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Oh, Young Hoon,Kang, Kyoung-Hee,Kwon, Mi Jeong,Choi, Jae Woo,Joo, Jeong Chan,Lee, Seung Hwan,Yang, Yung-Hun,Song, Bong Keun,Kim, Il-Kwon,Yoon, Ki-Hoon,Park, Kyungmoon,Park, Si Jae Published by Stockton Press on behalf of the Socie 2015 Journal of industrial microbiology & biotechnology Vol.42 No.11
<P>A whole-cell biocatalytic system for the production of cadaverine from L-lysine has been developed. Among the investigated lysine decarboxylases from different microorganisms, Escherichia coli LdcC showed the best performance on cadaverine synthesis when E. coli XL1-Blue was used as the host strain. Six different strains of E. coli expressing E. coli LdcC were investigated and recombinant E. coli XL1-Blue, BL21(DE3) and W were chosen for further investigation since they showed higher conversion yield of lysine into cadaverine. The effects of substrate pH, substrate concentrations, buffering conditions, and biocatalyst concentrations have been investigated. Finally, recombinant E. coli XL1-Blue concentrated to an OD600 of 50, converted 192.6??g/L (1317??mM) of crude lysine solution, obtained from an actual lysine manufacturing process, to 133.7??g/L (1308??mM) of cadaverine with a molar yield of 99.90??%. The whole-cell biocatalytic system described herein is expected to be applicable to the development of industrial bionylon production process.</P>
Production of 3-hydroxypropionic acid from glycerol by acid tolerant Escherichia coli.
Sankaranarayanan, Mugesh,Ashok, Somasundar,Park, Sunghoon Published by Stockton Press on behalf of the Socie 2014 Journal of industrial microbiology & biotechnology Vol.41 No.7
<P>The biological production of 3-hydroxypropionic acid (3-HP) has attracted significant attention because of its industrial importance. The low titer, yield and productivity, all of which are related directly or indirectly to the toxicity of 3-HP, have limited the commercial production of 3-HP. The aim of this study was to identify and select a 3-HP tolerant Escherichia coli strain among nine strains reported to produce various organic acids efficiently at high titer. When transformed with heterologous glycerol dehydratase, reactivase and aldehyde dehydrogenase, all nine E. coli strains produced 3-HP from glycerol but the level of 3-HP production, protein expression and activities of the important enzymes differed significantly according to the strain. Two E. coli strains, W3110 and W, showed higher levels of growth than the others in the presence of 25?g/L 3-HP. In the glycerol fed-batch bioreactor experiments, the recombinant E. coli W produced a high level of 3-HP at 460??10?mM (41.5??1.1?g/L) in 48?h with a yield of 31?% and a productivity of 0.86??0.05?g/L h. In contrast, the recombinant E. coli W3110 produced only 180??8.5?mM 3-HP (15.3??0.8?g/L) in 48?h with a yield and productivity of 26?% and 0.36??0.02?g/L h, respectively. This shows that the tolerance to and the production of 3-HP differ significantly among the well-known, similar strains of E. coli. The titer and productivity obtained with E. coli W were the highest reported thus far for the biological production of 3-HP from glycerol by E. coli.</P>
Ko, Kyong-Cheol,Rim, Soon-Ok,Han, Yunjon,Shin, Bong Seok,Kim, Geun-Joong,Choi, Jong Hyun,Song, Jae Jun Published by Stockton Press on behalf of the Socie 2012 Journal of industrial microbiology & biotechnology Vol.39 No.5
<P>A novel lipolytic enzyme was isolated from a metagenomic library after demonstration of lipolytic activity on an LB agar plate containing 1% (w/v) tributyrin. A novel esterase gene (estIM1), encoding a lipolytic enzyme (EstIM1), was cloned using a shotgun method from a pFosEstIM1 clone of the metagenomic library, and the enzyme was characterized. The estIM1 gene had an open reading frame (ORF) of 936 base pairs and encoded a protein of 311 amino acids with a molecular mass 34??kDa and a pI value of 4.32. The deduced amino acid sequence was 62% identical to that of an esterase from an uncultured bacterium (ABQ11271). The amino acid sequence indicated that EstIM1 was a member of the family IV of lipolytic enzymes, all of which contain a GDSAG motif shared with similar enzymes of lactic acid microorganisms. EstIM1 was active over a temperature range of 1-50°C, at alkaline pH. The activation energy for hydrolysis of p-nitrophenyl propionate was 1.04??kcal/mol, within a temperature range of 1-40°C. The activity of EstIM1 was about 60% of maximal even at 1°C, suggesting that EstIM1 is efficiently cold-adapted. Further characterization of this cold-adapted enzyme indicated that the esterase may be very valuable in industrial applications.</P>
Jung, Joon-Goo,Lee, Yong Jae,Velmurugan, Natarajan,Ko, Young-Joon,Lee, Hyang-Sim,Jeong, Ki Jun Published by Stockton Press on behalf of the Socie 2013 Journal of industrial microbiology & biotechnology Vol.40 No.7
<P>For effective control of foot-and-mouth disease (FMD), the development of rapid diagnostic systems and vaccines are required against its etiological agent, FMD virus (FMDV). To accomplish this, efficient large-scale expression of the FMDV VP1 protein, with high solubility, needs to be optimized. We attempted to produce high levels of a serotype O FMDV VP1 epitope in Escherichia coli. We identified the subtype-independent serotype O FMDV VP1 epitope sequence and used it to construct a glutathione S-transferase (GST) fusion protein. For efficient production of the FMDV VP1 epitope fused to GST (VP1e-GST), four E. coli strains and three temperatures were examined. The conditions yielding the greatest level of VP1e-GST with highest solubility were achieved with E. coli BL21(DE3) at 25?C. For high-level production, fed-batch cultures were conducted in 5-l bioreactors. When cells were induced at a high density and complex feeding solutions were supplied, approximately 11?g of VP1e-GST was obtained from a 2.9-l culture. Following purification, the VP1 epitope was used to immunize rabbits, and we confirmed that it induced an immune response.</P>
Jiang, Ling-Yan,Zhang, Yuan-Yuan,Li, Zhen,Liu, Jian-Zhong Published by Stockton Press on behalf of the Socie 2013 Journal of industrial microbiology & biotechnology Vol.40 No.10
<P>The experiments presented here were based on the conclusions of our previous proteomic analysis. Increasing the availability of glutamate by overexpression of the genes encoding enzymes in the L-ornithine biosynthesis pathway upstream of glutamate and disruption of speE, which encodes spermidine synthase, improved L-ornithine production by Corynebacterium glutamicum. Production of L-ornithine requires 2 moles of NADPH per mole of L-ornithine. Thus, the effect of NADPH availability on L-ornithine production was also investigated. Expression of Clostridium acetobutylicum gapC, which encodes NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, and Bacillus subtilis rocG, which encodes NAD-dependent glutamate dehydrogenase, led to an increase of L-ornithine concentration caused by greater availability of NADPH. Quantitative real-time PCR analysis demonstrates that the increased levels of NADPH resulted from the expression of the gapC or rocG gene rather than that of genes (gnd, icd, and ppnK) involved in NADPH biosynthesis. The resulting strain, C. glutamicum δAPRE::rocG, produced 14.84 g l?1 of L-ornithine. This strategy of overexpression of gapC and rocG will be useful for improving production of target compounds using NADPH as reducing equivalent within their synthetic pathways.</P>
Characteristics of bifunctional acidic endoglucanase (Cel5B) from Gloeophyllum trabeum.
Kim, Ho Myeong,Lee, Yoon Gyo,Patel, Darshan H,Lee, Kwang Ho,Lee, Dae-Seok,Bae, Hyeun-Jong Published by Stockton Press on behalf of the Socie 2012 Journal of industrial microbiology & biotechnology Vol.39 No.7
<P>The endoglucanase (Cel5B) from the filamentous fungus Gloeophyllum trabeum was cloned and expressed without a signal peptide, and alanine residue 22 converted to glutamine in Pichia pastoris GS115. The DNA sequence of Cel5B had an open reading frame of 1,077 bp, encoding a protein of 359 amino acid residues with a molecular weight of 47 kDa. On the basis of sequence similarity, Cel5B displayed active site residues at Glu-175 and Glu-287. Both residues lost full hydrolytic activity when replaced with alanine through point mutation. The purified recombinant Cel5B showed very high specific activity, about 80- to 1,000-fold and 13- to 70-fold in comparison with other endoglucanases and cellobiohydrolase, on carboxymethylcellulose and filter paper, respectively, at pH 3.5 and 55C. Cel5B displayed bifunctional characteristics under acidic conditions. The kinetic properties of the enzyme determined using a Lineweaver-Burk plot indicated that Cel5B is a catalytically efficient cellulolytic enzyme. These results suggest that Cel5B has high bifunctional endo- and exoglucanase activity under acidic conditions and is a good candidate for bioconversion of lignocellulose.</P>
Zhang, Haitao,Moon, Young Hwan,Watson, Brian J,Suvorov, Maxim,Santos, Elizabeth,Sinnott, Corinn A,Hutcheson, Steven W Published by Stockton Press on behalf of the Socie 2011 Journal of industrial microbiology & biotechnology Vol.38 No.8
<P>Saccharophagus degradans 2-40 is a marine gamma proteobacterium that can produce polyhydroxyalkanoates from lignocellulosic biomass using a complex cellulolytic system. This bacterium has been annotated to express three surface-associated 관-glucosidases (Bgl3C, Ced3A, and Ced3B), two cytoplasmic 관-glucosidases (Bgl1A and Bgl1B), and unusual for an aerobic bacterium, two cytoplasmic cellobiose/cellodextrin phosphorylases (Cep94A and Cep94B). Expression of the genes for each of the above enzymes was induced when cells were transferred into a medium containing Avicel as the major carbon source except for Bgl1B. Both hydrolytic and phosphorolytic degradation of cellobiose by crude cell lysates obtained from cellulose-grown cells were demonstrated and all of these activities were cell-associated. With the exception of Cep94B, each purified enzyme exhibited their annotated activity upon cloning and expression in E. coli. The five 관-glucosidases hydrolyzed a variety of glucose derivatives containing 관-1, (2, 4, or 6) linkages but did not act on any 관-linked glucose derivatives. All but one 관-glucosidases exhibited transglycosylation activity consistent with the formation of an enzyme-substrate intermediate. The biochemistry and expression of these cellobiases indicate that external hydrolysis by surface-associated 관-glucosidases coupled with internal hydrolysis and phosphorolysis are all involved in the metabolism of cellobiose by this bacterium.</P>
Choi, Oksik,Wu, Cheng-Zhu,Kang, Sun Young,Ahn, Jong Seog,Uhm, Tai-Boong,Hong, Young-Soo Published by Stockton Press on behalf of the Socie 2011 Journal of industrial microbiology & biotechnology Vol.38 No.10
<P>Biological synthesis of plant secondary metabolites has attracted increasing attention due to their proven or assumed beneficial properties and health-promoting effects. Phenylpropanoids are the precursors to a range of important plant metabolites such as the secondary metabolites belonging to the flavonoid/stilbenoid class of compounds. In this study, engineered Escherichia coli containing artificial phenylpropanoid biosynthetic pathways utilizing tyrosine as the initial precursor were established for production of plant-specific metabolites such as ferulic acid, naringenin, and resveratrol. The construction of the artificial pathway utilized tyrosine ammonia lyase and 4-coumarate 3-hydroxylase from Saccharothrix espanaensis, cinnamate/4-coumarate:coenzyme A ligase from Streptomyces coelicolor, caffeic acid O-methyltransferase and chalcone synthase from Arabidopsis thaliana, and stilbene synthase from Arachis hypogaea.</P>
Moon, Young Hwan,Madsen, Lee,Chung, Chang-Ho,Kim, Doman,Day, Donal F Published by Stockton Press on behalf of the Socie 2015 Journal of industrial microbiology & biotechnology Vol.42 No.2
<P>We have previously demonstrated the production of glucooligosaccharides via a fermentation of sucrose with Leuconostoc mesenteroides NRRL B-742 using sodium hydroxide (NaOH) to control the pH. Because NaOH is expensive, we sought to minimize the cost of our process by substituting hydrated lime and saccharate of lime (lime sucrate) in its place. The yield of glucooligosaccharides using either 5 % lime (41.4 ± 0.5 g/100 g) or 5 % lime sucrate (40.0 ± 1.4 g/100 g) were both similar to the NaOH control (42.4 ± 1.5 g/100 g). Based on this, it appears that the cost associated with pH control in our process can be reduced by a factor of approximately 2.4 using lime instead of NaOH. Because our chromatographic stage is based on a Ca(2+)-form resin to separate glucooligosaccharides, the use of lime not only negates the need for costly de-salting via ion-exchange (elimination of two ion-exchange sections) prior to separation, but also greatly reduces the resin regeneration cost.</P>
Le Vo, Tam Dinh,Ko, Ji-seun,Park, Si Jae,Lee, Seung Hwan,Hong, Soon Ho Published by Stockton Press on behalf of the Socie 2013 Journal of industrial microbiology & biotechnology Vol.40 No.8
<P>Gamma-aminobutyric acid (GABA) is a precursor of one of the most promising heat-resistant biopolymers, Nylon-4, and can be produced by the decarboxylation of monosodium glutamate (MSG). In this study, a synthetic protein complex was applied to improve the GABA conversion in engineered Escherichia coli. Complexes were constructed by assembling a single protein-protein interaction domain SH3 to the glutamate decarboxylase (GadA and GadB) and attaching a cognate peptide ligand to the glutamate/GABA antiporter (GadC) at the N-terminus, C-terminus, and the 233rd amino acid residue. When GadA and GadC were co-overexpressed via the C-terminus complex, a GABA concentration of 5.65 g/l was obtained from 10 g/l MSG, which corresponds to a GABA yield of 93 %. A significant increase of the GABA productivity was also observed where the GABA productivity increased 2.5-fold in the early culture period due to the introduction of the synthetic protein complex. The GABA pathway efficiency and GABA productivity were enhanced by the introduction of the complex between Gad and glutamate/GABA antiporter.</P>