• KSBB Journal
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• 제23권6호
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• pp.519-524
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• 2008

Monacolin-K는 Monascus sp.로부터 polyketide pathway를 통해 생합성 되는 이차대사산물로써 강력한 콜레스테롤 저하제로 알려져 있다. 본 연구에서는 monacolin-K의 생합성 경로에 대한 이해에 근거한 지속적인 rational screening을 통해 monacolin-K의 생산성을 향상시킬 수 있었는데 그 중에서 특히 monacolin-K 생합성에 관련된 전구체를 최적화된 생산배지에 첨가함으로써 monacolin-K 생산성이 대조군에 비해 눈에 띠게 증가하는 결과를 확인하였다. 황의 동화작용에서 cysteine이 여러 단계를 거쳐 S-adenosylmethionine (SAM)으로 전환된다는 연구결과와 더불어, SAM은 다양한 세포내에서 주된 methyl donor 역할을 하므로 monacolin-K 구조에 포함되어있는 많은 methyl기 역시 SAM으로부터 유래한다고 알려져 있다. 따라서 첨가한 cysteine이 SAM을 생합성하는데 이용된 것으로 보고 SAM을 생산균주 내에서 고농도로 생산한다면 monacolin-K 생산성이 증가할 것이라 기대하였다. 따라서 여러 균주에서 보고된 SAM synthetase 유전자를 cloning하여 생산균주 내로 도입함으로써 생산균주가 cysteine의 별도첨가 없이도 세포내에서 SAM을 고농도로 생산하도록 하여 monacolin-K의 생산성 향상을 꾀하고자 하였다. 이를 위해 염기서열이 밝혀진 균사형성 곰팡이인 Aspergillus nidulans로부터 SAM synthetase를 암호화하는 metK 유전자를 cloning하고 Monascus 유래의 gpdA promoter에 의해 발현되도록 하는 재조합 발현벡터 pBMmetK를 제작하였고 이를 생산균주 내로 도입하여 형질전환체와 대조군의 monacolin-K 생산성을 확인한 결과, 대조군에 비해 형질전환체에서 Monacolin-K 생산성이 약 3.3배가량 증가한 것을 관찰하였다. 이는 metK 유전자가 생산균주의 DNA 내로 삽입되어 안정적으로 발현됨으로써 세포내에서 많은 methyl 기를 제공함으로써 monacolin-K 생산성이 향상된 것으로 판단되며, 현재는 분자적 수준에서 이러한 형질전환체 내에서 metK 유전자의 발현 정도를 확인하는 중이다.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.80.1-80
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• 2003

Several plant and microbial genes that could confer disease resistance in transgenic rice plants are being cloned and characterized. We are currently constructing transgenic rice lines that overexpress the gene products, such as a galactinol synthase, a defensin, and a bacterial ACC deaminase. Subtractive hybridization of a rice cDNA library constructed from the Xanthomonas oryzae-infected ice leaves resulted in isolation of many inducible cDNA clones including a elongation factor EF2, a oryzain alpha, a catalase, a aldehyde dehydrogenase, a S-adenosylmethionine synthetase, a caffeic acid O-methyltransferase, a glyceraldehyde-3-phosphate dehydrogenase, a light-regulated protein, nKY transcription factors, and a nucleotide diphosphate kinase. Some genes among those may be useful genetic sources for construction of disease resistant transgenic rice. Full lengths of the rice OsFIERG and a rice oryzain genomic clones were cloned, and serial deletion fragments of the promoter regions of these genes were fused with GUS reporter gene in pCAMBIA1201, respectively. Promoter activities of these constructs will be examined upon various stresses and Pathogen infections to obtain the pathogen specific inducible-promoter. This work was supported by a grant from BioGreen 21 Program, Rural Development Administration, Republic of Korea.

• Molecules and Cells
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• 제37권10호
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• pp.727-733
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• 2014

Spinosyns A and D are potent ingredient for insect control with exceptional safety to non-target organisms. It consists of a 21-carbon tetracyclic lactone with forosamine and tri-Omethylated rhamnose which are derived from S-adenosyl-methionine. Although previous studies have revealed the involvement of metK1 (S-adenosylmethionine synthetase), rmbA (glucose-1-phosphate thymidylyltransferase), and rmbB (TDP-D-glucose-4, 6-dehydratase) in the biosynthesis of spinosad, expression of these genes into rational screened Saccharopolyspora spinosa (S. spinosa MUV) has not been elucidated till date. In the present study, S. spinosa MUV was developed to utilize for metabolic engineering. The yield of spinosyns A and D in S. spinosa MUV was $244mgL^{-1}$ and $129mgL^{-1}$, which was 4.88-fold and 4.77-fold higher than that in the wild-type ($50mgL^{-1}$ and $27mgL^{-1}$), respectively. To achieve the better production; positive regulator metK1-sp, rmbA and rmbB genes from Streptomyces peucetius, were expressed and co-expressed in S. spinosa MUV under the control of strong $ermE^*$ promoter, using an integration vector pSET152 and expression vector pIBR25, respectively. Here-with, the genetically engineered strain of S. spinosa MUV, produce spinosyns A and D up to $372/217mgL^{-1}$ that is 7.44/8.03-fold greater than that of wild type. This result demonstrates the use of metabolic engineering on rationally developed high producing natural variants for the production.

• Journal of Microbiology and Biotechnology
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• 제22권8호
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• pp.1127-1132
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• 2012

Nargenicin $A_1$ is a 28-membered polyketide macrolide, with antibacterial activity against methicillin-resistant Staphylococcus aureus, produced by Nocardia sp. CS682. In this study, the production of nargenicin $A_1$ was improved by enhancing the supply of different biosynthetic precursors. In Nocardia sp. CS682 (KCTC11297BP), this improvement was ~4.62-fold with the supplementation of 30 mM methyl oleate, 4.25-fold with supplementation of 15mM sodium propionate, and 2.81-fold with supplementation of 15 mM sodium acetate. In Nocardia sp. metK18 and Nocardia sp. CS682 expressing S-adenosylmethionine synthetase (MetK), the production of nargenicin $A_1$ was improved by ~5.57-fold by supplementation with 30 mM methyl oleate, 5.01-fold by supplementation with 15 mM sodium propionate, and 3.64-fold by supplementation with 15 mM sodium acetate. Furthermore, supplementing the culture broth of Nocardia sp. ACC18 and Nocardia sp. CS682 expressing the acetyl-CoA carboxylase complex (AccA2 and AccBE) with 30 mM methyl oleate, 15 mM sodium propionate, or 15 mM sodium acetate resulted in ~6.99-, 6.46-, and 5.58-fold increases, respectively, in nargenicin $A_1$ production. Our overall results showed that among the supplements, methyl oleate was the most effective precursor supporting the highest titers of nargenicin $A_1$ in Nocardia sp. CS682, Nocardia sp. metK18, and Nocardia sp. ACC18.

• Journal of Crop Science and Biotechnology
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• 제10권4호
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• pp.231-236
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• 2007

Differentially expressed genes(DEG) were identified in a rice variety, Sathi, an indica type showing high allelopathic potential against barnyardgrass(Echinochloa crus-galli(L.) Beauv. var. frumentaceae). Rice plants were grown with and without barnyardgrass and total RNA was extracted from rice leaves at 45 days after seeding. DEG full-screening was performed by $GeneFishing^{TM}$ method. The differentially expressed bands were re-amplified and sequenced, then analyzed by Basic Local Alignment Search Tool(BLAST) searching for homology sequence identification. Gel electrophoresis showed nine possible genes associated with allelopathic potential in Sathi, six genes(namely DEG-1, 4, 5, 7, 8, and 9) showed higher expression, and three genes(DEG-2, 3 and 6) showed lower expression as compared to the control. cDNA sequence analysis showed that DEG-7 and DEG-9 had the same sequence. From RT PCR results, DEG-6 and DEG-7 were considered as true DEG, whereas DEG-1, 2, 3, 4, 5, and 8 were considered as putative DEG. Results from blast-n and blast-x search suggested that DEG-1 is homologous to a gene for S-adenosylmethionine synthetase, DEG-2 is homologous to a chloroplast gene for ribulose 1,5-bisphosphate carboxylase large subunit, DEG-8 is homologous to oxysterol-binding protein with an 85.7% sequence similarity, DEG-5 is homologous to histone 2B protein with a 47.9% sequence similarity, DEG-6 is homologous to nicotineamine aminotransferase with a 33.1% sequence similarity, DEG-3 has 98.8% similarity with nucleotides sequence that has 33.1% similarity with oxygen evolving complex protein in photosystem II, DEG-7 is homologous to nucleotides sequence that may relate with putative serin/threonine protein kinase and putative transposable element, and DEG-4 has 98.8% similarity with nucleotides sequence for an unknown protein.

• 한국작물학회지
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• 제63권1호
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• pp.25-34
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• 2018

휴면성이 다른 백중밀, 금강밀, 우리밀 후숙종자의 ABA 농도에 따른 발아 및 배아에서의 단백질체 발현 특성을 조사한 결과는 다음과 같다. 1. 백중밀, 금강밀, 우리밀 등 3품종의 0, 10, 30 및 $50{\mu}M$ ABA에서의 평균 발아지수와 발아율은 각각 0.95와 98% 이상으로 통계적으로 유의한 차이가 없었다. 유아와 유근의 생장은 $0{\mu}M$ ABA보다 10, 30 및 $50{\mu}M$ ABA에서 생장이 크게 억제되었는데, ABA 농도가 높을수록 생장이 더 억제되었다. 2. 3품종 배아의 평균 ABA 함량은 $0{\mu}M$ ABA와 $50{\mu}M$ ABA에서 각각 0.78 ng/mg과 269.04 ng/mg으로서 농도에 따른 ABA 함량의 차이가 컸다. 3. $0{\mu}M$ ABA 처리구에 비하여 $50{\mu}M$ ABA 처리구에서 발현양이 증가한 단백질 spot (S1, S3, S4, S6, S15, S16, S17)은 7개였으며, 감소한 단백질 spot (S2, S5, S9, S10, S11, S12, S13, S14, S18)은 8개였고, 증가와 감소가 동시에 이루어진 단백질 spot (S2)은 1개였다. $50{\mu}M$ ABA에서만 검출된 단백질 spot (S7, S8)은 2개였다. 4. 각 단백질 spot의 $0{\mu}M$ ABA 처리구 양에 대한 $50{\mu}M$ ABA 처리구 양의 평균 배수 값(fold 값)이 1.5배 이상으로 증가한 단백질 spot은 S1 (globulin-3A), S6 (globulin-1 S allele), S16 (globulin-1 S allele), S17 (globulin-1 S allele) 등으로 모두 globulin류 단백질이었다. 또한 $50{\mu}M$ ABA에서만 확인된 단백질 spot인 S7 (globulin 3)과 S8 (globulin-1 S allele)도 globulin 단백질이었다. 5. 각 단백질 spot의 $0{\mu}M$ ABA 처리구 양에 대한 $50{\mu}M$ ABA 처리구 양의 평균 배수 값(fold 값)이 0.7 이하로 감소한 단백질 spot은 S10 (glutamine sysnthetase cytosolic isozyme), S12 (S-adenosylmethionine synthetase 2), S14 (isocitrate dehydrogenase NADP)이었다. 이상의 결과는 ABA에 의한 밀 유묘의 유아와 유근의 생장억제는 배아에서의 ABA 농도 증가, 그리고 이에 따른 배아의 glutamine 합성에 관여하는 다양한 효소의 발현 감소 및 메틸기공여물질의 감소와 이에 따른 메틸기 전이활성의 감소 등이 관여하고 있음을 의미한다. 한편 배아에서의 ABA에 의한 globulin 단백질의 증가는 배아 특이적 globulin의 일시적 합성 증가와 globulin 분해 효소의 활성 억제 등이 복합적으로 관여한 결과로 생각된다.