• KSBB Journal
• /
• v.19 no.4
• /
• pp.327-330
• /
• 2004

An inducible expression system of poly[(R)-3-hydroxybutyrate] (PHB) depolymerization was established in metabolically engineered Escherichia coli with the PHB biosynthesis genes. The Ralstonia eutropha PHB depolymerase gene was cloned in a vector system containing the PHB biosynthesis genes and expressed under inducible promoter. Recombinant E. coli harboring the PHB biosynthesis genes and depolymerase gene was first cultured for the accumulation of PHB, and then the depolymerase was expressed resulting in the degradation of accumulated PHB into (R)-3-hydroxybutyric acid (R3HB). R3HB could be produced with the concentration of 7.6 g/L in flask culture. Two different PHB biosynthesis genes from Alcaligenes latus and R. eutropha were compared for the production of R3HB. This strategy can be used for the production of enantiomerically pure (R)-hydroxycarboxylic acids with high concentration.

• KSBB Journal
• /
• v.10 no.3
• /
• pp.249-256
• /
• 1995

${\gamma}$-Glutamylcysteine production by the immobilized microbial system of recombinant Escherichia coli and yeast was investigated. ${\gamma}$-Glutamylcysteine was synthesized from L-glutamic acid and L-cysteine in the presence of ATP by the reaction catalyzed by ${\gamma}$-glutamylcysteine synthetase. An immobilized microbial cell system was developed for the efficient ${\gamma}$-glutamylcysteine production. Recombinant Escherichia coli and yeast were immobilized by alginate. Production of ${\gamma}$-glutamylcysteine was better with the recombinant Escherichia coli for both the synthesis of ${\gamma}$-glutamylcysteine and the ATP regeneration than the mixed system of recombinant Escherichia coli and yeast. The proper radio of recombinant Escherichia coli to yeast was experimentary observed to be 1:4 in the mixed system. Although the immobi1ized system had the slower reaction rate, its reaction stability was increased by 10%.

• 한국생물공학회:학술대회논문집
• /
• 2001.11a
• /
• pp.321-324
• /
• 2001

Two recombinant Escherichia coli strains, GCSC6576 harboring a plasmid pSYL107 containing the Ralstonia eutropha polyhyclroxyalkanoate (PHA) biosynthesis genes and a fadR atoC mutant LS5218 harboring a plasmid pJC4 containing the Alcaligenes latus PHA biosynthesis genes were compared for their ability to synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] from whey as the sale carbon source. With the pH-stat fed-batch culture of E. coli LS5218, 、 ,ve obtained a cell concentration, a P(3HB-co-3HV) concentration. a P(3HB-co-3HV) content, and a 3HV fraction of 31.8 g/L, 10.6 g/L, 33.4 wt%. and 6.26 mol%, respectively at 39 h.

• KSBB Journal
• /
• v.18 no.5
• /
• pp.404-407
• /
• 2003

Two recombinant Escherichia coli strains, GCSC6576 harboring a plasmid pSYL107 containing the Ralstonia eutropha polyhydroxyalkanoate (PHA) biosynthesis genes and a fadR atoC mutant LS5218 harboring a plasmid pJC4 containing the Alcaligenes latus PHA biosynthesis genes were compared for their ability to synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV) from whey. The 3HV fraction could be increased by acetic acid induction and oleic acid supplementation in flask cultures of recombinant E. coli GCSC6576. With the pH-stat fed-batch culture of recombinant E. coli LS5218, we obtained a cell concentration, a P(3HB-co-3HV) concentration, a P(3HB-co-3HV) content, and a 3HV fraction of 31.8 g/L, 10.6 g/L, 33.4%, and 6.26 mol%, respectively in 39 h.

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 1993.04a
• /
• pp.138-138
• /
• 1993

인슈린 유사체를 유전공학적 방법으로 생산하여 새로운 당뇨병 치료제로써의 가능성을 타진한다. 인슈린 B 사슬의 C 말단 아미노산 codon을 threonine 대신을 methionine을 지령하도록 하고 여기에 인슈린 A사슬을 지령하는 염기를 바로 부착시켜, 이를 대장균에서 발현시키므로써 외사슬 인슈린 선구체를 제조하고, 이를 분리 정제한 다음 취화브롬 으로 절단하므로서 ($B^{30}$-homoserine) 인슈린을 제조한다. 1. 외사슬 인슈린 전구체 유전자를 합성한 후 대장균의 발현 운반체내 e-galactosidase 유전자와 융합시켜 도입하므로서 재조합된 융합단백질을 생산하였다. 2. 재조합 대장균을 발효한 후 urea 융합단백질을 분리하고 DEAE-Sephacel과 Sephadex G-200을 이용하여 순수 정제하였다.

• Journal of Life Science
• /
• v.22 no.8
• /
• pp.1024-1033
• /
• 2012

A gene encoding a novel geranylgeranyl pyrophosphate (GGPP) synthase from Kocuria gwangalliensis has been cloned and expressed in Escherichia coli. The deduced amino acid sequence showed 59.6% identity with a putative GGPP synthase (CrtE) from K. rhizophila. An expression plasmid containing the crtE gene was constructed, and E. coli cells containing this plasmid produced a recombinant protein with a theoretical molecular mass of 41 kDa, corresponding to the molecular weight of GGPP synthase. Due to the lack of crtE, crtB, and crtI in E. coli, the biosynthesis of lycopene was only obtained when the plasmid pCcrtE was co-transformed into E. coli expressing the pRScrtBI-carrying carotenoid biosynthesis crtB and crtI genes, which were sub-cloned from Paracoccus haeundaensis. The biochemical studies on the expressed proteins were performed via HPLC. The results obtained from this study will provide a wider base of knowledge regarding the primary structure of CrtE cloned from K. gwangalliensis at the molecular level.

• Microbiology and Biotechnology Letters
• /
• v.44 no.1
• /
• pp.55-61
• /
• 2016

Sepiapterin, a precursor for tetrahydrobiopterin, is produced in higher mammals using guanosine triphosphate (GTP) as a biosynthetic intermediate. Four genes involved in GTP biosynthesis, namely those of guanosine monophosphate kinase (gmk), nucleoside diphosphate kinase (ndk), guanosine phosphate synthetase (guaA), and inosine-5'-monophosphate dehydrogenase (guaB), were expressed in sepiapterin-producing recombinant Escherichia coli BL21(DE3) to increase intracellular GTP concentration and to improve sepiapterin production concomitantly. Coexpression of gmk, ndk, guaA, and guaB, doubled the intracellular GTP concentration and increased the maximum sepiapterin concentration up to $126.1{\pm}19.3mg/l$ (an increase of 43% compared with control cells) in batch-cultivated recombinant E. coli.

• The Journal of Natural Sciences
• /
• v.16 no.1
• /
• pp.1-13
• /
• 2005

The angiotensin I-converting enzyme (ACE) inhibitor has anti-hypertensive effects and has long been used as prevention or remedy of hypertension. This study were carried out to produce and purify a new ACE inhibitor from recombinant E. coli and further elucidate its structure-function relationship. Recombinant pGEX-4T-3 containing ACE inhibitory peptide gene of Saccharomyces cerevisiae was transformed into E. coli BL21(DE3). Glutathione-S transferase (GST) fusion protein from E. Coli BL21(DE3) harboring the recombination pGEX-4T-3 was obtained and the ACE inhibitory peptide was purified with Sephadex G-25 column chromatography. The purified ACE inhibitory peptide was a novel decapeptide with sequence Tyr-Asp-Gly-Gly-Val-Phe -Arg-Val-Tyr-Thr which shows very low similarity to the other ACE inhibitory peptide sequence. The purified ACE inhibitor competitively inhibited ACE.

• KSBB Journal
• /
• v.10 no.5
• /
• pp.575-581
• /
• 1995

A plasmid pTTY2, containing the lipase-producing gene, was used to transform an E. coli phage lysogen, P90c/$\phi$434, into the lipase-producing lysogen, P90c/$\phi$434/pTTY2. After the overproduction of lipase by the isopropylthio-${\beta}$-D-galactoside induction, the prophage $\phi$434 in the chromosome of the host cell was induced by the milomycin C addition or ultraviolet irradiation to lyse the host cell. The optimum operating conditions, such as the isopropylthio-${\beta}$-D-galactoside induction period and the phage induction timing, were sought for the efficient cell lysis in the same fermenter. Effective cell lysis occurred at the earlier exponential growth phase with the isopropylthio-${\beta}$-D-galactoside induction period of 1 hour. The amount of the lipase production was qualitatively measured by the halo size in Luria-Bertani agar medium containing tributyrin and Rhodamine B plate.

• Microbiology and Biotechnology Letters
• /
• v.32 no.3
• /
• pp.243-248
• /
• 2004

Production of (R)-3-hydroxybutyric acid (R3HB) by fed-batch culture and continuous culture of metabolically engineered Escherichia coli harboring Ralstonia eutropha PHB biosynthesis and depolymerase genes was examined in a 30 1 pilot-scale fermentor. A new stable two-plasmid system, pBRRed containing the R. eutropha PHB depolymerase gene and pMCS 105 containing the R. eutropha PHB biosynthesis genes, was developed. Among a variety of E. coli strains harboring plasmids, recombinant E. coli XL-10 Gold (pBRRed, pMCS105) was able to produce R3HB with the highest efficiency in a batch culture. By the fed-batch culture of recombinant E. coli XL-10 Gold(pBRRed, pMCS 105) in a 30 1 fer-mentor, the final R3HB concentration was 22.4 g/l giving a productivity of 0.97 g/l-h. To produce R3HB to a high concentration with high productivity, a new strategy of fed-batch culture followed by a continuous culture was investigated. The maximum productivity and R3HB concentration were 5.06 g/l-h and 25.3 g/l, respectively. These results show that economical production of R3HB is possible by recombinant E. coli in large scale.