Inactivation of Pichia rhodanensis in relation to membrane and intracellular compounds due to microchip pulsed electric field (MPEF) treatment.

The effect of microchip pulsed electric field (MPEF) treatment on lethal and sublethal injury of Pichia rhodanensis (P. rhodanensis) were employed under 100-500 V for 20-100 pulses and the underlying mechanism of MPEF treatment was investigated as well. A 6.48 log10 reduction of P. rhodanensis was achieved at 500V for 80 pulse. The fluorescent staining with Propidium Iodide (PI) verified that the rate of sublethal injury cells maximum up to 27.2% under 200 V. MPEF can cause the damage of cell morphology and ultrastructure, meanwhile causing a decrease in cellular enzymes, antioxidant enzyme activity and cell membrane fluidity. The leakage of intracellular compounds (protein, nucleic acid, K+, Mg2+) and Ca2+-ATPase gradually increased as the growth of voltage, especially the proportion of protein in the supernatants increased from 2.0% to 26.4%. Flow cytometry analysis showed that MPEF has significant effect on membrane potential, but no obvious influence on non-specific esterase. MPEF can cause the changing of the secondary structure of protein, at the same time, double helix structure of DNA became loose and unwinding. These results provide a theoretical guidance for the widespread using of MPEF technology in the application of a non-thermal processing technique for food.

Cell-surface phytase on Pichia pastoris cell wall offers great potential as a feed supplement.

Cell-surface expression of phytase allows the enzyme to be expressed and anchored on the cell surface of Pichia pastoris. This avoids tedious downstream processes such as purification and separation involved with extracellular expression. In addition, yeast cells with anchored proteins can be used as a whole-cell biocatalyst with high value added. In this work, the phytase was expressed on the cell surface of P. pastoris with a glycosylphosphatidylinositol anchoring system. The recombinant phytase was shown to be located at the cell surface. The cell-surface phytase exhibited high activity with an optimal temperature at 50-55 degrees C and two optimal pH peaks of 3 and 5.5. The surface-displayed phytase also exhibited similar pH stability and pepsin resistance to the native and secreted phytase. In vitro digestibility test showed that P. pastoris containing cell-surface phytase released phosphorus from feedstuff at a level similar to secreted phytase. Yeast cells expressing phytase also provide additional nutrients, especially biotin and niacin. Thus, P. pastoris with phytase displayed on its surface has a great potential as a whole-cell supplement to animal feed.

A combination of heat treatment and Pichia guilliermondii prevents cherry tomato spoilage by fungi.

This study investigated the effectiveness of heat treatment and Pichia guilliermondii, either alone or in combination, to combat postharvest fungal spoilage in cherry tomato fruit. In vitro experiments demonstrated that heat treatment at 38 degrees C significantly inhibited mycelial growth of three different pathogens (Botrytis cinerea, Alternaria alternata and Rhizopus stolonifer Ehrenb). In vivo experiments unveiled that either heat treatment or P. guilliermondii reduced decay caused by these pathogens. Furthermore, a combination of heat treatment followed by the application of P. guilliermondii (H+P) provided the best efficacy in prevention of cherry tomato from fungal spoilage. Following, H+P treatment, electronic nose detected a reduction of volatility in cherry tomato fruit odor, an indicator of preserving fruit's freshness. Scanning electron microscopy unveiled that heat treatment at 38 degrees C for 24h inhibited hyphae growth and spore germination of R. stolonifer Ehrenb while P. guilliermondii multiplied rapidly on fruit wounds, and its cells had a strong capability of adhesion to the hyphae of R. stolonifer Ehrenb. However, heat treatment also seriously injured P. guilliermondii, therefore P. guilliermondii should be applied after heat treatment. A combination of heat treatment and P. guilliermondii is one of the most effective techniques at controlling postharvest fungal spoilage in cherry tomato fruit.

Pectin utilization by the methylotrophic yeast Pichia methanolica.

The methylotrophic yeast Pichia methanolica was able to grow on pectic compounds, pectin and polygalacturonate, as sole carbon sources. Under the growth conditions used, P. methanolica exhibited increased levels of pectin methylesterase, and pectin-depolymerizing and methanol-metabolizing enzyme activities. On the other hand, P. methanolica has two alcohol oxidase (AOD) genes, MOD1 and MOD2. On growth on pectin, the P. methanolica mod1Delta and mod1Deltamod2Delta strains showed a severe defect in the growth yield, although the mod2Delta strain could grow on polygalacturonate to the same extent as the wild-type strain. The expression of MOD1 was detected in pectin-grown cells, but the MOD2-gene expression detected by pectin was much lower than that of MOD1. Moreover, pectin could induce peroxisome proliferation in P. methanolica, like methanol and oleic acid. These findings showed that P. methanolica was able to utilize the methylester moiety of pectin by means of methanol-metabolic enzymes in peroxisomes, and that the functional AOD subunit for pectin utilization was Mod1p in P. methanolica.

Penicillium chrysogenum Pex5p mediates differential sorting of PTS1 proteins to microbodies of the methylotrophic yeast Hansenula polymorpha.

We have isolated the Penicillium chrysogenum pex5 gene encoding the receptor for microbody matrix proteins containing a type 1 peroxisomal targeting signal (PTS1). Pc-pex5 contains 2 introns and encodes a protein of approximately 75 kDa. P. chrysogenum pex5 disruptants appear to be highly unstable, show poor growth, and are unable to sporulate asexually. Furthermore, pex5 cells mislocalize a fluorescent PTS1 reporter protein to the cytosol. Pc-pex5 was expressed in a PEX5 null mutant of the yeast Hansenula polymorpha. Detailed analysis demonstrated that the PTS1 proteins dihydroxyacetone synthase and catalase were almost fully imported into microbodies. Surprisingly, alcohol oxidase, which also depends on Pex5p for import into microbodies, remained mainly in the cytosol. Thus, P. chrysogenum Pex5p has a different specificity of cargo recognition than its H. polymorpha counterpart. This was also suggested by the observation that Pc-Pex5p sorted a reporter protein fused to various functional PTS1 signals with different efficiencies.

Peroxisomicine A1 (plant toxin-514) affects normal peroxisome assembly in the yeast Hansenula polymorpha.

Previously we demonstrated that peroxisomicine A1 (T-514), a plant toxin isolated from Karwinskia species, has a deteriorating effect on the integrity of peroxisomes of methylotrophic yeasts. Here we describe two strains of Hansenula polymorpha, affected in the normal utilization of methanol as sole source of carbon and energy due to peroxisomicine A1 treatment. The two strains isolated (L17 and RV31) grew poorly on methanol, apparently due to malfunctioning of their peroxisomes. Moreover, the cells displayed a high peroxisome turnover rate. We argue that the peroxisomicine A1 induced phenotype of both strains is due to a genomic mutation. Strain L17 was functionally complemented after transformation with a H. polymorpha genomic library. The complementing 2.8 kb DNA fragment did not contain a well-defined ORF and led us to speculate that it may contain regulatory sequences that, when present in multiple copies in the cell, result in a change of expression of specific genes, thus causing restoration of normal methylotrophic growth.

Methanol metabolism in a peroxisome-deficient mutant of Hansenula polymorpha: a physiological study.

We have studied methanol-utilization in a peroxisome-deficient (PER) mutant of Hansenula polymorpha. In spite of the fact that in carbon-limited chemostat cultures under induced conditions the enzymes involved in methanol metabolism were present at wild-type (WT) levels, this mutant is unable to grow on methanol as a sole carbon and energy source. Addition of methanol to glucose-limited (SR = 12.5 mM) chemostat cultures of the PER mutant only resulted in an increase in yield when small amounts were used (up to 22.5 mM). At increasing amounts however, a gradual decrease in cell density was observed which, at 80 mM methanol in the feed, had dropped below the original value of the glucose-limited culture. This reduction in yield was not observed when increasing amounts of formate instead of methanol were used as supplements for the glucose-limited mutant culture and also not in WT cells, used as control in these experiments. The effect of addition of methanol to a glucose-limited PER culture was also studied in the transient state during adaptation of the cells to methanol. The enzyme patterns obtained suggested that the ultimate decrease in yield observed at enhanced methanol concentrations was due to an inefficient methanol-metabolism as a consequence of the absence of peroxisomes. The absence of intact peroxisomes results in two major problems namely i) in H2O2-metabolism, which most probably is no longer mediated by catalase and ii) the inability of the cell to control the fluxes of formaldehyde, generated from methanol. The energetic consequences of this metabolism, compared to the WT situation with intact peroxisomes, are discussed.