• 제목/요약/키워드: Intergeneric protoplast fusion

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Gliocladium virens 와 Trichoderma harzianum의 속간(屬間) 원형질체융합(原形質體融合) (Intergeneric protoplast fusion between Gliocladium virens and Trichoderma harzianum)

  • 신평균;조무제
    • 한국균학회지
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    • 제21권4호
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    • pp.323-331
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    • 1993
  • The protoplast formation and intergeneric protoplast fusion between Gliocladium virens and Trichoderma harzianum were attempted to obtain fusants. Protoplast formation was the most effective when the strains were treated with concentration of 5 mg/ml of Novozyme 234 and Cellulase at $25^{\circ}C$ for 3 hours in phosphate buffer, pH 6.5, supplemented with 0.6 M sorbitol as osmotic stabilizer. Auxotrophic mutants of G. virens G88 did not grow in minimal medium and benomyl resistant T. harzianum T95 from wild types, however, was selected by treatment with UV light as genetic marker to isolate fusants. When the intergeneric protoplast fusion between G. virens G88 and T. harzianum T95 was carried out using 30% PEG 4000 containing 10 mM $CaCl_{2}$, and 50 mM glycine (pH 8.5) as fusogenic agent at $25^{\circ}C$ for 10-15 min, the fusion frequency was $0.8{\times}10^{-4}$. Fusants obtained from intergeneric protoplast fusion were spontaneously segregated into va rious strains by continous culture on complete medium. Several intergeneric hybrids were classified into three types: parent-like hybrids, segregants, and recombinants.

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Studies on Intergeneric Protoplast fusion and L-Lysine Productivity

  • 이인선;조정일
    • 동아시아식생활학회지
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    • 제5권1호
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    • pp.93-99
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    • 1995
  • For the improvement of the L-lysine productivity of Brevibacterium flavum and Corynebacterium glutamicum, fusants were induced by interspecific protoplast fusion of Bacillus subtilis with C. glutamicum and B. flavum. The following results were obtained through protoplast formation of strains condition of protoplast fusion, characteristics of the fusants, and the productivity of lysine form starch. B. flavum BF-5 and C. glutamicum protoplasts were made by the treatment of 0.3unit/$m\ell$ of penicillin G at the early stationary growth phase for 2 hours followed by incubation with 10mg/$m\ell$ of lysozyme at 37$^{\circ}C$ for 120 min. When a mixture of the protoplast was treated with 30% PEG(M.W.6,000) solution containing 50mM CaCl2 at optimal conditions, the intergeneric fusion frequency between protoplasts of C. glutamicum CG-2 and B. subtilis BD 224 was 7.1${\times}$105. The genetic properties on the L-lysine producing fusants were compared with those of parental strains. As a results, the intergeneric fusants were completed in each auxotrophic requirement, resistances for S-(2-amino-ethyl)-L-cysteine and kanamycine were confirmed. And one of fusants selected, FBB-41 were found to be genetically stable fusants. The aspartokinase activity of FBB-41 strain increased than that of the parent strain.

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감자(Solanum tuberosum L.)와 담배 (Nicotiana tabacum L.)의 원형질체 배양 및 융합 (Culture and Fusion of Protoplasts from Potato (Solanum tuberosum L.) and Tobacco (Nicotiana tabacum L.))

  • 정상호
    • Journal of Plant Biology
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    • 제30권4호
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    • pp.287-298
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    • 1987
  • The regenerative capacities of protoplasts isolated from potato (Solamum tuberosum L.) tubers and tobacco (Nicotiana tabacum L.) mesophyll tissues were examined, and then their intergeneric protoplast fusion was carried out. The potato tuber-derived protoplasts proliferated into the calli some of which showed rudimentary shoot-like structures, which had not been attempted before from tubers, while the tobacco protoplasts were regenerated into the whole plants. Intergeneric protoplast fusion between potato and tobacco was carried out and the heteroplasmic fusion products were formed. The first cell division of some of them was observed after 5 days of culture.

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Intergeneric protoplast fusion between Bacillus pumilus and Cellulomonas fimi

  • Kim, D.M.;Lee, K.H.
    • 한국미생물생명공학회:학술대회논문집
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    • 한국미생물생명공학회 1986년도 추계학술대회
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    • pp.528.1-528
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    • 1986
  • Cellulose utilising hybrids between Cellulomonas fimi and Bacillus pumilus were isolated after PEG mediated protoplast fusion. 33% (w/v) PEG #6, 000 and 50mM $Ca^{++}$were optimum concentration. The intergeneric fusion frequency was 3.2$\times$10$^{-7}$ xtracellular CMCase and $\beta$-glucosidase activities were detected from one hydrid unlike only CMCase was detected from Cellulomonas fimi.

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Genetically Engineered Yeast by Heterologous Transformation and Intergeneric Two-Step Protoplast Fusion for Ethanol Fermentation

  • Kim, Young-Ho;Lee, Jae-Ran;Seu, Jung-Hwn
    • Journal of Microbiology and Biotechnology
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    • 제3권4호
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    • pp.232-237
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    • 1993
  • A strain of yeast which can convert starch directly to ethanol was developed by the intergeneric protoplast fusion between Schwanniomyces alluvius possessing $\alpha$ amylase as well as glucoamylase with debranching activity and FSC-14-75 which previously had been formed from a heterologous transformation and subsequent intergeneric protoplast fusion. Fusants were selected on minimal medium after protoplasts of auxotrophic mutant of S. alluvius fused with heat-treated protoplasts of FSC-14-75 in the presence of 30%(w/v) PEG and 20 mM $CaCl_2$. The fusion frequency was in the range of $10^{-6}$ order. All fusants tested were intermediate types of parental strains for carbon compound assimilation, and their cell volumes were approximately 1.1 times larger than FSC-14-75 and 1.8 times larger than S. alluvius. The fusants were unable to sporulate like FSC-14-75, while S. alluvius could sporulate. In flask scale the most promising fusant, FSCSa-R10-6, produced 7.83%(v/v) and 10.17%(v/v) ethanol from 15% and 20% of liquefied potato starch, respectively, indicating that the fermetation efficiency of each case increased 1.2 times and 1.6 times than that of FSC-14-75. The elution pattern on DEAE-cellulose chromatography showed that FSCSa-R10-6 has four distinct amylase peaks of which two peaks originated from S. alluvius and the other two from FSC-14-75. These results suggest that the enhanced fermentation efficiency of the fusant might be due to almost-complemented parental amylases.

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Rhizopus oryzae와 Aspergillus oryzae의 속간 원형질체융합 (Intergeneric Protoplast Fusion between Rhizopus oryzae and Aspergillus oryzae)

  • Lee, Soo-Youn;Jung, Sung-Won;Kim, Seong-Han;Lee, Yung-Nok
    • 미생물학회지
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    • 제31권3호
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    • pp.218-223
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    • 1993
  • Conditions for the release and regeneration of protoplasts form Rhizopus oryzae and intergeneric protoplast fusion between Rhizopus oryzae and Aspergillus oryzae were studied. High yields of protoplast fusion between Rhizopus oryzae and Aspergillus oxyzae were studied. High yield of protoplasts from young germilings of R. oryzae were obtained by using lytic enzymes containing chitosanase (3 mg/ml), chitinase (3 mg/ml) and Novozym 234 (5 mg/ml). 0.5M glucose was used as the osmotic stabilizer and optimum pH of buffer was determined to be pH 7.5-8.0. Under these conditions, protoplasts were formed after about 3-4 hrs incubation. Approximately, 1.0%-4.9% of these protoplasts were formed after about 3-4 hrs incubation. Approximately, 1.0%-4.9% of these protoplasts regenerated on solid medium with a soft agar overlay. We have also carried out protoplasts fusion between R. oryzae and A. oryzae and have succeeded in obtaining three types of intergeneric fusants. In these experiments, 35% PEG-4000 and 10 mM CaCl$_{2}$ were used as fsogenic agents, and auxotrophic properties were used as a genetic marker to select fusants. Complementation frequency be protoplasts fusion of A. oxyzae and R. oryzae was 4.4% * 10$^{-5}$ . The fusant strains of the first type were prototrophs showing an Aspergillus type morphology with dark-yellow sporulation, those of the second type were also Apergillus type morphology but showed no sporulation. And the strains of the third type stopped growing when fusion products grown on regeneration minimal medium were transferred to fresh minimal medium. The formation of fusion products was observed by fluorescent vital stains for complementary labelling of protoplats from R. oryzae and A. oryzae. Rhodamine 6G and fluorescein diacetate wer useful complementary vital stains of Rhizopus and Aspergillus protoplasts for visualization of requency and type (dicell, multicell) of fusion.

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Characterization of Alcohol Fermentation and Segregation of Protoplast Fusant of Saccharomyces cerevisiae and Pichia stipitis

  • YOON, GEE-SUN;TAE-SIK LEE;CHUL KIM;JIN-HO SEO;YEON-WOO RYU
    • Journal of Microbiology and Biotechnology
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    • 제6권4호
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    • pp.286-291
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    • 1996
  • A study was conducted to investigate the characteristics of segregation and alcohol fermentation of intergeneric fusants. The protoplast fusion of both Pichia stipitis CBS 5776 and Saccharomycess cerevisiae STV 89 was carried out. The fusion frequency was $5\times10^{-8}$ and among fusants selected, a fusant F5 showed the best results in ethanol production by sucrose and xylose fermentations. The performance of xylose fermentation by this fusant was better than that of P. stipitis CBS 5776 and fusant F5 exhibited sucrose fermentation patterns intermediate to the two parent strains. The fusant F5 was segregated into a pair of parental strains during the several culture passages. In the average, 91$%$ of colonies had a similar characteristics of P. stipitis while 7$%$ of colonies resembled S. cerevisiae. Only 2$%$ of colonies had the characteristics of the original fusants. At the sixth passage, all segregants resembled P. stipitis. From these results it is suggested that intergeneric protoplast fusion led to an integration of S. cerevisiae genes, rather than whole chromosomes, within the entire genome of P. stipitis.

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속간 원형질체 융합에 의한 섬유질 기질로부터 L-Lysine 생산균주 개발 -융합조건 및 융합체의 성질 - (Development of L-Lysine Producing Strains from Cellulosic Substrate by the Intergeneric Protoplast Fusion - Conditions for Fusion and Properties of Fusants-)

  • 성낙계;정덕화;박법규;정영철;전효곤
    • 한국미생물·생명공학회지
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    • 제16권3호
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    • pp.175-181
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    • 1988
  • 섬유소 기질로부터 L-lysine을 생산할 목적으로 Cellulomonas flavigena에 Brevibacterium flavum 및 Corynebacterium glutamicum을 각각 이속간 원형질체 융합을 하여 융합조건과 융합체의 성질을 조사하였다. 각각의 parental protoplast를 동량으로 혼합한 후 30% PEG 6000으로 3$0^{\circ}C$, 30분간 융합시킨 결과 1.9$\times$$10^{-6}$~2.1$\times$$10^{-6}$의 융합빈도를 얻었고, 134주의 융합체 중 유전적 안정화가 판명되고 CMC 기질로부터 L-lysine 생성능이 인정된 FCB 3 및 FCC 19를 최종 선별하였다. FCB 3 및 FCC 19는 친주보다 DNA 함량이 높을 분 아니라 G+C 함량도 융합전 친주의 G+C 총함량의 약 1/2에 해당하여 반수체로서 안정화되어 있었으며, 그리고 친주와 반대로 CMC 자화능이 있어 CMC로부터 L-lysine을 배지 중에 축적하였다.

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Brevibacterium flavum의 동종간 및 Corynebacterium glutamicum과의 이속간 원형질체 융합 및 개량균주의 L-Lysine 생합성의 대사제어 (Intraspecific Protoplast Fusion of Brevibacterium and Intergeneric Protoplast Fusion between Brevibacterium flavum and Corynebacterium glutamicum and the Metabolic Control of L-Lysine Biosynthesis in Improved Bacterial Strains)

  • 박청;임번삼;전문진
    • 한국미생물·생명공학회지
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    • 제15권2호
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    • pp.104-111
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    • 1987
  • Lysine 생산균주를 개량하기 위한 시도로서 Brevibacterium flavum ATCC 21528R 과 Brevibacterium flvum ATCC 21529S의 동종간 및 Brevibacterium flavum ATCC 21528R 과 Corynebacterium glutamicum ATCC 13058S 와 의 이속간 원형질체 융합을 실시하였다. 이들 균주들에 대한 원형질체 형성의 최적조건을 조사하고 재생과 융합에서의 plasma expander의 효과를 검토하였다. 융합주 No. CH23과 No. CH41은 최적 배양조건 하에서 L-lysine 생산성이 모균에서보다 각각 21%와 8.9% 향상된 것이었다. L-Lysine 생합성 회로상의 중심효소인 asparto-kinase를 포함한 주요효소의 활성을 측정하였고, 융합주 No. CH23과 No. CH41에서의 L-lysine 생합성 대사제어를 친주와 비교하였다.

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