Earticle

Heterologous protein production is becoming an important field in biotechnology as a source of many different kinds of proteins, including therapeutic proteins. Traditionally, these proteins were produced through the use of small model multi-cellular organisms, but these systems are giving away to microorganisms, such as the yeast P. pastoris, due to their rapid doubling time and lower cost in growth media. Additionally, the ease in which the proteins can be harvested makes P. pastoris and attractive host. We have constructed the genome-scale metabolic model of the methylotrophic yeast P. pastoris and incorporated additional biochemical reactions to represent the production of two proteins, human serum albumin (HSA) and human superoxide dismutase (hSOD). Using this metabolic model, we analyzed the yeast’ capacity for producing proteins and performed simulations to suggest metabolic engineering targets to improve production. [This work was supported by the Korean Systems Biology Research Project (20100002164) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea. Further support by the World Class University Program (R32-2008-000-10142-0) through the National Research Foundation of Korea funded by the MEST is appreciated.]

Resveratrol is a polyphenolic compound found in grapes, peanuts, several types of berries, and other plants, particularly red wine. It has several beneficial effects on human health, such as prevention of cancer and cardiovascular diseases, and antioxidative, antiviral, anti-inflammatory, estrogenic, and anti-aging effects. 4-coumarate:coenzyme A ligase gene (4CL1) from Arabidopsis thaliana and stilbene synthase gene (STS) from Arachis hypogaea were cloned to produce resveratrol in a food-grade yeast. And the clone was transformed into Saccharomyces cerevisiae W303-1A. A batch culture with 15.3 mg/l p-coumaric acid used as precursor resulted in the production of 3.1 mg/l resveratrol with 14.4 mol % yield. The resveratrol produced was unglycosylated and secreted into the culture medium. Deletion of the putative phenyl acrylic acid decarboxylase gene (PAD1) did not enhance resveratrol production.

Glycerol is a promising carbon source for production of reduced chemicals including succinic acid. To improve glycerol uptake and growth rate of a Mannheimia succiniciproducens strain for improved succinic acid production, genetic manipulations were done based on metabolic engineering strategies. Genes related to glycerol uptake pathways and biomass formations were selected for targets of metabolic engineering. Various combinations of target genes which are native or heterologous were co-overexpressed in the M. succiniciproducens strain. As a result, succinic acid production in the M. succiniciproducens strain was increased as well as cell growth rate. [This work was supported by the Korean Systems Biology Research Project (20100002164) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea. Further support by the World Class University Program (R32-2008-000-10142-0) through the National Research Foundation of Korea funded by the MEST is appreciated.]

DNA methylation at CpG dinucleotides residues is known as central mechanism to control gene expression. As aberrant DNA methylation is known to be genetic cause of many kinds of diseases, it is a strong candidate for biomarker for diagnosis.Although various technologies have been used to analyze DNA methylation, those methods are insufficient as a routine diagnosis tool because of potential error by incomplete conversion of DNA. Methylation sensitive (MS-) multiplex ligation-dependent probe amplification (MLPA) has a potential as a diagnosis tool, because it is a conversion-free method. Additionally, MS-MLPA can analyze multiple targets, which is essential for diagnosis of most diseases. But this method has limitations: designing a custom MLPA probe is more difficult due to the stuffer sequence, and only the limited target site can be analyzed due to the sequence specific restriction enzyme. In this study, we developed a novel DNA methylation analysis method using MS-MLPA, in which stuffer-free MLPA probes and McrBC enzyme are used for precise multiplex quantitative analysis. We tested our new method to detect the methylation statues of 4 tumor suppress genes.

Sesquiterpenes are a class of important natural products in pharmaceutics and cosmetics. Metabolic engineering is a promising technology for sesquiterpene production in Escherichia coli. Sesquiterpenes are derived from farnesyl diphosphate (FPP), a precursor to heme, quiniones, dolichols, etc. A direct way to increase sesquiterpene production is enhancement of FPP pool by overexpression of FPP synthase. However, the production is limited by the poor expression and inherent low capacity of plant sesquiterpene synthase in E. coli. Majority of FPP is dephosphorylated to generate farnesol by some endogenous phosphatases. Aiming to improve sesquiterpene production, we in-frame fused FPP synthase and sesquiterpene synthases including humulene synthase and santalene synthase. A dramatic increase in sesquiterpene production and reduction of farnesol production has been observed by overexpression of the fusion proteins in the engineered E. coli that imported a mevolonate pathway. Our results imply that spatial organization of sesquiterpene synthases has great potential to couple FPP metabolite to targeting sesquiterpene and thereby to improve sesquiterpene production. This work was supported by a grant (NRF-2010-C1AAA001-0029084) from the National Research Foundation, MEST; a grant from the Next-Generation BioGreen 21 Program (No. 2010-0000), Rural Development Administration, Republic of Korea; and BK21 program of Korea.

Bioengineers have been attempting to utilize variety of organisms and their enzymatic pathways to execute beneficial applications such as production of chemicals or treatment of pollutants. In recent years, design and implementation of synthetic pathways have been studied to enlarge the range and variety of biological pathways. Design of synthetic pathways needs a gene expression regulator module which is designable, tunable, extendable and modular.(1) RNA is one of promising candidates that meets aforementioned requirements for regulator module. There are numerous examples of RNA-mediated regulation of gene expression in nature. Moreover, engineers have produced various RNA-based regulators by mimicking their natural counterparts.(2) Due to its structural and functional versatility engineers expect to select or screen any RNA regulator with desired function from a pool of RNAs whose sequences are randomized. We have tried to select an RNA switch which controls expression level of transcriptionally fused downstream gene in response to amino acid L-tryptophan. L-tryptophan is an aromatic amino acid used as a diet supplement for animal feed or a treatment for depression. At first, previously selected L-tryptophan aptamer(3) was fused to tetA-sGFP fusion gene via randomized linker. Escherichia coli W3110 cells transformed with the vectors which harbor above gene construct were cultured under two different conditions for selection of L-tryptophan responsive RNA switch. After two rounds of selection(4) - positive and negative - we got several RNA switch candidates. We investigated performance of the switch candidates using fluorescence strength of sGFP.

Biodiesel is an interesting alternative energy source and is used as substitute for petroleum-based diesel. Microorganisms have been used for biodiesel production because of their significant environmental and economy of our society. This study was achieved by homologous expression in E. coli genes KR, ER, and KAS were predicted to encode enzymes relating in biosynthesis of Escherichia coli fatty acids. We have engineered more productive of long chain fatty acids than wild type strains and found some changes of UFA/SFA ratio in process of time. We also investigated quantitative relations of fatty acid contents and effects of various media for production of fatty acids. This approach offers a feasible means to improve the fatty acid composition of microdiesel and to produce biodiesel equivalents using engineered microorganisms in the near future.* KR: β-ketoacyl-ACP reductase, ER: enoyl-ACP reductase, KAS: β-ketoacyl-ACP synthase III

Clostridium beijerinckii is an anaerobic bacterium used for the fermentative production of acetone, butanol and ethanol. For the enhanced production of n-butanol, development of reporter system suitable to C. beijerinckii is needed. The lipase from C. beijerinckii NCIMB 8052 and Evoglow (aerobic &anaerobic fluorescent protein) were selected as model proteins for their easy detection. In order to test the reporter system, several promoters from C. acetobutylicum and C. beijerinckii were cloned upstream regions of the reporter gene. In this presentation, the detailed results will be presented.

In order to identify protein-protein specific interaction in bacterial cells, we designed an in vivo localization of protein complex in inclusion bodies, which could be visualized by tagged fluorescent proteins. First, we tested the possibility that fluorescent proteins could be actively displayed on inclusion bodies. Green or red fluorescence protein was translationally fused with cellulose binding domain (CBD) in E. coli. As a result, the fluorescent proteins mostly were expressed as inclusion bodies with their intrinsic fluorescent colors. In addition, the recombinant fluorescent particles seemed to be very stable and did not disappear even after cell lysis. Next, we fused leucine zipper proteinss (LZPs) with GFP-CBD and mRFP to make an LZP-GFP-CBD and LZP-mRFP, respectively. When mRFP and LZP-GFP-CBD were co-expressed in cells, mRFPs were dispersed in cytosol. However, in case of co-expression of LZP-mRFP and LZP-GFP-CBD, it was observed that mRFPs were co-localized with GFP inclusion bodies by the interaction of LZP motifs. Even after cells were disrupted by sonication, the interaction between LZP-mRFP and LZP-GFP-CBD was clearly observed in the form of stable particles. These results indicate that inclusion body-mediated fluorescence co-localization is useful for detecting and analyzing protein-protein interactions in living bacterial cells.

We constructed Candida antarctica lipase B 1422 (CalB1422, a mutated CalB with increased activity and secretion efficiency)-displaying yeast Saccharomyces cerevisiae by fusion of a-agglutinin anchor with the 5’å region of CalB1422. Cell-surface display of the CalB1422 fusion protein was confirmed by both p-nitrophenyl palmitate assay and flow cytometry after staining the cells with antibody against the CalB1422. CalB1422 fusion protein was observed with higher fluorescence intensity on the surface of the yeast than an yeast non-displayed CalB1422. The CalB1422 was displayed functionally on the cell surface of S. cerevisiae using the anchor protein a-agglutinin. These results suggested that CalB1422-displaying cells could be useful as a whole cell biocatalyst for the production of lipase-catalyzed products, such as enantioselective chemicals and biodiesel.

Sepiapterin is a precursor as well as a by-product of tetrahydrobiopterin (BH4) synthesis which is a well-known cofactor for aromatic amino acid hydroxylation and nitric oxide synthesis in higher animals. In this study, recombinant Escherichia coli strain harboring cyanobacterial GTP cyclohydrolase I (GCH1) and human 6-pyruvoyltetrahydropterin synthase (PTPS) genes was developed to produce sepiapterin. Optimum expression conditions for GCH1 and PTPS were 37C and 0.1 mM IPTG to obtain maximum sepiapterin concentration of 84.6(19.0) mg/l. Coexpression of guanosine monophosphate (gmk), IMP dehydrogenase (guaB), GMP synthetase (guaA), and nucleoside diphosphate kinase (ndk) related to GTP biosynthesis were attempted to increase sepiapterin production in recombinant E. coli. Coexpession of gmk, guaBA, ndk gave rise to highest sepiapterin concentration of 128.5(28.6) mg/ml. Simultaneous expression of guaBA did not increase sepiapterin production further compared to the control.

Isopentenol is a natural alcohol and one of the most simple terpenoid. It is clear colorless oil that is widely used in cosmetic and pharmaceutical industry. Recently, ispentenol is produced as 5-carbon alcohols from the genetically modified E. coli, which are worthy alternative to present hydrocarbon fuels. But mass production of isopentenol is hindered due to its toxicity to host strain, E. coli. Thus, it is desirable to achieve an isopentenol tolerance of host strain through genetic engineering. In our study we have developed a robust method of random mutagenesis and selection for rapid evolution of tolerant strain. To enable this procedure, we constructed temperature-sensitive plasmids that temporarily increase the mutation frequency of their hosts by 3000 to 5,000-fold. Under appropriate selection pressure, microorganisms transformed with mutator plasmids can be quickly evolved to exhibit new, complex traits. Subsequently, the evolved strains were returned to their normal low mutation rate by curing the cells of the mutator plasmids to prevent the accumulation of undesirable spontaneous mutations. By using this approach, we were able to increase the tolerance of E. coli DH5α strain to isoepentenol up to 1.25g/liter. Our work demonstrated an efficient method for rapid strain improvement based on in vivo mutagenesis.This work was supported by a grant (NRF-2010-C1AAA001-0029084) from the Natonal Research Foundation and a grant from 21C Frontier Microbial Genomics and Application Center Program, MEST, and BK21 program of Korea

On the basis of the hypothesis that high fat diets (HFD) can induce gender-different obesity development, we attempted to unravel the gender-difference at a molecular level. To this end, we performed proteomic analysis in plasma of rats fed a HFD by 2-DE combined with MALDI-TOF-MS, thereby analyzed differential expression patterns between male and female plasma proteins. When exposed the rats to HFD, the body weight gain and food intake of HFD-fed rats were higher than those of low fat diets (LFD) rats in both genders, where male rats gained approximately 41% more body weight than female rats. These rats were used for proteomic analysis and changed 31 proteins among 370 visualized spots were identified by PMF. Changed proteins in each group were classified into three groups which were composed of proteins with gender difference in LFD and/or HFD groups (category I), changed proteins by HFD in male and/or female groups (category II) and proteins with both gender difference in LFD and/or HFD groups as well as HFD effect in LFD and/or HFD groups (category III). As a result, 7 proteins of category I, 13 proteins of category II and 11 proteins of category III were found. These findings suggest that these proteins may contribute to the gender differences in obesity development directly or indirectly.

Metabolomics desire the gain of reproducible, reliable, and homogeneous biological data sets. Intracellular metabolite is a reflection of all the metabolic functions of an organism under any particular growth condition. For the development of experimental procedure, extraction of the metabolome to quantitative analysis of the metabolites is required. Klebsiella oxytoca was grown in chemostat culture so that cellular metabolism could be held in reproducible, steady-state conditions under a range of defined growth conditions, thus enabling sufficient replication of samples. In the absence of in situ methods capable of universally measuring metabolite pools, intracellular metabolite measurements need to be performed in vitro after extraction. This study indicates that 60% cold (-48 °C) methanol solution is the most appropriate method to quench metabolism. In this study, metabolites were extracted from Klebsiella oxytoca using six different commonly used procedures including acid or alkaline treatments, high-temperature extraction in the presence of ethanol or methanol or water and by lysis with chloroform-methanol. Intracellular metabolites were extracted by the six methods from cells grown under identical conditions. The intracellular metabolite profiles were generated using HPLC system coupled with a mass spectrometer.

1-Deoxynojirimycin (DNJ), a D-glucose analogue with a nitrogen atom substituting for the ring oxygen, is a strong inhibitor of the intestinal a-glucosidase. DNJ has several promising biological activities, such as antidiabetic, antitumor, and antiviral activities. Nevertheless, only limited amounts of DNJ is available because it can be extracted from some higher plants including the mulberry tree or purified from culture broth of several soil bacteria such as Streptomyces sp. and Bacillus sp. In our previous study, a DNJ producing bacterium, Bacillus subtilis MORI, has been isolated from a Korean traditional fermented food, Chungkookjang. Here, we report the identification of DNJ biosynthetic genes from the genomic DNA library of B. subtilis MORI strain constructed in E. coli, by selecting clones showing a-glucosidase inhibition activity. After DNA sequencing and series of subcloning, we were able to identify the DNJ biosynthetic operon with three genes encoding a putative transaminase, phosphatase, and oxidoreductase, respectively. Our results suggest the possibility to establish a large scale microbial DNJ overproduction system through genetic engineering and process optimization.

Retinoids are lipophilic isoprenoids molecules, which are defined as a class of substances comprising vitamin A and its natural and synthetic derivatives. Retinoids, being derived from beta-carotene, consist of a cyclic group and a linear chain with hydrophilic end group. They include retinol, retinal (retinyl aldehyde), retinoic acid, and retinyl esters, and also exist in other various structures differentiated by their functional cyclic or hydrophilic end groups. Retinoids are representative cosmetic active agents, which also have been known as effective pharmaceuticals for skin diseases. The cleavage of beta-carotene by BCM(D)O (E.C. 1.13.11.21 or E.C. 1.14.99.36) is an important step of retinoids synthesis. In order to produce retinal from beta-carotene, BCDO (beta-carotene dioxygenase) gene of uncultured marine bacterium was synthesized with codon optimization for E. coli expression and introduced to the beta-carotene producing strain. Therefore, we successfully produced high amount retinoids using recombinant E. coli. This work was supported by the Basic Research Program (Grant No. 2009-0084490) of MEST, and BK21 program of Korea.

The draft genome of a hexanoic acid-producing bacterium Megasphaera sp. BS-4 was obtained using GS-FLX titanium and 441,216,354 total base pairs data was generated from 1,213,411 reads. Both fragment library and paired-end library were used as sequencing templates and one-fouth plate was used to generate sequence reads for each library. Assembly was carried out using Newbler and the assembled genome was annotated using RAST. The assembled genome was consisted with 7 scaffolds and 82 contigs. The approximate size and coverage of the genome was 2.8 Mb and 155X respectively. The G+C content of the genome was 48.99%. The genome contained 2,713 ORFs, 5 rRNA genes and 53 tRNA genes. The longest contig was 384,514 bp. A total of 1966 genes were categorized based on COG. Among these genes, 240 genes were assigned to ‘mino acid transport and metabolism’(E). ‘arbohydrate transport and metabolism’(G) and ‘nergy production and conversion genes’(C) were 111 genes and 152 genes.

The four enzymatic reactions of the elongation cycle in fatty acid synthesis metabolism were catalyzed by β-ketoacyl-acyl carrier protein (ACP) synthase (KAS) (FabB, FabF and FabH), β-ketoacyl-ACP reductase (FabG), β-hydroxyacyl-ACP dehydrase (FabZ), and enoyl-ACP reductase (FabI). These enzymes play an essential role in the extension of fatty acid length. To investigate cellular long-chain fatty acids composition in E. coli K-12 MG1655 the overexpressed strains of the fabG::fabZ or fabG::fabZ::fabI or fabG::fabZ::fabI::fabH genes were constructed. The culture optimization was tried to observe enhanced production of long-chain fatty acids. The fatty acids content and chain length were analysed in the recombinant E. coli strains and wild-type by using GC/MS. The results indicated that developed strains produced more cellular total lipid and fatty acids, and altered long-chain fatty acids composition.

Since Escherichia coli requires homeostasis of internal pH in the range of 7.4 to 7.9, the pH response is important for cellular growth and survival under conditions of fluctuating pH [1,2]. The response and adaptation for pH change in E. coli strains were studied by proteome analysis. Comparison of their proteomes by separating two-dimensional gel electrophoresis following LC-MS/MS allowed us to identify 30 protein spots showing significantly altered levels in pH-dependent manner. The low pH responsive proteins included inner membrane proteins, membrane-associated proteins, outer membrane proteins and flagellum filament protein. This study shows that the different pH might induce drastic changes in composition and structure of E. coli membrane. [This work was supported by the Korean Systems Biology Research Grant (20090065571) of Ministry of Education, Science and Technology (MEST) to S.Y. Lee through the National Research Foundation (NRF), and the Basic Science Research Program (2010-0008826) from the NRF to M.-J. Han]

The emergence of antibiotic-resistant pathogens has rekindled interest toward the discovery of new antibiotics. Semi-synthetic or biosynthetic approach using microbe as a biocatalyst can be an efficient tool for the targeted modification of existing antibiotic chemical scaffolds to create the next generation of antibiotics as well as previously uncharacterized therapeutic agents. An attempt was made to expand the applicability of the unique bio-hydrogenation activity of Streptomyces venezuelae toward other unsaturated macrolide antibiotics (natural oligomycin A and semisynthetic tilmicosin) that possess the above mentioned identical catalytic scaffold, and the structure and antibiotic potential of two previously uncharacterized macrolides, 2,3-dihydro-oligomycin A and 10,11-dihydro-tilmicosin, isolated from a S. venezuelae culture supplemented with oligomycin A and tilmicosin, respectively, were evaluated.

L. mesenteroides has been isolated in sugar refining process and it shows strong sucrose-dependant metabolic features such as dextran and mannitol production. In this study, a proteomic analysis was performed to understand the carbohydrate metabolism of this species in glucose medium and to uncover its regulatory changes in fructose or sucrose medium. For this, a LC-MS/MS was used to analyze the intracellular protein levels of L. mesenteroides ATCC 8293 when cultured in 2% glucose-, 2% sucrose-, or 1% glucose and 1% fructose-MRS media. Shotgun proteomic results obtained from duplicate samples showed high correlation and reproducibility. Totally 516 proteins were detected with high confidence in each cultures. When it growth in different carbon sources, L. mesenteroides ATCC 8293 preferentially used carbohydrate metabolism related pathway. In the central metabolic pathway, most of the proteins are consistently expressed. It showed that central metabolic pathway related proteins of L. mesenteroides ATCC 8293 have homeostasis. Also, L. mesenteroides ATCC 8293 have incomplete TCA cycle. When look at the expression level of that was not related with growth of L. mesenteroides ATCC 8293.

C57BL/6J mice have been widely used as a diet-induced obesity model because they trigger common feature of human metabolic syndrome. In the present study, C57BL/6J male mice were fed either high fat diet (HFD) or normal diet (ND) during 24-week period, and analyzed age-dependent liver proteome of mice in two groups using 2-DE combined with MALDI-TOF-MS. Among identified proteins, up-regulated proteins were subdivided to early (during the first 4 weeks) and late (20~24 weeks) markers that played a role in diet-induced obesity development. Important early markers included ketohexokinase, prohibitin, and late markers included 75 kDa glucose-regulated protein, citrate synthase, selenium-binding liver protein. Of these, 75 kDa glucose-regulated protein has already been linked to obesity; however, prohibitin protein involved in obesity were identified for the first time in this study. In order to validate the proteomic results and gain insight into metabolic changes between the two groups, we further confirmed the expression pattern of some proteins of interest by Western blot analysis. Combined results of proteomic analysis with Western blot analysis revealed that anti-oxidant enzymes were progressively decreased whereas cytoskeletal proteins were time-dependently increased with a higher magnitude in HFD mice.

In agriculture, chitinolytic microorganisms have been widely used for biocontrol of fungal phytopathogens as cell wall of most fungal pathogens contains chitin to varying extent. Paenibacillus species are widely distributed in nature and have been reported for their biocontrol potential against bacteria and fungi owing to the production of cell wall degrading enzymes such as β-1,3-glucanases, cellulases, chitinases and proteases. This study indicated that plant growth promoting rhizobacterium P. polymyxa has higher chitinase activity compared to Bacillus thuringiensis which is the most widely used biopesticide and known primarily for its chitinase. A chitinase gene (chi45) from P. polymyxa was cloned, sequenced, and expressed in Escherichia coli. The gene consisted of an open reading frame of 1,248 nucleotides encoding a protein of 416 amino acids with a calculated molecular weight of 45,760 Da. Zymogram analysis indicated that the purified enzyme revealed the presence of the 45-kD chitinase. The purified chitinase was shown to inhibit the growth of Rhizoctonia solani, plant pathogenic fungus. Characterization of novel chitinase from P. polymyxa suggested that this enzyme is applicable for various industrial applications, including its use as biopesticide.

Fermentative production of hydrogen (H2) is still far from the industrial application since H2 production yield in terms of energy recovery is low, even though various approaches were attempted to improve H2 yield. For efficient energy recovery from glucose, co-production of H2 and ethanol is suitable strategy and this help to overcome the limitation of fermentative H2 production. In order to improve co-production yield of Escherichia coli, non-essential metabolic pathways competing with ethanol and H2 production were inactivated. While pentose phosphate (PP) pathway was enhanced by deleting Embden-Meyerhof pathway (△pgi or △pfkA) to generate excess NADPH which will be converted to NADH by soluble dehydrogenase (udhA), for additional ethanol production. However, developed strains with △pgi or △pfkA showed a negligible cell growth under fermentative condition. An adaptive evolution method was used to improve strain’ growth rate under anaerobic condition. While in these strains, majority of carbon consumed was secreted as pyruvate, intermediary metabolite. Studies to reduce pyruvate secretion are under progress with several approaches and surmounting this problem will allow to co-produce H2 and ethanol in a feasible scale.

Gamma-aminobutyric acid (GABA), a non-essential amino acid, is formed by decarboxylation of L-glutamate with the catalysis of glutamate decarboxylase (GAD, EC 4.1.1.15), a pyridoxal 5′-phosphate (PLP) dependent enzyme. In Escherichia coli, glutamate decarboxylase (GadB), together with GadC, a putative glutamate/GABA antiporter located downstream of gadB and cotranscribed with gadB, constitutes the gad acid resistance system, which controls the acidification of the cytosolic environment by decarboxylating an acidic substrate (glutamate) into a neutral compound (GABA) which would then be exported into the extracellular medium through the GadC. GadB has another isoform (GadA), which is 2100 kb away from GadB in the E.coli chromosome. In this study, GadA, GadB (52.7 kDa), GadC (55 kDa) were overexpressed in XL1-Blue by constructing four expression vectors containing GadA, GadB, GadA and GadC (GadA-GadC), GadB and GadC (GadB-GadC) respectively. In glutamate metabolic pathway of Escherichia coli, there is a gene called gabT coding for γ-aminobutyrate aminotransferase (2.6.1.19) which converts GABA into succinate semialdehyde and then reduces GABA concentration in the extracellular medium. Hence, this gene was knockouted to enhance GABA bioconversion efficiency in this work. The results show GABA concentrations in the extracellular medium containing MSG (monosodium glutamate) in cases of overexpression of GadA and GadC (GadA-GadC), GadB and GadC (GadB-GadC) are higher than the concentrations in cases of overexpression of only GadA and GadB respectively (about 84% GABA bioconversion rate after 48 hours under controlled conditions for GadA-GadC overexpression compared to 54% for GadA overexpression). Likewise, GABA concentrations in cases of mutated XL1-Blues are higher than the concentrations in the wild type (about 77% GABA bioconversion rate for GadA overexpression in mutated strain compared to 54% in the same overexpression manner in the wild type XL1-Blue). Therefore, by overexpression of GadA, GadB, GadC, and making mutated strains, the wild type XB1-Blues have been genetically engineered to perform conversion with high GABA levels in the extracellular medium from glutamate.

Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 is a lactic acid bacterium that converts pyruvate to L-(+)- and D-(-)-lactate by sterospecific enzymes designed as L-(+)- and D-(-)-lactate dehydrogenase (LDH), respectively. This study aims to determine the genes responsible for D-lactate formation in L. mesenteroides subsp. mesenteroides ATCC 8293, as well as to provide a basis for the realization of optically pure D-lactate production. We report the cloning and nucleotide sequence of the ldhD gene and LEUM_2043 which has not been annotated exactly from L. mesenteroides. The two genes had been cloned and expressed in Escherichia coli BL21(DE3) star with an inducible expression vector pETDuetTM-1 successfully. The two proteins, ldhD and LEUM_2043, had an apparent molecular mass of 36 kDa each on SDS-PAGE, and the LEUM_2043 was much more expression than ldhD. And the activity of ldhD and LEUM_2043 were similar. By now, the mechanism for D-lactate formation in L.mesenteroides has been characterized. Moreover, we extend our research to construct Leuconostoc strain which produces high level L-lactate and apply it as a starter culture.

The goal of this study was to increase fatty acid production by developing recombinant E. coli strains which fatty acid synthesis (FAS) were improved. The acetyl-CoA carboxylase (ACC) enzyme from accA, accB and accC genes catalyzes the addition of CO2 to acetyl-CoA to generate malonyl-CoA. The enzyme encoded by the fabD gene converts malonyl-CoA to malonyl-[acp], and the EC3.1.2.14 gene converts fatty acyl-ACP chains to long chain fatty acids. Therefore, over-expression of acc family genes and EC3.1.2.14 gene was expected to increase the productivity of fatty acids. All recombinant E. coli strains containing various gene combinations to enhance the enzymatic activities were developed using the pTrc99A vector. The in vitro metabolites, amount of total lipid and fatty acids produced were analyzed to observe the changes in metabolism by introducing five distinct genes. This result indicated that from recombinant E, coli which had EC3.1.2.14 gene, the amount of total lipid and fatty acids were produced much more than the wild type E. coli.

Guanosine-5’diphosphate (GDP)-L-fucose is an essential precursor for the synthesis of fucosyloligosaccharides, which are involved in various biological and pathological functions such as cell adhesion, inflammation, tumor metastasis and host immune response modulation. In this study, key genes for GDP-Lfucose production, encoding GDP-D-mannose-4,6-dehydratase (GMD) and GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase (W caG) from Escherichia coli were introduced into C. glutamicum. GDP-L-fucose production was attempted by combinatorial overexpression of mannose-6-phosphate isomerase (manA), phosphomanno-mutase (manB) and GTP-mannose-1-phosphate guanylyl-transferase (manC) genes, which are also involved in GDP-D-mannose biosynthesis. Batch fermentations using the recombinant C. glutamicum systems showed that overexpression of gmd, wcaG, manB, manC was the most effective for GDP-L-fucose production and gave a 5.8-fold enhancement in specific GDP-L-fucose content compared with the control strain overexpressing GMD and WcaG only. A maximal GDP-L-fucose concentration of 86.2 mg/l was achieved in a batch fermentation of the recombinant C. glutamicum overexpressing GMD, WcaG, ManB and ManC.

FK506 is the clinically important 23-membered macrocyclic immunosuppressive polyketide produced by a variety of Streptomyces species. The allyl moiety of FK506 is structurally unique amongst polyketides and critical for its potent biological activity. Here, we elucidated the detailed biosynthetic pathway of allylmalonyl-coenzyme A (CoA), from which the FK506 allyl group is derived, based on the comparison of three FK506 gene clusters, gene deletion, chemical complementation. Furthermore, the novel allyl group-modified FK506 analogs possessing improved neurite outgrowth activity were mutasynthesized. This work demonstrates the unique feature of FK506 biosynthesis in which a dedicated polyketide synthease (PKS) provides an atypical extender unit for main modular PKS, and a new tool for combinatorial biosynthesis of novel macrolide scaffolds as well as FK506 analogs.

Single nucleotide difference discrimination is important for field of genotyping including Single Nucleotide Polymorphism (SNP). Several technical approaches were applied for discriminating single nucleotide difference discrimination, ligation based technologies were broadly used which use DNA ligase and target specific probe set. Ligation based technology genotype the single nucleotide with ligation efficiency difference between perfectly matched probe and mismatched probe. If 3’' end of left probe oligo (LPO) hybridize perfectly with target site, LPO ligated with 5’' end of right probe oligo nucleotide (RPO). But in the case of mismatch between 3’' end of LPO and target site, ligation between LPO and RPO is still occurred in some case because of high reaction rate of DNA ligase, so it does not gives accurate information about genomic information of target site. We used commercially available alternative nucleobase such as 5’'-nitroindole, iso dC, iso dG in 5’' end of RPO to solve the problem by decreasing reaction rate of DNA ligase. By this approach, various matches and mismatch between 3’' end of LPO and target site was discriminated.