STUDY OF PHYSICOCHEMICAL AND ANTIOXIDATIVE
PROPERTIES OF YEAST MEMBRANE COMPONENTS
Michaela Drabkova, 3rd year of PGS
Supervisor: Prof. Ing. L. Omelka, DrSc.
Doc. RNDr. I. Márová, Ph.D.
Department of Food Chemistry and Biotechnology, Faculty of Chemistry, Brno University of
Technology, Purkynova 118, 612 00, Czech Republic, email: [email protected]
INTRODUCTION
Carotenoids are isoprenoid membrane-protective antioxidant pigments produced by plants, algae,
bacteria and fungi. They belong to the most widespread natural pigments with many important
biological activities. Production of carotenes, the great variety of natural carotenoids, is more than
100 million tons per year. They are produced by specific branch of common isoprenoid biosynthetic
pathway occuring in all types of organisms. With regard to different applications in food and feed
industry most attention is now being focused on the natural production of carotenoids by microbial
technology using yeast and/or bacteria.
There are two major ways to influence the microbial production of carotenoids: i) by
modification of cultivation conditions and ii) by construction of genetically modified
overproducers. Qualitative and quantitative changes in a cell metabolite complement can be induced
by environment, stress and other factors. Thus, identification of metabolic marker characteristics for
certain events provides important insight into the mechanisms of pathways occurring in the organism
and can also lead to the regulation of production of industrially significant metabolites. Especially in
microorganisms, production of metabolites is strongly influenced by various external factors.
Environmental stress surrounding of yeast cells evokes various changes in their behaviour in order to
survive under unfavourable conditions. Under stress, various specific compounds including lipidic
substances are overproduced (e. g. glycerol, phospholipids, carotenoids, ergosterol etc.). However,
more information is needed about regulation of production of these substances. Factors that influence
efficiency of natural carotenoid biosynthesis are important for commercial applications.
Carotenoids are membrane-bound lipid-soluble pigments, which can act as effective
antioxidants and scavenge singlet oxygen. In red yeast they probably act as adaptive and/or
protective mechanism against exogenous oxidative stress and UV-irradiation. Those compounds
are accumulated in particular cell organelles. It is not clear so far, whether carotenoids are present
in plasma membrane only or in other inner membrane systems as well as in cell wall. Also distribution of individual carotenoid derivatives in individual sub-cellular fractions has not been studied
yet. Moreover, significant changes of these parameters under exogenous stress could occur, which
could influence potential biotechnological use of red yeasts to industrial production of carotenoids.
In this work some techniques for isolation and separation of sub-cellular fractions (cell wall,
membrane fraction, cytosol) of red yeast cells grown in optimal conditions and under osmotic and
oxidative stress were tested. Further, analysis of carotenoids in these fractions as well as in
whole cells was done. Results of antioxidant properties of sub-cellular fractions were compared
with carotenoid composition and antioxidant activity of some standard carotenoids. The aim of this
work is to study, what is the distribution and trafficking of carotenoids in the cell and which
carotenoids are the main contributors of antioxidant activity in individual cell compartments.
Sborník soutěže Studentské tvůrčí činnosti Student 2006 a doktorské soutěže O cenu děkana 2005 a 2006
Sekce DSP 2005, strana 112
MICROORGANISMS
Yeast strains Rhodotorula glutinis CCY 20-2-26, Sporidiobolus salmonicolor CCY 19-4-8,
Phaffia rhodozyma CCY 77-1-1 and Saccharomyces cerevisiae CCY 21-4-88 were used.
Saccharomyces cerevisiae was used as comparative strain because of very high importance in
genetic engineering.
CULTIVATION CONDITIONS
Yeasts were cultivated on glucose medium aerobically with light at 28 °C to the exponential phase
of growth. Exogenous stress was induced by 2-5 % NaCl added into inoculation media and 2-5 mM
H2O2 added into production media.
Saccharomyces cerevisiae was cultivated on YPD medium anaerobicaly at 28 °C for 24 hours .
PREPARATION OF SPHEROPLASTS
Spheroplasts were prepared by incubation of yeast cells with some enzymes to softly disrupt
plasmatic membrane. Different concentrations of lyticase and glucuronidase, other detergents (e.g.
SDS, beta-merkaptoethanol, etc.) and osmotic lysis were used. Membrane fraction and cytosol were
separated by ultracentrifugation. Spheroplasts were detected by light-microscopy and with addition of
fluorescein dye by UV-microscopy. Further, spheroplasts were used for isolation of chromosomal
DNA from red yeasts. Isolation was performed in 1% low melting agarose plugs and analyzed then
by PFGE.
a) Spheroplasts of R. glutinis – Light microscopy
b) Bursting cells in isotonic environment
ISOLATION OF SUB-CELLULAR FRACTION
Sub-cellular fractions of cells were obtained by gradually separation using combination of
enzymes and detergents. Cell wall fraction (surface layer) was obtained using sonification followed
by ethanol precipitation. In selected fraction lipid profiles were analyzed using TLC. Levels of
carotenoids - lycopene, alpha-carotene, beta-carotene, torulen and phytoene were obtained from yeast
cells using acetone extraction and saponification by ethanolic KOH solution. The sample was
repeatedly extracted by diethylether, evaporated and dissolved into ethanol for HPLC. We analyzed
those samples using HPLC/MS. Antioxidant activity of individual sub-cellular fractions was tested
using ABTS Metod (Randox kit). Protein profiles under stress conditions were compared too. Those
were analyzed by PAGE-SDS.
Sborník soutěže Studentské tvůrčí činnosti Student 2006 a doktorské soutěže O cenu děkana 2005 a 2006
Sekce DSP 2005, strana 113
RESULTS AND DISCUSSION
The most important industrial red yeast Rhodotorula glutinis was used as tested strain for
identification of carotenoids and other membrane compounds. Dry biomass of these yeasts is used as
feed complement for improvement of appearance and nutritional properties of animal products (e. g.
the color of eggs and milk, the color of salmon meat).
This microorganism was chosen based on results of previous screening study on selected yeast
strains which produce carotenoids as natural pigments. Carotenogenic cultures were influenced by
several exogenic chemical and physical stress factors added into cultivation media to clear the role of
carotenoids in general stress response. We found that all types of stress conditions led to increased
production of beta-carotene according to phase of application or concentration of stress factor. Crossprotection and production of similar metabolites in various types of stress conditions suggests the
existence of an integrating mechanism that senses and responds to different forms of stress.
Preparation of individual sub-cellular fractions from red yeast cells was strongly complicated by
lipotrophic character of this strain. Especially preparation of spheroplasts was very difficult because of
rigid character of cell wall membrane and yields of membrane fractions were therefore very low.
Under stress conditions, about hundred proteins were overproduced by this strain. Expecting shock
proteins, it could be some enzymes that catalyze overproduction of stress metabolites including
carotenoids. Under both oxidative and osmotic stress pigments were overproduced, higher amount
was detected in surface cell structures (cell wall and plasma membrane). Ergosterol was found both
in cytosol and membrane lotions and its production under stress changed simultaneously with
carotenoid formation. Glycerol was detected above all in cytosol fraction and its production under
stress was inversely to carotenoid and ergosterol production.
As surprising finding can be noted high content of carotenoids found in upper cell wall fraction.
Presence of carotenoids, mainly beta-carotene, was detected in plasma membrane as well as in inner
membrane fraction. The highest antioxidant activity was found in surface structures.
This work was supported by project MSM 0021630501 H/'Czech Ministry of Education and by
project IAA400310506 f Grant Agency of the Academy of Sciences of the Czech Republic.
REFERENCES
.l. Marova I., Breierova E., Koci R., Friedl Z., Slovak B., Pokorna J.: Annals Microbiol. 54, 73
(2004).
2. Farina J. I., Molina O. E., Figueroa L. I.. C.: Journal of Applied Microbiology 96, 254-262 (2004)
3.Pardo M. Monteoliva L., Pla J. Sanchez M., Gil C. Nombela C.: Yeast 15, 459-472 (1999)
4. Misawa N. Shimada H.: J. Biotechnol. 59, 169 (1997).
5. Armstrong G. A., Hearst J. E.: FASEB J. 10, 228 (1996).
Sborník soutěže Studentské tvůrčí činnosti Student 2006 a doktorské soutěže O cenu děkana 2005 a 2006
Sekce DSP 2005, strana 114