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.

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.

Elevated production of the aromatic fragrance molecule, 2-phenylethanol, using Metschnikowia pulcherrima through both de novo and ex novo conversion in batch and continuous modes.

BACKGROUND: 2-phenylethanol (2PE) is a fragrance molecule predominantly used in perfumes and the food industry. It can be made from petrochemicals inexpensively, however, this is unsuitable for most food applications. Currently, the main method of production for the bio-derived compound is to extract the trace amounts found in rose petals, which is extremely costly. Potentially fermentation could provide an inexpensive, naturally sourced, alternative. RESULTS: In this investigation, 2PE was produced from the yeast Metschnikowia pulcherrima, optimised in flasks before scaling to 2 L batch and continuous operation. 2PE can be produced in high titres under de novo process conditions with up to 1500 mg L-1 achieved in a 2 L stirred bioreactor. This is the highest reported de novo titre to date, and achieved through high sugar loadings coupled with low nitrogen conditions. The process successfully ran in continuous mode also, with a concentration of 650 mg L-1 of 2PE being maintained. The 2PE production was further increased by the ex novo conversion of phenylalanine and semi-continuous solid phase extraction from the supernatant. Under optimal conditions 14 000 mg L-1 of 2PE was produced. CONCLUSIONS: The work presented here offers a novel route to naturally sourced 2PE through a scalable fermentation with a robust yeast highly suited to industrial biotechnology. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.