Earticle

Polyguluronate specific lyase (EC 4.2.2.11) from Streptomyces sp. ALG-5 was cloned in pColdI vector and characterized previously. We named the polyG-specific lyase as ALG5 lyase. Alg5 was engineered to improve the alginate degrading activity based on comparative homology modeling. The homology modeling of ALG5 lyase was constructed by using the 3D crystal structure (1UAI.pdb) of alginate lyase 2 from Corynebacterium sp. Aly1 as a template. ALG5 lyase belongs to polysaccharide lyase family 7 which the fold of the structure is β-jolly sandwich. ALG5 lyase had 8 β-strands (SA1-SA8)at upper layer and 7 β-strands (SB1-SB7) at lower layer. Active sites were formed with Arg72 of SA3, Gln117 of SA5, His119 of SA5, Tyr188 of SA4 and Tyr194 of SA5. In the case of water soluble protein, protein surface was known to be composed with hydrophilic amino acid residues whereas protein core was composed with hydrophobic amino acid residues. Stability of mutants was calculated with ProSa2003. Glu101 on SB4 was mutated to hydrophobic amino acid, Phe, Leu, Met, Ile and Val. When Glu101 was mutated to Leucine, the alginate degrading activity was increased about 5 fold than that of original ALG5 lyase.

Lignan, one of phytoestogens, has attracted great attention due to their estrogenic, anticarcinogenic, antioxidant effects. Among lignans, enterodiol (END) is converted by intestinal bacteria from the plant lignan secoisolariciresinol, which exist in flaxseed mainly. Among the 20 Actinomycetes strains examined, Streptomyces avermitilis MA-4680 and Nocardia facinica IFM10152 showed the highest hydroxylation activity for enterodiol. Reaction products profiling using GC/MS revealed that, five products of aliphatic and three of aromatic monohydroxylation were found from Streptomyces avermitilis MA-4680, whereas only two aromatic monohydroxylated products were detected from Nocardia facinica IFM10152.To identify which cytochrome P450s are related to hydroxylation reaction toward enterodiol, we constructed 27 P450s from Nocardia facinica IFM10152 in pET vector and putidaredoxin (camB) and putidaredoxin reductase (camA) from Pseudomonas putida in pET duet vector to use different antibiotic system. P450s are co-expressed with camA and camB as redox partner in E.coli. Eventually, we found that CYP154 were able to catalyze hydroxylation reaction of enterodiol at ortho position in vivo. Finally, we purified CYP154, camA and camB by using Ni-NTA his tag purification system and confirmed hydroxylation activity with enterodiol.

Much interest has been recently focused on the production of large quantities of hydrogen, due to its potential importance in our economy and needs in the petroleum and chemical industries. Formate dehydrogenase H (FDH-H) from Escherichia coli containing selenocysteine that oxidizes formate to carbon dioxide with the release of a hydrogen, is a component of the anaerobic formate hydrogen lyase complex of E. coli. In this approach, the fdhF gene was subcloned into expression vector, pET-22b(+), and a 6xHis tag was fused to FDH-H at the C-terminus and overexpressed in E. coli. However, overexpression of FDH-H in E. coli resulted in the formation of inclusion body. Several efforts including low temperature for induction and optimization of inducer concentration were tried to improve the functional expression of FDH-H.

Osteoarthritis (OA) is the most common rheumatic pathology. A major objective for OA research is development of early diagnostic strategies which include a proteomic technology. Synovial fluid (SF) is a logical potential compartment for OA biomarkers because it is derived directly from the diseased site and the systemic circulation. To enrich relatively lower-abundant proteins in the SF sample, the efficient and reliable preparation of SF samples is very critical in proteome analysis. In this study, we evaluated the pattern of SF proteins on 2 DE-PAGE as a function of sample enrichment and protein loading. Removal of albumin and IgGs and hyaluronidase treatment improve the detection of protein spots on 2DE-PAGE.

Actinomycetes are important industrial microorganisms and a rich source of soluble cytochrome P450 hydroxylase (CYP) enzymes, which play critical roles in the bioactivation and detoxification of a wide variety of metabolite biosynthesis and xenobiotic transformation. Sebekia benihana is belonging to a rare actinomycetes family, capable of hydroxylation at the position of 4th N-methyl leucine of cyclosporin A (CyA), one of the most commonly-prescribed immunosuppressive drugs, leading to display different biological activity spectrum such as loss of immunosuppressive activities yet retaining hair growthstimulating side effect. In order to improve this regio-selective CyA hydroxylation in S. benihana, previously-identified several secondary metabolite up-regulatory genes from Streptomyces coelicolor and S. avermitilis were heterologously overexpressed in S. benihana. Among tested, SCO4967 encoding a conserved hypothetical protein significantly stimulated region-specific CyA hydroxylation in S. benihana, implying that some common regulatory systems functioning in both biosynthesis and bioconversion of secondary metabolite might be present in different actinomycetes species.

Succinic acid has been of interest with its various uses as a precursor for industrially useful chemicals, pharmaceuticals, and biopolymers. Native succinic acid producers including Mannheimia succiniciproducens were found to produce phosphoenolpyruvate (PEP) carboxykinase as a main catalyst for the carboxylation of PEP yielding oxaloacetate, a key intermediate for succinic acid1. Hence, we employed the PEP carboxykinase from M. succiniciproducens to a metabolically engineered E. coli strain which generated succinic acid with a high yield by increasing PEP pool under the anaerobic condition. In addition to the introduction of the enzyme, we also constructed the genetic environment to amplify the effect of PEP carboxykinase. Consequently, we observed the advantages of using PEP carboxykinase originated from M. succiniciproducens in a non-natural succinic acid producer and developed enhanced succinic acid producing E. coli strain in the aspect of both productivity and yield while reducing byproducts. [This work was supported by the Genome-Based Integrated Bioprocess Development Project of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea (#20100002111). Further supports by WCU (World Class University) program by MEST (R32-2008-000-10142-0) are appreciated.]

Zymomonas mobilis ZM4 is famous for producing ethanol from various carbon sources, including glucose, fructose, and sucrose, via the Entner-Doudoroff pathway1. To further enhance its metabolic performance for industrial application, systems metabolic engineering is required. Thus, the genome-scale metabolic model of Z. mobilis is reconstructed based on its annotated genes, literature, physiological and biochemical information to systematically analyze the metabolic characteristics of this bacterium under various range of genotypic and environmental conditions. Physiological features of Z. mobilis were then examined using constraints-based flux analysis, and the substrate utilization range of Z. mobilis was expanded to pentose sugar metabolism by introducing additional metabolic pathways to Z. mobilis. Finally, double gene knock-out simulations were performed to suggest a strategy for overproducing succinic acid. This reconstructed metabolic model will allow better understanding of Z. mobilis metabolism and consequently designing metabolic engineering strategies for various biotechnological applications. [This work was supported by the Korean Systems Biology Research Project (20100002164) of the Ministry of Education, Science and Technology (MEST). Further support by the World Class University Program (R32-2009-000-10142-0) through the National Research Foundation of Korea funded by the MEST is appreciated.]

L-isoleucine, one of branched chain amino acids, has been considered an industrially valuable product since it is employed as a component of pharmaceuticals, cosmetics and dietary products as well as animal feed additives. Escherichia coli, the host organism of this study, tightly regulates L-isoleucine biosynthetic pathway by operating complex regulatory mechanisms, including feedback inhibitions and transcriptional regulations. In this study, we removed all the negative regulations that hamper the production of L-isoleucine, and overexpressed the genes encoding enzymatic reactions and exporters in order to improve the host strain’s L-isoleucine-producing capability. The approaches described in this study are a good example of rationally engineering microorganisms for the enhanced production of L-isoleucine, and they can also be generally applicable to development of strains for the efficient production of other valuable bioproducts. [This work was supported by the Korean Systems Biology Research Project (20100002164) and WCU (World Class University) program (R322009000101420) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea.]

Heterologous protein production in microorganisms has been gaining momentum as tools and techniques for genetic manipulation become more sophisticated. Traditionally, heterologous proteins have been produced in small model multi-cellular organisms. However, due to the time and resources used to care for these ‘factories’, alternate and simpler systems are sought. Yeasts have been the preferred unicellular system for eukaryotic studies and the methylotrophic yeast Pichia pastoris has gained much attention as a host, particularly for proteins with therapeutic values. Here, the genome-scale metabolic model is presented and the analysis of P. pastoris capabilities in producing proteins is investigated. The production of two proteins, human serum albumin (HSA) and human superoxide dismutase (hSOD), were analyzed and metabolic engineering targets were suggested to improve their production. Using genome-scale metabolic model, potential phenotypes can be screen in a high-throughput manner, accelerating the development of strategies for engineering improved strains. [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, by the Austrian Science Fund (FWF), project I37-B03, and the Austrian Research Promotion Agency (program FHplus)]

Economical viability and natural potential as a carbon source have motivated engineering Mannheimia succiniciproducens for the production of succinic acid through glycerol fermentation1. The methods of rational approaches and adaptive evolution were used to improve the ability of utilization of glycerol as a carbon source in M. succinicproducens. The performance of engineered strains such as PALK-glpA, PALK-glpAcrp, PALK -glpFKA, and PALK-sldABdhaK was better than that of control strain in batch fermentation experiments Above all, in fed-batch fermentation, the most productive strain harboring C.acetobutylicum ATCC824 glycerol 3-phosphate dehydrogenase, and M.succiniciproducens cAMP receptor protein increased both concentration and productivity of succinic acid. Surprisingly, expression of the genes encoding the glycerol dehydrogenase and dihydroxyacetone kinase from G.oxydans was able to improve the rate of glycerol utilization and the production of succinic acid significantly as previously reported in E.coli. [This work was supported by the Genome-Based Integrated Bioprocess Development Project (20100002111) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea. Further support by WCU (World Class University) program by MEST (R322009000101420) is appreciated.]

Streptomyces peucetius industrial mutant was recursively mutated to be able to overproduce doxorubicin (DXR) than S. peucetius ATCC27952. Previously we identified wblA, a global antibiotic down-regulator, using a Streptomyces interspecies DNA microarray analysis. Also we confirmed that disruption of wblA ortholog in S. peucetius industrial mutant resulted in an approximately 70% increase in DXR productivity than those of S. peucetius industrial mutant strain. Deletion of DXR gene cluster from the chromosomes of S. peucetius industrial mutant and wblA-disrupted S. peucetius OIM strains enable to verify possibility of heterologous expression of polyketide synthase (PKS) gene cluster as a host. A subset of S. coelicolor actinorhodin (act) biosynthetic genes (act Ⅰ-orf1, actⅠ-orf2, actⅠ-orf3, actⅢ, actⅦ, actⅣ) for aloesaponarinⅡ, a highly fluorescence anthraquinone polykeitde derived from one acetyl-CoA and seven malonyl-CoA extender units, and actinorhodin cluster activator gene (actⅡ-orf4) was successfully cloned to express in both S. peucetius OIM△DXR and S. peucetius △wblA△DXR strains.

Recently, recombinant Streptomyces venezuelae has been established as a heterologous host for microbial production of flavanones and stilbenes, a class of plant-specific polyketides. In the present work, we expanded the applicability of the S. venezuelae system to the production of more diverse plant polyketides including flavones and flavonols. A plasmid with the synthetic codon-optimized flavones synthase I gene from Petroselium crispum was introduced to S. venezuelae DHS2001 bearing a deletion of the native pikromycin polyketide synthase gene, and the resulting strain generated flavones from exogenously fed flavanones. In addition, a recombinant S. venezuelae mutant expressing a codon-optimized flavanone 3β-hydroxylase gene from Citrus siensis and a flavonol synthase gene from Citrus unshius also successfully produced flavonols.

Gene therapy is considered as a potential remedy for treating diseases. Therefore, safe and efficient gene delivery materials have been investigated. Gene delivery vectors are categorized into two groups: viral and non-viral. Though viral vectors are most effective, safety concerns are limitations on clinical cure. However, non-viral vectors have improved safety and manufacturing capacity. Here, we report the recombinant mussel adhesive protein (MAP) as a synthetic DNA delivery vehicle. Recombinant MAPs are biocompatible, biodegradable proteins for underwater adhesion. Because of the plenty of basic amino acid composition, they were considered as a possible carrier of DNA. After DNA binding assay, mouse NIH3T3 cells were transfected with pEGFP by MAP. The transfection efficiency was compared with LipofectamineTM 2000, the widespread transfection agent. Optimized transfection condition will achieve better capability in transferring DNA into model cell line.

Anthracycline antibiotics including doxorubicin and darubicin are clinically important anticancer agents. It has been reported that these drugs intercalate into DNA and interact with topoisomerase II-DNA complex. Epirubicin is a semisynthetic analogue of doxorubicin displaying reduced cardiotoxic and equal antitumor property when compared with doxorubicin. In order to enhance epirubicin production in S. venezuelae system, we assayed four glycosyltransferases and eight 4-ketoreductases from various Streptomyces species. The results of this assay suggested that glycosyltransferase AknS and 4-ketoreeductase AvrE are the best enzymes among the glycosyltransferases and 4-ketoreeductases tested.

Klebsiella oxytoca stains are excellent 2,3-butanediol producers. To use this strain in industry area, 2,3-butanediol must be produced more than now. In order to achieve a high production, the budC gene of acetoin reductase was overexpressed by inserting the budC gene, which is ligated to puc18K with a kanamycin resistance, into K. oxytoca KTCC 1685 and K. oxytoca ATCC 43863. Byproducts like lactic acid, acetoin, acetic acid and ethanol reduce the amounts of 2,3-butanediol. Among of them, lactic acid especially affects the fermentation efficiency for 2,3-butanediol production. Two K. oxytoca mutants deficient in lactic acid formation were successfully constructed by knocking out the ldhA gene coding for lactate dehydrogenase of them. After fermentation studies of mutant stains, the result showed that 2,3-butanediol production was enhanced compared with the wild type strains.

The bacterial genus Streptomyces has long been appreciated for its ability to produce various kinds of medically important secondary metabolites, such as antibiotics, anti-tumour agents, immunosuppressants and enzyme inhibitors.Tautomycetin (TMC), which is produced by Streptomyces sp. CK4412, is a novel activated T cell-specific immunosuppressive and anti-cancer compound with an ester bond linkage between a terminal cyclic anhydride moiety and a linear polyketide chain bearing an unusual terminal alkene.Previously, we isolated and characterized the entire TMC biosynthetic gene cluster from Streptomyces sp. CK4412. Sequence information analysis of an 110kb DNA region revealed multi-modular type I polyketide synthases, type II thioesterase, various proteins for dialkylmaleic anhydride biosynthesis, regulatory proteins and several tailoring enzymes. Although both TMC and its close structural relative called Tautomycin (TM) compounds have similar inhibitory activities against two major serine/threonine protein phosphatise, PP1 and PP2A, the different linear polyketide moiety present in TMC and TM is believe to play a critical role because only TMC exhibits an immunosuppressant activity as well as a higher PP1 selectivity. To increase our understanding about the functional specificity, the targeted gene inactivation of various biosynthetic genes involved in the formation of a linear TMC polyketide moiety was performed, leading to the generation of several novel TMC analogs with different polyketide terminal region.

Clostridium acetobutylicum is a model organism for butanol production. C. acetobutylicum M5 is a degenerate derivative of C. acetobutylicum ATCC824. Loss of solvent production in this strain can be complemented by over-expression of adhE1-ctfAB. This strain, M5 (pIMP1E1AB), produced butanol with reduced amounts of other solvents relative to the wild-type strain C. acetobutylicum ATCC824. A high butanol selectivity of 0.84 was achieved. In this study, we used a proteomics approach to examine changes in global protein expression between solventogenic C. acetobutylicum ATCC824 and degeneratenonsolventogenic C. acetobutylicum M5. In order to further understand M5 (pIMP1E1AB) strain, we also compared protein expression patterns to those in the wild type ATCC824 strain, both in acidogenic and solventogenic phases. [This work was supported by the Ministry of Knowledge Economy grant funded by the Korea government (#10030795). Further supports by the GS-Caltex and the BioFuelChem].

Mannheimia succiniciproducens MBEL55E, a gram-negative facultative capnophilic rumen bacterium, produces mainly succinate by metabolizing a wide range of carbon sources. Of those, fermentation studies have shown that M. succiniciproducens produces equal or higher titers of succinic acid using sucrose rather than the other carbon sources. Hence, we identified genes involved in the sucrose transport and utilization. The sucrose-utilizing phenotype was examined both for the parent strain and the deletion mutant of each gene. The corresponding enzymes were further characterized by measuring enzyme activities. As a consequence, it was revealed that M. succiniciproducens uses a sucrose phosphotransferase system (PTS), sucrose 6-phosphate hydrolase, and a fructose PTS for the transport and utilization of sucrose. Strategies evolved from this characterization of the sugar metabolism were also suggested in detail. [This work was supported by the Genome-Based Integrated Bioprocess Development Project (20100002111) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea (NRF). Further support by World Class University program (R322009000101420) by MEST through NRF is appreciated.]

For the last decade short noncoding RNAs are discovered to play an important role in genetic regulations. Among them, regulatory small RNAs (sRNA) in Escherichia coli repress translation initiation by hybridizing with a ribosome binding site in mRNA to prevent ribosome binding and by facilitating the degradation of mRNA. Such regulatory small RNAs could provide another way of genetic regulation in synthetic biology so that genetic circuits could be controlled precisely in concert with protein-based regulations. We could construct efficient customized sRNAs for DsRed2 repression and also constructed three different sRNAs for mRNAs of LuxR, AraC, and KanR without cross-reactivity. As a proof of concept, we constructed a temperaturesensitive genetic circuit controlled by artificial sRNA and the circuit operated successfully. The results represent that gene expression can be fine-tuned by designed small RNAs. The possibility of designing small regulatory RNAs opens a new way of regulation in synthetic biology, and which is expected to facilitate the development of precisely regulated synthetic circuits. [This work was supported by the Korean Systems Biology Research Project (20090065571) of the Ministry of Education, Science and Technology (MEST) through the National Research Foundation of Korea (NRF). Further supports by the World Class University Program (R32-2008-000-10142-0) of the MEST.]

LK8 protein is the 38th kringle fragment of human apoliprotein consisting of 86 amino acids with strong anti-angiogenesis activity. Invertase is a glycoprotein that catalyzes the hydrolysis of sucrose into fructose and glucose. With these two model proteins, we investigated the effect of the various folding or secretory pathway associated proteins in Saccharomyces cerevisiae. First, to establish the stable and efficient expression system, LK8 and invertase expression cassettes containing the galactose-induced promoter and mating factor-α signal sequence were constructed and introduced into S. cerevisiae 2805 by δ-integration. Second, to facilitate protein maturation and exit from ER, several folding accessories, Pdi1P, Sec23p and Trx2p were coexpressed with LK8 or invertase. Pdi1p, Sec23p and Trx2p increased the secretion of LK8 from 18.1 mg/L to 23.7, 20.7 and 19.3 mg/L, respectively. In Pdi1p, Sec23p and Trx2p coexpressed strains, the invertase activity in media was 70.2, 65.7 and 47.1 U/mL, respectively, whereas the control strain showed 47.5 U/mL of activity. Sec23p was effective in both cases as well as Pdi1p, a well-known protein folding facilitator.

Hydrogen production in biological way can be one of solutions for energy crisis. Previously, we successfully demonstrate to produce biohydrogen by expressing [NiFe]-hydrogenase in recombinant E. coli. Although [NiFe]-hydrogenase showed relatively high oxygen-tolerance compare to other hydrogenases, still biohydrogen production efficiency was low. In this work, we introduce proteorhodopsin, light acceptor for prokaryote working as proton pump, into [NiFe]-hydrogenase expressing recombinant E. coli for using light energy to produce biohydrogen. Proteorhodopsin need a chemical named retinal for working as proton pump by using light energy but E. coli does not have a pathway to synthesize it. In order to give E. coli a ability to synthesize retinal, we express five foreign genes expressing enzymes for retinal synthesize pathway. By introducing light accepting system into E. coli expressing [NiFe]-hydrogenase, we could get enhanced biohydrogen productivity.

One of the key issues in bioethanol production is a negative effect of ethanol on cell growth and ethanol production. In this study, the genes conferring ethanol tolerance on Sacchaomyces cerevisiae were identified through the yeast genomic library screening, and then the selected genes were used for strain development. By the screening of the S. cerevisiae S288C overexpression genomic library in a medium with 8% ethanol, the strains harboring each of PAU15, CMD1 and CIN5 genes were selected based on rapid growth rate relative to the control strain. In fed-batch fermentation in a media with 100 g/L glucose, however, overexpression of PAU15, CMD1, CIN5 did not improve ethanol production. To verify the reason for such inconsistency of the batch fermentation in 8% ethanol stress and the fed-batch fermentation, real-time PCR was performed. The transcriptional levels of the PAU15, CMD1 and CIN5 genes sharply increased during the early exponential phase, and then decreased in the stationary phase. The result implies that the PAU15, CMD1 and CIN5 gene products are not involved in the cell activity recovery in the gradual ethanol stress but effective in abrupt ethanol stress.

The objective of this research is to enhance the hydrogen productivity in fermentative hydrogen producing bacterium; E. asburiae SNU-1 by genetic manipulation. In certain formate concentration and pH, formate hydrogen lyase (FHL) decomposes formate to hydrogen and CO2. This FHL is controlled by fhlA transcription activator and hycA repressor. Consequently, to increase the FHL, the aim is the overexpression of fhlA gene and deletion of hycA gene. From results of SDS-PAGE and Western-blot, we confirmed the fhlA overexpression in E. asburiae SNU-1. hycA gene was disrupted by Red/ET recombination and it was confirmed by antibiotics resistance tests and colony PCR where several primer combinations were used. We tested the hydrogen productivity in PYG growth medium and in formate production medium using ΔhycA and fhlA overexpressed E. asburiae SNU-1, and found that fhlA overexpression has specific hydrogen production enhancing effect by 36.89% and 56.28%, respectively. But unfortunately ΔhycA shows reduction in hydrogen productivity. These results show that the overexpression of fhlA has hydrogen production enhancing effect and hence anticipate its use in hydrogen production systems.

Tautomycetin (TMC), which is produced by Streptomyces sp. CK4412, is a novel activated T cell-specific immunosuppressive and anti-cancer compound with an ester bond linkage between a terminal cyclic anhydride moiety and a linear polyketide chain bearing an unusual terminal alkene. Especially TMC needed 100-fold lower concentrations than cyclosporine A as immunosuppressant. Previously, we isolated and characterized the entire TMC biosynthetic gene cluster from Streptomyces sp. CK4412. Within the TMC biosynthetic gene cluster, we found and identified two putative hydroxylase genes, tmcG and tmcR, through in silico sequence comparison. TmcG was proposed as function of 3'-carbon hydroxyase, and tmcR was expected as 5-carbon oxygenase which modified TMC structure. Here we disrupted and over expressed tmcG, tmcR to understand each genes function. Moreover we constructed double knock-out mutant such as ΔtmcGΔtmcR. We found that those mutants produced novel TMC-like compounds through HPLC analysis. These modified compounds were shown different antifungal and protein phosphatase activity. The detailed result will be further discussed.

In previous study, we successfully utilized Streptomyces interspecies DNA microarray analysis system to identify novel regulatory genes associated with secondary metabolite overproduction in S. peucetius Doxorubicin (DXR) overproducing industrial strain [1]. Especially wblA, a whiB-like putative transcription factor gene, and SCO1712, a tetR-family transcriptional regulatory gene, were identified as strong antibiotic biosynthetic down-regulators in both S. coelicolor as well as S. peucetius species. Moreover, these two strong antibiotic down-regulators were specifically deleted in the S. coelicolor wild-type chromosome, resulting S. coelicolor △wblA△SCO1712 double knock-out (KO) mutant which shown significantly higher actinorhodin (Act) productions [2]. To further increase our understanding about the genetic nature of Streptomyces antibiotic overproduction, a comparative transcriptome analysis was conducted again using S. coelicolor whole genome chip between S. coelicolor wild-type and a S. coelicolor △wblA△SCO1712 double KO mutant. As a result, the double KO strain was found to express the Act biosynthetic cluster genes stronger than the wild-type. Also, various gene expression signatures were obtained by deletion of wblA and SCO1712. 875 genes showed a two-fold or greater change in expression at two or more time points and 14 genes showed non-change in expression pattern at most of time points. First, in case of putative wblA &SCO1712 dependent genes, expression pattern was confirmed in wild-type and double KO mutant using RT-PCR. And these genes were individually over-expression into S. coelicolor to verity the biological functions of these putative targets, so we selected some putative effective antibiotics-relating regulators in M145 wild-type. Second, SCO5426 relating regulation of carbon flux was found among wblA & SCO1712 independent genes [3]. So SCO5426 was disrupted in S. coelicolor △wblA△SCO1712 double KO mutant additionally and Act productivity was increased than double KO mutant. This additional transcriptomics-driven approach in a double KO mutant led to the identification of previously-unidentified novel wblA &SCO1712 dependent or independent regulatory genes related to secondary metabolite overproduction.

Polyene macrolides are mostly produced by Streptomycetes and some other soil bacteria and exert their fungicidal action via interaction with membrane sterols, resulting in the formation of highly organized hydrophilic channels, through which small molecules and ions can leak out. Using the genomics-guided polyene screening method, a rare actinomycetes called Pseudonocardia autotrophica was previously identified to contain a functionally-clustered nystatin-like biosynthetic genes and to produce a presumably novel polyene compound named NPP. Many previous researches revealed that modification of the polyene structure through engineering of its biosynthetic genes is a promising strategy for the production of novel polyene derivatives suitable for potential pharmaceuticals. In our research, we have obtained new derivatives by engineering of genes related with both early and late stages of NPP biosynthetic pathways. This biosynthetic modification approach is already yielding sufficient amount of novel materials for testing the toxicity and bioactivity, thus opening possibilities for discovery of drug-leads for development of effective and safe pharmaceuticals.

Metabolomics require the gain of reproducible, robust, 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 robust experimental procedure, extraction of the metabolome to quantitative analysis of the metabolites is required. E. coli was grown in chemostat culture so that cellular metabolism could be held in reproducible, steady-state conditions under a range of precisely defined growth conditions, thus enabling sufficient replication of samples. This investigation indicates that 60% cold (-40°C) methanol solution is the most appropriate method to quench metabolism. In this study, metabolites were extracted from E. coli using six different commonly used procedures including acid or alkaline treatments, high-temperature extraction in the presence of ethanol or methanol, lysis with chloroform-methanol, and extraction by hot water. 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 an ion trap mass spectrometer equipped with a turbo-ion spray source.

Phosphomannomutase (ManB), whose main function is the conversion of mannose-6-phosphate to mannose-1-phosphate, is involved in biosynthesis of GDP-mannose for numerous processes such as synthesis of structural carbohydrates, production of alginates and ascorbic acid, and post-translational modification of proteins in prokaryotes and eukaryotes. ManB from Streptomyces coelicolor was shown to have both phosphomannomutase and phosphoglucomutase activities. Deletion of manB in S. coelicolor caused a dramatic increase in actinorhodin (ACT) production in the low-glucose Difco nutrient (DN) medium and slight decrease of sporulation in complex medium (R5-). Experiments involving complementation of the manB deletion showed that increased ACT production in DN media was due to blockage of phosphomannomutase activity rather than phosphoglucomutase activity. These results provide useful information for the role of phosphomannomutase in S. coelicolor on antibiotic production and sporulation.

To examine the expression of bovine growth hormone (BGH) gene in Pleurotus eryngii, which belongs to the family of oyster myshrooms. Growth hormone is responsible for a variety of regulatory effects on growth and various metabolic processes including nitrogen, lipid, mineral, and carbohydrate metabolism. The bGH gene was amplified using the designed primers and subcloned into binary expression vector pPEV, which contains the CaMV 35S promoter and nopaline synthase (nos) terminator. Recombinant expression vector (pPEVbGH) was introduced in P. eryngii via Agrobacterium tumefaciens-mediated transformation. Transformed mycelium was confirmed by PCR amplification and sequencing. The mycelium harboring pPEVbGH was grown at 25℃ for 7 days in MCM (mushroom complete medium) containing 0.2% bactotryptone, 0.2% yeast extract, 2% glucose, 0.05% MgSO4, 0.1% K2HPO4, and 0.046% KH2PO4, and then the expression levels of bGH protein were analyzed by SDS-PAGE and western blot. The bGH protein was produced to considerable levels in the range of 26.3% to 27.2% of total mycelial proteins and MW of the bGH was approximately 21 kDa as expected. Futhermore, bGH protein was immunologically detected by western blot using polyclonal anti-bGH antibody. Interestingly, transformed mycelium of P. eringii showed significant increase in mycelial growth rate (approximately 2.5 fold than the control). Finally, this fungal expression system was also thought to be effective for expression and production of functional proteins.

The entire biosynthetic gene cluster for Tautomycetin (TMC), a potent T-cell immunosuppressive compound that are superior to cyclosporine A both in vitro and in vivo. TMC produced by soil microorganism Streptomyces sp. CK4412 has earlier been investigated, cloned and sequenced[1]. TMC has a unique structure, containing an ester bond linkage between a terminal cyclic anhydride moiety and a linear polyketide chain bearing an unusual terminal alkene. Here we propose two putative genes which participate a pathway to synthesize both dialkylmaleic anhydride moiety and terminal alkene as dehydratase enzyme. Within the TMC biosynthetic gene cluster, we found two putative dehydratase enzyme tmcM and tmcQ. A 1.5Kb gene tmcM and 1.4kb gene tmcQ both are located downstream of the PKS tmcB gene. In silico database comparisons revealed that TmcM function as L-carnitine dehydratase and another putative dehydratase enzyme TmcQ is a member of the MmgE/PrpD protein family. New TMC analogues characterized from inactivation of two genes, tmcM and tmcQ in Streptomyces sp. CK4412, resulting from this it constructed mutant strains △tmcM and △tmcQ respectively. The importance of the function by TmcM and TmcQ will be further studied.