• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.644-648
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• 2005

Dextranase (${\alpha}$-1,6-D-glucan-6-glucanogydrolase:E.C. 3.2.1.11) catalyzes the hydrolysis of ${\alpha}$-(1.6) linkages of dextran. A lsd1 gene encoding an extracellular dextranase was isolated from the genomic DNA of L. starkeyi. The lsd11 gene is a synthetic dextranase (lsd1) after codon optimization for gene expression with Pichia pastoris system. A open reading frame of lsd11 gene was 1827 bp and it was inserted into the pPIC3.5K expression vector. The plasmid linearized by Sac I was integrated into the 5'AOX region of the chromosomal DNA of P. pastoris. The lsd11 gene fragment encoding a mature protein of 608 amino acids with a predicted molecular weight of 70 kDa, was expressed in the methylotrophic yeast P. pastoris by controling the alcohol oxidase-1 (AOX1) promoter. The recombinant lds11 was optimized by using the shake-flask expression and upscaled using fermentation technology. More than 9.8 mg/L of active dextranase was obtained after induction by methanol. The optimum pH of LSD11 was found to be 5.5 and the optimum temperature $28^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• 제14권1호
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• pp.90-96
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• 2004

A synthetic defined medium, fortified with amino acids, was developed for the stable production of the kringle fragments of human apolipoprotein(a) (apo(a)), rhLK68. Using a complex rich medium containing yeast extract and a high-cell-density fed-batch culture, the expression level of rhLK68 reached 17% of the total cellular protein, which corresponded to $5\;g\;l^{-1}$ of the culture. To replace the complex media with chemically defined media, several amino acids that positively affect cell growth and gene expression were chosen by a statistical method. The various combinations of the selected amino acids were tested for its fortifying effect on a minimal defined medium. When glutamine only was added, the overall expression level of rhLK68 reached 93% of the complex rich medium increasing the specific expression level by 22.4% and decreasing the cell growth by 24%. Moreover, the addition of glutamine resulted in a 2-fold increase in the concentration of rhLK68 in the culture broth, compared with the minimal defined medium. The synthetic defined media developed in this study could be generally applied to high-cell-density cultures of the recombinant Escherichia coli BL21(DE3), especially for the production of therapeutic proteins that require a strict quality control of the culture media and fermentation processes.

• Korean Chemical Engineering Research
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• 제55권2호
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• pp.237-241
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• 2017

아세토인(acetoin)은 식품과 화학산업에서 플랫폼 물질로 이용되며 산업적으로 다양한 응용이 가능한 물질이다. 본 연구에서는 대사공학(metabolic engineering)을 이용하여 아세토인의 생산량이 증가한 재조합 Klebsiella pneumoniae를 구축하였다. 우선 2,3-부탄디올(2,3-butanediol)생산을 위해 제작되었던 재조합 K. pneumoniae (KMK-05)에서 두 가지 2,3-butanediol dehydrogenase (budC, dhaD)를 유전체에서 제거하여 아세토인 생산량을 늘리고, 전사인자 중 하나인 AcoK를 제거하여 아세토인을 분해하는 효소의 발현량을 줄였다. 그리고 NADH oxidase를 발현시켜 세포 내 산화 환원 균형(redox balance)을 맞춰 대사흐름을 개선하였다. 이렇게 대사공학을 통해 구축된 재조합 Klebsiella pneumoniae(KJW-03-nox)로 아세토인 생산량과 수율을 높였고, 36시간 동안의 유가식 배양을 진행하여 51 g/L의 아세토인 농도와 최대 생산성 2.6 g/L/h을 달성하였다.

• 농업과학연구
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• 제44권1호
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• pp.30-39
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• 2017

This study was performed to produce succinic acid from biomass by developing mutants of Cellulomonas flavigena in which the succinate dehydrogenase gene (sdh) is deleted. For development of succinate producing mutants, the upstream and downstream regions of sdh gene from C. flavigena and antibiotic resistance gene (neo, bla) were inserted into pKC1139, and the recombinant plasmids were transformed into Escherichia coli ET12567/pUZ8002 which is a donor strain for conjugation. C. flavigena was conjugated with the transformed E. coli ET12567/pUZ8002 to induce the deletion of sdh in chromosome of this bacteria by double-crossover recombination. Two mutants (C. flavigena H-1 and H-2), in which sdh gene was deleted in the chromosome, were constructed and confirmed by PCR. To estimate the production of succinic acid by the two mutants when the culture broth was fermented with biomass such as CMC, xylan, locust gum, and rapeseed straw; the culture broth was analyzed by HPLC analysis. The succinic acid in the culture broth was not detected as a fermentation products of all biomass. One of the reasons for this may be the conversion of succinic acid to fumaric acid by sdh genes (Cfla_1014 - Cfla_1017 or Cfla_1916 - Cfla_1918) which remained in the chromosomal DNA of C. flavigena H-1 and H-2. The other reason could be the conversion of succinyl-CoA to other metabolites by enzymes related to the bypass pathway of TCA cycle.

• BMB Reports
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• 제40권1호
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• pp.22-28
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• 2007

MyoD, expressed in skeletal muscle lineages of vertebrate embryo, is one of muscle-specific basic helix-loop-helix (bHLH) transcription factors, which plays a key role in the determination and differentiation of all skeletal muscle lineages. In this study, a cDNA of grass carp MyoD was cloned and characterized from total RNA of grass carp embryos by RT-PCR. The full-length cDNA of grass carp MyoD is 1597 bp. The cDNA sequence analysis reveals an open reading frame of 825 bp coding for a protein of 275 amino acids, which includes a bHLH domain composed of basic domain (1-84th amino acids) and HLH domain (98-142th amino acids), without signal peptide. Then the MyoD cDNA of grass carp was cloned to yeast expression vector pPICZ$\alpha$A and transformed into P. pastoris GS115 strain, the recombinant MyoD protein with a molecular weight of about 31KD was obtained after inducing for 2d with 0.5% methanol in pH 8.0 BMGY medium, and the maximum yield was about 250 mg/L in shaking-flask fermentation. The results were expected to benefit for further studies on the crystal structure and physiological function of fish MyoD.

• Journal of Applied Biological Chemistry
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• 제62권2호
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• pp.157-165
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• 2019

Di- and tri-methylation of lysine 4 on histone H3 (H3K4me2 and H3K4me3, respectively) are epigenetic markers of active genes. Complex associated with Set1 (COMPASS) mediates these H3K4 methylations. The involvement of COMPASS activity in secondary metabolite (SM) biosynthesis was first demonstrated with an Aspergillus nidulans cclA knockout mutant. The cclA knockout induced the transcription of two cryptic SM biosynthetic gene clusters, leading to the production of the cognate SM. Monascus spp. are filamentous fungi that have been used for food fermentation in eastern Asia, and the pigment Monascus azaphione (MAz) is their main SM. Monascus highly produces MAz, implying that the cognate biosynthetic genes are highly active in transcription. In the present study, we examined how COMPASS activity modulates MAz biosynthesis by inactivating Monascus purpureus cclA (Mp-cclA) and swd1 (Mp-swd1). For both ${\Delta}Mp-cclA$ and ${\Delta}Mp-swd1$, a reduction in MAz production, accompanied by an abated cell growth, was observed. Suppression of MAz production was more effective in an agar culture than in the submerged liquid culture. The fidelity of the ${\Delta}Mp-swd1$ phenotypes was verified by restoring the WT-like phenotypes in a reversion recombinant mutant, namely, trpCp: Mp-swd1, that was generated from the ${\Delta}Mp-swd1$ mutant. Real-time quantitative Polymerase chain reaction analysis indicated that the transcription of MAz biosynthetic genes was repressed in the ${\Delta}Mp-swd1$ mutant. This study demonstrated that MAz biosynthesis is under the control of COMPASS activity and that the extent of this regulation is dependent on growth conditions.

• KSBB Journal
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• 제15권5호
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• pp.489-496
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• 2000

고농도 유전자 재조합 대장균을 이용하여 pyruvate dehy-drogenase complex-E2 특이성 인간 모노클론 항체의 Fab 부분을 효율적으로 생산하기 위해 회분식, 이단 연속식, 반 유 가식, two-stage cyclic fed-batch 동 여러 가지 배양 방법이 조사되었다. 먼저 플라스미드 안정성 문제를 극복하기 위해 growth stage와 production stage를 분리하는 two-phase 회분식 배양과 이단 연속식 시스템을 시도하였다 그 결과 two-phase 회분식 배양보다는 이단 연속식 배양에서의 세포농도와 항체 생산성이 우수하였다 또한 이단 연속식 배양에서의 세포 성 장과 항처l 생산성은 용존산소를 제어한 경우가 그렇지 않은 경우보다 월등하게 높았다. 그리고 plasmid 안정성에 있어서 는 실험기간 내에 거의 100%를 유지하여 높은 안정도를 보 여주었다. 유가식 공정에 적합한 공급 배지로 변형된 M9 배 지가 최적배지로 선정되었고 이 배지 중 최적의 CjN 비율을 조사한 결파 2:3으로 결정되었다. 반 유가식 시스템에서 constant feeding 전략을 사용할 경우 최적 공급속도는 $0.6g/\ell/hr$이었다. 또한 pulse에 의해 공급배지를 공급할 경우에는 총 공급 량이 같을 경우 소량으로 자주 공급해 주는 것이 공급배지를 한꺼번에 많은 양을 공급해주는 것 보다 바람직하였다. 여러 가지 feeding 전략을 조사해 본 결과 linear feeding 방법이 가장 효과적이었다. 하지만 linear feeding 방법마저도 고농도 세포배양에 한계가 있었기 때문에 pH-stat 방법을 이용한 two-stage cyclic fed-batch 시스템을 시도하여 $54 g/\ell$의 세포 를 얻을 수 있었다. 따라서 이 방법이 일단 생산성 향상을 위한 세포의 고농도 배양에는 조사한 여러 배양 시스템 중에 가장 효율적인 시스템임올 알 수 있었다 하지만 이 시스템 에서 포도당을 낮은 level로 유지할 수 있었으나, 초산의 과도한 축적으로 항체 생산성의 향상은 예상에 비해 크지 않았다.

• BMB Reports
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• 제37권3호
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• pp.282-291
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• 2004

Phytases catalyze the release of phosphate from phytic acid. Phytase-producing microorganisms were selected by culturing the soil extracts on agar plates containing phytic acid. Two hundred colonies that exhibited potential phytase activity were selected for further study. The colony showing the highest phytase activity was identified as Aspergillus niger and designated strain 113. The phytase gene from A. niger 113 (phyI1) was isolated, cloned, and characterized. The nucleotide and deduced amino acid sequence identity between phyI1 and phyA from NRRL3135 were 90% and 98%, respectively. The identity between phyI1 and phyA from SK-57 was 89% and 96%. A synthetic phytase gene, phyI1s, was synthesized by successive PCR and transformed into the yeast expression vector carrying a signal peptide that was designed and synthesized using P. pastoris biased codon. For the phytase expression and secretion, the construct was integrated into the genome of P. pastoris by homologous recombination. Over-expressing strains were selected and fermented. It was discovered that ~4.2 g phytase could be purified from one liter of culture fluid. The activity of the resulting phytase was 9.5 U/mg. Due to the heavy glycosylation, the expressed phytase varied in size (120, 95, 85, and 64 kDa), but could be deglycosylated to a homogeneous 64 kDa species. An enzymatic kinetics analysis showed that the phytase had two pH optima (pH 2.0 and pH 5.0) and an optimum temperature of $60^{\circ}C$.

• 생명과학회지
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• 제17권9호통권89호
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• pp.1248-1254
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• 2007

Kluyveromyces marxianus 유래의 exoinulinase (INU1) 분비 서열과 GAL10 promoter를 이용하여 Clostridium thermocellum endoglucanase A gene (celA)의 과발현과 분비 성능 검증연구를 수행하였다. 자체 분비 서열을 가지는 pYEG- CT1 과 INU1 분비 서열을 가지는 pYInu-CT1, 두개의 plasmid는 S. cer-evisiae SEY2102와 S. cerevisiae 2805에 각각 형질전환시켜 ur-acil이 결핍된 배지에서 선별하였다. celA gene 자체 분비 서열보다 INU1 분비 서열을 사용했을 때 발현량과 분비효율은 각각 약 $18{\sim}22%$ 와 11% 향상되었다. 이 중 361 unit/l의 발현율과 89%의 plasmid 안정성, 그리고 70%의 분비효율을 보인 S. cerevisiae 2805/pYinu-CT1 효모 형질전환체를 cellolose를 효과적으로 분해하는 재조합 효모 생균체로 선별하였다. Galactose 배지내에서 S. cerevisiae 2805/ pYInu-CT1의 발효조 회분배양 결과, 총발현량과 분비효율은 각각 418 unit/l 와 73% 로 나타났다. 또한 분비된 endoglucanase A는 분자량 100kDa 이상에서 활성 밴드를 보여, N-linked 당쇄부가에 의해 상당한 비율의 당쇄가 부가됨을 추측할 수 있었다.

• 한국식품과학회지
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• 제31권1호
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• pp.224-230
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• 1999

$_L-Lysine$ 발효산업에 이용되고 있는 B. lactofermentum의 L-lysine 생합성은 succinylase 경로와 dehydrogenase 경로를 통하여 일어난다. 특히 lysine 생산 균주에 부가적으로 존재하는 dehydrogenase 경로는 lysine 생합성에 있어서 필수적인 경로로 작용하며 이때 meso-DAP-dehydrogenase (DDH)를 암호화하는 ddh gene이 관여한다. 그러므로 B. lactofermentum의 lysine 발효에 있어서 ddh gene의 over expression에 의한 lysine 생성량을 비교 조사하기 위하여, shuttle vector pEB1 및 pJC1으로 ddh gene을 삽입하여 재조합 plasmid pRK1 및 pRK31을 구축하였고 이를 B. lactofermentum으로 도입시켜 DDH 활성을 측정한 결과 pRK1을 함유한 균주는 대조균주보다 7배 정도, pRK31을 함유한 균주는 14배 정도 증가하였다. 또한 Shuttle vector를 함유한 대조균주와 재조합 plasmid를 함유한 균주간의 성장 비교에서는 서로 비슷한 수준을 나타내었으며 플라스크 배양에서 lysine 생성량의 비교 분석에서는 재조합 plasmid를 함유한 균주의 경우 48시간 이후부터 대조균주보다 lysine 생성량이 증가하기 시작하여 72시간때에는 최대치를 나타내었으며 그 이후는 오히려 감소하였다. 최대치를 나타낸 72시간 때의 lysine 생성량은 대조균주가 4.38g/L를 나타내었으며 pRK1 및 pRK31을 함유한 균주는 각각 5.34g/L 및 5.21 g/L이었다. 이상의 결과로 미루어 볼 때 B. lactofermentum내에서 ddh gene의 증폭에 의한 lysine 생성량은 pRK1 및 pRK31에서 대조균주보다 각각 20% 및 19% 증가하였다. 또한 발효조 배양에서의 lysine 생성량도 재조합 균주가 대조균주보다 23% 정도 증가를 나타내어 B. lactofermentum에서 ddh gene의 증폭으로 lysine 생성량이 증가하였음을 확인하였다.