Reduction of chromate and carotene-synthesizing activity of selenite-resistant mutants of the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma)

Aim. The yeast P. rhodozymà is a perspective microbial producer of carotenoid pigment astaxanthin with a high antioxidant power. The aim of the work was to study the ability of the selenite-resistant strains of this yeast to reduce chrome(VI) compounds, as well as to analyze the relations between synthesis of carotenoids, resistance to selenite and chromate-reducing activity of P. rhodozymà. Methods. The yeast cells were grown at standard conditions for this species. The residual chromate content in cultural liquid was determined colorimetrically using diphenylcarbazide. The carotenoid content was determined after extraction of the pigments from the previously permeabilized cells by organic solvents. Results. The selected selenite-resistant mutants of the yeast P. rhodozymà revealed the different combinations of the phenotypes related with tolerance/sensitivity to chromate and selenite, as well as ability to reduce chromate. Conclusions. The obtained results give reasons for suggesting that pathways of detoxification of chromate and selenite by the yeast P. rhodozyma are different, although run through a common reductive type. The isolated mutant strains would be served as the useful models to study relations between homeostasis of Se and Cr oxyanions and biosynthesis of carotenes.

Introduction.Due to wide industrial application and distinguishing oxidizing abilities, chromium(VI) compounds are considered to be dangerous environment pollutants.Chromates (CrO 4 2-) and bichromates (Cr 2 O 7 2-) are water soluble and capable of penetrating biological membranes.In microorganisms, in the yeasts in particular, this process is mediated by sulphate transporters, the products of SUL1 and SUL2 genes [1], which is the basis for selection of mutants impaired in sulphate transportation related to chromate resistance [2].After penetrating a cell, Cr(VI) compounds react with intercellular proteins, nucleic acids, and other cell components, causing mutagenic and carcinogenic effects [3,4].
Liv ing cells are ca pa ble of intracellular re duc tion of Cr(VI) to Cr(III).This pro cess is re lated to de tox i fi cation, as it was shown that Cr(III) com pounds are 100-fold less toxic for bac te ria and fungi com pared to Cr(VI) com pounds.Intracellular re duc tion may take place via non-en zy matic and en zy matic path ways.Ascor bic acid, glutathione, and cysteine are ef fi cient re duc ers of Cr(VI) to Cr(III) at phys i o log i cal con ditions.The mech a nisms of en zy matic re duc tion of chromates are well stud ied in bac te ria and may func tion both at aer o bic and an aer o bic con di tions.For in stance, Cr(VI)-re sis tant strains of Enterobacter cloacae re duce chromates in an aer o bic con di tions, us ing Cr(VI) as elec tron ac cep tors.In bac te ria, caus ing aer o bic chromate re duc tion (Pseu do mo nas, Aeromonas, etc.), this process is catalyzed by NADH-and NADP(H)-dependent reductases [5,6].
As for eukaryotic mi cro or gan isms, yeasts in par ticu lar, it is yet to be de ter mined which re duc tion sys temei ther en zy matic/non-en zy matic one or intra-/extracellular one -plays a de ci sive role in chromate-de tox i fi ca tion pro cesses.Be sides, there are no data on the abil ity of biotechnologycally prom is ing yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) to detoxicate and remediate chromates.Astaxanthin, the main carotenoid pig ment, syn the sized by the men tioned yeast, is no ta ble for its pow er ful an tiox i dant ac tiv ity that is 500 times higher than that of a-tocopherol, which is the high est among all the known ca rot en oids [7][8][9].Due to this fact the yeast X. dendrorhous is con sid ered to be a prom is ing source in pro duc tion of phar ma co log i cal prep a ra tions for can cer pre ven tion, im prove ment of im mune response, and protection from the activity of free radicals.
At the same time, con sid er able dif fer ences in redox-po ten tials of astaxanthin (E 0 = 0.75 V) and chromate (E 0 = 1.33 V) [10,11] al low the as sump tion on in creased ac tiv ity of car o tene-syn the siz ing yeast X. dendrorhous re lated to chromate re duc tion.In or der to in crease re duc tion de tox i fi ca tion po ten tial of astaxanthin-syn the siz ing yeast we per formed a se lec -tion of mu tants of the yeast P. rhodozyma, resistant to selenite [12].
The aim of this work was the in ves ti ga tion on reduc tion fea tures of sel e nite-re sis tant strains of car otene-syn the siz ing yeast P. rhodozyma re lated to chromium(VI) com pounds and the anal y sis of the in ter re lation be tween the level of carotenoid syn the sis, re sis tance to sel e nite, and ability of chromium reduction.
Ma te ri als and Meth ods.The ob jects of our in vesti ga tion were wild-type strains of X. dendrorhous (P.rhodozyma) NRRL Y-10921 from the col lec tion of mi cro or gan isms of the In sti tute of Cell Bi ol ogy, NAS of Ukraine, and mu tants of P. rhodozyma, ob tained by us, which dem on strated re sis tance to so dium sel e nite (sit strains).
The se lec tion of spon ta ne ous sit-mu tants of X. dendrorhous was per formed col lect ing the yeast colo nies, grown on agarose me dium in the pres ence of 7.5 mM Na 2 SeO 3 .
The ex po nen tial growth cells (the first 24 hours) were used in ex per i ments.A ster ile so lu tion of po tassium chromate was added to cells' sus pen sion with the con cen tra tion of 0.5 mg/ml with sub se quent in cu ba tion at t = 22°C and aer a tion.The bio mass was de ter mined us ing op tic den sity of cells' sus pen sion at the wavelength of 540 nm with sub se quent cal cu la tion into ab solute dry bio mass of cells in accordance to the calibration curve.
The re sis tance of mu tant P. rhodozyma strains to so dium sel e nite was an a lyzed by ap ply ing cells' suspen sion (0.2 mg/ml) on agarose me dium with the range of sel e nite con cen tra tion from 1 to 30 mM with the sub se quent study of growth ki net ics in 2 days grow ing at t = 22°C.
The con tent of ca rot en oids was de ter mined af ter permeabilization of yeast cells by dimethyl sulphoxide with sub se quent ex trac tion of ca rot en oids into hexane-ethyl ac e tate mix tures [13].Dur ing the in cu ba tion of yeast cells with chromate, the con cen tra tion of re sidual chromate in the cul tural liq uid was de ter mined by means of colorimetric diphenylcarbaside method [6].All the ex per i ments were repeated three times.
Re sults and Dis cus sion.There are nu mer ous lit era ture data on the abil ity of sul phate-re duc ing cells of mi cro or gan isms to re duce Cr(VI) [14].As selenate and sul phate are of the same re duc tion sys tem, char ac terized by mu tual com pet i tive ness [15,16], one may extend this anal ogy and as sume that com pounds of chromium and se le nium may also have com mon en zy matic mech a nisms of re duc tion.Fig. 1 dem on strates the results of test ing ini tial and de riv a tive sit-mu tants of X. dendrorhous, se lected via pos i tive se lec tion in the me dium with 7.5 mM of po tas sium selenite, concerning their resistance to selenite.
Iso lated mu tant strains of P. rhodozyma showed dif fer ent de grees of re sis tance to this toxic fac tor.Wild-type strain of P. rhodozyma NRRL Y-10921 did not grow at 6 mM Na 2 SeO 3 , while six sit-strains out of ten were ac tively grow ing at this con cen tra tion of sodium sel e nite.Even in case of five-fold in crease in Na 2 SeO 3 con cen tra tion in agarose me dium (30 mM), there still were three strains -sit9, sit11, and sit15, the growth of which was steady; sit9 and sit11 showed the high est re sis tance to selenite.
The abil ity of sel e nite-re sis tant strains of car otene-syn the siz ing yeast X. dendrorhous to re duce chromium(VI) com pounds was stud ied via mon i tor ing of growth and con tent of re sid ual chromate in the cul tural liq uid for 6 days of yeast in cu ba tion (ini tial con cen tration -0.5 mg/ml) with 0.9 mM of chromate.Fig. 2 demon strates the re sults of the ex per i ment on the 4 th day of yeast incubation.
It was re vealed that iso lated mu tants with dif fer ent level of re sis tance to sel e nite dem on strate dif fer ent toler ance to chromate, both in the abil ity to grow in the pres ence of chromate and in the level of its re duc tion.For in stance, two sel e nite re sis tant strains sit14 and sit16 re duced all the chromate and had the high est biomass in crease -5.40 and 5.36 mg/ml, re spec tively.The strains with re main ing 3-22% chromate in their cul ture (sit4, sit5, sit8) were char ac ter ized by more in ten sive growth com pared to strains, re duc ing about a half of added chromate (sit9, sit11, sit13, sit15).The cor re lation anal y sis of growth ac tiv ity data and val ues of re sidual con tent of chromate for tested strains re vealed clear neg a tive cor re la tion be tween these pa ram e ters (R = -0.825).Thus, the strains, re duc ing chromate the most ef fi ciently (lower level of re sid ual Cr(VI) in the cultural liquid) usually grow better in the presence of Cr(VI).
The study on car o tene-syn the siz ing ac tiv ity of inves ti gated P. rhodozyma strains re vealed in hi bi tion of this pro cess in the pres ence of high con cen tra tions of chromate in the in cu ba tion mix ture.The cells did not have any col our un til chromate con cen tra tion in the medium de creased to app.0.2 mM, which caused ac tive growth of yeast and syn the sis of ca rot en oids.Therefore, chromate in hib its the pro cess of carotenoid synthe sis along with growth inhibition.
Fig. 3 dem on strates the growth ki net ics and pro cess of chromate re duc tion for some sit-mu tants of the yeast.Strains sit15 and sit16 showed the high est ac tiv ity in their growth and chromate re duc tion, be sides, chromate dis ap peared from the cul tural liq uid sooner -in 4 days of in cu ba tion, while con cen tra tion of chromate on the 6 th days of in cu ba tion for strains sit9 and sit11 was decreased only by half and amounted to 0.4-0.5 mM at the end of ex per i ment.Strains sit9 and sit11 were also no ta ble for the slow est growth rate.Growth in ten si fi cation of the rest of mu tants co in cided with chromate decrease in the cul tural liq uid; strains sit8, sit13 and sit5, wild-type strain re duced all the chromate on the 5 th and 6 th days of experiment, respectively.
There fore, the anal y sis of abil ity to re duce chromate of mu tant strains with the high est re sis tance to sel e nite (sit9, sit11, sit15) showed that chromate in the con cen tra tion of 0.9 mM in hib its their growth consid er ably; at the same time they re duce it much worse com pared to strains sit14 and sit16, re sis tance to sel enite of which is less ex pressed.It may tes tify to a dif ference in re duc tion path ways of these two an ions.Our pre vi ous works on the model of flavinogenous yeast Pichia guilliermondii [17] dem on strated that mu tants, re sis tant to sel e nite, are char ac ter ized by the abil ity of ac cu mu lat ing el e men tary se le nium (Se 0 ) in cells, while re duc tion de tox i fi ca tion of chromate in this yeast results in ac cu mu la tion of Cr(III) biocomplexes in the cultural liquid [6,18].
Thus, dif fer ent na ture of end prod ucts of re duc ing toxic oxianions of chro mium and se le nium and dif ferences in their lo cal iza tion may re sult in am big u ous inter re la tion be tween re sis tance phe no types to sel e nite and chromate.It is also pos si ble that in creased abil ity to reductive de tox i fi ca tion of sel e nite in sel e nite-re sis tant strains of X. dendrorhous re sults in the in crease in the pool of a very toxic rad i cal Cr(V) -the in ter me di ate of Cr(VI) re duc tion.We in tend to ver ify this hy poth e sis via mon i tor ing of Cr(V) gen er a tion in the cul tures of sit-mu tants us ing EPR-spec tros copy, as we have done be fore for chromate-re sis tant strains of flavinogenous yeast P. guilliermondii [20,21].
We cul ti vated yeast cells for 4 days and on ob taining the max i mal level of carotenoid syn the sis [19] we de ter mined to tal con tent of ca rot en oids and bio mass increase for wild-type strains and mu tants (Fig. 4).
The ma jor ity of sel e nite-re sis tant strains syn thesized less ca rot en oids than the ini tial strain, which produced 327 µg/g of dry bio mass, and the co ef fi cient of de crease in car o tene-syn the siz ing ac tiv ity for mu tants was fluc tu at ing from 1.1 to 2.5.Only strain sit16 produced ca rot en oids on the level, sim i lar to that of wild-type strain.The anal y sis of in ter re la tion be tween re sis tance to so dium sel e nite and the con tent of ca rot enoids, pro duced by the tested strains, re vealed that mutants sit9, sit11, sit15 are the most re sis tant to so dium sel e nite and they syn the size ap prox i mately twice less ca rot en oids com pared to the wild-type strain.This fact may tes tify to the ab sence of di rect cor re la tion be tween the level of carotenogenesis and ability to selenite detoxification.
We have an a lyzed the re sis tance of ob tained sit-mu tants of the yeast P. rhodozyma to sel e nite (Fig. 1), com pared ob tained re sults to the data on their abil ity to re duce chromate (Fig. 2), and di vided all the in ves ti gated strains into three groups ac cord ing to pheno type char ac ter is tics (Table ).
It turned out that there are prac ti cally all the com bina tions of phe no types re lated to re sis tance/sen si tiv ity to chromate and sel e nite which tes ti fies to the ab sence of def i nite de pend ence be tween re sis tance to oxianions of chromium and selenium.
Con clu sions.The re sults ob tained al low the assump tion that the path ways of chromate and sel e nite de tox i fi ca tion in the yeast P. rhodozyma are dif fer ent though these pro cesses take place ac cord ing to a common reductive type.The ab sence of def i nite de pendence be tween the re sis tance to chromate and sel e nite and the abil ity of the yeast to syn the size ca rot en oids tes ti fies to the ne ces sity of search ing for bio chem i cal and ge netic mech a nisms, re spon si ble for these processes.Ob tained mu tants with dif fer ent com bi na tion of phe no types of re sis tance to se le nium and chro mium oxianions will serve a con ve nient model in the study on in ter re la tion be tween ho meo sta sis of the men tioned elements and biosynthesis of carotenoids with a high antioxidant activity.
The au thors would like to ex press their sin cere grati tude to the Sci en tific Re search Com mit tee (Po land) for fi nan cial sup port, pro vided in the frame work of Polish-Ukrai nian grant "Mu tants of non-con ven tional yeast for ef fi cient bioremediation of chromate and pu rifi ca tion of in dus trial sew age".

Fig. 1
Fig. 1 Test for resistance of the cells of wild-type strain of P. rhodozyma NRRL Y-10921 and mutant sit-strains to sodium selenite

Fig. 3
Fig. 3 Growth kinetics (a) and chromium reduction (b) for wild-type strain of the yeast P. rhodozyma NRRL Y-10921 and mutants, resistant to selenite.
Phenotype parameters of resistance of P. rhodozyma sit-mutants to selenite and chromate Fig.4Growth and synthesis of carotenoids for wild-type strain of P. rhodozyma NRRL Y-10921 and mutants, resistant to sodium selenite, in the medium without the addition of the latter: red columns -carotenoids; green columns -biomass Ã. È.