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Gulevich A.Y.,Research Institute for Genetics and Selection of Industrial Microorganisms | Skorokhodova A.Y.,Research Institute for Genetics and Selection of Industrial Microorganisms | Sukhozhenko A.V.,Research Institute for Genetics and Selection of Industrial Microorganisms | Debabov V.G.,Research Institute for Genetics and Selection of Industrial Microorganisms
Journal of Biotechnology | Year: 2017

Enantiomers of 3-hydroxybutyric acid (3-HB) can be used as the chiral precursors for the production of various optically active fine chemicals, including drugs, perfumes, and pheromones. In this study, Escherichia coli was engineered to produce (S)-3-HB from glucose through the inverted reactions of the native aerobic fatty acid β-oxidation pathway. Expression of only specific genes encoding enzymes responsible for the conversion of acetyl-CoA to acetoacetyl-CoA, reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA and subsequent hydrolysis of 3-hydroxybutyryl-CoA to 3-HB was directly upregulated in an engineered strain. The operation of multiple turns of the inverted fatty acid β-oxidation was precluded by the deletion of gene encoding enzyme that catalyse the terminal stage of the respective cycle. While the overexpression of the C-acetyltransferase gene enabled 3-HB biosynthesis through the inverted fatty acid β-oxidation, the efficient conversion of glucose to the target product was achieved resulting from the additional overexpression of the gene encoding appropriate termination thioesterase II. The engineered strain synthesised the (S)-stereoisomer of 3-HB with an enantiomeric excess of more than 99%. Under microaerobic conditions, up to 9.58 g/L of enantiopure (S)-3-HB was produced from glucose, with a yield of 66% of the theoretical maximum. © 2017 Elsevier B.V.


Fertig E.J.,Johns Hopkins University | Ding J.,Dana-Farber Cancer Institute | Favorov A.V.,Johns Hopkins University | Favorov A.V.,Research Institute for Genetics and Selection of Industrial Microorganisms | And 2 more authors.
Bioinformatics | Year: 2010

Summary: Coordinated Gene Activity in Pattern Sets (CoGAPS) provides an integrated package for isolating gene expression driven by a biological process, enhancing inference of biological processes from transcriptomic data. CoGAPS improves on other enrichment measurement methods by combining a Markov chain Monte Carlo (MCMC) matrix factorization algorithm (GAPS) with a threshold-independent statistic inferring activity on gene sets. The software is provided as open source C++ code built on top of JAGS software with an R interface. © The Author 2010. Published by Oxford University Press. All rights reserved.


Kulakovskiy I.V.,RAS Engelhardt Institute of Molecular Biology | Boeva V.A.,Research Institute for Genetics and Selection of Industrial Microorganisms | Boeva V.A.,French Institute of Health and Medical Research | Boeva V.A.,MINES ParisTech | And 2 more authors.
Bioinformatics | Year: 2010

Summary: ChIP-Seq data are a new challenge for motif discovery. Such a data typically consists of thousands of DNA segments with base-specific coverage values. We present a new version of our DNA motif discovery software ChIPMunk adapted for ChIP-Seq data. ChIPMunk is an iterative algorithm that combines greedy optimization with bootstrapping and uses coverage profiles as motif positional preferences. ChIPMunk does not require truncation of long DNA segments and it is practical for processing up to tens of thousands of data sequences. Comparison with traditional (MEME) or ChIP-Seq-oriented (HMS) motif discovery tools shows that ChIPMunk identifies the correct motifs with the same or better quality but works dramatically faster. © The Author 2010. Published by Oxford University Press. All rights reserved.


Gulevich A.Y.,Research Institute for Genetics and Selection of Industrial Microorganisms | Skorokhodova A.Y.,Research Institute for Genetics and Selection of Industrial Microorganisms | Sukhozhenko A.V.,Research Institute for Genetics and Selection of Industrial Microorganisms | Shakulov R.S.,Research Institute for Genetics and Selection of Industrial Microorganisms | Debabov V.G.,Research Institute for Genetics and Selection of Industrial Microorganisms
Biotechnology Letters | Year: 2012

The basic reactions of the clostridial 1-butanol biosynthesis pathway can be regarded to be the inverted reactions of the fatty acid β-oxidation pathway. A pathway for the biosynthesis of fuels and chemicals was recently engineered by combining enzymes from both aerobic and anaerobic fatty acid β-oxidation as well as enzymes from other metabolic pathways. In the current study, we demonstrate the inversion of the entire aerobic fatty acid β-oxidation cycle for 1-butanol biosynthesis. The constructed markerless and plasmidless Escherichia coli strain BOX-3 (MG1655 lacI QattB-P trc-ideal-4-SD φ10-adhE(Glu568Lys) attB-P trc-ideal-4-SD φ10-atoB attB-P trc-ideal-4-SD φ10-fadB attB-P trc-ideal-4-SD φ10-fadE) synthesises 0. 3-1 mg 1-butanol/l in the presence of the specific inducer. No 1-butanol production was detected in the absence of the inducer. © 2011 Springer Science+Business Media B.V.


Medvedeva Y.A.,Research Institute for Genetics and Selection of Industrial Microorganisms | Fridman M.V.,Research Institute for Genetics and Selection of Industrial Microorganisms | Oparina N.J.,RAS Engelhardt Institute of Molecular Biology | Malko D.B.,Research Institute for Genetics and Selection of Industrial Microorganisms | And 4 more authors.
BMC Genomics | Year: 2010

Background: Recently, it has been discovered that the human genome contains many transcription start sites for non-coding RNA. Regulatory regions related to transcription of this non-coding RNAs are poorly studied. Some of these regulatory regions may be associated with CpG islands located far from transcription start-sites of any protein coding gene. The human genome contains many such CpG islands; however, until now their properties were not systematically studied.Results: We studied CpG islands located in different regions of the human genome using methods of bioinformatics and comparative genomics. We have observed that CpG islands have a preference to overlap with exons, including exons located far from transcription start site, but usually extend well into introns. Synonymous substitution rate of CpG-containing codons becomes substantially reduced in regions where CpG islands overlap with protein-coding exons, even if they are located far downstream from transcription start site. CAGE tag analysis displayed frequent transcription start sites in all CpG islands, including those found far from transcription start sites of protein coding genes. Computational prediction and analysis of published ChIP-chip data revealed that CpG islands contain an increased number of sites recognized by Sp1 protein. CpG islands containing more CAGE tags usually also contain more Sp1 binding sites. This is especially relevant for CpG islands located in 3' gene regions. Various examples of transcription, confirmed by mRNAs or ESTs, but with no evidence of protein coding genes, were found in CAGE-enriched CpG islands located far from transcription start site of any known protein coding gene.Conclusions: CpG islands located far from transcription start sites of protein coding genes have transcription initiation activity and display Sp1 binding properties. In exons, overlapping with these islands, the synonymous substitution rate of CpG containing codons is decreased. This suggests that these CpG islands are involved in transcription initiation, possibly of some non-coding RNAs. © 2010 Medvedeva et al; licensee BioMed Central Ltd.


Skorokhodova A.Y.,Research Institute for Genetics and Selection of Industrial Microorganisms | Gulevich A.Y.,Research Institute for Genetics and Selection of Industrial Microorganisms | Morzhakova A.A.,Research Institute for Genetics and Selection of Industrial Microorganisms | Shakulov R.S.,Research Institute for Genetics and Selection of Industrial Microorganisms | Debabov V.G.,Research Institute for Genetics and Selection of Industrial Microorganisms
Biotechnology Letters | Year: 2013

Two different approaches to activate the glyoxylate bypass in model Escherichia coli K-12 strains for succinate biosynthesis during dual-phase fermentation in minimal glucose media were examined. Inactivation of IclR and FadR, the transcriptional regulators of the aceBAK operon, were insufficient for the involvement of the glyoxylate bypass in anaerobic succinate biosynthesis by strains grown aerobically under glucose-abundant conditions. In contrast, the strains that constitutively expressed the aceEF-lpdA operon coding for the pyruvate dehydrogenase complex could partially synthesise succinate anaerobically via the glyoxylate bypass, even in the presence of intact regulators. The results suggest that the intensive acetyl-CoA formation in the strains constitutively expressing pyruvate dehydrogenase matches the physiological conditions that favour the activation of the glyoxylate bypass. © 2012 Springer Science+Business Media Dordrecht.


Lavrov K.V.,Research Institute for Genetics and Selection of Industrial Microorganisms | Yanenko A.S.,Research Institute for Genetics and Selection of Industrial Microorganisms
Russian Journal of Genetics | Year: 2013

The gene for new Rhodococcus erythropolis TA37 acylamidase, which possesses unique substrate specificity, has been cloned and expressed in E. coli. Substrates for this enzyme are not only simple amides, such as acetamide and propionamide, but also N-substituted amides, such as 4′-nitroacetanilide. The 1431-bp gene was expressed in E. coli BL21 (DE3) cells on pET16b plasmid under the control of a promoter of the φ{symbol} 10 gene from the T7 phage. The molecular mass of recombinant acylamidase in E. coli was 55 kDa, which corresponded to that of native acylamidase from Rhodococcus erythropolis TA37. Recombinant acylamidase was able to hydrolize N-substituted amides. A search of a nucleotide database and multiple alignment revealed that acylamidase belonged to the Amidase protein family PF01425, but its nucleotide and amino acid sequences differed significantly from those of the described amidases. © 2013 Pleiades Publishing, Inc.


Kurochkina V.B.,Research Institute for Genetics and Selection of Industrial Microorganisms | Sklyarenko A.V.,Research Institute for Genetics and Selection of Industrial Microorganisms | Berezina O.V.,Research Institute for Genetics and Selection of Industrial Microorganisms | Yarotskii S.V.,Research Institute for Genetics and Selection of Industrial Microorganisms
Applied Biochemistry and Microbiology | Year: 2013

The review describes two major groups of α-amino acid ester hydrolases (AEHs)-enzymes with a similar active center structure, which determines their unique specificity to esters containing an amino group in the α position to the carbonyl. The first group comprises microbial AEHs of the β-lactam acylase type. Technical biocatalysts based on this group of enzymes are used for the production of semi-synthetic amino-β-lactam antibiotics. The second AEH group includes eukaryotic valacyclovirases, which activate in vivo a number of antiviral and anticancer prodrugs. The directed activity of these enzymes is used for the development of target pharmaceutical preparations for the therapy of viral and oncological diseases. The review summarizes and compares the available data on the structure and properties, substrate specificity, and the kinetic parameters of enzymes of these two groups. Experiments identifying the AEH active site and providing the molecular basis for the unique specificity of these enzymes are discussed. The data from the available scientific and patent publications concerning the aminopenicillin and aminocephalosporin synthesis catalyzed by β-lactam acylase AEHs are reviewed and systematized. © 2013 Pleiades Publishing, Inc.


Kulakovskiy I.V.,RAS Engelhardt Institute of Molecular Biology | Belostotsky A.A.,Research Institute for Genetics and Selection of Industrial Microorganisms | Kasianov A.S.,RAS Engelhardt Institute of Molecular Biology | Esipova N.G.,RAS Engelhardt Institute of Molecular Biology | And 3 more authors.
Bioinformatics | Year: 2011

Motivation: Modern experimental methods provide substantial information on protein-DNA recognition. Studying arrangements of transcription factor binding sites (TFBSs) of interacting transcription factors (TFs) advances understanding of the transcription regulatory code. Results: We constructed binding motifs for TFs forming a complex with HIF-1α at the erythropoietin 3'-enhancer. Corresponding TFBSs were predicted in the segments around transcription start sites (TSSs) of all human genes. Using the genome-wide set of regulatory regions, we observed several strongly preferred distances between hypoxia-responsive element (HRE) and binding sites of a particular cofactor protein. The set of preferred distances was called as a preferred pair distance template (PPDT). PPDT dramatically depended on the TF and orientation of its binding sites relative to HRE. PPDT evaluated from the genome-wide set of regulatory sequences was used to detect significant PPDT-consistent binding site pairs in regulatory regions of hypoxia-responsive genes. We believe PPDT can help to reveal the layout of eukaryotic regulatory segments. © The Author 2011. Published by Oxford University Press. All rights reserved.


PubMed | Research Institute for Genetics and Selection of Industrial Microorganisms
Type: | Journal: Journal of biotechnology | Year: 2017

Enantiomers of 3-hydroxybutyric acid (3-HB) can be used as the chiral precursors for the production of various optically active fine chemicals, including drugs, perfumes, and pheromones. In this study, Escherichia coli was engineered to produce (S)-3-HB from glucose through the inverted reactions of the native aerobic fatty acid -oxidation pathway. Expression of only specific genes encoding enzymes responsible for the conversion of acetyl-CoA to acetoacetyl-CoA, reduction of acetoacetyl-CoA to 3-hydroxybutyryl-CoA and subsequent hydrolysis of 3-hydroxybutyryl-CoA to 3-HB was directly upregulated in an engineered strain. The operation of multiple turns of the inverted fatty acid -oxidation was precluded by the deletion of gene encoding enzyme that catalyse the terminal stage of the respective cycle. While the overexpression of the C-acetyltransferase gene enabled 3-HB biosynthesis through the inverted fatty acid -oxidation, the efficient conversion of glucose to the target product was achieved resulting from the additional overexpression of the gene encoding appropriate termination thioesterase II. The engineered strain synthesised the (S)-stereoisomer of 3-HB with an enantiomeric excess of more than 99%. Under microaerobic conditions, up to 9.58g/L of enantiopure (S)-3-HB was produced from glucose, with a yield of 66% of the theoretical maximum.

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