• 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.

• 한국산학기술학회논문지
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• 제10권7호
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• pp.1766-1772
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• 2009

본 연구에서는 한국인 아동의 우식치아로부터 S. mutans를 분리하고, acid stress하에서 분리한 S. mutans의 내산성 능력과 관련된 단백질을 규명하고자 하였다. 2D gel electrophoresis를 수행한 결과, acid stress동안 elongation factor Ts, hypothetical protein, putative amino acid ABC transporter, adenylate kinase, fructokinase, Putative 40K cell well protein precursor, peptide deformylase, shikimate 5-dehydrogenase, mannose-6-phosphate isomerase, threonine synthase, putative dTDP-glucose-4,6-dehydratase의 발현량이 뚜렷이 증가하였으며 이들 단백질은 acid stress에 관여하는 단백질들로 추정된다.

• 한국버섯학회지
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• 제10권3호
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• pp.120-128
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• 2012

표고균사에서 갈변시기를 조절하고 확인할 수 있는 유전공학적 시스템을 개발하여 톱밥재배용 표고균주를 조기에 선별할 수 있도록 표고균사가 갈변되는 동안 균사상태에서 특이적으로 발현되는 유전자를 분리하기 위하여 갈변되지 않은 균사와 갈변이 완전히 이루어진 균사에서 differential display를 실시하였다. 그 결과 이들 균사로부터 특이적으로 발현되는 두 개의 1.6kb와 1.2kb의 cDNA clone을 선발하여 염기서열을 분석하였다. 이 중 1.6kb의 cDNA단편은 Dugenia polichroa로부터 분리된 microsatellites 유전자와 100%의 상동성을 나타냈다. 그러나 1.2kb의 cDNA 단편은 3'부위에 poly A tail과 5 부위에 partial open reading frame를 가지고 있어 이를 primer로 제작한 후 갈변되지 않은 균사와 갈변이 이루어진 균사에서 RT-PCR을 실시하여 본 결과 갈변이 되지 않은 백색의 균사에서 발현이 확인되었다. 1.2kb의 cDNA 단편의 5' 부위의 염기서열 분석은 110개의 아미노산으로 구성된 partial open reading frame으로 나타났다. 이 유전자를 DNASIS database에서 상동성을 비교해 본 결과 Arabidopsis thaliata에서 분리된 dTDP-glucose 4,6-dehydratases 유전자와 DNA 수준에서는 66.7%, 아미노산 수준에서는 69.2%의 높은 상동성을 나타내었다. 갈변에 관련된 특이 유전자(BCR gene)를 확인하였다. 이 유전자는 산화 stress에 대해 저항성을 나타내는 기능을 가진 것으로 알려져 있어 표고 균사가 갈변될 때 repressor로서 작용할 것으로 생각된다. 따라서 이 유전자를 BCR (Brown Color Repressor) 유전자라고 명명하였다.

• Journal of Microbiology and Biotechnology
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• 제27권7호
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• pp.1345-1358
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• 2017

The impact of overproduction of a heterologous protein on the metabolic system of host Lactococcus lactis was investigated. The protein expression profiles of L. lactis IL1403 containing two near-identical plasmids that expressed high- and low-level of the green fluorescent protein (GFP) were examined via shotgun proteomics. Analysis of the two strains via high-throughput LC-MS/MS proteomics identified the expression of 294 proteins. The relative amount of each protein in the proteome of both strains was determined by label-free quantification using the spectral counting method. Although expression level of most proteins were similar, several significant alterations in metabolic network were identified in the high GFP-producing strain. These changes include alterations in the pyruvate fermentation pathway, oxidative pentose phosphate pathway, and de novo synthesis pathway for pyrimidine RNA. Expression of enzymes for the synthesis of dTDP-rhamnose and N-acetylglucosamine from glucose was suppressed in the high GFP strain. In addition, enzymes involved in the amino acid synthesis or interconversion pathway were downregulated. The most noticeable changes in the high GFP-producing strain were a 3.4-fold increase in the expression of stress response and chaperone proteins and increase of caseinolytic peptidase family proteins. Characterization of these host expression changes witnessed during overexpression of GFP was might suggested the metabolic requirements and networks that may limit protein expression, and will aid in the future development of lactococcal hosts to produce more heterologous protein.