[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.

[The concentration dynamics of inorganic polyphosphates during the cephalosporin C synthesis by Acremonium chrysogenum].

The contents of five fractions of energy-rich inorganic polyphosphates (polyPs), ATP, and H(+)-ATPase activity in the plasma membrane were determined in a low-activity cephalosporin C (cephC) producer Acremonium chrysogenum ATCC 11550 and selected highly efficient producer strain 26/8 grown on glucose or a synthetic medium providing for active synthesis of this antibiotic. It was shown that strain 26/8 on the synthetic medium produced 26-fold higher amount of cephC as compared with strain ATCC 11550. This was accompanied by a drastic decrease in the cell contents of ATP and the high-molecular-weight fractions polyP2, polyP3, and polyPS with a concurrent increase in the low-molecular-weight fraction polyP1. These data suggest that polyPs are involved in the cephC synthesis as a source of energy. H(+)-ATPase activity insignificantly changed at both low and high levels of cephC production. This confirms the assumption that A. chrysogenum has other alternative antibiotic transporters in addition to cefT. The obtained results can be used for optimizing commercial-scale cephC biosynthesis.

[Various physico-chemical and kinetic properties of metalloproteinase from bacteriolytic lysoamidase preparation]. / Nekotorye fiziko-khimicheskie i kineticheskie svoistva metalloproteinazy bakterioliticheskogo preparata lizoamidazy.

The amino acid composition of metalloproteinase was determined. It was shown that the enzyme is made up of four cysteinyl residues which makes it distinct from other known neutral metalloproteinases from Bacillus brevis, Bacillus subtilis and thermolysine that are devoid of cysteinyl residues. The inhibiting effect of amino acids and some di- and tripeptides on the metalloproteinase activity was studied. The pH-dependence of the Michaelis constant (pKm0) of native and diethylpyrocarbonate-modified metalloproteinase (pKm) suggests that the enzyme active center contains two imidazole groups of histidine with pK alpha 1 = 6.75 +/- 0.1 and pK alpha 2 = 5.4 +/- 0.1. The experimental results are compared to those obtained with other microbial metalloproteinases.