The Oleaginous Yeast Metschnikowia pulcherrima Displays Killer Activity against Avian-Derived Pathogenic Bacteria.

Metschnikowia pulcherrima is a non-conventional yeast with potential to be used in biotechnological processes, especially those involving low-cost feedstock exploitation and biocontrol applications. The combination of traits that supports these industrial applications in M. pulcherrima also makes it an attractive option to study in the context of livestock health. In this study, we examined the specific interactions between M. pulcherrima and multiple avian pathogenic bacteria. We tested individual bacteria-yeast interactions and bacterial combinations in both solid and liquid media and in variable nutrient environments. Across multiple isolates of M. pulcherrima, we observed different levels of antimicrobial activity, varying from supporting the growth of competing bacteria through suppression and bacterial killing, and we found that these responses varied depending on the bacterial strains and media. We identified multiple molecular routes, including proteins produced by M. pulcherrima strains, that acted to control these microbial interactions. Furthermore, protein screening revealed that M. pulcherrima strains were induced to produce proteins specifically when exposed to bacterial strains, suggesting that fine-tuned mechanisms allow M. pulcherrima to function as a potential lynchpin in a microbial community.

Variation among Metschnikowia pulcherrima Isolates for Genetic Modification and Homologous Recombination.

Metschnikowia pulcherrima is a non-conventional yeast with the potential to be used in biotechnological processes, especially involving low-cost feedstock exploitation. However, there are a lack of tools for researching it at a molecular level and for producing genetically modified strains. We tested the amenability to genetic modification of ten different strains, establishing a transformation protocol based on LiAc/PEG that allows us to introduce heterologous DNA. Non-homologous integration was broadly successful and homologous recombination was successful in two strains. Chemical inhibition of non-homologous end joining recombination had a modest effect on the improvement of homologous recombination rates. Removal of selective markers via flippase recombinase was successful across integrated loci except for those targeted to the native URA3 locus, suggesting that the genome sequence or structure alters the efficacy of this system.

Semi-continuous pilot-scale microbial oil production with Metschnikowia pulcherrima on starch hydrolysate.

BACKGROUND: Heterotrophic microbial oils are potentially a more sustainable alternative to vegetable or fossil oils for food and fuel applications. However, as almost all work in the area is conducted on the laboratory scale, such studies carry limited industrial relevance and do not give a clear indication of what is required to produce an actual industrial process. Metschnikowia pulcherrima is a non-pathogenic industrially promising oleaginous yeast which exhibits numerous advantages for cost-effective lipid production, including a wide substrate uptake, antimicrobial activity and fermentation inhibitor tolerance. In this study, M. pulcherrima was fermented in stirred tank reactors of up to 350 L with 250-L working volume in both batch and semi-continuous operation to highlight the potential industrial relevance. Due to being food-grade, suitable for handling at scale and to demonstrate the oligosaccharide uptake capacity of M. pulcherrima, enzyme-hydrolysed starch in the form of glucose syrup was selected as fermentation feedstock. RESULTS: In batch fermentations on the 2-L scale, a lipid concentration of 14.6 g L-1 and productivity of 0.11 g L-1 h-1 were achieved, which was confirmed at 50 L (15.8 g L-1; 0.10 g L-1 h-1). The maximum lipid production rate was 0.33 g L-1 h-1 (daily average), but the substrate uptake rate decreased with oligosaccharide chain length. To produce 1 kg of dry yeast biomass containing up to 43% (w/w) lipids, 5.2 kg of the glucose syrup was required, with a lipid yield of up to 0.21 g g-1 consumed saccharides. In semi-continuous operation, for the first time, an oleaginous yeast was cultured for over 2 months with a relatively stable lipid production rate (around 0.08 g L-1 h-1) and fatty acid profile (degree of fatty acid saturation around 27.6% w/w), and without contamination. On the 250-L scale, comparable results were observed, culminating in the generation of nearly 10 kg lipids with a lipid productivity of 0.10 g L-1 h-1. CONCLUSIONS: The results establish the importance of M. pulcherrima for industrial biotechnology and its suitability to commercially produce a food-grade oil. Further improvements in the productivity are required to make M. pulcherrima lipid production industrial reality, particularly when longer-chain saccharides are involved.

Snf2 controls pulcherriminic acid biosynthesis and antifungal activity of the biocontrol yeast Metschnikowia pulcherrima.

Metschnikowia pulcherrima synthesises the pigment pulcherrimin, from cyclodileucine (cyclo(Leu-Leu)) as a precursor, and exhibits strong antifungal activity against notorious plant pathogenic fungi. This yeast therefore has great potential for biocontrol applications against fungal diseases; particularly in the phyllosphere where this species is frequently found. To elucidate the molecular basis of the antifungal activity of M. pulcherrima, we compared a wild-type strain with a spontaneously occurring, pigmentless, weakly antagonistic mutant derivative. Whole genome sequencing of the wild-type and mutant strains identified a point mutation that creates a premature stop codon in the transcriptional regulator gene SNF2 in the mutant. Complementation of the mutant strain with the wild-type SNF2 gene restored pigmentation and recovered the strong antifungal activity. Mass spectrometry (UPLC HR HESI-MS) proved the presence of the pulcherrimin precursors cyclo(Leu-Leu) and pulcherriminic acid and identified new precursor and degradation products of pulcherriminic acid and/or pulcherrimin. All of these compounds were identified in the wild-type and complemented strain, but were undetectable in the pigmentless snf2 mutant strain. These results thus identify Snf2 as a regulator of antifungal activity and pulcherriminic acid biosynthesis in M. pulcherrima and provide a starting point for deciphering the molecular functions underlying the antagonistic activity of this yeast.