[Cephalosporin-Acid Synthetase of Escherichia coli Strain VKPM B-10182: Genomic Context, Gene Identification, Producer Strain Production].

An enzyme of cephalosporin-acid synthetase produced by the E. coli strain VKPM B-10182 has specificity for the synthesis of ß-lactam antibiotics of the cephalosporin acids class (cefazolin, cefalotin, cefezole etc.). A comparison of the previously determined genomic sequence of E. coli VKPM B-10182 with a genome of the parent E. coli strain ATCC 9637 was performed. Multiple mutations indicating the long selection history of the strain were detected, including mutations in the genes of RNase and ß-lactamases that could enhance the level of enzyme synthesis and reduce the degree of degradation of the synthesized cephalosporin acids. The CASA gene--a direct homolog of the penicillin G-acylase gene--was identified by bioinformatics methods. The homology of the gene was confirmed by gene cloning and the expression and determination of its enzymatic activity in the reaction of cefazolin synthesis. The CASA gene was isolated and cloned into the original expression vector, resulting in an effective E. coli BL2l(DE3) pMD0107 strain producing CASA.

Comparative gene expression profiling reveals key changes in expression levels of cephalosporin C biosynthesis and transport genes between low and high-producing strains of Acremonium chrysogenum.

Transcript levels of several key genes responsible for cephalosporin C (CPC) biosynthesis and transport have been determined using qPCR analysis of Acremonium chrysogenum strains differing more than 100-fold in the levels of CPC production. The expression of genes involved in the final steps of CPC production was significantly increased in the high-producing RNCM F-4081D strain compared to the wild-type ATCC 11550 strain. Different dynamics in the course of cultivation was observed for the genes known to be involved in the transport of CPC intermediates between subcellular compartments. Overall, comparative expression analysis showed balanced and fine-tuned expression of the genes responsible for CPC biosynthesis and transport in the genetically selected A. chrysogenum RNCM F-4081D strain, reflecting its capacity to overcome known CPC biosynthesis "bottlenecks" and produce CPC of high yield and purity.

[Functional characteristic of the CefT transporter of the MFS family involved in the transportation of beta-lactam antibiotics in Acremonium chrysogenum and Saccharomyces cerevisiae].

Vectors for the expression of the CefT transporter of the MFS family in Acremonium chrysogenum--a producer of beta-lactam antibiotic cephalosporin C--and in Saccharomyces cerevisiae as a fusion with the cyan fluorescent protein (CFP) have been created. The subcellular localization of the CefT-CFP hybrid protein in yeast cells has been investigated. It was shown that the CefT-CFP hybrid protein is capable of complementation of the qdr3, tpo 1, and tpo3 genes encoding for orthologous MFS transporters of Saccharomycetes, making the corresponding strains resistant to spermidine, ethidium bromide, and hygromycin B. High-yield strain VKM F-4081D of A. chrysogenum, expressing the cefT-cfp fusion, was obtained by an agrobacteria conjugated transfer. It was also shown that the constitutive expression of cefT in A. chrysogenum VKM F-4081D led to a change in the biosynthetic profiles of cephalosporin C and its precursors. This resulted in a 25-35% decrease in the finite product accumulated in the cultural liquid with a simultaneous increase in the concentration of its intermediators.

[Chromosomal polymorphism of Acremonium chrysogenum strains producing cephalosporin C].

Using pulse electrophoresis in controlled homogenous electric field we conducted molecular karyotyping of highly-productive and laboratory strains of Acremonium chrysogenum generating antibiotic cephalosporin C (cefC). Differences in size of several chromosomes of highly active strain CB26/8 compared to the wild-type strain ATCC 11550 were revealed. It was shown that chromosomal polymorphism in the highly active strain was not associated with alteration of localization and copy number ofcephalosporin C biosynthesis and transport genes. A cluster of "early" cefC biosynthesis genes is located on chromosome VI (4.4 Mb); a cluster of the "late genes", on chromosome II (2.3 Mb). Both clusters are presented as a single copy perA. chrysogenum genome in the wild-type and in CB26/8 producer strains. Based on comparative analysis of laboratory and industrial cefC producers, a karyotype scheme for A. chrysogenum strains of various origins was designed.

[Genetic transformation of the mycelium fungi Acremonium chrysogenum].

The system of transformation of heterologous genes under the method of agrobacterial transfer into Acremonium chrysogenum ATCC 11550 wild-type strains, natural producents of beta-lactam antibiotic cephalosporin C, and strains highly producing cephalosporin C 26/8 revealed by the multistage selection on its basis were developed. Vectors for agrobacterial transformation of A. chrysogenum containing expression cassettes of genes encoding resistance to geneticin (G418) and bleomicin (Zeocin) antibiotics under control of Ashbya gossypii and Saccharomyces cerevisiae TEF1 promoters were constructed. A comparable assessment of agrotransformation methods while co-cultivating fungi and agrobacterial cells on filters and in deep culture was conducted. Transformants, selected by resistance to geneticin and bleomicin, were characterized by PCR and Southern blot analyses.

[Purification and properties of recombinant Erwinia carotovora L-asparaginase expressed in E.coli cells]. / Vydelenie, ochistka i nekotorye svoistva rekombinantnoi L-asparaginazy Erwinia carotovora, ékspressirovannoi v kletkakh E. coli.

The method of purification Erwinia carotovora recombinant L-asparaginase, expressed in E.coli, including ultrasonic disintegration of biomass, fractionation ammonium sulfate and column chromatography on CM- or SP-Sepharose has been developed. According to SDS-PAAGE the enzyme preparation was homogeneous, its specific activity and yield consist respectively about 620 IU/mg of protein and 75%. Physical-chemical and structural properties of recombinant Erwinia carotovora L-asparaginase are similar to the enzymes from the wild strains Erwinia carotovora and recombinant L-asparaginase Erwinia chrysanthemi.

[Heterologous expression of eukaryotic CYP450. 1. Heterologous expression of cytochrome P450 2B4 using groups with various affinity in E. coli]. / Geterologichnaia ékspressiia éukarioticheskikh CYP450. 1. Geterologichnaia ékspressiia tsitokhroma P450 2B4 c razlichnymi kontsevymi affinnymi gruppami v shtammakh E. coli.

The expression levels of cytochrome P450 2B4 variants with N- and C-terminal modifications were compared and some of the enzymatic characteristics of recombinant proteins studied. Following C-terminal hybrids for CYP2B4 gene were constructed: 1) with intein-chitin binding domain cassette 2) with hexahistidine tag. These modifications were combined with P450 2B4 glutathione-S-transferase N-terminal fusions [Pernecky S.J., et. al., (1995) Arch. Biochem. Biophys., 318, 446-456]. The obtained constructs provided for the synthesis of full-length protein products in E. coli cells with holoenzyme yield at the levels of 200-1000 nmoles/l of the bacterial culture. Partial in vivo proteolysis was observed for C-terminal fusions with intein moiety despite the presence of glycine aminoacid residue at the junction of two proteins. The principle inapplicability of standard purification scheme for isolation of P450 2B4-intein fusions is demonstrated, since the P450 domain is inactivated at 40 mM DTT concentrations. The recombinant full-length CYP 2B4 with C-terminal oligohistidine tail was expressed under the control of T7 promoter and purified using immobilized metal-ion chelating chromatography. The C-terminal hexahistidine tag does not affect the catalytic properties of recombinant enzyme in 7-pentoxyresorufin O-dealkylation reaction.

[Cloning of the human cytochrome p450 2B6 gene in Escherichia coli cells]. / Klonirovanie gena tsitokhroma p450 2B6 cheloveka v kletkakh Escherichia coli.

Human cytochrome P450 2B6 gene from plasmid pUC9, carrying cytochrome CYP2B6 cDNA was cloned into eucariotic expression vector pcDNA 3.1 (+). Cytochrome P450 2B6 gene in recombinant plasmid pcDNA 3.1 (+)/CYP 2B6 was expressed in E. coli cells. The expression of catalytically active recombinant protein was 60-100 nm per liter of the culture medium.

[Technical means for the segmental perfusion of the small intestine]. / Tekhnicheskie sredstva dlia segmentarnoi perfuzii tonkoi kishki

One of the most exact methods of diagnosing disturbed absorption is perfusion of the small intestine. To make pertinent investigations a special perfusion probe manufactured of a roentgen-contrast rubber and a peristaltic pump for introducing a perfusion solution into the lumen of the intestine are proposed. A procedure used in conducting the examination is described and a formula for determining the absorption rate is given.