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.

[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.

[Structure peculiarities of cell walls of Acremonium chrysogenum--an autotroph of cephalosporin C].

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.

[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.

[A study of steroid hydroxylation activity of Curvularia lunata mycelium].

It has been demonstrated that the mycelium of Curvularia lunata at the end of the logarithmic growth phase displays a maximal 11-hydroxylase activity towards cortexolone (4-6 g/l) used for transformation as a microcrystalline suspension in phosphate buffer. The mycelium at a later stage of fungal growth displays an elevated 14-hydroxylase activity, necessary for generation of 14-hydroxyandrostenedione. The effects of different forms of substrate added to the reaction mixture, age and concentration of mycelium, and fungal clones tolerant to salts of heavy metals (0.35-0.5%) were studied to remove the side 14-hydroxylation, accompanying the main cortexolone transformation. Mycelia of the fungal clones tolerant to Co2+ and Cu2+ displayed a weak hydroxylase activity or its complete absence and an elevated content of melanin, the biosynthesis of which is intensified under adverse conditions. The results obtained suggest that the transformation of steroids by the studied C. lunata strain is a detoxication of foreign compounds.

[Biotechnological grounds for the regulation of the component composition of cephalosporins in cultures of Acremonium chrysogenum]. / Biotekhnologischeskie osnovy regulirovaniia komponentnogo sostava tsefalosporinov pri kul'tivirovanii Acremonium chrysogenum.

The dynamics of cephalosporine C and desacetylcephalosporine C alterations in cultures of Acremonium chrysogenum 309A under different conditions was studied. It was shown expedient to determine the fermantation period by the data of HPLC. Genetic predisposition of the strain used to production of both cephalosporine C and desacetylcephalosporine C is discussed. With changing the cultivation procedure, medium active acidity and cultivation time it is possible to change the biosynthesis pathway.

[Effect of lysine on cephalosporin biosynthesis and morphogenesis in Acremonium chrysogenum]. / Vliianie lizina na biosintez tsefalosporina i morfogenez Acremonium chrysogenum.

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.

[Effect of carbohydrates in the culture medium on amino acid composition of a cephalosporin C producer]. / Vliianie uglevodov sredy na sostav aminokislot produtsentov tsefalosporina C.

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.

[Effect of carbohydrate catabolic repression on the accumulation and intracellular distribution of methionine in Acremonium chrysogenum--the producer of cephalosporin C]. / Vliianie uglevodnoi katabolitnoi repressii na nakoplenie i vnutrikletochnoe raspredelenie metionina u Acremonium chrysogenum--produtsenta tsefalosporina C.

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.

[Correlation of cephalosporin C synthesis and proteolytic enzymes in a differentiating culture of Acremonium chrysogenum (Cephalosporum acremonium) mutants]. / Korreliatsiia sinteza tsefalosporina C i proteoliticheskikh fermentov v differentsiruiushcheisia kul'ture mutantov Acremonium chrysogenum (Cephalosporium acremonium).

The effect of the growth conditions and composition of the nutrient medium on the synthesis of cephalosporin C, alkaline exoproteases and cell differentiation was studied in various strains of Acremonium chrysogenum. It was observed that the changes in the above processes occurred simultaneously and depended on the aeration rate, temperature, illumination level and concentrations of methionine and soybean meal. Close correlation between the synthesis of alkaline exoproteases, cephalosporin C and formation of secondary structures in the strains of A. chrysogenum was shown.