Achieving a high-density oleaginous yeast culture: Comparison of four processing strategies using Metschnikowia pulcherrima.

Microbial lipids have the potential to displace terrestrial oils for fuel, value chemical, and food production, curbing the growth in tropical oil plantations and helping to reduce deforestation. However, commercialization remains elusive partly due to the lack of suitably robust organisms and their low lipid productivity. Extremely high cell densities in oleaginous cultures are needed to increase reaction rates, reduce reactor volume, and facilitate downstream processing. In this investigation, the oleaginous yeast Metschnikowia pulcherrima, a known antimicrobial producer, was cultured using four different processing strategies to achieve high cell densities and gain suitable lipid productivity. In batch mode, the yeast demonstrated lipid contents more than 40% (w/w) under high osmotic pressure. In fed-batch mode, however, high-lipid titers were prevented through inhibition above 70.0 g L-1 yeast biomass. Highly promising were a semi-continuous and continuous mode with cell recycle where cell densities of up to 122.6 g L-1 and maximum lipid production rates of 0.37 g L-1 h-1 (daily average), a nearly two-fold increase from the batch, were achieved. The findings demonstrate the importance of considering multiple fermentation modes to achieve high-density oleaginous yeast cultures generally and indicate the limitations of processing these organisms under the extreme conditions necessary for economic lipid production.

Analytical impact of Metschnikowia pulcherrima in the volatile profile of Verdejo white wines.

Most wine aroma compounds, including the varietal fraction, are produced or released during wine production and derived from microbial activity. Varietal aromas, typically defined as terpenes and thiols, have been described as derived from their non-volatile precursors, released during wine fermentation by different yeast hydrolytic enzymes. The perception of these minority aroma compounds depends on the chemical matrix of the wine, especially on the presence of majority aroma compounds, such as esters or higher alcohols. Strategies aiming to reduce the production of these masking flavors are on the spotlight of enology research as a way to better define varietal standard profiles for the global market. Using a natural white must from Verdejo variety (defined as a thiol grape variety), here we describe the analytical and sensorial impact of using, in sequential inoculations, a selected strain of Metschnikowia pulcherrima, in combination with two different Saccharomyces cerevisiae strains. An increase in the levels of the thiol 4-MSP (4-methyl-4-sulfanylpentan-2-one) over its sensory threshold, together with a decrease in higher alcohol production, was observed when M. pulcherrima was used. This has an important impact on these wines, making them fruitier and fresher, always preferred by the sensory panel.

Purification and molecular characterization of a Metschnikowia saccharicola killer toxin lethal to a crab pathogenic yeast.

The marine yeast strain Metschnikowia saccharicola DD21-2, isolated from sediments in the Yalu River, produces a killer toxin with a lethal effect on Metschnikowia bicuspidate strain WCY, a pathogenic yeast strain that infects crabs. In this study, the killer toxin was purified and characterized. After sequential purification, the purity of the killer toxin was increased 72.2-fold over the purity of the yeast cell culture supernatant. The molecular weight of the purified killer toxin was 47.0 kDa. The optimal pH and temperature for killing activity were 5.5°C and 16°C, respectively. The killing activity was stable over a pH range of 4.0-6.5 and temperature range of 0°C-40°C. The purified killer toxin was only effective against toxin-sensitive integral cells and had no killing effect on the protoplasts of toxin-sensitive cells. When exerting the killing effect, the toxin bind to a cell wall receptor of the treated strain, disrupted cell wall integrity and eventually caused death. The amino acid sequence identified by mass spectroscopy indicated that the purified killer toxin might be a protein kinase, but did not show ß-1,3-glucanase activity, consistent with the laminarin hydrolysis results. These findings provide a basis for disease prevention and control in marine aquaculture.

Characterisation of the enzymatic properties of MpAPr1, an aspartic protease secreted by the wine yeast Metschnikowia pulcherrima.

BACKGROUND: MpAPr1, encoding an acid protease from the wine yeast Metschnikowia pulcherrima IWBT Y1123, was previously isolated and shown to display potential activity against casein and grape proteins. However, its characterisation remained partial. RESULTS: MpAPr1 was cloned into the pGAPZαA vector and transformed into Komagataella pastoris X33 for heterologous expression. After verification of activity, the enzyme properties were characterised. Protease activity within the concentrated supernatant was retained over a pH range of 3.0 to 5.0 and between 10 °C and 50 °C. Optimal conditions for protease activity were found at 40 °C and pH 4.5. Activity was mostly unaffected by the presence of metal ions with the exception of Cu2+ and Ni2+ . Furthermore, proteolytic activity was retained in the presence of sugar and ethanol. pH and temperature conditions for MpAPr1 expression in K. pastoris were optimised. Purification was achieved by means of cation exchange chromatography and kinetic parameters (Km and Vmax ) were determined. MpAPr1 activity against grape proteins was confirmed, but the extent of the degradation was dependent on the nature of these proteins and the environmental conditions. CONCLUSION: Overall, the results suggest that MpAPr1 could be applied in food biotechnology processes such as winemaking. © 2017 Society of Chemical Industry.

A new method for monitoring the extracellular proteolytic activity of wine yeasts during alcoholic fermentation of grape must.

The existing methods for testing proteolytic activity are time consuming, quite difficult to perform, and do not allow real-time monitoring. Proteases have attracted considerable interest in winemaking and some yeast species naturally present in grape must, such as Metschnikowia pulcherrima, are capable of expressing this activity. In this study, a new test is proposed for measuring proteolytic activity directly in fermenting grape must, using azocasein, a chromogenic substrate. Several yeast strains were tested and differences in proteolytic activity were observed. Moreover, analysis of grape must proteins in wines revealed that protease secreted by Metschnikowia strains may be active against wine proteins.

"This is not an apple"-yeast mutualism in codling moth.

The larva of codling moth Cydia pomonella (Tortricidae, Lepidoptera) is known as the worm in the apple, mining the fruit for food. We here show that codling moth larvae are closely associated with yeasts of the genus Metschnikowia. Yeast is an essential part of the larval diet and further promotes larval survival by reducing the incidence of fungal infestations in the apple. Larval feeding, on the other hand, enables yeast proliferation on unripe fruit. Chemical, physiological and behavioral analyses demonstrate that codling moth senses and responds to yeast aroma. Female moths are attracted to fermenting yeast and lay more eggs on yeast-inoculated than on yeast-free apples. An olfactory response to yeast volatiles strongly suggests a contributing role of yeast in host finding, in addition to plant volatiles. Codling moth is a widely studied insect of worldwide economic importance, and it is noteworthy that its association with yeasts has gone unnoticed. Tripartite relationships between moths, plants, and microorganisms may, accordingly, be more widespread than previously thought. It, therefore, is important to study the impact of microorganisms on host plant ecology and their contribution to the signals that mediate host plant finding and recognition. A better comprehension of host volatile signatures also will facilitate further development of semiochemicals for sustainable insect control.

Isolation and characterization of new Metschnikowia pulcherrima strains as producers of the antimicrobial pigment pulcherrimin.

Metschnikowia pulcherrima is a highly effective biocontrol yeast due to its pigment pulcherrimin that accumulates in the cells and in the growth medium. Three different strains of M. pulcherrima were isolated from local grapes. The yeast isolates were characterized on the basis of their biochemical, physiological and ITS1-5.8 s rDNA-ITS2 region. Based on the obtained results, the M. pulcherrima isolates were identified as new strains of M. pulcherrima. Strong antagonistic activities of the M. pulcherrima strains on the human pathogens Proteus vulgaris, Escherichia coli, Candida albicans, Candida parapsilosis, Candida krusei, and Trichosporon mucoides were determined. In addition, antagonistic effects of these M. pulcherrima strains were also tested against Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Trichoderma spp., Paecilomyces spp., and Bipolaris spp. and it was shown that the three different strains of M. pulcherrima also have an antagonistic effect on the growth of these fungal species at different extents. This study showed that all three strains of M. pulcherrima produce the same amount of the pigment pulcherrimin, but their antimicrobial activities on different microorganisms show important variations.