Enrico Berardi

Pichia angusta is an effective biocontrol yeast against postharvest decay of apple fruit caused by Botrytis cinerea and Monilia fructicola

The efficacy of eight isolates of Pichia angusta against three common postharvest pathogens of apple fruit was evaluated for the first time. All tested strains showed significant biocontrol activity against both Botrytis cinerea and Monilia fructicola, whereas efficacy against Penicillium expansum was poor. A leucine-auxotrophic mutant had no significant biocontrol activity against brown rot of apple, while the addition of 0.6-1.2 g L(-1) leucine in the fruit wound fully restored the biocontrol activity of this mutant against M. fructicola. Given the extremely well-developed classical and molecular genetics, the availability of genomic libraries, and its complete genomic sequence, this species can serve to elucidate the mechanisms related to biocontrol capacity.

Evolution of sourdough microbiota in spontaneous sourdoughs started with different plant materials.

The preparation of sourdough in bakeries may include the use of inocula, e.g. fruits, flowers or rumen cuts to accelerate the process of selection of suitable microorganisms. The aim of this work was to investigate the effect of these inocula on the microbial evolution in sourdoughs. First, the microbiota of nineteen traditional sourdoughs that were initially started with diverse inocula was identified. Second, de novo sourdoughs were started with plant materials and the evolution of sourdough microbiota was investigated by culture, and by high-resolution melting curve quantitative PCR (HRM-qPCR). This study developed a new protocol for HRM-qPCR analysis of yeast microbiota in sourdough, and indicates this independent culture method suitable for characterization of yeasts. Microbiota of traditional sourdoughs were largely independent from the use of inoculum, however, Acetobacter spp. were identified only in sourdoughs started with apple flowers or apple pulp. In de novo sourdoughs started with plant materials, microbiota rapidly stabilized, and were characterized by Lactobacillus sanfranciscensis, Lactobacillus plantarum, Lactobacillus graminis, or Lactobacillus rossiae, and Saccharomyces cerevisiae as dominant species. Competition experiments revealed that the ecological fitness of L. plantarum, L. graminis, and L. rossiae in wheat or rye malt sourdoughs was lower when compared to L. sanfranciscensis, demonstrating that their presence in de novo sourdoughs reflects dispersal limitation. In conclusion, establishment of microbiota in de novo sourdoughs is dispersal limited. This study provides scientific support for the artisanal practice to inoculate de novo sourdoughs with flowers, berries, or related plant material.

Microbiological characterisation and volatiles profile of model, ex-novo, and traditional Italian white wheat sourdoughs

The interplay of sourdough microbiology and generated volatile compounds that define its sensory characteristics was studied. In order to detail the flavour generating potential of microorganisms, eight single-strain dough fermentations were studied, four of them never investigated before. Moreover, for the first time, both ex-novo and traditional wheat sourdoughs were investigated and compared to chemically acidified dough. HS-SPME-GC-MS was used to sample and analyse volatile compounds, some of which have never been detected before in sourdoughs. Alcohols, esters, carbonyl compounds, and acids mainly characterised the volatile profiles. Different sourdough microbiota resulted in different volatile profiles. PCA indicated that samples could be clustered according to their specific microbiota. Production of aroma compounds was strain-specific, confirming previous findings. This study can contribute to the management of desirable features and differentiate specialty products, as well as selecting new, suitable, sourdoughs after microbial screening.

Mutations affecting the expression of the MOX gene encoding peroxisomal methanol oxidase in Hansenula polymorpha.

Abstract In this study, aimed at identifying genetic factors acting positively upon the MOX gene, we report the isolation and characterisation of several methanol utilisation-defective (Mut−) mutants of Hansenula polymorpha. These fall into 12 complementation groups, eight of which show significant reductions in alcohol (methanol) oxidase activity in methanol. Three of these groups, identifying the MUT3, MUT5 and MUT10 loci, exhibit extremely low levels of MOX promoter activity, not only in methanol medium, but also during growth in glycerol or methylamine. We suggest that these loci play a significant role in the derepression of the MOX gene expression. One of these genes (MUT10) also seems to be involved in the utilisation of carbon sources other than methanol, and it is apparent that the same gene plays some role in the biogenesis or in the enlargement of the peroxisome. Three other genes (MUT7, MUT8 and MUT9) appear to be involved in peroxisome biogenesis, whereas most other mutants harbour lesions that leave the peroxisome biogenesis and proliferation unaffected.

Nitrate reduction and the isolation of Nit- mutants in Hansenula polymorpha.

Hansenula polymorpha (syn. Pichia angusta) is able to grow on nitrate as sole nitrogen source. Nitrate reductase (NR) assays, optimized in crude extracts from nitrate-grown cells, revealed that NR preferentially used NADPH, but also used NADH, as electron donor and required FAD for maximum activity. NR activity was present in nitrate-grown and nitrite-grown cells, and was absent in cells grown in ammonium, glutamate and methylamine. Addition of reduced nitrogen compounds to nitrate-grown cells led to loss of NR activity, even if added with nitrate. Under nitrogen starvation, NR activity was not observed; however, following growth on nitrate, NR activity is maintained in the absence of nitrate. Increases but not decreases in NR activity were dependent on protein synthesis. Conditions for chlorate selection were optimized, and Nit- (nitrate-) mutants were isolated. Some of these mutants showed reduced or absent NR activity. Sixty-one NR- mutants revealed the monogenic recessive nature of their lesions and were grouped in 10 complementation classes. These mutants will be used in gene cloning experiments aimed at identifying structural and regulatory elements involved in the first step of nitrate reduction.

Monitoring of peroxisome induction and degradation by flow cytometric analysis of Hansenula polymorpha cells grown in methanol and glucose media : cell volume, refractive index and FITC retention

Cell refractive index has been used to monitor peroxisome behaviour in the yeast Hansenula polymorpha by means of flow cytometry. Peroxisomes are inducible organelles which may occupy a large fraction of the cell volume when yeast cells are growing in methanol media. These organelles harbour a catalase that decomposes the hydrogen peroxide produced in methanol oxidation by alcohol oxidase, a peroxisomal enzyme whose subunits are arranged to form a regular crystalloid. Peroxisomes undergo a degradation process mediated by vacuoles whenever they and their enzymes become metabolically redundant (e.g. during growth on glucose). Flow cytometric analyses of side scattered light (depending on cell volume, morphology and structure) and fluorescein isothiocyanate retention (due to the vacuole) were made on two wild-type strains of H. polymorpha during exponential growth in glucose and methanol media and during nutritional shifts from one carbon source to the other. The same parameters were also analysed for a mutant strain only partially repressed by glucose. We show that both the parameters are substrate-dependent and appear to reflect peroxisome development in the cells. The data reported correlate well with the known cytological and biochemical data, showing the possibility of using flow cytometry, a fast and sensitive technique, to analyse the dynamics of peroxisome proliferation and degradation in response to environmental as well as genetic factors.

A genetically improved wine yeast

SummaryMost of 31 hybrids, obtained by fusion between the petite mutant of a nonsporous strain ofSaccharomyces cerevisiae SS-1090 and a Kar mutant K1 killer ofS. cerevisiae adenine and uracil auxotrophic, proved to be enologically as useful as the parent strain, and in some cases more so. In addition, all of them possessed killer factor, rendering them potentially more competitive against wild yeasts. Most of them also proved to be highly sporous.

ALG2, the Hansenula polymorpha isocitrate lyase gene

To set the basis for molecular and cellular studies of the glyoxylate cycle in methylotrophic yeasts, we isolated and characterized ALG2, the Hansenula polymorpha isocitrate lyase gene. Complementation work and sequence analysis revealed an ORF of 1458 nucleotides, encoding a 486 amino acid protein with a predicted molecular mass of 54.9 kDa. This protein is shorter than the Saccharomyces cerevisiae and Candida tropicalis ICLs, lacks a PST1 signal and possesses a PTS2‐like signal. The transcriptional regulation of ALG2 mRNA levels by carbon source is mainly achieved by glucose repression–derepression, whereas ethanol induction plays only a minor role. We present evidence indicating that, in H. polymorpha, neither isocitrate lyase activity nor the ALG2 gene product are necessary for C1‐peroxisome degradation triggered by ethanol. Therefore, the involvement of glyoxylate in degradation, as described by Kulachkovsky et al. ( 1997 ) for Pichia methanolica, does not necessarily apply to all methylotrophic yeasts. The relevant nucleotide sequence has been deposited at GenBank (Accession No. AF373067.1). Copyright

Genetics and Molecular Biology of Methylotrophic Yeasts

At least 31 yeasts are able to utilize methanol as a sole carbon source (J.A. Barnett, pers. comm.). There is now much interest in these organisms for basic biological and ecological studies and practical applications. The development of genetic tools is advanced enough that three species are amenable to genetic analysis and recombinant DNA systems for them are known. Details of the methylotrophic yeasts are available (Table 14.1), including reviews on their genetics and molecular biology (Cregg 1986; Sibirny et al. 1988; Sudbery and Gleeson 1989). We discuss here current knowledge of fundamental and biotechnological problems.

Interaction of Yna1 and Yna2 Is Required for Nuclear Accumulation and Transcriptional Activation of the Nitrate Assimilation Pathway in the Yeast Hansenula polymorpha

A few yeasts, including Hansenula polymorpha are able to assimilate nitrate and use it as nitrogen source. The genes necessary for nitrate assimilation are organised in this organism as a cluster comprising those encoding nitrate reductase (YNR1), nitrite reductase (YNI1), a high affinity transporter (YNT1), as well as the two pathway specific Zn(II)2Cys2 transcriptional activators (YNA1, YNA2). Yna1p and Yna2p mediate induction of the system and here we show that their functions are interdependent. Yna1p activates YNA2 as well as its own (YNA1) transcription thus forming a nitrate-dependent autoactivation loop. Using a split-YFP approach we demonstrate here that Yna1p and Yna2p form a heterodimer independently of the inducer and despite both Yna1p and Yna2p can occupy the target promoter as mono- or homodimer individually, these proteins are transcriptionally incompetent. Subsequently, the transcription factors target genes containing a conserved DNA motif (termed nitrate-UAS) determined in this work by in vitro and in vivo protein-DNA interaction studies. These events lead to a rearrangement of the chromatin landscape on the target promoters and are associated with the onset of transcription of these target genes. In contrast to other fungi and plants, in which nuclear accumulation of the pathway-specific transcription factors only occur in the presence of nitrate, Yna1p and Yna2p are constitutively nuclear in H. polymorpha. Yna2p is needed for this nuclear accumulation and Yna1p is incapable of strictly positioning in the nucleus without Yna2p. In vivo DNA footprinting and ChIP analyses revealed that the permanently nuclear Yna1p/Yna2p heterodimer only binds to the nitrate-UAS when the inducer is present. The nitrate-dependent up-regulation of one partner protein in the heterodimeric complex is functionally similar to the nitrate-dependent activation of nuclear accumulation in other systems.