The effect of various carbon compounds on the synthesis of alcohol oxidase in a medium with methanol was studied in the wild type strain of Pichia pinus as well as in gcrl and ecrl mutants defective in glucose and ethanol repression of methanol metabolic enzymes, respectively. Compounds repressing the synthesis of alcohol oxidase in the wild type strain were divided into four groups. Repression of alcohol oxidase by compounds of the first group (glucose, fructose, mannose, galactose, L-sorbose and xylose) was impaired only in the gcrl mutant and that by compounds of the second group (ethanol, acetate, 2-oxoglutarate and erythritol) only in the ecrl mutant. Repression by compounds of the third group (malate, dihydroxyacetone) was not impaired in both these regulatory mutants and that by compounds of the fourth group (succinate, fumarate, L-arabinose, sorbitol, salicin, xylitol and cellobiose) was partially reduced in both gcrl and ecrl strains.Mutationgcrl causes a significant decrease in phosphofructokinase activity. It also led to a six-to seven-fold increase in intracellular pools of glucose-6-phosphate and fructose-6-phosphate and to a two-fold decrease in the intracellular pool of fructose-1,6-bisphosphate. In ecrl strains, a decrease in 2-oxoglutarate dehydrogenase activity accompanied by an increase in activities of NAD-and NADP-dependent isocitrate dehydrogenases and NAD-and NADP-dependent glutamate dehydrogenases was demonstrated. The intracellular pool of 2-oxoglutarate was increased 2.5-fold in ecrl strains. Genes GCRl and ECRI are not linked.The mechanisms of catabolite repression of alcohol oxidase in methylotrophic yeasts are discussed. KEY WORDS -Pichiapinus; alcohol oxidase; catabolite repression; metabolic regulation; methanol. (USSR) 50,242-248. 22,584-592. ( U S S R ) 13,322-329. 24, 1-82.

The modified asparaginase Was79 was derived from the recombinant wild-type L-asparaginase of Wolinella succinogenes. The Was79 contains the amino acid substitutions V23Q and K24T responsible for the resistance to trypsinolysis and the N-terminal heparin-binding peptide KRKKKGKGLGKKR responsible for the binding to heparin and tumor K562 cells in vitro. When tested on a mouse model of Fischer lymphadenosis L5178Y, therapeutic efficacy of Was79 was significantly higher than that of reference enzymes at all single therapeutic doses used (125-8000 IU/kg). At Was79 single doses of 500-8000 IU/kg, the complete remission rate of 100 % was observed. The Was79 variant can be expressed intracellularly in E. coli as a less immunogenic formyl-methionine-free form at high per cell production levels.

A collection ofmutants of Pichiapinus which are unable to grow on ethanol but retain the ability to grow on glucose and methanol. was obtained. Genetic and biochemical analysis of these strains revealed mutations in seven nuclear gcnes affecting activities of isocitrate lyase (icll), malate synthase (mlsl), phosphoenolpyruvate carboxykinase @ckl), 'malic' enzyme (n7ddI) and acetyl-CoA synthetase (acsl, acs2 and acs3). All mutations except acsl-acs3 have no effect on the activities of other enzymes involved in Cz metabolism. Mutations acsl, acs2 and acs3 have a pleiotropic action, leading to partial reduction in activities of isocitrate lyase and malate synthase. Ethanol-induced repression of the synthesis of the methanol oxidative enzymes, alcohol oxidase and catalase, is not impaired in these seven mutant classes. On the other hand. C2 compound-induced inactivation of alcohol oxidase and catalase is impaired in mutants acsl, acs2, ucs3 and icll. It was suggested that glyoxylate and acetate (or acetate precursors) act as low molecular weight effectors, 'swilching on' inactivation and repression, respectively, of alcohol oxidase and catalase in the medium containing ethanol or acetate.

The expression of a hybrid gene encoding an alpha-factor prepro leader peptide-miniproinsulin (MPI) fusion [MPI is the same as the LysArg human insulin precursor described by Thim et al. (1986)] was tested in a series of isogenic yeast strains to investigate the influence of some genetic and physiological factors on heterologous production in yeast. We found that: (i) an MF alpha 1 gene disruption in haploid cells, as well as MF alpha 1 gene product expression in diploid cells, do not affect the MPI secretion level; (ii) under conditions of exogenous leucine availability, MPI production is hindered by leucine auxotrophy (a leu2 mutation); (iii) rho- mutations increase the per-cell MPI yield approximately three-fold; (iv) the MPI yield is apparently dependent on the pH of the culture medium: the higher the external pH, the larger the per-cell MPI yield.

Genetic mechanisms of switching the mating type locus MAT1 in the homothallic yeast Pichia pinus were studied. By analysis of mutations affecting MAT1 complex structural and functional organization of this locus was shown. The existence of two functional regions in MAT1 is postulated. Region I controls mating ability of haploid cells, determines the neutrality of heterozygous cells and regulates the work of Region II. Region II controls meiosis and/or sporulation in the cultures heterozygous for Region I as well as controls switching MAT1 ⇋ MAT1α in haploid cells.