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.

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.

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.

Hansenula polymorpha NMR2 and NMR4, two new loci involved in nitrogen metabolite repression.

In the yeast Hansenula polymorpha (Pichia angusta) nitrate assimilation is tightly regulated and subject to a dual control: nitrogen metabolite repression (NMR), triggered by reduced nitrogen compounds, and induction, elicited by nitrate itself. In a previous paper [Serrani, F., Rossi, B. and Berardi, E (2001) Nitrogen metabolite repression in Hansenula polymorpha: the nmrl-l mutation. Curr. Genet. 40, 243-250], we identified five loci (NMR1-NMR5) involved in NMR, and characterised one of them (NMR1), which likely identifies a regulatory factor. Here, we describe two more mutants, namely nmr2-1 and nmr4-1. The first one possibly identifies a regulatory factor involved in nitrogen metabolite repression by various nitrogen sources alternative to ammonium. The second one, apparently involved in ammonium assimilation, probably has sensor functions.

New yeast expression platforms based on methylotrophic Hansenula polymorpha and Pichia pastoris and on dimorphic Arxula adeninivorans and Yarrowia lipolytica - a comparison.

Yeasts combine the ease of genetic manipulation and fermentation of a microbial organism with the capability to secrete and to modify proteins according to a general eukaryotic scheme. Yeasts thus provide attractive platforms for the production of recombinant proteins. Here, four important species are presented and compared: the methylotrophic Hansenula polymorpha and Pichia pastoris, distinguished by an increasingly large track record as industrial platforms, and the dimorphic species Arxula adeninivorans and Yarrrowia lipolytica, not yet established as industrial platforms, but demonstrating promising technological potential, as discussed in this article.

The NII2 gene of Hansenula polymorpha is involved in nitrite assimilation.

To establish a basis for genetic and molecular studies of nitrite assimilation in the methylotrophic yeast Hansenula polymorpha, we isolated and characterised six nitrite-negative mutants still capable of growing on nitrate. Gene isolation work yielded the NII2 gene, encoding a membrane protein homologous to the Saccharomyces cerevisiae Pho86p. Sequence analysis revealed an ORF of 860 bp encoding a 286-amino-acid protein with a predicted molecular mass of 32.8 kDa. This protein is shorter than its S. cerevisiae homologue, and is predicted to lack an ER-retention signal. Cell suspension work revealed that the null mutant is unable to take up nitrite from the medium.

Hypoxia abolishes transience of the heat-shock response in the methylotrophic yeast Hansenula polymorpha.

The heat-shock response is conserved amongst practically all organisms. Almost invariably, the massive heat-shock protein (Hsp) synthesis that it induces is subsequently down-regulated, making this a transient, not a sustained, stress response. This study investigated whether the heat-shock response displays any unusual features in the methylotrophic yeast Hansenula polymorpha, since this organism exhibits the highest growth temperature (49-50 degrees C) identified to date for any yeast and grows at 47 degrees C without either thermal death or detriment to final biomass yield. Maximal levels of Hsp induction were observed with a temperature upshift of H. polymorpha from 30 degrees C to 47-49 degrees C. This heat shock induces a prolonged growth arrest, heat-shock protein synthesis being down-regulated long before growth resumes at such high temperatures. A 30 degrees C to 49 degrees C heat shock also induced thermotolerance, although H. polymorpha cells in balanced growth at 49 degrees C were intrinsically thermotolerant. Unexpectedly, the normal transience of the H. polymorpha heat-shock response was suppressed completely by imposing the additional stress of hypoxia at the time of the 30 degrees C to 49 degrees C temperature upshift. Hypoxia abolishing the transience of the heat-shock response appears to operate at the level of Hsp gene transcription, since the heat-induced Hsp70 mRNA was transiently induced in a heat-shocked normoxic culture but displayed sustained induction in a culture deprived of oxygen at the time of temperature upshift.

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 C(1)-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).

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.

Temperature profile ofCandida cacaoi SS-2566.

Candida cacaoi SS-2566 was found to have a dissociative temperature profile and thermotolerant growth yield behaviour. With temperature-independent growth rates up to 42°C, its utilization at relatively high temperature for the production of cells (SCP, SCL) and/or metabolites may well be of interest, particularly in view of its hydrocarbon utilization.