Selective production of acetone during continuous synthesis gas fermentation by engineered biocatalyst Clostridium sp. MAceT113.

AIMS: To engineer acetogen biocatalyst capable of fermenting synthesis gas blend to acetone as the only liquid carbonaceous product. METHODS AND RESULTS: The metabolic engineering comprised inactivation of phosphotransacetylase via integration of a cassette comprising synthetic genes erm(B), thiolase and HMG-CoA synthase. Acetaldehyde dehydrogenase was inactivated via integration of a cassette consisting of synthetic genes cat, HMG-CoA lyase and acetoacetate decarboxylase. The engineered biocatalyst Clostridum sp. MAceT113 lost production of 253 mmol l(-1) ethanol and 296 mmol l(-1) acetate and started producing 1.8 mol l(-1) acetone in single-stage continuous syngas fermentation. CONCLUSIONS: The acetone concentration in culture broth is economical for bulk manufacture because it is about twenty times of that achieved with known acetone-butanol-ethanol fermentation of sugars. SIGNIFICANCE AND IMPACT OF THE STUDY: The process shows the opportunity to produce acetone from synthesis gas at concentrations comparable with production of acetone from products of petroleum cracking. This is the first report on elimination of acetate and acetaldehyde production and directing carbon flux from Acetyl-CoA to acetone via a non-naturally occurring in acetogen acetone biosynthesis pathway identified in eukaryotic organisms.

[The preparative method for 2-fluoroadenosine synthesis].

The preparative method for the synthesis of 2-fluoroadenosine starting from commercially available guanosine was developed. It included the intermediate formation of 2-amino-6-azido-9-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)purine, which was isolated exclusively in the tetrazolo[5,1-i]-form {5-amino-7-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)-7H-tetrazolo[5,1-i]purine}. The latter compound was converted by the Schiemann reaction to 6-azido-2-fluoro-9-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)purine, which was isolated at an 80% yield after careful optimization of the process. The IR and 1H NMR spectroscopy data indicated the 6-azido-2-fluoropurine structure of the aglycone. The catalytic reduction of the azido group in 6-azido-2-fluoro-9-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)purine to the amino moiety and the subsequent deacetylation by the routine procedure resulted in 2-fluoroadenosine at a total yield of 74%.

[15-19-mer synthetic oligoribonucleotide as a model of the translation initiation segment of the phage fr replicase gene]. / 15-19-zvennye sinteticheskie oligoribonukleotidy kak modeli initsiatornogo uchastka transliatsii gena replikazy faga fr.

Automatic solid-phase synthesis of 15-19-meric oligoribonucleotides was carried out using 5'-O-dimethoxytrityl-2'-O-(2-tetrahydropyranyl)- [or 2'-O-(2-tetrahydrofuranyl)-] N-acylribonucleoside-3'-O-(methyl-N, N-diisopropyl)phosphoramidite synthons and 1-H-tetrazole as an activator. Comparative analysis of the template activity of the oligoribonucleotides synthesized, which are models of the translation initiation region of the replicase gene of MS2 and fr phages, showed that the minimal active fragment of RNA is a 16-mer containing the initiation AUG codon of the gene, a short spacer, a Shine-Dalgarno domain, and the 5'-terminal AUGA sequence with a functionally important termination AUG codon.

[The characteristics of the accumulation of metals in the hair of children]. / K voprosu ob osobennostiakh nakopleniia nekotorykh metallov v volosakh detei.

Gender- and colour-associated quantitative features of accumulation of zinc, copper, nickel, lead, molybdenum and chromium have been studied in the hair of children aged 3-4 yr residing in different districts of Kiev. Inverse relationship was found between accumulation of lead and zinc, zinc and copper; direct proportionality between copper and lead, chromium and molibdenum. The levels of nickel detected in 34% of the hair samples did not permit establishing the pattern of this metal relations to other metallic elements.

Complete nucleotide sequence of the group I RNA bacteriophage fr.

We report the complete nucleotide sequence of the group I RNA bacteriophage fr. The entire genome consists of 3575 nucleotides, six nucleotides more than the only other sequenced group I representative, MS2. The greatest divergence between these phages occurs in the 5' terminal region of the A gene, while the lysis-replicase gene overlap, the coat gene and the central region of the replicase gene are highly conserved. Overall sequence homology between fr and MS2 is 77%. Here, we present a general comparison between the two phages. In the accompanying paper we use phylogenetic sequence comparison between MS2 and fr to deduce the secondary structure at the 3' untranslated region.

Affinity labelling of Escherichia coli ribosomes with a benzylidene derivative of AUGU6 within initiation and pretranslocational complexes.

Affinity labelling of E. coli ribosomes with the 2',3'-O-[4-(N-2-chloroethyl)-N-methylamino]benzylidene derivative of AUGU6 was studied within the initiation complex (complex I) obtained by using fMet-tRNAMetf and initiation factors and within the pretranslocational complex (complex II) obtained by treatment of complex I with the ternary complex Phe-tRNAPhe.GTP.EF-Tu. Both proteins and rRNA of 30 S as well as 50 S subunits were found to be labelled. Sets of proteins labelled within complexes I and II differ considerably. Within complex II, proteins S13 and L10 were labelled preferentially. On the other hand, within complex I, multiple modification is observed (proteins S4, S12, S13, S14, S15, S18, S19, S20/L26 were found to be alkylated) despite the single fixation of a template in the ribosome by interaction of the AUG codon with fMet-tRNAMetf.

[Primary structure of a fragment of cDNA from phage fr]. / Pervichnaia struktura fragmenta kDNK faga fr.

The nucleotide sequence of a 1392 bp fragment of phage fr cDNA has been determined. The fragment contains 3'-terminal part of the A-protein gene, the complete coat protein gene, and beginning of the replicase gene. A comparison between the sequences of the corresponding genes and regulatory regions from the phage fr and MS2 genomes reveals 320 base changes.

[Study of the mRNA-binding region of ribosomes at different steps of translation. II. Affinity modification of Escherichia coli ribosomes by benzylidene derivative of AUGU6 in the 70S initiation complex]. / Issledovanie mRNK-sviazyvaiushchei ioblasti ribosomy na raznykh étapakh transliatsii. II. Affinnaia modifikatsiia ribosom Escherichia coli benzilidenovym proizvodnym AUGU6 v 70S komplekse initsiatsii.

2',3'-O-(4-[N-(2-chloroethyl)-N-methylamino]) benzylidene derivative of AUGU6 was used for identification of the proteins in the region of the mRNA-binding centre of E. coli ribosomes. This derivative alkylated ribosomes (preferentially 30S ribosomal) with high efficiency within the 70S initiation complex. In both 30S and 50S ribosomal subunits proteins and rRNA were modified. Specificity of the alkylation of ribosomal proteins and rRNA with the reagent was proved by the inhibitory action of AUGU6. Using the method of two-dimensional electrophoresis in polyacrylamide gel the proteins S4, S12, S13, S14, S15, S18, S19 and S20/L26 which are labelled by the analog of mRNA were identified.

Novel human leukocyte interferon subtype and structural comparison of alpha interferon genes.

In the cDNA library of virus-induced human leukocytes a novel subtype of IFN-alpha gene has been identified and sequenced, named IFN-alpha-N. A comparison of nucleotide sequences within the genes coding for different subtypes of human leukocyte interferon (IFN-alpha) has revealed the natural hybrid structure of individual alpha-IFNs-H,B,F, and N. Certain regions of the genes for IFN-alpha-H,B,F,N show a homology with one of the two structurally distinct groups of leukocyte interferons--either IFN-alpha-A,D or IFN-alpha-C,C1 utilized as reference standards.

[Study of the ribosome mRNA-binding region during different stages of translation. I. Functional activity of mRNA analogs, AUGU6 and its benzylidene derivatives, in ribosome-dependent protein synthesis]. / Issledovanie mRNK-sviazyvaiushchei oblasti ribosomy na raznykh étapakh transliatsii. I. Funktsional'naia aktivnost' analogov mRNK--AUGU6 i ego benzilidenovykh proizvodnykh--v ribosomnozavisimom belkovom sinteze.

Hexaribouridylic acid, prepared by digestion of poly(U) with cobra venom endonuclease, and trinucleotide AUG synthesized chemically by triester approach were joined by RNA-ligase to yield a nonaribonucleotide AUGU6 bearing the initiation codon at its 5'-terminus. 2',3'-O-(4-[N-(2-chloro(or hydroxy) ethyl-N-Methylamino])- benzylidene residues were introduced at the 3'-terminus of oligonucleotide AUGU6 and its benzylidene derivatives AUGU6CHRCl or AUGU6CHROH were obtained. The mRNA analogs synthesized were tested for their template activity in the formation of 70S initiation complex. AUGU6, AUGU6CHRC1 and AUGU6CHROH were shown to stimulate factor-dependent binding of fMet-tRNA to ribosomes. The effect of benzylidene fragment on the template activity of AUGU6CHROH in the course of translation process was studied. It was shown that AUGU6CHROH stimulates synthesis of di- and tripeptides with the same efficiency as AUGU6.

The regulatory region of phage fr replicase cistron. III. Initiation activity of specific fr RNA fragments.

RNA fragments from phage fr covering the complete or part of the replicase cistron initiation region have been used as templates in the formation of a ribosomal initiation complex in vitro. The results so obtained together with our earlier findings in a similar approach applied to fragments of the structurally related RNA from phage MS2 have allowed us to pinpoint the boundaries of the replicase cistron initiation region on phage RNA. A structural model of the above initiation region has been provided which shows that besides the minimal initiation region (comprises the Shine-Dalgarno sequence and initiator AUG), the flanking regions are also involved and are responsible for additional interactions with the ribosome. The flanking regions possibly contribute to the stability of specific contact between the ribosome and template realized by the minimal initiation region.

[Regulatory region of fr phage replicase cistron. II. Isolation and structure of specific fr RNA fragments]. / Reguliatornyi uchastok tsistrona replikazy faga fr. II. Vydelenie i struktura spetsificheskikh fragmentov RNK fr.

A new set of short RNA templates has been prepared for functional studies in initiation of translation in vitro. Number of individual RNA fragments which contain complete or part of the initiatory region of phage fr replicase cistron were isolated from complex fr RNA--fr coat protein. Their primary structure were determined by using standard fingerprint technique and rapid gel sequencing. Secondary structure of several RNA fragments and their binding activity with phage fr and MS2 coat proteins has been also studied.

[Preparation of RNAase-free E. coli ribosomes active in initiation of protein biosynthesis]. / Vydelenie aktivnykh v initsiatsii biosinteza belka ribosom E. coli, svobodnykh ot primesei RNKaz.

The level of contaminating RNAases in the main components of the protein biosynthesis initiation system, the initiation factors and ribosomes of E. coli, was studied. It was shown that the ribosomes are the major source of contaminating RNAases. A simple procedure for purification of ribosomes active in initiation including Sephadex G-200 gel-filtration of unwashed ribosomes in a 1.5 M NH4Cl-containing buffer was developed.