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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.

On the base of previously developed microbial models high effective scheme for screening of inhibitors of sterol biosynthesis (ISB) is proposed. It is based on cultivation of halophilic bacteria Halobacterium salinarum (former Halobacterium halobium), possessing mevalonate pathway of sterol biosynthesis, and cultivation of fungus Acremonium fusidioides (former Fusidium coccineum), that is producer of steroid antibiotic fusidin (fusidic acid), which biosynthesis has great similarity (with coincidence of its initial steps till squalene formation) to cholesterol biosynthesis in human organism. In H. salinarum model ISB are revealed as compounds that inhibit test-culture growth, whereas in A. fusidioides test-system they are revealed as compounds that strongly reduce fusidin production without any visible influence on producer's growth. Mevalonate that is one of the crucial intermediates of sterol biosynthesis remove inhibition induced by many microbial metabolites that is the evidence of their action at early stages of sterol biosynthetic pathway, including HMG-CoA reductase step. Both test-systems are developed as micromethod and could be easily mechanized due to miniaturization of microbiological procedures, cultivation in sterile 96-well plates and usage of automatic micropipettes and dispensers. Effectiveness of both test-systems, as well as their sensitiveness, laboriousness and ability to give false-positive or false-negative results in ISB screening work is compared. The proposed scheme of screening of ISB includes microbial models at early steps of screening procedures and Hep G2 test-system at the late step. The preliminary screening of microbial metabolites possessing antifungal activity at initial step is compulsory. Miniaturization and mechanization of microbial processes and purification of producers' culture broth with micro- and ultrafiltration are under consideration as well.

Alterations of cell walls of Acremonium chrysogenum occurring at intensive synthesis of cephalosporin C has been studied. It is shown, using electron microscopy, that the cell wall of the cells ofATCC 11550 strain ("wild" type) became looser and thicker during growth. The cell wall of the cells of strain 26/8 (hyperautotroph of cephalosporin C) considerably degraded by the end of the stationary phase. Biochemical analysis has shown that these alterations entailed decrease of the proteins' content covalently or noncovalently linked with the polysaccharides of cell walls of both strains. An increase of sensitivity of cell walls of the strain-superproducer to an activity of lytic enzymes of chitinase, laminarinase, proteinase K, and lyticase preparation has been observed during the growth, but this increase has not been found in the case of "wild" type strain. The obtained results evidence to the structure failure of the cell wall of A. chrysogenum entailing the intensive creation of antibiotic.

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.

It has been found out, that contamination of marine and river water and soil with nitrates significantly influence the viability of microscopic fungi living there. This influence was positive when the nitrates content was moderate (from 0.2 to 2 g/l), extremely high concentrations of nitrates (20 g/l) lead to the inhibition of fungi growth and to their death.

Influence of the quality of the fats used on their utilization in the process of cephalosporin C fermentation and accumulation was studied. A decrease in the level of all the fractions of the fatty acids was observed during the fermentation process. The antibiotic yield with the use of oxidized fats was lower. Treatment of the fats with gaseous nitrogen prevented their oxidation. It was supposed that the decreased yields of the antibiotic were associated with the influence of the oxidized fats on the biosynthetic processes.

A bireplicone plasmid pSU901,4.6 kb in length, was constructed on the basis of plasmid pUC19 and the pstIB fragment, 1.9 kb in length, from mitochondrial DNA of A. chrysogenum. Based on the hybrid plasmid pSU901 and kanamycin resistance determinant, an autonomically replicating vector for A. chrysogenum, a culture producing cephalosporin C, is being constructed.

The influence of various preservation conditions of viability and antibiotic activity of Acremonium chrysogenum 281A and 305A strains producing cephalosporin C was studied. Cryogenization of the culture in the form of suspension of the vegetative mycelium in 20 per cent glycerol solution at a rate of 1 degree/min showed to be advantageous over lyophilization and L-drying. Cryogenization under such conditions provided rather high viability of the culture and preservation of its initial antibiotic activity for the period of its storage for at least 1.5 years in liquid nitrogen at a temperature of -196 degrees C.

The attitude of the cephalosporin C-producing organism to various sources of carbon and nitrogen was studied. Carbohydrates such as maltose, starch and sucrose and nitrogen sources such as mineral ((NH4)2SO4 + KNO3) and organic nitrogen (asparagine) may be successfully used for the culture growth and antibiotic biosynthesis. The use of the mineral nitrogen necessitates additional regulation of pH during the cultivation process. The effect of pH on cephalosporin C biosynthesis was studied and its optimal values were found. It was shown that methyloleate played no specific role in cephalosporin C biosynthesis. It is an additional source of carbon which may be replaced by whale oil. It was demonstrated that the 1st stage of assimilation of inorganic sulfate, i.e. its reduction was blocked in C. acrimonium 218A. The study on the problem of phases in production of cephalosporin C showed that unlike the penicillin-producing organism C. acremonium had no capacity for the antibiotic biosynthesis at the early developmental stages irrespective of the cultivation conditions.

Exposure of Cephalosporium acremonium, strain 1435, to N-nitrozo-N-methylbiuret resulted in a changed variation coefficient with respect to two quantitative features--the antibiotic production and proteolytic activity. Correlation between the variation coefficient and mutagen exposure time was different for every feature. Positive correlation was found in variation with respect to the antibiotic production and proteolytic activity in populations of various Cephalosporium acremonium strains chosen with regard to one or two of the above features. The level and form of the correlation in variation of the above features in populations changed during selection. Selection according to the two quantitative features resulted in an increased correlation coefficient.

The effect of exogenic lysine on production of cephalosporin by auxotrophic and prototrophic strains of A. chrysogenum and their development was studied. It was shown that lysine added to the medium at a concentration of 0.5 or 1 mg/ml partially eliminated the effect of catabolic repression by glucose and had a stimulating effect on production of cephalosporin in the presence of sucrose. Both phenomena were most pronounced in the prototrophic strain 309-A. High concentrations of lysine (10 mg/ml) inhibited cephalosporin biosynthesis more intensively in the auxotrophic strain 291-A than in the prototrophic strain 309-A. Marked differences in the character of the culture development in media supplemented with 1 or 10 mg/ml of lysine were also observed.

Dependence of the content of intracellular methionine and its distribution between the fraction of the high-molecular intracellular compounds and the cytoplasmic amino acid pool in the cephalosporin C-producing organism A. chrysogenum on the content of the carbohydrate source in the medium was studied. In the presence of both glucose and sucrose accumulation of methionine in the cells was observed prior to the antibiotic production. With the use of 35S- or 2(14)C-methionine it was shown that glucose had no repressing effect on methionine transport by the cells of different age (24 and 72 hours). In the presence of glucose the higher levels of 35S- or 2(14)C-methionine were detected in the fraction of the low-molecular compounds of the cells within the first 36 hours of the culture growth. Apparently, the intracellular methionine was used to a larger extent for protein synthesis rather than for construction of the antibiotic molecule.

Parameters providing qualitative estimation of the cephalosporin C-producing organism seed material were studied. Before inoculation of the fermentation medium the seed material should have the maximum respiration intensity, maximum specific growth rate and not less than 15 per cent of the biomass.

Among 24 fungal strains belonging to the genera Aspergillus, Acremonium and Verticillium, eight strains synthesized proteases with the coagulating activity. The most active strains producing such proteases were found among Aspergillus species. The fibrinolytic activity was detected in 12 strains of the fungi. Proteases with the fibrinolytic activity differed in their sensitivity to plasma inhibitors and the least sensitive proteases were found in A. niger and A. flavus. Some fungal strains synthesized proteases with the fibrinolytic activity exerting an indirect action. Such enzymes activated the conversion of blood profibrinolysin into fibrinolysin in a manner similar to that of staphylokinase, urokinase and trypsin.

There were observed two pathways of glutamic acid formation in two strains of Acremonium chrysogenum differing in the production levels of cephalosporin C. The pathway involving glutamate dehydrogenase is known. The other pathway involved amination catalyzed by glutamine synthetase. Activity of both the enzymes during intensive synthesis of the antibiotic was higher in the highly productive strain. Under conditions of limited nitrogen content in the medium production of glutamate during the antibiotic biosynthesis depended on glutamine synthetase. When there was an excess of nitrogen in the medium the main role in production of glutamic acid at the phase of cephalosporin synthesis was played by the other enzyme i. e. glutamate dehydrogenase. By the dynamics the curve of the glutamate dehydrogenase activity correlated with that of the antibiotic production.

Spruce and ash wood were subjected to acid hydrolysis. The hydrolysates were paper chromatographed for the presence of saccharides. The following monosaccharides were detected: D-xylose, D-mannose, D-glucose, D-galactose and L-arabinose. The monosaccharides and L-rhamnose in addition in the form of concentrated solutions were used as part of lactose in production of penicillin V. Similarly, the whole amounts of D-glucose and saccharose were substituted in biosynthesis of cephalosporin C. With regard to the results it follows that hydrolysates of the conifers and especially of the hardwood can substitute part of the fermentation medium components in production of penicillin V and cephalosporin C.

A scheme for automatic impulse foam breaking during biosynthesis of cephalosporin C in 0.63 m3 pilot plant fermenters was developed and tested. It was shown that the use of an aerodynamic foam breaker did not limit the antibiotic biosynthesis by oxygen and promoted stabilization of the foam level in the apparatus without chemical antifoamers and increased air supply.

The intracellular and extracellular amino acid composition of an auxotrophic methionine-deficient strain of Acremonium chrysogenum was studied in respect of the content of various carbohydrates in the fermentation medium. In the presence of glucose, an intensive involvement of exogenous DL-methionine into the cell metabolism was observed at earlier stages than in the presence of dextran or succrose. The total content of intracellular amino acids was lower in the cells grown on the medium with glucose. The production of cephalosporin C depended on the intracellular content of methionine, glutamic acid and lysine.

Dependence of the final results of cephalosporin biosynthesis on the parameters of the operating conditions and the design of fermentors was shown. The value of the specific power input for agitation had a significant effect on the biosynthetic process. An equation for estimating the final results of the biosynthetic process by the values of the operating conditions and the fermentor design parameters is presented.