenty mutants were chosen to be tested for their resistance or sensitivity against four antifungal agents. Out of them, four isolates were selected on the basis of their response to antifungal agents and their productivities of carboxymethylcellulase (CMCase) and &beta;-glucosidase to be introduced into intraspecific protoplast fusion experiments using two different methods (PEG and electrofusion). Three crosses were carried out among the selected four isolates. Results showed that, the number of fusants obtained after electrofusion were more than those obtained after polyethylene glycol (PEG) method. In addition, high productivities of CMCase and &beta;- glucosidase were obtained after electrofusion in the three crosses. The applied protoplast electrofusion method proved to be a good and effective method for obtaining&nbsp;T.&nbsp; harzianum&nbsp;fusants with higher productivity of &beta;- glucosidase enzyme. &nbsp; Key words:&nbsp;Trichoderma, protoplast fusion, electroporation, &beta;-glucosidase.

Biochemical and molecular characterization of wild-type and fused protoplasts of Beauveria bassiana and Metarhizium anisopliae

Protoplasts were isolated from two isolates each of Beauveria bassiana and Metarhizium anisopliae using lysing enzymes. Intra- and intergeneric protoplast fusion has been carried out using 40% polyethylene glycol. The fused protoplasts of B. bassiana and M. anisopliae have been regenerated on Czapek-Dox agar media, and a total of four fusants were selected for further studies. An increase in proteinase and chitinase enzyme activity was recorded with all fusants as compared to the wild-type isolates. To understand the nature of recombination process, random amplification of polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) were carried out on genomic DNA of fused and wild-type isolates. The present study demonstrates the scope and significance of the protoplast fusion technique as a rapid consistent method for identification of B. bassiana and M. anisopliae fused and wild-type isolates based on the banding pattern of RAPD and RFLP that can be reliably used ahead for further applications on these species.

Chitinolytic enzyme production and genetic improvement of a new isolate belonging to Streptomyces anulatus

Thirty bacterial isolates were obtained from different sources and sites at Jeddah, Saudi Arabia, on chitin agar medium; 9 of the 30 isolates were cultured in liquid medium containing chitin as sole carbon and nitrogen sources. Isolate SM21, which was isolated from shrimp shells, showed the best growth and chitinase production in liquid medium. According to its morphological, physiological and biochemical characteristics, SM21 belongs to the genus Streptomyces and was identified as Streptomyces anulatus SM21. Identification was confirmed using 16S rDNA analysis. The chitinase enzyme was precipitated with 80% NH4SO4 and purified using DEAE-cellulose ion exchange chromatography followed by Sephadex G-100 gel filtration. The molecular weight determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis was 28 kDa. Genetic improvement using the protoplast fusion technique was carried out between the identified Streptomyces isolate and Streptomyces coelicolor SM1. These two species, which have different resistance profiles to streptomycin and tetracycline (400 μg/ml and 10 μg/ml, respectively), were used in an intraspecific protoplast fusion using PEG 6000. The percentage of real protoplasts that could regenerate successfully was 71% for S. coelicolor SM1 and 80% for S. anulatus SM21. Out of three recombinant fusants obtained, one (named Fu3) showed higher chitinase production compared to both parents (5 fold increase).

Mutagenesis and inter-specific protoplast fusion between Trichoderma koningii and Trichoderma reesei for biocontrol improvement

The present work aimed to apply mutagenesis and inter-specific protoplast techniques of two locally isolated Trichoderma spp. to enhancement their biocontrol abilities against some important plant fungal pathogens which cause root-rot and damping-off diseases that attacking different crops. A combination of UV-light and nitrate treatment induced seven mutants (T. koningii, 3 mutants and T. reesei, 4 mutants). The inter-specific protoplast fusion between T. koningii and T. reesei gave fusion frequency reached 1.25% and thirteen fusants were isolated. In vitro bioassay of the parental Trichoderma spp., their selected mutants and fusants indicated the effective of both T. koningii and T. reesei in suppressive the tested fungal pathogens. On the other hand, most of the selected mutants and fusants showed superiority in their antagonistic activity against these pathogens than their parental strains. the superiority in biological control activities of the selected mutants or fusants than their parents against the tested pathogens may be due to the effect of the genetic treatments (i.e. mutagenesis an protoplasts fusion) on their genetic back ground to be varied that allow to change in genetic control of antifungal metabolites production to be more effective. The results indicated that mutation and protoplast fusion techniques are successful tools to enhance the antagonistic effects of Trichoderma species against several fungal plant pathogens.

Identification of protoplast fusion strain Fhhh by randomly amplified polymorphic DNA

A functional strain Fhhh was constructed through protoplast fusion of three parental strains (Phanerochaete chrysosporium, Saccharomyces cerevisiae and native bacterium YZ) to improve the degradation efficiency of purified terephthalic acid wastewater. Randomly amplified polymorphic DNA (RAPD) and scanning electron microscope (SEM) analysis were applied to identify and confirm the fusant Fhhh through phenotypic and genetic relationship. The result of SEM analysis demonstrated that the cell shape of fusant Fhhh differed from all three parental strains. RAPD analysis of 40 arbitrary primers generated a total of 1,135 bands. The genetic similarity indices between Fhhh and parental strains Phanerochaete chrysosporium (PC), Saccharomyces cerevisiae (SC) and native bacterium (YZ) were 34.01%, 33.16%, and 35.97%, respectively. The targeted-gene PCR results showed that Fhhh inherited the DNA fragments of mnp and lip genes from parental strain PC and FLO1 gene fragment from parental strain SC. Our results suggested protoplast fusion technique may be considered as a promising technique in environmental pollution control.

Genetic analysis of protoplast fusant Xhhh constructed for pharmaceutical wastewater treatment

In order to analyse genetic relationships between functional strain Xhhh previously constructed through protoplast fusion for pharmaceutical wastewater treatment and its parents, random amplification polymorphic DNA (RAPD) and polymerase chain reaction (PCR) were used to investigate genetic similarities among the strains based on genome and functional genes analyses. A total of 739 clear and consistent bands were produced in the RAPD fingerprint analysis with 40 primers. The genetic similarity indices between Xhhh and parental strains PC ( Phanerochaete chrysosporium), SC ( Saccharomyces cerevisiae) and XZ (native bacterium Bacillus sp.) were 36.21%, 37.73% and 37.48%, respectively. With PCR amplification and DNA sequencing, Xhhh was found containing functional genes of mnp and lip from PC, FLO1 from SC and 16S rDNA fragments from XZ. Experimental results of genetic analyses were in accordance with Xhhh biochemical and phenotypic characteristics, and protoplast fusion technique is considered as a promising technique in environmental pollution control.

Generation of recombinants strains to cellulases production by protoplast fusion between Penicillium echinulatum and Trichoderma harzianum

Nowadays, cellulase importance is growing mainly due to its potential utilization in developing second-generation ethanol technology. Thus, considerable effort has been made to obtain new genotypes with improved capacity to produce efficient and cost-effective enzyme complexes for cellulose hydrolysis. The majority of the genetically manipulated strains that produce higher amounts of cellulases come from of the wild Trichoderma reesei QM6A. Such as T. reesei cellulolytic mutants, Penicillium echinulatum mutants also produce high titles of cellulases (filter paper activities—FPA), and its enzymatic complex effectively hydrolyses cellulose. In the present work, we obtained genetic variability with the purpose of enhancing cellulase production by protoplast fusion between P. echinulatum and T. harzianum. The resulting fusants, who were all morphologically like the P. echinulatum parental, were initially characterized by RAPD, and the results showed that almost all amplified DNA bands showed correlation with P. echinulatum DNA bands. Some of new selected strains presented higher FPA and β-glucosidases activities when compared to the parental strains. One particular clone – BP2 – showed values of FPA activities around of 2 UI ml −1, in submerged cultures and all selected strains showed faster and higher secretion of cellulases in solid-state cultures, in comparison to the parental strain of P. echinulatum. Altogether, at the same time that our results show increased cellulase secretion by P. echinulatum strains, they also show that protoplast fusion techniques associated with an efficient selection process could be a useful tool in genetic improvement to generate better cellulose-producing strains aimed at economically hydrolyzing cellulose with a view to second-generation ethanol.

Potential of Verticillium lecanii (Lecanicillium spp.) hybrid strains as biological control agents for soybean cyst nematode: Is protoplast fusion an effective tool for development of plant-parasitic nematode control agents?

Verticillium lecanii is a potentially useful biological control agent (BCA) for soybean cyst nematode (SCN), Heterodera glycines. The objective of this study was to screen hybrid strains of V. lecanii, derived from protoplast fusion, for effective control agents of SCN, and to investigate whether the protoplast fusion technique is an effective tool for development of nematode control agents. Three parental strains (Vertalec®, Mycotal®, and B-2) and their 162 hybrid strains were screened in greenhouse pot tests. Some of these hybrid strains suppressed damage on soybean plants and reduced the density of SCN in the soil. In particular, one hybrid strain, AaF42, was observed to reduce the nematode egg density by 93.2% as compared with the control. Furthermore, this strain significantly reduced the cyst and egg density as compared with the parental strains. In conclusion, some of the hybrid strains exhibited enhanced biocontrol efficacy by protoplast fusion. Therefore, the protoplast fusion technique may be a potentially valuable tool for developing nematode-antagonistic fungi as BCA.

Formation of interspecies fusants of Agaricus bisporus and Agaricus bitorquis mushroom by protoplast fusion.

Interspecies fusants are formed between Agaricus bisporus and Agaricus bitorquis by protoplast fusion technique. Protoplasts were isolated and regenerated by using Novozyme 234 lytic enzyme. Twenty slow growing isolates were separated from the protoplast regenerated colonies, which were assumed as homokaryons (putative homokaryons). These twenty isolates were subjected to growth rate, colony morphology and spawn run studies for screening of true homokaryons. Antifungal markers were developed for selection of fusants.

Protoplast fusion technique in Tolypocladium inflatum for increasing Cyclosporine production

Protoplast fusion technique in Tolypocladium inflatum for increasing Cyclosporine production

Morphological, cytological and molecular characterization of a novel symmetric somatic hybrid between N. tabacum and N. glauca

By using the protoplast fusion technique, we have obtained 44 regenerated plants, phenotypically different and distinct from their parents, among which we have identified a fertile symmetric somatic hybrid, designated as TG-32, between N. tabacum var. Gexin No.1 and N. glauca. The morphology, fertility, chromosome number and nuclear constitution of the somatic hybrid have been studied in detail. Unlike other asymmetric interspecific somatic hybrids, the chromosome number of the symmetric somatic hybrid is 72, equal to the sum of chromosomes of both parents. The TG-32 plant has flowers similar to those of N. tabacum, but with petals similar to those of N. glauca. Interestingly the offspring of TG-32 vary in seed production ability with temperature, and produce more seeds under a relatively low temperature. Two SCAR markers were used to evaluate genetic variability and structure. The hybrid amplified the expected fragment, but the parents showed only one of two markers. This experimental result supports the hypothesis of the co-existence of two parental genomes in the somatic hybrid.

A Pilot Study on the Biological Treatment of PTA Wastewater with Functional Strain Fhhh

The functional strain Fhhh, constructed with the protoplast fusion technique, was applied to PTA wastewater treatment at a NJYZ pilot plant. The maximum chemical oxygen demand (COD) removal efficiency and effluent suspended solids obtained were 97% and 59 mg L−1, respectively, at a hydraulic retention time of 1.3 days and an F/M rate of 1.35 g COD g SS−1 day−1. The specific degradation rate arrived at 1.31 day−1 in the bioreactor after a 180-day operation, which is much higher than corresponding data of other related references. The addition of Mn2+, Cu2+, Zn2+, and SeO3 2− significantly stimulated the MnP exprssion, and a positive correlation was established between the improvement of MnP expression and the removal efficiencies of COD, tereogtgaleuc acid, p-toluic acid, and 4-carboxybenzaldehyde in purified terephthalic acid wastewater. The effluent water quality was also improved significantly as long as the COD:TN:TP ratio was optimally maintained around 100:1.16:0.22; the best growth and flocculation performance were obtained with a specific growth rate of 0.133 day−1 and sludge volume index of 136 mL g−1.