[Metabolic engineering of yeast Hansenula polymorpha for construction of efficient ethanol producers].

Until recently, the methylotrophic yeast was not considered as a potential producer of biofuels, particularly of ethanol from lignocellulosic hydrolysates. The first work published 10 years ago reveals the ability of thermotolerant methylotrophic yeast Hansenula polymorpha to ferment xylose--one of the main sugars of lignocellulosic hydrolysates, which has made these yeast promising organism for high temperature alcoholic fermentation. Such feature of the H. polymorpha can be used in the implementation of potentially effective process of simultaneous saccharification and fermentation (SSF) of raw materials. SSF allows combining enzymatic hydrolysis of raw materials with the conversion of produced sugars into ethanol: enzymes hydrolyze polysaccharides to monomers, which are immediately consumed by microorganisms-producers of ethanol. However, the efficiency of alcoholic fermentation of major sugars realized after hydrolysis of lignocellulosic raw materials, and especially xylose, by wild strains of H. polymorpha requires significant improvement. In this review the main results of metabolic engineering of H. polymorpha for the construction of improved producers of ethanol from xylose, starch, xylan, and glycerol, as well as strains with increased tolerance to high temperature and ethanol are represented.

[Molecular mechanisms of peroxisome biogenesis in yeasts].

Peroxisomes contain oxidases generating hydrogen peroxide, and catalase degrading this toxic compound. Another characteristic function of each eukaryotic peroxisome, from yeast to man, is fatty acid beta-oxidation. However, in peroxisomes a variety of other metabolic pathways are located. In fungi, peroxisomes contain enzymes involved in catabolism of unusual carbon and nitrogen sources (methanol, purines, D-amino acids, pipecolynic acid, sarcosine, glycolate, spermidine etc) as well as biosynthesis of lysine in yeasts and penicillin in mycelial fungi. Impairment of peroxisomal structure and functions causes many human disorders. The similar defects have been identified in yeast mutants defective in peroxisomal biogenesis. Peroxisomal biogenesis is actively studied during last two decades using uni- and multicellular model systems. It was observed that many aspects of peroxisomal biogenesis and proteins involved in this process display striking similarity between all eukaryotes, from yeasts to humans. Yeast is a convenient model system for this kind of research. Current review summarizes data on molecular events of peroxisomal biogenesis, functions of peroxine proteins, import of peroxisomal matrix and membrane proteins and on mechanisms of peroxisomedivision and inheritance.

[The microbial synthesis of flavin nucleotides: a review].

Recent data on the synthesis and hydrolysis of flavin nucleotides in yeast and bacteria and the regulation of this process are summarized. Specific examples are provided and the prospects of the use of genetically modified microorganisms for the industrial manufacturing of flavin mononucleotide and flavin dinucleotide are considered.

[Construction of flavocytochrome b2-overproducing strains of the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta)].

L-Lactate cytochrome c oxidoreductase (flavocytochrome b2, FC b2) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker's yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b2 producers with overexpression of the H. polymorpha CYB2 gene, encoding FC b2. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (gcr1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b2 activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b2 producer characterized by a sixfold increased (to 3 micromol min(-1) mg(-1) protein in cell-free extract) activity of the enzyme.

[Molecular mechanisms of insertional mutagenesis in yeasts and mycelium fungi].

Random insertional mutagenesis is an efficient tool for studying molecular mechanisms of many genetically determined processes. An improved variant of this method is REMI (Restriction Enzyme Mediated Integration) mutagenesis. In this method, the insertion cassette is introduced into the recipient cell together with restriction endonuclease. As a result, the REMI cassette insertion occurs in sites recognized by the restriction enzyme. The use of restriction endonucleases enhances transformation rate and provides cassette insertion in virtually any locus. A mutation is tagged by the insertion cassette, which can be identified by isolating the REMI cassette together with the flanking genomic DNA regions. The review describes general requirements to REMI. The mechanisms of REMI mutagenesis are surveyed with special reference to yeast Saccharomyces cerevisiae. Special attention is given to the development and use of REMI for other lower eukaryotes (yeasts and mould fungi). Drawbacks of the method and perspectives of its use are discussed.

[The pleiotropic nature of rib80, hit1, and red6 mutations affecting riboflavin biosynthesis in the yeast Pichia guilliermondii].

The yeast Pichia guilliermondii is capable of riboflavin overproduction under iron deficiency. The rib80, hit1, and red6 mutants of this species, which exhibit impaired riboflavin regulation, are also distinguished by increased iron concentrations in the cells and mitochondria, morphological changes in the mitochondria, as well as decreased growth rates (except for red6) and respiratory activity. With sufficient iron supply, the rib80 and red6 mutations cause a 1.5-1.8-fold decrease in the activity of such Fe-S cluster proteins as aconitase and flavocytochrome b2, whereas the hitl mutation causes a six-fold decrease. Under iron deficiency, the activity of these enzymes was equally low in all of the studied strains.

[Cloning of the GSH1 and GSH2 genes complementing the defective biosynthesis of glutathione in the methylotrophic yeast Hansenula polymorpha]. / Klonirovanie genov GSH1 i GSH2 komplementiruiushchikh defect biosinteza glutationa u metilotrofnykh drozhzhei Hansenula polymorpha.

The cloning of 7.2- and 9.6-kbp fragments of the methylotrophic yeast Hansenula polymorpha DNA restored the wild-type phenotype Gsh+ in the glutathione-dependent gsh1 and gsh2 mutants of this yeast defective in glutathione (GSH) synthesis because of a failure of the gamma-glutamylcysteine synthetase reaction. The 9.6-kbp DNA fragment was found to contain a 4.3-kbp subfragment, which complemented the Gsh- phenotype of the gsh2 mutant. The Gsh+ transformants of the gsh1 and gsh2 mutants, which bear plasmids pG1 and pG24 with the 7.2- and 4.3-kbp DNA fragments, respectively, had a completely restored wild-type phenotype with the ability to synthesize GSH and to grow in GSH-deficient synthetic media on various carbon sources, including methanol, and with acquired tolerance to cadmium ions. In addition, the 4.3-kbp DNA fragment borne by plasmid pG24 eliminated pleiotropic changes in the gsh2 mutants associated with methylotrophic growth in a semisynthetic (GSH-supplemented) medium (poor growth and alterations in the activity of the GSH-catabolizing enzyme gamma-glutamyltransferase and the methanol-oxidizing enzyme alcohol oxidase).

[Cellular microbiosensors for methanol and ethanol determination on the basis of pH-sensitive field transistors]. / Kletochnye mikrobiosensory na osnove pH-chuvstvitel'nykh polevykh tranzistorov dlia opredeleniia metanola i étanola.

Cellular sensors for methanol and ethanol determination were developed using immobilized mutant cells of methylotrophic yeasts Hansenula polymorpha and Pichia pinus (able to extrude protons in the presence of alcohol) and pH-sensitive field effect transistors (pH-SFETs). The intact cells of yeasts were immobilized in Ca-alginate gel to obtain a biomembrane. The minimal detectable response was obtained to approximately 0.5 mM of methanol and ethanol, a linear dependence of biosensor's response on the logarithmic alcohol concentration was observed in the range from 5 to 100 mM for both types of alcohol. The prospects for application of biosensors to determine alcohols in the analytical practice are discussed.

[Quantitative photometric analysis of ethanol using purified alcohol oxidase and mutant cells of methylotrophic yeast]. / Kil'kisnyi fotometrychnyi analiz etanolu z vykorystanniam ochyshchenoï alkohol'oksydazy ta mutantnykh klityn metylotrofnykh drizhdzhiv.

The photometric method is suggested for ethanol analysis by means of purified alcohol oxidase isolated from a wild strain of methylotrophic yeast Hansenula polymorpha. Three different chromogenic systems for peroxidative oxidation together with alcohol oxidase reaction have been used. For the first time the intact cells of the catalase-negative mutant as a source of alcohol oxidase are employed in the photometric alcohol assay. The prospects for application of the latter method for practical purposes are discussed.

[The effect of citrate and dihydroxyacetone on methanol oxidation in Hansenula polymorpha]. / Vliianie tsitrata i dioksiatsetona na okislenie metanola u drozhzhei Hansenula polymorpha.

Citrate and dihydroxyacetone inhibited [14C] incorporation from radioactive methanol to CO2 by washed cells of methylotrophic yeast Hansenula polymorpha grown in the media containing mixture of methanol with citrate or dihydroxyacetone, respectively. These results are discussed in connection with the earlier hypothesis on participation of the tricarboxylic acid cycle in the energy supply of the methylotrophic growth.

[The nature of methanol-induced acidification of the medium by products of metabolism of methylotrophic yeasts]. / O prirode indutsiruemogo metanolom zakisleniia sredy produktami metabolizma metilotrofnykh drozhzhei.

The washed cells of Hansenula polymorpha and Pichia pinus grown on the medium with methanol rapidly acidify the medium during incubation with the mentioned alcohol or formaldehyde. It is found that proton extrusion is coupled with formate anion efflux. Acidification is proved to be energy-dependent process since it is inhibited by respiration poisons, uncouplers of oxidative phosphorylation, and by ATPase inhibitors.

[A method of quantitative determination of alcohol oxidase and catalase in yeast colonies]. / Metod kolichestvennogo opredeleniia alkogol'oksidazy i katalazy v drozhzhevykh koloniiakh.

A rapid, simple and highly reproducible method is developed for qualitative determination of alcohol oxidase and catalase in yeast colonies using digitonin as a permeabilizing agent. The method permits finding differences in regulation of catalase by ethanol in methylotrophic and nonmethylotrophic yeasts.

[Genetic control of riboflavin biosynthesis in Pichia guilliermondii yeasts. The detection of a new regulator gene RIB81]. / Geneticheskii kontrol' biosinteza riboflavina u drozhzhei Pichia guilliermondii. Obnaruzhenie novogo reguliatornogo gena RIN81.

The properties of mutants resistant to 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)-isoalloxazine (MTRY) were studied. The mutants were isolated from a genetic line of Pichia guilliermondii. Several of them were riboflavin overproducers and had derepressed flavinogenesis enzymes (GTP cyclohydrolase, 6.7-dimethyl-8-ribityllumazine synthase) in iron-rich medium. An additional derepression of these enzymes as well as derepression of riboflavin synthase occurred in iron-deficient medium. The characters "riboflavin oversynthesis" and "derepression of enzymes" were recessive in mutants of the 1st class, or dominant in those of the 2nd class. The hybrids of analogue-resistant strains of the 1st class with previously isolated regulatory mutants ribR (novel designation rib80) possessed the wild-type phenotype and were only capable of riboflavin overproduction under iron deficiency. Complementation analysis of the MTRY-resistant mutants showed that vitamin B2 oversynthesis and enzymes' derepression in these mutants are caused by impairment of a novel regulatory gene, RIB81. Thus, riboflavin biosynthesis in P. guilliermondii yeast is regulated at least by two genes of the negative action: RIB80 and RIB81. The meiotic segregants which contained rib80 and rib81 mutations did not show additivity in the action of the above regulatory genes. The hybrids of rib81 mutants with natural nonflavinogenic strain P. guilliermondii NF1453-1 were not capable of riboflavin oversythesis in the iron-rich medium. Apparently, the strain NF1453-1 contains an unaltered gene RIB81.

[Selection and mutant properties of Pichia guilliermondii yeasts with derepressed GTP cyclohydrolase, the enzyme of the 1st step in flavinogenesis]. / Selektsiia i svoistva mutantov drozhzhei Pichia guilliermondii s derepressirovannym fermentom pervogo etapa flavinogeneza--GTF-tsiklogidrolazoi.

A positive method is proposed for selecting Pichia guilliermondii mutants with derepressed GTP cyclohydrolase. Mutants with the incompletely blocked gene RIB2 were used as parent strains; these can grow in a medium without riboflavin (RF) only if the enzyme is derepressed as the result of iron deficiency in cells. Strains growing in a medium without RF at the optimal supply of cells with iron were selected as regulatory mutants. The mutants accumulated 6,7-dimethylpterin in high concentrations and a small amount of RF in the medium and in the cells. The activity of GTP cyclohydrolase rather than that of RF synthase increased in the mutants; the activity of RF kinase and FAD pyrophosphorylase was not elevated. Hybrids produced by crossing the regulatory mutants with wild type strains did not accumulate 6,7-dimethylpterin in the medium and the activity of the GTP cyclohydrolase did not increase; this is indicative of the negative regulation for the expression of the structural gene for GTP cyclohydrolase. The authors propose a model for the regulation of GTP cyclohydrolase and RF synthase at the gene level involving iron ions as a corepressor.

[Activity of the enzyme of the 2d step of flavinogenesis, 2,5-diamino-6-hydroxy-4-ribosylaminopyrimidine-5'-phosphate reductase, in Pichia guilliermondii yeasts]. / Ob aktivnosti fermenta vtorogo étapa flavinogeneza 2,5-diamino-6-oksi-4-ribozilaminopirimidin-5'-fosfatreduktazy u drozhzhei Pichia guilliermondii.

The activity of 2,5-diamino-6-hydroxy-4-ribosylaminopyrimidine-5'-phosphate reductase, an enzyme of the second step of flavinogenesis, was studied in the yeast Pichia guilliermondii using a new fluorimetric technique. The activity of the enzyme was found in extracts of the wild-type strain and the genotype rib mutants but not in extracts of the FM 1-4 (rib 2) mutant. Interallelic complementation was revealed in the gene rib 2. The activity of the enzyme was identical in extracts of the cells grown in the medium with either a high or a low iron content which synthesized riboflavin at a low or a high rate, respectively. Therefore, the synthesis of this enzyme in P. guilliermondii is not regulated with iron.

[Riboflavin transport in mutant of Pichia guilliermondii yeast with damaged glucose uptake]. / Transport riboflavina u mutanta drozhzhei Pichia guilliermondii s narushennym pogloshcheniem glukozy.

The mutant of P. guilliermondii yeast MS-50 which had lost the ability to grow in a glucose-containing medium but not in the presence of sucrose or maltose was selected. The mutant has a damaged glucose uptake from the medium. The riboflavin permease activity of the MS1-50 mutant is not impaired; glucose inhibits the riboflavin uptake by this strain. Thus, riboflavin permease does not promote glucose transport into yeast cells.

[Increase in yeast and bacterial sensitivity to inhibitors and riboflavin as affected by high sulfate and phosphate concentrations]. / Uvelichenie chuvstvitel'nosti drozhzhei i bakterii k nekotorym ingibitoram i riboflavinu pod vozdeistviem vysokikh kontsentratsii sul'fata i fosfata.

Cultivation of the yeast Pichia guilliermondii in a medium with a high content of sulfate or phosphate ions (0.6 M and higher) increased its susceptibility to actinomycin D and 7-methyl-8-trifluoromethyl 10-(1'-D-ribityl)isoalloxazin, and analog of riboflavin, and decreased the requirement of the riboflavin-dependent mutant P7 in exogenous vitamin B2. The protoplasts of the yeast were also very susceptible to actinomycin D when they were incubated in a medium with a high sulfate concentration. Sulfate and phosphate ions elevated the susceptibility to actinomycin D in the following yeasts, apart from P. guilliermondii: Pichia pinus, Saccharomyces cerevisiae, Torulopsis candida, hansenula polymorpha, Schwanniomyces occidentalis, Candida utilis and Candida tropicalis. The growth of Escherichia coli was also very susceptible to actinomycin D when the bacterium was cultivated in medium with an elevated phosphate concentration (0.2 M). High phosphate or sulfate concentrations can be used in experiments aimed at studying the effect of transcription inhibitors (actinomycin D, 8-hydroxyquinoline) on the induction of alpha-glucosidase in P. guilliermondii.

[Overproduction of riboflavin in mutants of Pichia guilliermondii yeasts resistant to 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)isoalloxazine]. / Sverkhsintez riboflavina u mutantov drozhzhei Pichia gluilliermondii, rezistentnykh k 7-metil-8-triftometil-10-(1'-D-ribitil)izoalloksazinu.

Mutants resistant to 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)isoalloxazine were selected from the yeast Pichia guilliermondii, strain MS14-A10, possessing a multiple sensitivity to antibiotics and antimetabolites. A lot of such mutants displayed an elevated flavinogenic activity. The investigation of the properties of three mutants with the highest flavinogenic activity, viz. RZ4, RZ7 and RZ11, has shown that their capability for riboflavin overproduction does not stem from disordered regulation of the de novo purine biosynthesis, from the damaged transport of iron ions into the cell, or from changes in the allosteric properties of GTP cyclohydrolase. A twofold increase in the specific activities of GTP cyclohydrolase and riboflavin synthase was observed in the two mutants, RZ4 and RZ11. These data suggest that the mechanism for repression of the synthesis of flavinogenic enzymes, in which iron ions are involved, is impaired in these mutants. The reason for riboflavin overproduction in the mutant RZ7 remains obscure.

[Genetic classification of riboflavin-dependent mutants of Pichia guilliermondii yeasts]. / Geneticheskaia klassifikatsiia riboflavinzavisimykh mutantov drozhzhei Pichia guilliermondii.

114 riboflavinless mutants were selected from the genetic line of Pichia guilliermondii yeast. By means of accumulation test the mutants were divided into five biochemical groups. In genetic experiments seven complementation classes were found among 106 mutants. The strains of the I biochemical group, accumulating no specific products, corresponded to complementation class rib1; II group, accumulating 2,4,5-triaminopyrimidine - to the class rib2; III group, accumulating 2,6-dihydroxy-4-ribitylaminopyrimidine - to the class rib3; the mutants of the IV group, accumulating 2,6-dihydroxy-5-amino-4-ribitylaminopyrimidine, were divided into three complementation classes rib4, rib5 and rib6; the mutants of the V group, acculumating 6,7-dimethyl-8-ribityllumazine, corresponded to the class rib7. Two mutants of the IV biochemical group within complementation classes rib4 and rib5 were detected could not grow in the medium with diacetyl without riboflavin. Intragenic complementation was found within classes rib6 and rib7. No linkage between mutations of different complementation classes was detected.