Proteomic analysis of the carotenogenic yeast Xanthophyllomyces dendrorhous

Abstract

Background: The yeast Xanthophyllomyces dendrorhous is used for the microbiological production of the antioxidant carotenoid astaxanthin. In this study, we established an optimal protocol for protein extraction and performed the first proteomic analysis of the strain ATCC 24230. Protein profiles before and during the induction of carotenogenesis were determined by two-dimensional polyacrylamide gel electrophoresis and proteins were identified by mass spectrometry.

Results: Among the approximately 600 observed protein spots, 131 non-redundant proteins were identified. Proteomic analyses allowed us to identify 50 differentially expressed proteins that fall into several classes with distinct expression patterns. These analyses demonstrated that enzymes related to acetyl-CoA synthesis were more abundant prior to carotenogenesis. Later, redox- and stress-related proteins were up-regulated during the induction of carotenogenesis. For the carotenoid biosynthetic enzymes mevalonate kinase and phytoene/squalene synthase, we observed higher abundance during induction and/or accumulation of carotenoids. In addition, classical antioxidant enzymes, such as catalase, glutathione peroxidase and the cytosolic superoxide dismutases, were not identified.

Conclusions: Our results provide an overview of potentially important carotenogenesis-related proteins, among which are proteins involved in carbohydrate and lipid biosynthetic pathways as well as several redox- and stress-related proteins. In addition, these results might indicate that X. dendrorhous accumulates astaxanthin under aerobic conditions to scavenge the reactive oxygen species (ROS) generated during metabolism.

Figure 1

Growth and pigment production in X. dendrorhous. Growth was measured by the absorbance at 560 nm (shown on a log scale), which is represented by the squares and solid line. The means ± SD of the values obtained from four independent cultures are shown. The vertical arrows indicate the harvest times for the assays (24, 70 and 96 h, which corresponded to lag, late exponential and stationary growth phases, respectively). The solid line represents the total carotenoids. The asterisk indicates the induction of carotenoid biosynthesis.

Figure 2

Representative 2D gel of soluble proteins of X. dendrorhous. Protein profile in stationary growth phase. The image was obtained with PDQuest software ver. 7.1.1. The ID numbers were manually added and correspond to all non-redundant proteins identified by MALDI-TOF MS.

Figure 3

Relative intensities of multiple spots for X. dendrorhous proteins in MM-glucose. The growth phases are represented by different colors. A. Multi-spot proteins that exhibited essentially the same general pattern of variation. B. Multi-spot proteins that were regulated in different ways. The axis numbers correspond to the SSP spot identifications generated by PDQuest software. The y axis scale (× 10³) corresponds to the normalized spot intensity. To normalize, the spot intensities were divided by the total density of valid spots and then multiplied by 10⁶. Finally, the normalized values from replicates of 24-h, 70-h and 96-h were averaged. Asterisks represent p < 0.01 and circles represent p < 0.05.

Figure 4

Classification of identified proteins by cellular function. A. Pie chart showing the functional classifications of the identified proteins based on annotations from the KEGG and Swiss-Prot/TrEMBL protein databases. B. Proteins involved in metabolism (49%) were subdivided according to pathway modules in the KEGG database. Percentages were calculated by dividing the number of proteins in the group by the total number of proteins identified and then multiplying by 100.

Figure 5

Fold changes of differentially expressed proteins. Proteins with more than two-fold changes (see Table 1) were plotted according to their fold change in exponential phase (left graph) or stationary phase (right graph) relative to their abundance in lag phase. The proteins are colored by their functional classification and grouped by fold change (shown on the x-axis of each graph). SSP spot identification was performed using PDQuest software.