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A system for the production of mutant recombinant human alpha-fetoprotein (rhAFPO) lacking the glycosylation site has been engineered in the yeast Pichia pastoris. A strain of the methylotrophic yeast Pichia pastoris GS 115/pPICZ?A/rhAFP0, which produces unglycosylated rhAFPO and secretes it to the culture medium, has been constructed. Optimization and scale-up of the fermentation technology have resulted in an increase in the rhAFP0 yield to 20 mg/L. A scheme of isolation and purification of biologically active rhAFP0 has been developed. The synthesized protein has the antitumor activity, which is analogous to the activity of natural human embryonic alpha-fetoprotein.

Penicillium canescens strain F178 is a natural producer of beta-galactosidase and endo-1,4-beta-xylanase. The transcription of genes bgaS and xylA, coding for these proteins, is subject to carbon catabolite repression. The system for selective isolation of regulatory mutants in P. canescens is developed. Two strains from the mutant collection are studied in details. It is shown that both mutations can be complement by creA gene of P. canescens, encoding global regulator of carbon catabolite repression in filamentous fungi. creA(-) alleles contain frameshift mutations in C-domain of CreA. Gene xylA is derepressed in mutants at transcription level in the presence of D-glucose. A transcription of creA gene in mutants is also derepressed proving effect of autoregulation for this gene.

Four novel genes of the enzymes of the endoxylanase (EC 3.2.1.8) families found in the mycelial fungus Penicillium canescens have been cloned. The xylB, xylC, and xylD genes encode endoxylanases of glycosyl hydrolase family 11; the xylEgene, those of family 10. In the promoter region of the xylB, xylC, and xylD genes, the binding sequences for the protein activator of xylanolytic gene transcription have been found; the promoter region of the xylB gene contains the binding sequences for the catabolite repression protein. Since the TATAA sequence, which is an element of the minimal eukaryotic promoter, has not been found in the promoter region of the xylC gene, in contrast to those of the xylB and xylD genes, it may be assumed that this gene is silent. Comparative phylogenetic analysis has shown that the cloned genes are highly homologous to some endoxylanase genes of mycelial fungi of the genera Penicillium and Aspergillus. However, within the species P. canescens, they exhibit a low homology both within and between families, and they diverge into different branches of the phylogenetic tree, which suggest divergence of the genes of this group at an early stage of evolution.

Penicillium canescens strain F178 is a natural producer of beta-galactosidase and endo-1,4-beta-xylanase. Tanscription of genes bgaS and xylA coding for these proteins is subject to carbon catabolite repression which proceed mainly in filamentous fungi by transcriptional repressor CreA. creA gene of P. canescens was cloned. It was demonstrated that creA transcription is also subject to carbon catabolite repression. CreA protein remains intranuclear independently of nature of carbon source and glucose concentration in culture medium. In vitro experiments confirm availability of four CreA-binding sites in bgaS promoter, four sites in xylA promoter and one such site in creA promoter.

A heterologous protein expression in the fungus Penicillium canescens is described for the first time. The fungal strains producing Trametes hirsuta laccase under control of a highly efficient promoter of the P. canescens gene bgaS has been constructed. These strains efficiently transcribe the T. hirsuta 072 laccase gene with a correct intron splicing. Activity of the secreted heterologous laccase in the culture liquid reaches 3 U/ml, accounting for 98% of the total laccase activity, which demonstrates a high efficiency ofheterologous secretion. The synthesized P. canescens laccase has the same molecular weight as the enzyme produced by T. hirsuta 072.

The gene egl3 of the filamentous fungus Penicillium canescens endo-1,4-beta-glucanase, belonging to family 12 glycosyl hydrolases, was cloned and sequenced. The gene was expressed in P. canescens under the control of the strong promoter of gene bgaS, coding for beta-galactosidase of this fungus, and efficient endoglucanase producer strains were obtained. The recombinant protein was isolated from the culture liquid of the producer strain EGL3-13 and purified to homogeneity; its specific activity was 31.7 IU; molecular weight, 26 kDa; and pH and temperature optimums, 3.2 and 54 degrees C, respectively. The Km and Vm values for CMC hydrolysis were determined; they amounted to 17.1 g/1 and 0.31 microM/(mg s), respectively.

The fungus Penicillium canescens strain F178 (VKPM) and its niaD- mutant exhibited an increased capability of synthesizing extracellular enzymes beta-galactosidase (70-80 U/ml) and xylanase (100 U/ml). The synthesis was induced by arabinose and its catabolite, arabitol. A deficiency in arabitol dehydrogenase, leading to arabitol accumulation in the cell, was detected in the chain of reactions of arabinose catabolism. The increased synthesis of beta-galactosidase and xylanase in P. canescens is accounted for by (1) cellular accumulation of the inducer (arabitol) at low concentrations of arabinose in the medium and (2) prevalence of induction over repression.

The induction of the synthesis of secreted enzymes endo-1,4-beta-xylanase (EC 3.2.1.8) and beta-galactosidase (EC 3.2.1.23) in the original and recombinant Penicillium canescens strains has been studied. In all producer strains, the synthesis of these enzymes was induced by arabinose and its metabolite arabitol. The two enzymes differed in the concentration of arabinose required for the induction: the synthesis of beta-galactosidase was most pronounced at 1 mM, whereas maximum synthesis of endo-1,4-beta-xylanase was observed at 5 to 10 mM. An increase in the number of endo-1,4-beta-xylanase copies in the high-copy-number strain of the fungus suppressed the synthesis of beta-galactosidase; the synthesis of endo-1,4-beta-xylanase in the high-copy-number recombinant producing beta-galactosidase was affected to a lesser extent. The amount of the enzymes synthesized did not depend on the saccharide used as a sole source of carbon for growing the mycelium prior to its transfer to the inducer-containing medium.

The complete gene xylA that encodes endo-1,4-beta-xylanase secreted by Penicillium canescens was cloned and sequenced. The coding region of the gene is separated by eight introns. The protein comprises 302 amino acids of the mature protein and 25 amino acids of the signal peptide. The xylanase of P. canescens belongs to the glycosyl hydrolase family 10. Nucleotide sequences for binding catabolite repression protein CREA and transactivator protein were detected in the promoter region. A set of multicopy strains displaying a seven-eightfold increase in xylanase yield was obtained. The fraction of xylanase in most productive strains amounted to 30-50% of the total secreted protein.

A system for introduction of plasmids into industrial producers of antibiotics chlortetracycline and bialaphos using intergeneric conjugation of Escherichia coli and Streptomyces was developed. Low level stability of inheritance of autonomously replicating DNA in recipient strains was shown. Site-specific integration of the conjugative-integrative vector pTO1 provided stable plasmid maintenance within the chromosomes of Streptomyces aureofaciens and S. hygroscopicus. Phenomenon of disturbance in differentiation and antibiotic production, resulting from pTO1 integration into S. hygroscopicus chromosome, was discovered.

Yeast strains belonging to the genera Candida and Hansenula were shown to differ in their susceptibility to the action of protein antibiotics produced by the yeasts Williopsis and Zygowilliopsis. This finding can be used as an additional criterion for yeast identification.