Novel functional elements in the genome of E. coli
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S528 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 [P-S.45] A cell-free protein synthesis system derived from the extract of Streptomyces venezuelae KY Lee ∗ , YC Kwon, KH Lee, JY Byun, HN Kim, DM Kim Chungnam National University, Republic of Korea Keywords: Cell-free protein synthesis system; Streptomyces venezuelae; Glycosyltransferase; metagenomic protein expression Cell-free protein synthesis technology provides an excellent platform for rapid generation and analysis of proteins. Most of the bacterial cell-free protein synthesis systems currently being used are prepared from the cell extract of E.coli, mainly due to relatively simple procedures and high yield of protein production. However, as in the case of in vivo expression, many of the heterologous pro- teins expressed in the E.coli cell-free synthesis system often form insoluble aggregates. In the present study, we have developed a cell-free protein synthesis system derived from the extract of Strep- tomyces venezuelae. When 5 different glycosyltransferases were examined as model proteins, the proteins expressed in the extracts from Streptomyces showed substantially higher solubility of the expressed proteins than in the E.coli system. We expect that the cell-free protein synthesis systems derived from non-E.coli strains would expand the option for cell-free expression of functional pro- teins, including those from various metagenomes. doi:10.1016/j.jbiotec.2010.09.852 [P-S.46] Screening of optimal ribosome binding sites (RBSs) for high- level protein expression in a cell-free protein synthesis system derived from Streptomyces venezuelae JY Park ∗ , YC Kwon, KH Lee, HN Kim, KY Lee, DM Kim Chungnam National University, Republic of Korea Keywords: Cell-free protein synthesis system; Streptomyces venezuelae; Randomize; Ribosome binding sites (RBSs) In recent years, cell-free protein synthesis system has drawn a renewed attention as a powerful tool for deciphering genetic information. Most of the present cell-free protein synthesis sys- tems are derived from the extract of E.coli cells. Similar to the case of in vivo expression of heterologous proteins in E.coli cells, cell- free synthesis systems prepared from E.coli cells often suffer poor solubility/activity of the expressed proteins, which rationalize the development of cell-free protein synthesis system from various origins other than E.coli. Streptomyces strains are well conceived as the source of numerous enzymes of industrial implications. Therefore, a cell-free protein synthesis system derived from Strep- tomyces will be useful for exploring the genetic resources of the same species. In this study, we attempted to engineer the RBS for optimal expression of target proteins in a cell-free protein synthesis system derived from Streptomyces venezuelae. In the experiments using EGFP as a reporter protein, the 5 -UTR sequences were randomized and the relative fluorescence intensity of the cell-free synthesized EGFP was measured to determine the relative efficiency of transla- tion. Through the expression analysis of several hundreds of variant genes, we were able to select a number of RBS sequences that led to substantially higher yield of downstream structural genes. doi:10.1016/j.jbiotec.2010.09.853 [P-S.47] Towards a yeast cell cycle hybrid model: global transcriptional response analysis to the dosage of the FAR1 gene encoding apro- tein involed in setting the cells size at S phase initiation Laura Gotti ∗ , Stefano Busti, Giovanni Felici, Lilia Alberghina, Paola Bertolazzi, Marco Vanoni Dept. of Biotechnology and Bioscience, University of Milano- Bicocca, Italy 2 Istituto di Analisi dei Sistemi ed Informatica “A Ruberti” IASI-CNR Roma, Italy Keywords: FAR1 gene; Cln3; G1/S transition The FAR1 gene encodes an 830 residue bifunctional protein, whose major function is inibtion of cyclin-dependent kinase complexes involved in the G1/S transition. FAR1 transcription is maximal between mitosis and early G1 phase. Enhanced FAR1 tran- scription is necessary but not sufficient for the pheromone-induced G1 arrest, since FAR1 overexpression itself does not trigger cell cycle arrest. Besides its well established role in the response to pheromone, recent evidences suggest that Far1 may also regulate the mitotic cell cycle progression: in particular, it has been proposed that Far1, together with the G1 cyclin Cln3, may be part of a cell sizer mecha- nism that controls the entry into S phase. Far1 is an unstable protein throughout the cell cycle except during G1 phase. Far1 levels peak in newborn cells as a consequence of a burst of synthetic activity at the end of the previous cycle, and the amounts per cell remains roughly constant during the G1 phase. Phosphorylation (at serine 87) by Cdk1-Cln complexes primes Far1 for ubiquitin-mediated proteolysis. By coupling a genome-wide transcriptional analysis of FAR1- overexpressing and far1 cells grown in ethanol- or glucose- supplemented minimal media with a range of phenotypic analysis, we show that FAR1 overexpression not only coordinately increases RNA and protein accumulation, but induces strong transcriptional remodelling, metabolism being the most affected cellular property, suggesting that the Far1/Cln3 sizer regulates cell growth either directly or indirectly by affecting metabolism and pathways known to modulate ribosome biogenesis. Consistetly FAR1 overexpres- sion affects sensitivity of yeast cells to the Tor-inhibiting drug rapamycin. doi:10.1016/j.jbiotec.2010.09.854 [P-S.48] Novel functional elements in the genome of E. coli K.S. Shavkunov ∗ , M.N. Tutukina, O.N. Ozoline Institute of Cell Biophysics RAS, Russian Federation Keywords: E. coli; transcription regulation; promoter “island” A computer-based annotation of newly sequenced genomes has long been considered as the most informative part of their initial analysis. Improvement of such approaches often allows reveal- ing completely unexpected patterns in the genomes of even the well-studied organisms. Thus, using promoter finder PlatProm we discovered a new type of genomic regions in the most stud- ied model organism - E.coli. These regions have high density of potential promoter-like sites (promoter “islands”) and efficiently interact with RNA polymerase in vivo, but intensity of RNA syn- thesis from these loci appeared to be much less than might be expected. Keeping in mind a possibility of functional interference between overlapping promoter sites, we select promoter “island”
Transcript of Novel functional elements in the genome of E. coli
S528 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
[P-S.45]
A cell-free protein synthesis system derived from the extract ofStreptomyces venezuelae
KY Lee ∗, YC Kwon, KH Lee, JY Byun, HN Kim, DM Kim
Chungnam National University, Republic of KoreaKeywords: Cell-free protein synthesis system; Streptomycesvenezuelae; Glycosyltransferase; metagenomic protein expression
Cell-free protein synthesis technology provides an excellentplatform for rapid generation and analysis of proteins. Most of thebacterial cell-free protein synthesis systems currently being usedare prepared from the cell extract of E.coli, mainly due to relativelysimple procedures and high yield of protein production. However,as in the case of in vivo expression, many of the heterologous pro-teins expressed in the E.coli cell-free synthesis system often forminsoluble aggregates. In the present study, we have developed acell-free protein synthesis system derived from the extract of Strep-tomyces venezuelae. When 5 different glycosyltransferases wereexamined as model proteins, the proteins expressed in the extractsfrom Streptomyces showed substantially higher solubility of theexpressed proteins than in the E.coli system. We expect that thecell-free protein synthesis systems derived from non-E.coli strainswould expand the option for cell-free expression of functional pro-teins, including those from various metagenomes.
doi:10.1016/j.jbiotec.2010.09.852
[P-S.46]
Screening of optimal ribosome binding sites (RBSs) for high-level protein expression in a cell-free protein synthesis systemderived from Streptomyces venezuelae
JY Park ∗, YC Kwon, KH Lee, HN Kim, KY Lee, DM Kim
Chungnam National University, Republic of KoreaKeywords: Cell-free protein synthesis system; Streptomycesvenezuelae; Randomize; Ribosome binding sites (RBSs)
In recent years, cell-free protein synthesis system has drawna renewed attention as a powerful tool for deciphering geneticinformation. Most of the present cell-free protein synthesis sys-tems are derived from the extract of E.coli cells. Similar to the caseof in vivo expression of heterologous proteins in E.coli cells, cell-free synthesis systems prepared from E.coli cells often suffer poorsolubility/activity of the expressed proteins, which rationalize thedevelopment of cell-free protein synthesis system from variousorigins other than E.coli. Streptomyces strains are well conceivedas the source of numerous enzymes of industrial implications.Therefore, a cell-free protein synthesis system derived from Strep-tomyces will be useful for exploring the genetic resources of thesame species.
In this study, we attempted to engineer the RBS for optimalexpression of target proteins in a cell-free protein synthesis systemderived from Streptomyces venezuelae. In the experiments usingEGFP as a reporter protein, the 5′-UTR sequences were randomizedand the relative fluorescence intensity of the cell-free synthesizedEGFP was measured to determine the relative efficiency of transla-tion. Through the expression analysis of several hundreds of variantgenes, we were able to select a number of RBS sequences that ledto substantially higher yield of downstream structural genes.
doi:10.1016/j.jbiotec.2010.09.853
[P-S.47]
Towards a yeast cell cycle hybrid model: global transcriptionalresponse analysis to the dosage of the FAR1 gene encoding apro-tein involed in setting the cells size at S phase initiation
Laura Gotti ∗, Stefano Busti, Giovanni Felici, Lilia Alberghina, PaolaBertolazzi, Marco Vanoni
Dept. of Biotechnology and Bioscience, University of Milano- Bicocca,Italy2 Istituto di Analisi dei Sistemi ed Informatica “A Ruberti” IASI-CNRRoma, ItalyKeywords: FAR1 gene; Cln3; G1/S transition
The FAR1 gene encodes an 830 residue bifunctional protein,whose major function is inibtion of cyclin-dependent kinasecomplexes involved in the G1/S transition. FAR1 transcription ismaximal between mitosis and early G1 phase. Enhanced FAR1 tran-scription is necessary but not sufficient for the pheromone-inducedG1 arrest, since FAR1 overexpression itself does not trigger cell cyclearrest.
Besides its well established role in the response to pheromone,recent evidences suggest that Far1 may also regulate the mitoticcell cycle progression: in particular, it has been proposed that Far1,together with the G1 cyclin Cln3, may be part of a cell sizer mecha-nism that controls the entry into S phase. Far1 is an unstable proteinthroughout the cell cycle except during G1 phase. Far1 levels peakin newborn cells as a consequence of a burst of synthetic activityat the end of the previous cycle, and the amounts per cell remainsroughly constant during the G1 phase. Phosphorylation (at serine87) by Cdk1-Cln complexes primes Far1 for ubiquitin-mediatedproteolysis.
By coupling a genome-wide transcriptional analysis of FAR1-overexpressing and far1� cells grown in ethanol- or glucose-supplemented minimal media with a range of phenotypic analysis,we show that FAR1 overexpression not only coordinately increasesRNA and protein accumulation, but induces strong transcriptionalremodelling, metabolism being the most affected cellular property,suggesting that the Far1/Cln3 sizer regulates cell growth eitherdirectly or indirectly by affecting metabolism and pathways knownto modulate ribosome biogenesis. Consistetly FAR1 overexpres-sion affects sensitivity of yeast cells to the Tor-inhibiting drugrapamycin.
doi:10.1016/j.jbiotec.2010.09.854
[P-S.48]
Novel functional elements in the genome of E. coli
K.S. Shavkunov ∗, M.N. Tutukina, O.N. Ozoline
Institute of Cell Biophysics RAS, Russian FederationKeywords: E. coli; transcription regulation; promoter “island”
A computer-based annotation of newly sequenced genomes haslong been considered as the most informative part of their initialanalysis. Improvement of such approaches often allows reveal-ing completely unexpected patterns in the genomes of even thewell-studied organisms. Thus, using promoter finder PlatPromwe discovered a new type of genomic regions in the most stud-ied model organism - E.coli. These regions have high density ofpotential promoter-like sites (promoter “islands”) and efficientlyinteract with RNA polymerase in vivo, but intensity of RNA syn-thesis from these loci appeared to be much less than might beexpected. Keeping in mind a possibility of functional interferencebetween overlapping promoter sites, we select promoter “island”
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S529
associated with the appY gene for thorough experimental analy-sis.
Three overlapping and one individual DNA fragments, con-taining multiple promoters were used for in vitro and in situexperiments. All of them form open complexes as testified bygel-shift and permanganate footprinting. One of the complexes inintergenic part of DNA fragment apparently corresponds to appYpromoter. Single-round transcription assay confirmed transcrip-tion activity from this promoter in vitro and revealed anotherproducts (∼305 nt) generated from the internal promoter in anti-sense direction. However synthesis of appY-mRNA has not beenregistered by primer extension from the total RNA, thus testifyinglow transcription of appY. Activity of its promoter and processivityof RNA synthesis has been further studied using gfp-expression sys-tem on pET28b-eGFP. The data obtained unambiguously indicatedexclusively high activity of appY promoter per se and low transcrip-tion efficiency within coding sequence of appY gene, thus assumingnew type of expression regulation.
The displayed phenomenon obviously should be taken intoaccount for optimizing prokaryotic metabolism in biotechnology.
doi:10.1016/j.jbiotec.2010.09.855
[P-S.49]
Enhancing myogenic commitment in adult stem cells by Magic-F1 recombinant protein and PCL scaffold
F Ronzoni 2,∗, S Conte 2, D Galli 2, S Duim 1, F D’Angelo 3, SMartino 3
1 Translational Cardiomyology SCIL K.U.Leuven, Belgium2 Department of Experimental Medicine, Section of Human Anatomy,Università di Pavia, Italy3 Department of Experimental Medicine and Biochemical Sciences, Sec-tion of Biochemistry and Molecular Biology, University of Perugia, ItalyKeywords: stem cells; scaffold; skeletal muscle; recombinant pro-tein
Hepatocyte Growth Factor (HGF) is a pleiotropic cytokine ofmesenchymal origin that mediates cell proliferation, survival,motility and morphogenesis. Adult myogenic stem cells (satel-lite cells) express both HGF and Met, its high affinity receptor.Following muscle injury, autocrine HGF-Met stimulation plays akey role in promoting activation and early division of satellitecells. Magic-F1 (Met-Activating Genetically Improved ChimericFactor-1) is an HGF-derived, engineered protein with two Met-binding domains. Magic-F1 protects myogenic precursors againstapoptosis, increasing their fusion ability and enhancing musculardifferentiation (Cassano et al., 2008). Hemizygous and homozygousMagic-F1 transgenic mice displayed constitutive muscular hyper-trophy, improved running performance and accelerated muscleregeneration following injury. Skeletal muscle tissue engineeringrepresents an attractive approach to overcome problems asso-ciated with autologous transfer of muscle tissue and provides avalid alternative in muscle regeneration enhancement. One of thesuitable biomaterial for these studies is polycaprolactone (PCL),a biodegradable polyester processed by electrospinning (Riboldiet al., 2005). This material shows appropriate characteristics forskeletal muscle: it is biocompatible and elastic to accommodatecontractile function. Importantly, PCL is widely used for the pro-duction of implantable prosthesis, such as synthetic ligaments,bone and tendons. PCL scaffold membranes did not show anytoxic residuals and exhibited satisfactory mechanical characteris-tics. Magic-F1 recombinant protein positively affected fusion indexof satellite cells cultivated on PCL scaffolds. Here we show that
satellite cells have an impairment to colonize the PCL scaffolds, butthen robustly proliferate and fuse on PCL scaffolds. The results ofthis study provide significant evidence of the suitability of elec-trospun PCL membranes as scaffolds for skeletal muscle tissueengineering.
This work was supported in part by: the Italian Ministry ofUniversity and Scientific Research (grant n. 2005067555 003);CARIPLO 2009-2012 Rif. 2008.2005.
References
Cassano, et al., 2008. Magic-factor 1, a partial agonist of Met, induces muscle hyper-trophy by protecting myogenic progenitors from apoptosis. PLoS One. 3 (9),e3223.
Riboldi, et al., 2005. Electrospun degradable polyesterurethane membranes: poten-tial scaffolds for skeletal muscle tissue engineering. Biomaterials. 26 (22),4606–4615.
doi:10.1016/j.jbiotec.2010.09.856
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Identification of genetic alterations in the glucoamylaseAspergillus awamori gene
Fabiana Carina Pavezzi ∗, Heloiza Ferreira Alves-Prado, HenriqueFerreira, Paula M.M. Martins, Eleni Gomes, Roberto da Silva
UNESP, BrazilKeywords: thermostable; glucoamylase; hydrolyzes; recombinant
Glucoamylase is an enzyme of great importance to the fer-mentation and food industries for saccharification of starch. Ithydrolyzes �-1,4 and �-1,6 glycosidic bonds starch resulting inthe production of glucose. The main use of enzyme is relatedto the production of glucose. The glucoamylase is produced bya variety of microorganisms, in which Aspergillus and Rhizopushave been considered as being the most important for industrialapplication. Glucoamylase efficiently catalyzes the saccharifica-tion reaction within a relatively narrow range of temperatures,because its catalytically active conformation changes at high tem-peratures. In recent years, there has been a keen interest in theproduction of heterologous proteins by recombinant DNA tech-nology, and numerous approaches have successfully been takento improve the thermostability of glucoamylase by site-direct andrandom mutagenesis. Therefore, the development of a more ther-mostable glucoamylase would greatly contribute to the starchsaccharification process. In this work a thermostable mutant glu-coamylase of Aspergillus awamori was expressed by the yeastSaccharomyces cerevisiae and characterized genetically. The Plas-mid YEpPM18 containing the mutated glucoamylase gene wasisolated from the transformed yeast. Competent Escherichia coliDH 5� was transformed by electroporation with this preparation;then ampicillin-resistent transformants were selected. A singletransformant colony of pPM18/DH5� was inoculated into LB +Ampicillin; plasmid DNA containing the mutant gene was pro-duced overnight and then purified using the Gene JetTM PlasmidMiniprep kit. The glucoamylase gene from the mutant was geneti-cally characterized by DNA sequencing. From the mutant sequence,a total of 4 mutations were identified, all of which were identifiedin the catalytic domain. One mutation was silent, whereas threeresulted in amino acid replacement: Ser54→Pro, Thr314→Ala andHis415→Tyr. These alterations contributed to a significant increasein thermostability when compared to the wild-type enzyme.
Financial support: Fapesp
doi:10.1016/j.jbiotec.2010.09.857
