Melkina O.E.,State Research Institute of Genetics and Selection of Industrial Microorganisms |
Goryanin I.I.,Moscow Institute of Physics and Technology |
Zavilgelsky G.B.,State Research Institute of Genetics and Selection of Industrial Microorganisms
Russian Journal of Genetics | Year: 2017
The effects of histone-like protein H-NS on transcription of promoters of the Quorum Sensing regulated operons from marine luminescent mesophilic bacterium Aliivibrio fischeri and psychrophilic Aliivibrio logei, as well as from pathogenic Pseudomonas aeruginosa, are studied. In the present work, the plasmids carrying DNA fragments with the promoters Pr1f (upstream of the luxICDABEG operon from A. fischeri), Pr1l (upstream of the luxCDABEG operon from A. logei), Pr2l (upstream of luxI gene from A. logei), PluxCf (upstream of luxC gene from A. fischeri), and PlasI (upstream of lasI gene from P. aerugenosa) are used. In these plasmids, promoter-operator regions are transcriptionally fused to the reporter genes cassette luxCDABE from Photorhabdus luminescens. Here we have shown that the transcription of the QS-regulated promoters in E. coli hns::kan cells increases 100 to 1000 times. Furthermore, transcription of the QS-regulated promoters in E. coli hns+ cells increases 10 to 100 times in the cells transformed with the plasmid carrying gene ardA ColIb-P9 encoding DNA mimic antirestriction protein ArdA, inhibitor of the type I restriction-modification systems. © 2017, Pleiades Publishing, Inc.
Naumov G.I.,State Research Institute of Genetics and Selection of Industrial Microorganisms
Microbiology (Russian Federation) | Year: 2013
The review deals with the early studies of Saccharomyces paradoxus (syn. S. cerevisiae var. tetrasporus) yeast. The data demonstrate strong evidence that, in contrast to the well-known cultivated Saccharomyces yeasts (baker, wine, spirits, and beer yeast), wild Saccharomyces yeasts are often found in natural habitats, such as exudate and leaf litter of trees, decaying wood, soil, and insect intestines. These yeasts form a potentially valuable gene pool for research and breeding programs. © 2013 Pleiades Publishing, Ltd.
Savrasova E.A.,Ajinomoto Co. |
Kivero A.D.,Ajinomoto Co. |
Shakulov R.S.,State Research Institute of Genetics and Selection of Industrial Microorganisms |
Stoynova N.V.,Ajinomoto Co.
Journal of Industrial Microbiology and Biotechnology | Year: 2011
Microbiological synthesis of higher alcohols (1-butanol, isobutanol, 2-methyl-1-butanol, etc.) from plant biomass is critically important due to their advantages over ethanol as a motor fuel. In recent years, the use of branched-chain amino acid (BCAA) biosynthesis pathways together with heterologous Ehrlich pathway enzyme system (Hazelwood et al. in Appl Environ Microbiol 74:2259-2266, 2008) has been proposed by the Liao group as an alternative approach to aerobic production of higher alcohols as new-generation biofuels (Atsumi et al. in Nature 451:86-90, 2008; Atsumi et al. in Appl Microbiol Biotechnol 85:651-657, 2010; Cann and Liao in Appl Microbiol Biotechnol 81:89-98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769-5775, 2008; Shen and Liao in Metab Eng 10:312-320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471-479, 2009). On the basis of these remarkable investigations, we re-engineered Escherichia coli valine-producing strain H-81, which possess overexpressed ilvGMED operon, for the aerobic conversion of sugar into isobutanol. To redirect valine biosynthesis to the production of alcohol, we also-as has been demonstrated previously (Atsumi et al. in Nature 451:86-90, 2008; Atsumi et al. in Appl Microbiol Biotechnol 85:651-657, 2010; Cann and Liao in Appl Microbiol Biotechnol 81:89-98, 2008; Connor and Liao in Appl Environ Microbiol 74:5769-5775, 2008; Shen and Liao in Metab Eng 10:312-320, 2008; Yan and Liao in J Ind Microbiol Biotechnol 36:471-479, 2009)-used enzymes of Ehrlich pathway. In particular, in our study, the following heterologous proteins were exploited: branched-chain 2-keto acid decarboxylase (BCKAD) encoded by the kdcA gene from Lactococcus lactis with rare codons substituted, and alcohol dehydrogenase (ADH) encoded by the ADH2 gene from Saccharomyces cerevisiae. We show that expression of both of these genes in the valine-producing strain H-81 results in accumulation of isobutanol instead of valine. Expression of BCKAD alone also resulted in isobutanol accumulation in the culture broth, supporting earlier obtained data (Atsumi et al. in Appl Microbiol Biotechnol 85:651-657, 2010) that native ADHs of E. coli are also capable of isobutanol production. Thus, in this work, isobutanol synthesis by E. coli was achieved using enzymes similar to but somewhat different from those previously used. © 2010 Society for Industrial Microbiology.
Naumov G.I.,State Research Institute of Genetics and Selection of Industrial Microorganisms |
Naumova E.S.,State Research Institute of Genetics and Selection of Industrial Microorganisms
Russian Journal of Genetics | Year: 2010
Molecular and genetic analyses revealed that the distillers race XII, which is an ancestor of Saccharomyces cerevisiae Peterhof and Gatchina genetic lines, has three polymeric β-fructosidase genes: SUC2, SUC5, and SUC8. The latter gene located on the X chromosome was identitied in this work for the first time. The presence of the single SUC2 gene in yeasts used in the international project on sequencing of the S. cerevisiae genome is discussed. © Pleiades Publishing, Ltd 2010.
Debabov V.G.,State Research Institute of Genetics and Selection of Industrial Microorganisms
Applied Biochemistry and Microbiology | Year: 2015
In this review, existing succinic acid manufacturing methods are reviewed. The economic status and the prospects for chemical and microbiological means of biosuccinic acid production are also presented. Possible approaches to lower production costs via improvements in known technologies, as well as the designing of new methods, are considered. The prospects for developing biosuccinic acid markets are discussed. © 2015, Pleiades Publishing, Inc.
Gordeeva T.L.,State Research Institute of Genetics and Selection of Industrial Microorganisms |
Borschevskaya L.N.,State Research Institute of Genetics and Selection of Industrial Microorganisms |
Sineoky S.P.,State Research Institute of Genetics and Selection of Industrial Microorganisms
Journal of Microbiological Methods | Year: 2010
Gene synthesis technologies provide a powerful tool for increasing protein expression through codon optimization and gene modification. Here we describe an improved PCR-based gene synthesis technology, which is accurate, simple and cheap. The improved PCR-based gene synthesis (IPS) method consists of two steps. The first one is the synthesis of 300-400. bp fragments by PCR reaction with Pfu DNA polymerase from 60-mer and 30-mer oligonucleotides with a 15. bp overlap. The second one is assembling of fragments from the first step into the full-length gene by PCR reaction. Using this approach, we have successfully synthesized a modified phytase gene with 1256. bp in length with optimal codons for expression in Pichia pastoris. P. pastoris strain that expressed the modified phytase gene (phyA-mod) showed a 50% increase in phytase activity level. In addition, we propose an inexpensive method for error correction, based on overlap-extension PCR (OE-PCR). © 2010 Elsevier B.V.
Sklyarova S.A.,State Research Institute of Genetics and Selection of Industrial Microorganisms |
Mironov A.S.,State Research Institute of Genetics and Selection of Industrial Microorganisms
Russian Journal of Genetics | Year: 2014
We studied the regulation of the Bacillus subtilis ypaA gene-encoding riboflavin-transporter by FMN riboswitch. Using translational fusions of the leader region of wild-type ypaA gene with the lacZ-reporter gene in the leader region we showed that in vivo ypaA gene expression decreased more than 10-fold in the presence of endogenous FMN. Introduction of two nucleotide substitutions providing stabilization of the sequester hairpins results in almost complete repression of reporter gene expression. Using toeprint assay in vitro it has been shown that FMN presence inhibits the formation of the 30S initiation complexint the ypaA gene leader mRNA. Our results support the model of ypaA gene regulation whereby FMN binding with the ypaA gene leader sequence results in translation suppression through the sequestering of the SD-sequence. © 2014 Pleiades Publishing, Inc.
Shatalin K.,New York University |
Shatalina E.,New York University |
Mironov A.,State Research Institute of Genetics and Selection of Industrial Microorganisms |
Nudler E.,New York University
Science | Year: 2011
Many prokaryotic species generate hydrogen sulfide (H2S) in their natural environments. However, the biochemistry and physiological role of this gas in nonsulfur bacteria remain largely unknown. Here we demonstrate that inactivation of putative cystathionine β-synthase, cystathionine γ-lyase, or 3-mercaptopyruvate sulfurtransferase in Bacillus anthracis, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli suppresses H2S production, rendering these pathogens highly sensitive to a multitude of antibiotics. Exogenous H2S suppresses this effect. Moreover, in bacteria that normally produce H2S and nitric oxide, these two gases act synergistically to sustain growth. The mechanism of gas-mediated antibiotic resistance relies on mitigation of oxidative stress imposed by antibiotics.
Debabov V.G.,State Research Institute of Genetics and Selection of Industrial Microorganisms
Russian Journal of Genetics | Year: 2015
Microorganism producer strains are the basis of industrial biotechnology. Their properties determine the economical parameters of the production. Methods of rational design (metabolic engineering) and combinatorial methods of mutagenesis and selection (laboratory evolution, adaptive evolution, protein and genomic shuffling) are used for the construction of microorganism strains. Combination of these methods is frequently used. Modern strains usually do not contain plasmids and markers of drug resistance. All changes are introduced into the chromosome by the methods of homologous and site-specific recombination. The sum of such approaches is called recombineering. Gene expression is carried out at the optimal level under the control of promoters of a certain power (frequently regulated). Knowledge of a complete genomic sequence is almost a mandatory condition for the use of methods of metabolic engineering. Bioinformatics significantly assists in the selection of enzymes and the search for necessary genes and metabolic reactions. Measurement of metabolic fluxes largely assists in the construction of strains. The current level of science makes it possible to construct metabolic pathways de novo in strains for the production of chemicals and biofuel. Carbon dioxide has potential as a raw material for microbiological industry; therefore, the study of CO2 fixation by acetogens and electrogens is a promising direction of studies. © 2015, Pleiades Publishing, Inc.
Berezina O.V.,State Research Institute of Genetics and Selection of Industrial Microorganisms |
Zakharova N.V.,State Research Institute of Genetics and Selection of Industrial Microorganisms |
Brandt A.,TU Munich |
Yarotsky S.V.,State Research Institute of Genetics and Selection of Industrial Microorganisms |
And 3 more authors.
Applied Microbiology and Biotechnology | Year: 2010
A Lactobacillus brevis strain with the ability to synthesize butanol from glucose was constructed by metabolic engineering. The genes crt, bcd, etfB, etfA, and hbd, composing the bcs-operon, and the thl gene encode the enzymes of the lower part of the clostridial butanol pathway (crotonase, butyryl-CoA-dehydrogenase, two subunits of the electron transfer flavoprotein, 3-hydroxybutyryl-CoA dehy-drogenase, and thiolase) of Clostridium acetobutylicum. They were cloned into the Gram-positive/Gram-negative shuttle plasmid vector pHYc. The two resulting plasmids pHYc-thl-bcs and pHYc-bcs (respectively, with and without the clostridial thl gene) were transferred to Escherichia coli and L. brevis. The recombinant L. brevis strains were able to synthesize up to 300 mg l-1 or 4.1 mM of butanol on a glucose-containing medium. A L. brevis strain carrying the clostridial bcs-operon has the ability to synthesize butanol with participation of its own thiolase, aldehyde dehydrogenase, and alcohol dehydrogenase. The particular role of the enzymes involved in butanol production and the suitability of L. brevis as an n-butanol producer are discussed. © Springer-Verlag 2010.