Bacterial communities during the period of massive under-ice dinoflagellate development in Lake Baikal

Taxonomic diversity of Lake Baikal bacteria during the period of massive under-ice development of dinoflagellate Gymnodinium baicalense was studied. During the ice-covered period in 2013, both the abundance and biomass of G. baicalense were several orders of magnitude higher than the values for previous years, the maximum values were 8.9 × 10⁶ cells/L and 405 g/m³, respectively. The taxonomic structure of bacterial communities was determined using the data obtained by 454 pyrosequencing (Roche) with Mothur 1.19.0. Predominance of three phyla was revealed: Bacteroidetes, Proteobacteria, and Actinobacteria. Massive dinoflagellate development resulted in a considerable decrease in the richness and diversity of bacterial communities compared to the results of the earlier long-term studies.     

Comparison of bioethanol production from cultivated versus wild <Emphasis Type="Italic">Gracilaria verrucosa</Emphasis> and <Emphasis Type="Italic">Gracilaria gigas</Emphasis>

The seaweed genus Gracilaria is a potential candidate for the production of bioethanol due to its high carbohydrate content. Gracilaria is abundant throughout the world and can be found in both wild and cultivated forms. Differences in the ecological factors such as temperature, salinity, and light intensity affecting wild and cultivated specimens may influence the biochemical content of seaweeds, including the carbohydrate content. This study aimed to investigate the proximate composition and potential bioethanol production of wild and cultivated G. gigas and G. verrucosa. Bioethanol was produced using separate hydrolysis fermentation (SHF), employing a combination of enzymatic and acid hydrolysis, followed by fermentation with Saccharomyces cerevisiae ATCC 200062. The highest carbohydrate content was found in wild G. gigas. The highest galactose and glucose contents (20.21 ± 0.32 and 9.70 ± 0.49 g L^?1, respectively), as well as the highest production of bioethanol (3.56 ± 0.02 g L^?1), were also found in wild G. gigas. Thus, we conclude that wild G. gigas is the most promising candidate for bioethanol production. Further research is needed to optimize bioethanol production from wild G. gigas. Domestication of wild G. gigas is a promising challenge for aquaculture to avoid overexploitation of this wild seaweed resource.     

Rare triterpene glycoside of ginseng (ginsenoside malonyl-Rg<Subscript>1</Subscript>) detected in plant cell suspension culture of <Emphasis Type="Italic">Panax japonicus</Emphasis> var. <Emphasis Type="Italic">repens</Emphasis>

This paper reports for the first time about the detection and identification of ginsenoside malonyl-Rg₁ (the rare 20(S)-protopanaxatriol-type ginsenoside) in the biomass of plant cell suspension culture of Japanese ginseng (Panax japonicus C.A. Mey. var. repens). Ginsenosides were analyzed by means of high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC-ESI-MS) in positive-ion mode. Malonyl-Rg1 was identified as a result of interpretation of MS spectra obtained upon fragmentation of protonated molecular ion ([M + H]⁺) of this compound in an ionization source. Chromatographic analysis and MS spectra showed that the cells of P. japonicus var. repens cultivated in vitro contain several isomers of malonyl-Rg₁. Thus, we ascertained for the first time that, in addition to malonyl ginsenosides of 20(S)-protopanaxadiol group, the plant cell culture of ginseng P. japonicus var. repens can accumulate glycosides of 20(S)-protopanaxatriol group acylated with a malonic acid residue. The obtained results showed that, in the cells of ginseng cultivated in vitro for a long time (for 10 years and more), the assortment of secondary metabolites (ginsenosides) may be as wide as in intact plants.     

Molecular cytogenetic characterization of <Emphasis Type="Italic">Triticum timopheevii</Emphasis> chromosomes provides new insight on genome evolution of <Emphasis Type="Italic">T. zhukovskyi</Emphasis>

Triticum timopheevii (2n = 4x = 28, GGA^tA^t) is a tetraploid wheat formerly cultivated in western Georgia. The natural allopolyploid Triticum zhukovskyi is a hexaploid taxon originated from hybridization of T. timopheevii with cultivated einkorn T. monococcum (2n = 2x = 14, A^mA^m). Karyotypically T. timopheevii and T. zhukovskyi differ from other tetraploid and hexaploid wheats and were assigned to the section Timopheevii of the genus Triticum L. Triticum timopheevii and T. zhukovskyi are resistant to many fungal diseases and therefore could potentially be utilized for wheat improvement. We were aiming to precisely identify all T. timopheevii chromosomes and to trace the evolution of T. zhukovskyi. For this, we developed a set of molecular cytogenetic landmarks based on eleven DNA probes. Each chromosome can now be characterized by two to eight probes. The pTa-535 sequence allows the identification of all A^t-genome chromosomes, whereas G-genome and some A^t-genome chromosomes can be identified using (GAA/CTT) _n and pSc119.2 probes. The probes pAesp_SAT86, pAs1, Spelt-1, Spelt-52 and 5S and 45S rDNA can be applied as additional markers to discriminate particular chromosomes or chromosomal regions. The distribution of (GAA/CTT) _n , pTa-535 and pSc119.2 DNA probes on T. timopheevii chromosomes is distinct from other tetraploid wheats and can therefore be used to track individual chromosomes in introgression programs. Our study confirms the origin of T. zhukovskyi from hybridization of T. timopheevii with T. monococcum; however, we show that the emergence was accompanied by changes involving mostly A^t-genome chromosomes. This may be due to the presence of two closely related A-genomes in the T. zhukovskyi karyotype.     

Gas exchange,carbon balance and stomatal traits in wild and cultivated rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) genotypes

Carbon balancing within the plant species is an important feature for climatic adaptability. Photosynthesis and respiration traits are directly linked with carbon balance. These features were studied in 20 wild rice accessions Oryza spp., and cultivars. Wide variation was observed within the wild rice accessions for photosynthetic oxygen evolution or photosynthetic rate (A), dark (R _d), and light induced respiration (LIR) rates, as well as stomatal density and number. The mean rate of A varied from 10.49 μmol O₂ m^?2 s^?1 in cultivated species and 13.09 μmol O₂ m^?2 s^?1 in wild spp., The mean R _d is 2.09 μmol O₂ m^?2 s^?1 and 2.31 μmol O₂ m^?2 s^?1 in cultivated and wild spp., respectively. Light induced Respiration (LIR) was found to be almost twice in wild rice spp., (16.75 μmol O₂ m^?2 s^?1) compared to cultivated Oryza spp., Among the various parameters, this study reveals LIR and A as the key factors for positive carbon balance. Stomatal contribution towards carbon balance appears to be more dependent on abaxial surface where several number of stomata are situated. Correlation analysis indicates that R _d and LIR increase with the increase in A. In this study, O. nivara (CR 100100, CR 100097), O. rufipogon (IR 103404) and O. glumaepatula (IR104387) were identified as potential donors which could be used in rice breeding program. Co-ordination between gas exchange and patchiness in stomatal behaviour appears to be important for carbon balance and environmental adaptation of wild rice accessions, therefore, survival under harsh environment.     

Production of <Emphasis Type="Italic">trans</Emphasis>-10,<Emphasis Type="Italic">cis</Emphasis>-12-conjugated linoleic acid using permeabilized whole-cell biocatalyst of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Objective

To improve the production of trans-10,cis-12-conjugated linoleic acid (t10,c12-CLA) from linoleic acid in recombinant Yarrowia lipolytica.

Results

Cells of the yeast were permeabilized by freeze/thawing. The optimal conditions for t10,c12-CLA production by the permeabilized cells were at 28 °C, pH 7, 200 rpm with 1.5 g sodium acetate l^?1, 100 g wet cells l^?1, and 25 g LA l^?1. Under these conditions, the permeabilized cells produced 15.6 g t10,c12-CLA l^?1 after 40 h, with a conversion yield of 62 %. The permeabilized cells could be used repeatedly for three cycles, with the t10,c12-CLA extracellular production remaining above 10 g l^?1.

Conclusion

Synthesis of t10,c12-CLA was achieved using a novel method, and the production reported in this work is the highest value reported to date.

     

Polysaccharide-based silver nanoparticles synthesized by <Emphasis Type="Italic">Klebsiella oxytoca</Emphasis> DSM 29614 cause DNA fragmentation in <Emphasis Type="Italic">E. coli</Emphasis> cells

Silver nanoparticles (AgNPs), embedded into a specific exopolysaccharide (EPS), were produced by Klebsiella oxytoca DSM 29614 by adding AgNO₃ to the cultures during exponential growth phase. In particular, under aerobic or anaerobic conditions, two types of silver nanoparticles, named AgNPs-EPS^aer and the AgNPs-EPS^anaer, were produced respectively. The effects on bacterial cells was demonstrated by using Escherichia coli K12 and Kocuria rhizophila ATCC 9341 (ex Micrococcus luteus) as Gram-negative and Gram-positive tester strains, respectively. The best antimicrobial activity was observed for AgNPs-EPS^aer, in terms of minimum inhibitory concentrations and minimum bactericidal concentrations. Observations by transmission electron microscopy showed that the cell morphology of both tester strains changed during the exposition to AgNPs-EPS^aer. In particular, an electron-dense wrapped filament was observed in E. coli cytoplasm after 3 h of AgNPs-EPS^aer exposition, apparently due to silver accumulation in DNA, and both E. coli and K. rhizophila cells were lysed after 18 h of exposure to AgNPs-EPS^aer. The DNA breakage in E. coli cells was confirmed by the comparison of 3-D fluorescence spectra fingerprints of DNA. Finally the accumulation of silver on DNA of E. coli was confirmed directly by a significant Ag⁺ release from DNA, using the scanning electrochemical microscopy and the voltammetric determinations.     

Diversity of <Emphasis Type="Italic">A</Emphasis> mating type in <Emphasis Type="Italic">Lentinula edodes</Emphasis> and mating type preference in the cultivated strains

Diversity of A mating type in Lentinula edodes has been assessed by analysis of A mating loci in 127 strains collected from East Asia. It was discovered that hypervariable sequence region with an approximate length of 1 kb in the A mating locus, spanning 5′ region of HD2-intergenic region-5′ region of HD1, could represent individual A mating type as evidenced by comprehensive mating analysis. The sequence analysis revealed 27 A mating type alleles from 96 cultivated strains and 48 alleles from 31 wild strains. Twelve of them commonly appeared, leaving 63 unique A mating type alleles. It was also revealed that only A few A mating type alleles such as A1, A4, A5, and A7 were prevalent in the cultivated strains, accounting for 62.5% of all A mating types. This implies preferred selection of certain A mating types in the process of strain development and suggests potential role of A mating genes in the expression of genes governing mushroom quality. Dominant expression of an A mating gene HD1 was observed from A1 mating locus, the most prevalent A allele, in A1-containing dikaryons. However, connections between HD1 expression and A1 preference in the cultivated strains remain to be verified. The A mating type was highly diverse in the wild strains. Thirty-six unique A alleles were discovered from relatively small and confined area of mountainous region in Korean peninsula. The number will further increase because no A allele has been recurrently observed in the wild strains and thus newly discovered strain will have good chances to contain new A allele. The high diversity in small area also suggests that the A mating locus has evolved rapidly and thus its diversity will further increase.     

Interspecific variation in extracellular polysaccharide content and colony formation of <Emphasis Type="Italic">Microcystis</Emphasis> spp. cultured under different light intensities and temperatures

Single cells of five different Microcystis species (M. ichthyoblabe, M. viridis, M. flos-aquae, M. wesenbergii, and M. aeruginosa) were batch-cultured at different temperatures and light intensities: (a) 25 °C and 50 μmol photons m^?2 s^?1 (control culture); (b) 25 °C and 10 μmol photons m^?2 s^?1; and (c) 15 °C and 50 μmol photons m^?2 s^?1. The extracellular polysaccharide content was significantly higher in treatments b and c than in the control treatment. All Microcystis species existed as single cells under the control treatment but formed colonies in treatments b and c. All of the colonies were irregular with indistinct margins. M. ichthyoblabe, M. viridis, M. flos-aquae, and M. wesenbergii formed colonies with similar morphologies and their cells were loosely aggregated. In contrast, M. aeruginosa formed denser colonies with no distinct holes. The colony morphologies differed from the classic morphology of M. ichthyoblabe field-grown colonies but resembled that of small colonies found in Lake Taihu (Yangtze Delta Plain, China) during early spring. This indicates that field- and laboratory-grown colonies are governed by similar formation processes. We suggest that in laboratory and field environments, M. ichthyoblabe (or M. flos-aquae) colonies are representative of small colonies formed from single Microcystis cells, whereas the morphology of older colonies evolves to resemble M. wesenbergii and M. aeruginosa colonies.     

<Emphasis Type="Italic">Cytisus aeolicus</Emphasis> Guss. ex Lindl. <Emphasis Type="Italic">in vitro</Emphasis> cultures and genistin production

Cytisus aeolicus Guss. ex Lindl. (Fabaceae family, subfamily Faboideae) is an endangered endemic species of the Aeolian Islands, Sicily. In vitro multiplication of C. aeolicus shoots was described in this work and cell cultures were established from cotyledons and hypocotyls to investigate their potential production of isoflavones. Aseptically germinated seeds, cultivated on LS modified basal medium, gave the initial explants used both to induce axillary propagation and callus cultures. The LS (Linsmaier and Skoog) basal medium, supplemented with 0.1 mg L^?1 of 6-benzylaminopurine were used to induce axillary propagation. The callus induction was performed using the basal medium added with 5 mg L^?1 2,4-dichlorophenoxy acetic acid and 5 mg L^?1 kinetin (control medium). Basal medium was also added with 2000 mg L^?1 casein hydrolysate (CH) or 900 mg L^?1myo-inositol (MI). C. aeolicus callus cultures on CH and MI media produced an unique compound, the isoflavone genistein 7-O-ß-D-glucopyranoside (genistin), which has not previously been isolated from wild plants. Callus cultures grown on the medium containing myo-inositol produced the greatest amount of genistin. C. aeolicus tissue culture procedures could provide suitable plant material both for germplasm preservation (by micropropagation) and for biotechnological selective isoflavone production (by callus culture).     

Cloning of <Emphasis Type="Italic">rec</Emphasis>A gene of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> and phenotypic complementation of <Emphasis Type="Italic">Escherichia coli</Emphasis> recombinant deficient strain

Nucleotide and amino acid sequences of Corynebacterium glutamicum recA genes, from GenBank, were compared in silico. On the basis of the identity found between sequences, two degenerate primers were designed on the two sides of the deduced open reading frame (ORF) of the recA gene. PCR experiments, for amplifying the recA ORF region, were done. pGEM^®-T Easy vector was selected to be used for cloning PCR products. Then recA ORF was placed under the control of Escherichia coli hybrid trc promoter, in pKK388-1 vector. pKK388-1 vector, containing recA ORF, was transformed to E. coli DH5α ΔrecA (recombinant deficient strain), in an attempt to phenotypically complement it. Ultraviolet (u.v.) exposure experiments of the transformed and non-transformed E. coli DH5α ΔrecA cells revealed tolerance of transformed cells up to dose 0.24 J/cm², while non-transformed cells tolerated only up to dose 0.08 J/cm². It is concluded that phenotypic complementation of E. coli DH5α ΔrecA with recA ORF of C. glutamicum, could be achieved and RecA activity could be restored.     

Taxonomic composition of bacteria associated with cultivated mollusks <Emphasis Type="Italic">Crassostrea lugubris</Emphasis> and <Emphasis Type="Italic">Perna viridis</Emphasis> and with the water of the Gulf of Nha Trang lagoon,Vietnam

One hundred and four strains of heterotrophic bacteria have been isolated and characterized from two species of bivalve mollusks cultivated in the Gulf of Nha Trang (Vietnam) and from the water of a mariculture farm. The isolates have been identified on the basis of morphological, physiological, biochemical, and chemotaxonomic properties, as well as by the content of G+C bases in DNA. In the microflora of mollusks, Vibrio alginolyticus was predominant; the pathogenic species V. harveyi and V. splendidus were found as well. Staphylococci and bacilli occupied the second place in abundance after vibrios. In addition, coryneforms and enterobacteria, as well as Pseudomonas spp. and Pseudoalteromonas spp., were revealed. The composition of the water microflora was more diverse as compared with the microflora of mollusks. In the water, Bacillus spp., Vibrio spp., and Pseudomonas spp. were predominant. Brevibacterium spp. and other coryneform bacteria, as well as enterobacteria, occurred in significant amounts. In addition, Pseudoalteromonas spp., Marinococcus sp., Halobacillus sp., Shewanella sp., Sulfitobacter sp., and bacteria of the CFB cluster were noticed. The presence of pathogenic and conditionally pathogenic bacterial species in the water and mollusks is probably the reason for the high death rate of cultivated animals at the mariculture farm.     

The C-terminus of DSX<Superscript>F5</Superscript> protein acts as a novel regulatory domain in <Emphasis Type="Italic">Bombyx mori</Emphasis>

The doublesex gene regulates the somatic sexual development of Bombyx mori by alternatively splicing into sex-specific splice forms. In our previous study, the splice form Bmdsx ^F7 , which encodes the BmDSX^F5 protein, was found to be expressed in a female-specific manner and to contain a novel C-terminus. In this study, we aimed to investigate the role of this C-terminus. Two transgenic lines, L1 and L2, were constructed to ectopically express Bmdsx ^F7 in males. Phenotype and W chromosome-specific polymerase chain reaction (PCR) analysis showed that developmental abnormalities and sex reversal did not occur. Moreover, the sex ratio was also normal. Quantitative PCR revealed that the expression levels of SP1 and Vg were upregulated in the fat body of transgenic males. Additionally, the expression level of PBP was downregulated in the antenna of transgenic males. The results suggested that the C-terminus of BmDSX^F5 functioned as a regulatory domain during regulation of downstream target gene expression and that BmDSX^F5 participated in the sexual development of somatic cells together with other DSX proteins in B. mori.     

Inactivation of bone morphogenetic protein 1 (<Emphasis Type="Italic">Bmp1</Emphasis>) and tolloid-like 1 (<Emphasis Type="Italic">Tll1</Emphasis>) in cells expressing type I collagen leads to dental and periodontal defects in mice

Bone morphogenetic protein 1 (BMP1) and tolloid-like 1 (TLL1) belong to the BMP1/tolloid-like proteinase family, which cleaves secretory proteins. The constitutive deletion of the Bmp1 or Tll1 genes causes perinatal or embryonic lethality in mice. In this study, we first studied the β-galactosidase activity in mice in which an IRES-lacZ-Neo cassette was inserted in the intron of either the Bmp1 or the Tll1 gene; the β-galactosidase activities were used to reflect the expression of endogenous Bmp1 and Tll1, respectively. Our X-gal staining results showed that the odontoblasts in the tooth and cells in the periodontal ligament express both Bmp1 and Tll1. We then created Bmp1 ^flox/flox and Tll1 ^flox/flox mice by removing the IRES-lacZ-Neo cassette. By breeding 2.3 kb Col1a1-Cre mice with the Bmp1 ^flox/flox and Tll1 ^flox/flox mice, we further generated Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice in which both Bmp1 and Tll1 were inactivated in the Type I collagen-expressing cells. We employed X-ray radiography, histology and immunohistochemistry approaches to characterize the Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice. Our results showed that the molars of the Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice had wider predentin, thinner dentin and larger pulp chambers than those of the normal controls. The dentinal tubules of the molars in the Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice appeared disorganized. The level of dentin sialophosphoprotein in the molars of the 6-week-old Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice was lower than in the normal controls. The periodontal ligaments of the Col1a1-Cre;Bmp1 ^flox/flox ;Tll1 ^flox/flox mice were disorganized and had less fibrillin-1. Our findings indicate that the proteinases encoded by Bmp1 and Tll1 genes play essential roles in the development and maintenance of mouse dentin and periodontal ligaments.     

Protective Effects of <Emphasis Type="Italic">Mekabu</Emphasis> Aqueous Solution Fermented by <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Sanriku-SU7 on Human Enterocyte-Like HT-29-luc Cells and DSS-Induced Murine IBD Model

Most wakame Undaria pinnatifida, a brown algae, products are made from the frond portion. In this study, the polysaccharide content and antioxidant property of aqueous extract solutions (AESs) of the four parts (frond: wakame, stem of the frond: kuki-wakame, sporophyll: mekabu, and kuki-mekabu) of wakame were investigated. Polysaccharide content was high in both the wakame and mekabu. Superoxide anion (O₂ ^?) radical-scavenging capacities were high in the mekabu. These AESs could be fermented by Lactobacillus plantarum Sanriku-SU7. The O₂ ^? radical-scavenging activity of the kuki-wakame, mekabu, and kuki-mekabu were increased by the fermentation. Fermented mekabu clearly showed a protective effect on human enterocyte-like HT-29-luc cells and in a mouse model of dextran sodium sulphate-induced inflammatory bowel disease (IBD). These results suggest that the mekabu fermented by L. plantarum Sanriku-SU7 has anti-IBD effect related to O₂ ^? radical-scavenging.     

Laboratory and simulated-field bioassays for assessing mixed cultures of <Emphasis Type="Italic">Lysinibacillus sphaericus</Emphasis> against <Emphasis Type="Italic">Aedes aegypti</Emphasis> (Diptera: Culicidae) larvae resistant to temephos

Aedes aegypti (L.) is the main vector of tropical diseases such as dengue, chikungunya and Zika. Due to the overuse of insecticides, Ae. aegypti resistant populations have increased. Biological control with Lysinibacillus sphaericus (Ahmed) has been used against Culex sp. and Anopheles sp. Although Ae. aegypti is refractory to the binary toxin of L. sphaericus spores, vegetative cells have been shown to be effective against Ae. aegypti larvae. In this work, the effect of L. sphaericus vegetative cells on Ae. aegypti temephos-resistant larvae was assessed under lab and simulated field conditions. L. sphaericus caused about 90% mortality of insecticide-resistant Ae. aegypti larvae under simulated field conditions. Likewise, Ae. aegypti larvae were more sensitive to mixed cultures of L. sphaericus than to individual strains; then, the most effective mixed culture exhibited an LC₅₀ of 1.21 × 10⁵ CFU/mL with Rockefeller larvae and 8.04 × 10⁴ CFU/mL with field-collected larvae. Additionally, we found that mixed cultures composed of two L. sphaericus strains were more effective than a culture formed by the three strains. Our results suggest that mixed cultures comprising L. sphaericus vegetative cells could be useful for controlling temephos-resistant populations of Ae. aegypti, as evidenced by the effectiveness demonstrated under laboratory and simulated field conditions.     

The dependence of intracellular ATP level on the nutrition mode of the acidophilic bacteria <Emphasis Type="Italic">Sulfobacillus thermotolerans</Emphasis> and <Emphasis Type="Italic">Alicyclobacillus tolerans</Emphasis>

The dynamics of the ATP pool in the aerobic spore-forming acidothermophilic mixotrophic bacteria Sulfobacillus thermotolerans Kr1^T and Alicyclobacillus tolerans K1^T were studied in the course of their chemolithoheterotrophic, chemoorganoheterotrophic, and chemolithoautotrophic growth. It was established that, during mixotrophic growth, the maximum ATP concentrations in the cells of S. thermotolerans Kr1 and A. tolerans K1 were 3.8 and 0.6 nmol/mg protein, respectively. The ATP concentrations in sulfobacilli and alicyclobacilli during organotrophic growth were 2.2 and 3.1 nmol/mg protein, respectively. In the cells of the obligately heterotrophic bacterium Alicyclobacillus cycloheptanicus 4006^T, the maximum ATP concentration was several times higher and reached 12.3 nmol/mg protein. During lithotrophic growth, the maximum values of the ATP concentration in the cells of S. thermotolerans Kr1 and A. tolerans K1 were 0.3 and <0.1 nmol/mg protein, respectively; in the cells of the autotrophic bacterium Acidithiobacillus ferrooxidans TFBk, the ATP content was about 60–300 times higher (17.0 nmol/mg protein). It is concluded that low ATP content is among the possible causes of growth cessation of S. thermotolerans Kr1 and A. tolerans K1 under auto-and heterotrophic conditions after several culture transfers.     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) 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 b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. 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 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ 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 b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.