Invasive fungal infection caused by Blastobotrys mokoenaii in an immunocompromised patient with acute myeloid leukemia: A case report.

Blastobotrys is a genus of rare yeast that is increasingly recognized as a cause of fungal infections in humans. However, there have been no reports of fungal infections in humans caused by Blastobotrys mokoenaii. We describe a case of invasive fungal infection (IFI) caused by B. mokoenaii in an immunocompromised patient with acute myeloid leukemia (AML). A 46-year-old man with relapsed/refractory AML underwent a second allogeneic peripheral blood hematopoietic stem cell transplantation (allo-PBSCT) during remission. The patient had prolonged neutropenia and received systemic steroid therapy for graft-versus-host disease before the second allo-PBSCT. Uncommon yeast was isolated from the blood cultures obtained on day 4. We initially suspected that the uncommon yeast was Trichosporon spp. based on its morphology. However, unlike Trichosporon spp., in vitro antifungal susceptibility tests showed that this yeast isolate was resistant to micafungin, caspofungin, voriconazole, itraconazole, and fluconazole. We performed DNA sequencing and identified it as B. mokoenaii. B. mokoenaii was persistently isolated from blood cultures taken during combination therapy with liposomal amphotericin B and voriconazole. The patient died of multiorgan failure on day 24. B. mokoenaii can cause severe IFI in immunocompromised patients; however, it may not be correctly identified by routine clinical microbiology testing in a hospital laboratory and DNA sequencing is useful for diagnosis.

Characterization of a new Blastobotrys navarrensis strain indicates that it is not a later synonym of Blastobotrys proliferans.

The species Blastobotrys navarrensis Sesma and Ramirez was delineated based on the description of the single strain CBS 139.77T. Based on its phenotypic similarities to Blastobotrys proliferans, B. navarrensis CBS 139.77T was later considered a synonym of B. proliferans. In the present study, we isolated the yeast strain IST 508 (=PYCC 8784=CBS 16671) from the soil surrounding an olive tree in Ferreira do Alentejo, Portugal. The phylogenetic analysis of D1/D2 domain and ITS sequences from strain IST 508 indicates that is closely related to B. navarrensis and B. proliferans. Although strain IST 508 differs from B. navarrensis CBS 139.77T by 14 substitutions and 20 indels (6.6 % divergence) in the ITS sequence, no divergence was detected at the level of D1/D2 domain, mitochondrial small subunit rDNA, and cytochrome oxidase II sequences. On the other hand, strains IST 508 and CBS 139.77 differ from B. proliferans NRRL Y-17577T by eight substitutions (1.4 % divergence) in the D1/D2 domain sequence, by 16 substitutions (2.7 % divergence) in the cytochrome oxidase II sequence, and by 16 substitutions (3.7 % divergence) in the mitochondrial small subunit rDNA sequence. Due to the high number of variable phenotypic tests in B. proliferans and B. navarrensis, strains from the two species are difficult to distinguish. Contrasting with what is described for other Blastobotrys species, no differences were detected at the level of micromorphology between the two species. Nevertheless, based on the molecular differences between the two strains, CBS 139.77 and IST 508, and B. proliferans NRRL Y-17577T and their phylogenetic analysis, strains CBS 139.77 and IST 508 are from B. navarrensis and this species should be considered as an independent species and not a later synonym of B. proliferans. We propose an emended description of B. navarrensis.

Proteogenomics Study of Blastobotrys adeninivorans TMCC 70007-A Dominant Yeast in the Fermentation Process of Pu-erh Tea.

Blastobotrys adeninivorans plays an essential role in pile-fermenting of Pu-erh tea. Its ability to assimilate various carbon and nitrogen sources makes it available for application in a wide range of industry sectors. The genome of B. adeninivorans TMCC 70007 isolated from pile-fermented Pu-erh tea was sequenced and assembled. Proteomics analysis indicated that 4900 proteins in TMCC 70007 were expressed under various culture conditions. Proteogenomics mapping revealed 48 previously unknown genes and corrected 118 gene models predicted by GeneMark-ES. Ortho-proteogenomics analysis identified 17 previously unidentified genes in B. adeninivorans LS3, the first strain with a sequenced genome among the genus Blastobotrys as well. More importantly, five species specific genes were identified from TMCC 70007, which could serve as a barcode for strain typing and were applicable for fermentation process protection of this industrial species. The datasets generated from tea aqueous extract culture not only increased the proteome coverage and accuracy but also contributed to the identification of proteins related to polyphenols and caffeine, which were considered to change greatly during the microbial fermentation of Pu-erh tea. This study provides a proteome perspective on TMCC 70007, which was considered to be an important strain in the production of Pu-erh tea. The systematic proteogenomics analysis not only made a better annotation on the genome of B. adeninivorans TMCC 70007 as previous proteogenomics study but also provided solution for fermentation process protection on valuable industrial species with species specific genes uniquely identified from proteogenomics study.

Yeasts of the Blastobotrys genus are promising platform for lipid-based fuels and oleochemicals production.

Strains of the yeast genus Blastobotrys (subphylum Saccharomycotina) represent a valuable biotechnological resource for basic biochemistry research, single-cell protein, and heterologous protein production processes. Species of this genus are dimorphic, non-pathogenic, thermotolerant, and can assimilate a variety of hydrophilic and hydrophobic substrates. These can constitute a single-cell oil platform in an emerging bio-based economy as oleaginous traits have been discovered recently. However, the regulatory network of lipogenesis in these yeasts is poorly understood. To keep pace with the growing market demands for lipid-derived products, it is critical to understand the lipid biosynthesis in these unconventional yeasts to pinpoint what governs the preferential channelling of carbon flux into lipids instead of the competing pathways. This review summarizes information relevant to the regulation of lipid metabolic pathways and prospects of metabolic engineering in Blastobotrys yeasts for their application in food, feed, and beyond, particularly for fatty acid-based fuels and oleochemicals. KEY POINTS: • The production of biolipids by heterotrophic yeasts is reviewed. • Summary of information concerning lipid metabolism regulation is highlighted. • Special focus on the importance of diacylglycerol acyltransferases encoding genes in improving lipid production is made.

Pulmonary infection secondary to Blastobotrys raffinosifermentans in a cystic fibrosis patient: Review of the literature.

BACKGROUND: The genus Blastobotrys consists of at least 20 species. Disease in humans has been reported with B adeninivorans, B raffinosifermentans, B proliferans and B serpentis, mostly in immunocompromised patients and those with cystic fibrosis. OBJECTIVE: We report a lung infection secondary to B raffinosifermentans in a cystic fibrosis patient successfully treated with isavuconazole and review the literature of invasive infections caused this genus. We also evaluated clinical isolates in our laboratory for species identification and antifungal susceptibility. METHODS: Phylogenetic analysis was performed on a collection of 22 Blastobotrys isolates in our reference laboratory, and antifungal susceptibility patterns were determined for nine clinically available antifungals against 19 of these isolates. RESULTS: By phylogenetic analysis, 21 of the 22 isolates in our collection were identified as B raffinosifermentans and only 1 as B adeninivorans. Most were cultured from the respiratory tract, although others were recovered from other sources, including CSF and blood. Isavuconazole, caspofungin and micafungin demonstrated the most potent in vitro activity, followed by amphotericin B. In contrast, fluconazole demonstrated poor activity. The patient in this case responded to isavuconazole treatment for breakthrough infection due to B raffinosifermentans that was cultured from pleural fluid while on posaconazole prophylaxis post-bilateral lung transplantation for cystic fibrosis. CONCLUSIONS: Blastobotrys species are rare causes of infections in humans and primarily occur in immunocompromised hosts. In our collection, the majority of isolates were identified as B raffinosifermentans. To our knowledge, this is the first report of successful treatment of such an infection with isavuconazole.

Anti-quorum sensing and antibiofilm activities of Blastobotrys parvus PPR3 against Pseudomonas aeruginosa PAO1.

The bacterial cell communication also termed as Quorum sensing (QS) system was involved in the expression of several virulence traits during Pseudomonas infection. The attenuating of this bacterial cell communication system is an attractive approach for the management of bacterial infections without the complication of resistance development. In this respect, the marine environment has gained significant attention due to its biodiversity and as a source of novel bioactive compounds. The present study aimed to screening effective QS inhibitors from marine associated fungal species for QS inhibitors. Twelve morphologically distinct fungal isolates were isolated from the wood of Avicennia marina from marine ecosystem. The anti-QS potential of fungal crude extract from was investigated in biosensor strain and test bacterium, Chromobacterium violaceum and Pseudomonas aeruginosa PAO1, respectively. Promising anti-QS activity was observed in the crude extract of one of the fungal isolate and identified by molecular characterization using internal transcribed spacer (ITS) region as Blastobotrys parvus PPR3. The anti-virulence and antibiofilm effects of ethyl acetate fractions from PPR3 against P. aeruginosa PAO1 were evaluated. The fungal metabolites responsible for the anti-QS activity of fungal crude extract was identified using gas chromatography-mass spectrometry (GC-MS). Furthermore, molecular docking studies were performed to understand the interaction of bioactive compounds with as receptors of P. aeruginosa PAO1. The crude extract of PPR3 showed reduction in different virulence traits of P. aeruginosa PAO1 such as production of pyocyanin, elastase, protease, chitinase, swimming and swarming motility, biofilm formation, exopolysaccharide production and alginate production at different sub-MIC concentrations. Interaction of bioactive metabolites with LasR and RhlR receptors of P. aeruginosa PAO1 was reported. The findings of the present study suggested that metabolites of B. parvus PPR3 interfere with QS system of P. aeruginosa PAO1 and alters the production of virulence factors.

Blastobotrys baotianmanensis sp. nov. and Blastobotrys xishuangbannaensis f.a., sp. nov., two novel yeast species associated with insects and rotting wood.

Five yeast strains were isolated from the gut of the groundbeetle Pterostichus gebleri and rotting wood, which were collected from two different localities in China. These strains were identified as representing two novel species of the genus Blastobotrys through comparison of sequences in the D1/D2 domains of the LSU rRNA gene and other taxonomic characteristics. Blastobotrys baotianmanensis sp. nov. produces two to three spherical ascospores per ascus, and is most closely related to the type strains of B. elegans, B. capitulata, B. arbuscula, and an undescribed species represented by strain BG02-7-20-006A-3-1. Blastobotrys baotianmanensis sp. nov. differed from these strains by 3.6-8.4 % divergence (21-46 substitutions and 0-4 gaps) in the D1/D2 sequences. Blastobotrys xishuangbannaensis f.a., sp. nov. is closely related to B. nivea, B. elegans and B. aristata but the formation of ascospores was not observed on various sporulation media, and it differed from its relatives by 6.2-8.5 % divergence (34-43 substitutions and 2-6 gaps) in the D1/D2 sequences. The holotype of Blastobotrys baotianmanensis sp. nov. is NYNU 1581 and the holotype of Blastobotrys xishuangbannaensis f.a., sp. nov. is NYNU 181030.

Blastobotrys bombycis sp. nov., a d-xylose-fermenting yeast isolated from the gut of the silkworm larva Bombyx mori.

The gut of insects harbors a yeast community that is still poorly understood. Here, a novel species of the ascomycetous genus Blastobotrys is proposed based on a yeast strain isolated from the larval gut of the silkworm Bombyx mori (Order Lepidoptera). The novel species is closely related to Blastobotrys aristata and Blastobotrys elegans on the basis of the results of molecular phylogenetic analyses. A preliminary screening revealed that it produces 1.5 g l-1 ethanol by fermenting 5 % d-xylose. The novel species, that represents the first report, to our knowledge, of yeast isolation from silkworms, is described as Blastobotrys bombycis sp. nov. (type strain RAAB001T=CBS 15274T=PYCC 8105T=MCC 1427T; MycoBank accession number MB 825095).

Blastobotrys persicus sp. nov., an ascomycetous yeast species isolated from cave soil.

Two strains (AHD129-1T and AHD129-2) of a new anamorphic yeast species were isolated from Mejare cave soil samples of Abdanan, Ilam, Iran. Nucleotide divergence in the D1/D2 domain of the large subunit (LSU) rRNA, and internal transcribed spacer (ITS) genes suggest that the two strains can be assigned to the Trichomonascus/Blastobotrys clade. A maximum likelihood tree based on sequences of the D1/D2 domain revealed that the new species is closely related to the species Trichomonascus ciferrii, Candida allociferrii, and Candida mucifera. The new species could be distinguished from the closely related species by its ability to grow at 42 °C and the inability to assimilate D-arabinose and D-mannitol. The name B. persicus sp. nov. is proposed for the new anamorphic species. The type strain of B. persicus is AHD129-1T = IBRC-M30238T = CBS 14259T, and the Mycobank number is MB 819148.

Biosynthesis of pyruvic acid from glucose by Blastobotrys adeninivorans.

The ability of taxonomically different yeasts to synthesize pyruvic acid (PA) from glucose was studied. The study showed that many yeasts are able to produce PA from glucose under the condition of growth limitation by thiamine. This ability was found in the yeast Blastobotrys adeninivorans for the first time. The production (oversynthesis) of PA in this yeast can be explained by disturbance in the function of thiamine-dependent pyruvate dehydrogenase. Namely, the partial inhibition of this enzyme brings about the excretion of PA from the yeast cells. Due to incomplete inhibition of pyruvate dehydrogenase, the formation of acetyl-CoA continues, although at a lower level, maintaining the synthesis of α-ketoglutaric acid (KGA) in the tricarboxylic acid (TCA) cycle. KGA is no longer oxidized in the TCA cycle, because thiamine limitation inhibits α-ketoglutarate dehydrogenase. As a result, KGA is excreted from the yeast cells as a byproduct of PA oversynthesis. Furthermore, the increased level of KGA in the yeast cells inhibits NAD-dependent isocitrate dehydrogenase in the TCA cycle and enhances the production and excretion of citric acid, another byproduct of PA oversynthesis. During cultivation in a fermentor, the strain Blastobotrys adeninivorans VKM Y-2677 produced 43.2 g l(-1) PA from glucose with a product yield (YPA) of 0.77 g PA/g glucose. The proportion of PA to byproducts was 18:1 for KGA and 8:1 for citric acid.

Novel polyurethane-degrading cutinase BaCut1 from Blastobotrys sp. G-9 with potential role in plastic bio-recycling.

Environmental pollution caused by plastic waste has become global problem that needs to be considered urgently. In the pursuit of a circular plastic economy, biodegradation provides an attractive strategy for managing plastic wastes, whereas effective plastic-degrading microbes and enzymes are required. In this study, we report that Blastobotrys sp. G-9 isolated from discarded plastic in landfills is capable of depolymerizing polyurethanes (PU) and poly (butylene adipate-co-terephthalate) (PBAT). Strain G-9 degrades up to 60% of PU foam after 21 days of incubation at 28 â„ƒ by breaking down carbonyl groups via secretory hydrolase as confirmed by structural characterization of plastics and degradation products identification. Within the supernatant of strain G-9, we identify a novel cutinase BaCut1, belonging to the esterase family, that can reproduce the same effect. BaCut1 demonstrates efficient degradation toward commercial polyester plastics PU foam (0.5 mg enzyme/25 mg plastic) and agricultural film PBAT (0.5 mg enzyme/10 mg plastic) with 50% and 18% weight loss at 37 â„ƒ for 48 h, respectively. BaCut1 hydrolyzes PU into adipic acid as a major end-product with 42.9% recovery via ester bond cleavage, and visible biodegradation is also identified from PBAT, which is a beneficial feature for future recycling economy. Molecular docking, along with products distribution, elucidates a special substrate-binding modes of BaCut1 with plastic substrate analogue. BaCut1-mediated polyester plastic degradation offers an alternative approach for managing PU plastic wastes through possible bio-recycling.

Acceptor properties of "carbon nanotubes-redox-active polymer based on bovine serum albumin modified with ferrocenecarboxaldehyde" composite for creating a BOD biosensor with Blastobotrys adeninivorans BKM Y-2677 yeast.

The possibility of using a composite material based on bovine serum albumin (BSA) covalently bonded with ferrocenecarboxaldehyde and containing carbon nanotubes (CNT) for the immobilization of Blastobotrys adeninivorans BKM Y-2677 (B. adeninivorans) yeast is discussed. The optimal ratio of ferrocenecarboxaldehyde to BSA for the redox-active polymer synthesis is 1:2, since the heterogeneous electron transfer constant is 0.45 ± 0.01 s-1. When carbon nanotubes (CNTs) are added to this polymer, the heterogeneous electron transfer constant increases: at a CNT specific density of 2.5 µg/mm2, it reaches a maximum value of 0.55 ± 0.01 s-1. The addition of CNTs into the conducting system leads to increasing of the rate constant of interaction redox species with B. adeninivorans yeast by an order: the rate constant of interaction between B. adeninivorans yeast and electroactive particles in a redox-active polymer is 0.056 ± 0.005 dm3/g × s and in a composite material based on CNTs is 0.51 ± 0.02 dm3/g × s. The yeast specific density at the electrode of 0.1 mg/mm2 and electrolyte pH of 6.2 was chosen as the working value for the receptor system operation. Immobilized in a composite material, yeast oxidizes a wider range of substrates compared with a similar receptor element based on the ferrocene mediator. The biosensors formed on the basis of hybrid polymers have a high sensitivity with a lower limit of determined concentrations of 1.5 mg/dm3 with an assay time of 5 min and a high correlation (R = 0.9945) with the results of the standard method for determining biochemical oxygen demand (BOD) in nine real surface water samples of the Tula region.

A Two-Mediator System Based on a Nanocomposite of Redox-Active Polymer Poly(thionine) and SWCNT as an Effective Electron Carrier for Eukaryotic Microorganisms in Biosensor Analyzers.

Electropolymerized thionine was used as a redox-active polymer to create a two-mediated microbial biosensor for determining biochemical oxygen demand (BOD). The electrochemical characteristics of the conducting system were studied by cyclic voltammetry and electrochemical impedance spectroscopy. It has been shown that the most promising in terms of the rate of interaction with the yeast B. adeninivorans is the system based on poly(thionine), single-walled carbon nanotubes (SWCNT), and neutral red (kint = 0.071 dm3/(g·s)). The biosensor based on this system is characterized by high sensitivity (the lower limit of determined BOD concentrations is 0.4 mgO2/dm3). Sample analysis by means of the developed analytical system showed that the results of the standard dilution method and those using the biosensor differed insignificantly. Thus, for the first time, the fundamental possibility of effectively using nanocomposite materials based on SWCNT and the redox-active polymer poly(thionine) as one of the components of two-mediator systems for electron transfer from yeast microorganisms to the electrode has been shown. It opens up prospects for creating stable and highly sensitive electrochemical systems based on eukaryotes.

Cutinase ACut2 from Blastobotrysraffinosifermentans for the Selective Desymmetrization of the Symmetric Diester Diethyl Adipate to the Monoester Monoethyl Adipate.

Monoethyl adipate (MEA) is a highly valuable monoester for activating resistance mechanisms and improving protective effects in pathogen-attacked plants. The cutinase ACut2 from the non-conventional yeast Blastobotrys (Arxula) raffinosifermentans (adeninivorans) was used for its synthesis by the desymmetrization of dicarboxylic acid diester diethyl adipate (DEA). Up to 78% MEA with 19% diacid adipic acid (AA) as by-product could be synthesized by the unpurified ACut2 culture supernatant from the B. raffinosifermentans overexpression strain. By adjusting pH and enzyme concentration, the selectivity of the free ACut2 culture supernatant was increased, yielding 95% MEA with 5% AA. Selectivity of the carrier immobilized ACut2 culture supernatant was also improved by pH adjustment during immobilization, as well as carrier enzyme loading, ultimately yielding 93% MEA with an even lower AA concentration of 3-4%. Thus, optimizations enabled the selective hydrolysis of DEA into MEA with only a minor AA impurity. In the up-scaling, a maximum of 98% chemical and 87.8% isolated MEA yield were obtained by the adsorbed enzyme preparation with a space time yield of 2.6 g L-1 h-1. The high monoester yields establish the ACut2-catalyzed biosynthesis as an alternative to existing methods.

Development of a Vector Set for High or Inducible Gene Expression and Protein Secretion in the Yeast Genus Blastobotrys.

Converting lignocellulosic biomass into value-added products is one of the challenges in developing a sustainable economy. Attempts to engineer fermenting yeasts to recover plant waste are underway. Although intensive metabolic engineering has been conducted to obtain Saccharomyces cerevisiae strains capable of metabolising pentose sugars mainly found in hemicellulose, enzymatic hydrolysis after pretreatment is still required. Blastobotrys raffinosifermentans, which naturally assimilates xylose and arabinose and displays numerous glycoside hydrolases, is a good candidate for direct and efficient conversion of renewable biomass. However, a greater diversity of tools for genetic engineering is needed. Here, we report the characterisation of four new promising promoters, a new dominant marker, and two vectors for the secretion of epitope tagged proteins along with a straightforward transformation protocol. The TDH3 promoter is a constitutive promoter stronger than TEF1, and whose activity is maintained at high temperature or in the presence of ethanol. The regulated promoters respond to high temperature for HSP26, gluconeogenic sources for PCK1 or presence of xylose oligomers for XYL1. Two expression/secretion vectors were designed based on pTEF1 and pTDH3, two endogenous signal peptides from an α-arabinanase and an α-glucuronidase, and two epitopes. A heterologous α-arabinoxylan hydrolase from Apiotrichum siamense was efficiently secreted using these two vectors.

A new lipase (Alip2) with high potential for enzymatic hydrolysis of the diester diethyladipate to the monoester monoethyladipate.

Several putative lipase genes from the genome of the yeast Blastobotrys (Arxula) raffinosifermentans (adeninivorans) LS3 were overexpressed in the yeast itself and screened for the desymmetrization of the dicarboxylic acid diester diethyl adipate (DEA) into the monoester monoethyl adipate (MEA). MEA can serve as a monomeric spacer group for functional polymers used in medical chemistry and dental applications. The selected lipase Alip2-c6hp was intracellularly located. After overexpression of the corresponding gene, it was purified and biochemically characterized using p-nitrophenyl butyrate as the substrate for standard activity tests. In fed-batch cultivation with constructed yeast strain B. raffinosifermentans G1212/YRC102-Alip2-c6h for large scale production of the Alip2-c6hp biocatalyst enzyme activities up to 674 U L-1 were reached. Several tested diesters were hydrolyzed selectively to monoesters. Under optimized conditions, the purified enzyme Alip2p-c6h converted 96 % of the substrate DEA to MEA within 30 min incubation, whereby only 1.6 % of the unwanted side-product adipic acid (AA) was formed. At room temperature the dicarboxylic acid esters diethyl malonate (DEM), diethyl succinate (DES), dimethyl adipate (DMA) and dimethyl suberate (DMSub) were completely hydrolyzed to their corresponding monoesters. A high yield of 87 % and 25 % could also be achieved with the dioldiesters 1,4-diacetoxybutane (DAB) and diacetoxyhexane (DAH). In conclusion the potential of the lipase Alip2-c6hp expressed in B. raffinosifermentans is very promising for selective hydrolysis of DEA to MEA as well as for the production of other monoesters.

Characterization of Catechol-1,2-Dioxygenase (Acdo1p) From Blastobotrys raffinosifermentans and Investigation of Its Role in the Catabolism of Aromatic Compounds.

Gallic acid, protocatechuic acid, catechol, and pyrogallol are only a few examples of industrially relevant aromatics. Today much attention is paid to the development of new microbial factories for the environmentally friendly biosynthesis of industrially relevant chemicals with renewable resources or organic pollutants as the starting material. The non-conventional yeast, Blastobotrys raffinosifermentans, possesses attractive properties for industrial bio-production processes such as thermo- and osmotolerance. An additional advantage is its broad substrate spectrum, with tannins at the forefront. The present study is dedicated to the characterization of catechol-1,2-dioxygenase (Acdo1p) and the analysis of its function in B. raffinosifermentans tannic acid catabolism. Acdo1p is a dimeric protein with higher affinity for catechol (K M = 0.004 ± 0.001 mM, k cat = 15.6 ± 0.4 s-1) than to pyrogallol (K M = 0.1 ± 0.02 mM, k cat = 10.6 ± 0.4 s-1). It is an intradiol dioxygenase and its reaction product with catechol as the substrate is cis,cis-muconic acid. B. raffinosifermentans G1212/YIC102-AYNI1-ACDO1-6H, which expresses the ACDO1 gene under the control of the strong nitrate-inducible AYNI1 promoter, achieved a maximum catechol-1,2-dioxygenase activity of 280.6 U/L and 26.9 U/g of dry cell weight in yeast grown in minimal medium with nitrate as the nitrogen source and 1.5% glucose as the carbon source. In the same medium with glucose as the carbon source, catechol-1,2-dioxygenase activity was not detected for the control strain G1212/YIC102 with ACDO1 expression under the regulation of its respective endogenous promoter. Gene expression analysis showed that ACDO1 is induced by gallic acid and protocatechuic acid. In contrast to the wild-type strain, the B. raffinosifermentans strain with a deletion of the ACDO1 gene was unable to grow on medium supplemented with gallic acid or protocatechuic acid as the sole carbon source. In summary, we propose that due to its substrate specificity, its thermal stability, and its ability to undergo long-term storage without significant loss of activity, B. raffinosifermentans catechol-1,2-dioxygenase (Acdo1p) is a promising enzyme candidate for industrial applications.

A kinetic approach to the formation of two-mediator systems for developing microbial biosensors as exemplified by a rapid biochemical oxygen demand assay.

This work proposes a method of forming a microorganism-mediator(s) receptor system, in which the rates of separate stages of mediator bioelectrocatalysis are used as the basis for the development of biosensors for the biochemical oxygen demand (BOD) rapid assay. In the presence of a ferrocene mediator, the yeast Blastobotrys adeninivorans was shown to enable oxidation of a larger range of substrates as compared with other investigated microorganisms-bacteria Escherichia coli and yeast Ogataea polymorpha. The rate constants of the interaction of the yeast B. adeninivorans with nine compounds, electron transfer mediators, were determined; the best mediator for these microorganisms was found to be neutral red (k int = 0.681 ± 0.009 dm3/g s). Neutral red possesses a high rate of interaction with the ferrocene mediator (14,200 ± 100 dm3/mol s) shown earlier to be the most promising acceptor of electrons at a carbon paste electrode (0.4 ± 0.1 cm/s). These features enabled the formation of a two-mediator ferrocene-neutral red system to be used in a biosensor. A two-mediator-based biosensor had a higher sensitivity (the lower limit of detected BOD concentrations, 0.16 mg/dm3) than that of a one-mediator system based on neutral red and ferrocene. Analysis of ten samples from surface water reservoirs showed the combination of ferrocene, neutral red and the yeast B. adeninivorans to enable the data that highly correlated (R = 0.9693) with those of the standard method.

Towards Biological Control of Aspergillus carbonarius and Botrytis cinerea in Grapevine Berries and Transcriptomic Changes of Genes Encoding Pathogenesis-Related (PR) Proteins.

Grapevine bunch rot, caused by Botrytis cinerea and Aspergillus carbonarius, causes important economic losses every year in grape production. In the present study, we examined the plant protective activity of the biological control agents, Paenibacillus alvei K165, Blastobotrys sp. FP12 and Arthrobacter sp. FP15 against B. cinerea and A. carbonarius on grapes. The in vitro experiments showed that strain K165 significantly reduced the growth of both fungi, while FP15 restricted the growth of A. carbonarius and FP12 was ineffective. Following the in vitro experiments, we conducted in planta experiments on grape berries. It was shown that K165, FP12 and FP15 reduced A. carbonarius rot severity by 81%, 57% and 37%, respectively, compared to the control, whereas, in the case of B. cinerea, the only protective treatment was that with K165, which reduced rot by 75%. The transcriptomic analysis of the genes encoding the pathogenesis-related proteins PR2, PR3, PR4 and PR5 indicates the activation of multiple defense responses involved in the biocontrol activity of the examined biocontrol agents.

Optimization of Culture Medium for the Growth of Candida sp. and Blastobotrys sp. as Starter Culture in Fermentation of Cocoa Beans (Theobroma cacao) Using Response Surface Methodology (RSM).

BACKGROUND AND OBJECTIVE: Inoculation of starter culture in cocoa bean fermentation produces consistent, predictable and high quality of fermented cocoa beans. It is important to produce healthy inoculum in cocoa bean fermentation for better fermented products. Inoculum could minimize the length of the lag phase in fermentation. The purpose of this study was to optimize the component of culture medium for the maximum cultivation of Candida sp. and Blastobotrys sp. MATERIALS AND METHODS: Molasses and yeast extract were chosen as medium composition and Response Surface Methodology (RSM) was then employed to optimize the molasses and yeast extract. RESULTS: Maximum growth of Candida sp. (7.63 log CFU mL-1) and Blastobotrys sp. (8.30 log CFU mL-1) were obtained from the fermentation. Optimum culture media for the growth of Candida sp., consist of 10% (w/v) molasses and 2% (w/v) yeast extract, while for Blastobotrys sp., were 1.94% (w/v) molasses and 2% (w/v) yeast extract. CONCLUSION: This study shows that culture medium consists of molasses and yeast extract were able to produce maximum growth of Candida sp. and Blastobotrys sp., as a starter culture for cocoa bean fermentation.