Temperature-sensitive mutants of a thermotolerant yeast, Hansenula polymorpha

[J. Ferment. Technol., Vol. 66, No. 4, 455-459. 1988]

Note

Temperature-Sensitive Mutants of a Thermotolerant Yeast, Hansenula polymorpha KAZUYOSHI OHTA*, SUSHILACHANDRANIWIJEYARATNE, and SHINSAKUHAYASHIDA

Departmentof AgriculturalChemistry,Facultyof Agriculture, Kyushu University, Higashi-ku, Fukuoka 812, Japan Temperature-sensitive mutants (TS-1 and TS-7) of a thermotolerant yeast, Hansenula polymorphaCK-1, were isolated. The mutants were unable to grow at 50°C, the maximum growth temperature of the wild type. Mutants TS-1 and TS-7 grown at 20°C showed 33 and 50% viahilities after 6 h of incubation at 50°C, respectively. Mutant TS-1 showed little variation of the degree of fatty acid unsaturation (1.26-1.28/mol) and mutant TS-7 had an almost constant sterol/phospholipid molar ratio (0.31-0.34) at 20, 30 and 40°C, although the wild type bad a decrease of the degree of fatty acid unsaturation from 1.56 at 20°C to 1.30 at 40°C and an increase of the sterol/phosphofipid molar ratio from 0.26 at 20°C to 0.54 at 40°C.

A thermotolerant yeast, Hansenula polymorpha CK-1, can grow in synthetic medium at temperatures of 15 to 50°C and its optimum temperature for growth is 40°C.1) Unlike thermophilic yeasts, which were defined by their minimum temperature for growth at 20°C and above, the thermotolerant yeast contained linoleic ( 1 8 : 2 ) and linolenic (18 : 3) acids in addition to oleic (18 : 1) acid. An increase in the growth temperature from 20 to 50°C reduced the proportion of 18 : 3 acid. Furthermore, the amounts of sterols (mainly ergosterol) relative to the highly unsaturated phospholipids increased in cells grown at 40 and 50°C. Accordingly, it was suggested that such changes in lipid composition were necessary for growth of this yeast at high temperatures. Subsequent work2) showed that a nystatin-resistant mutant of H. po~morpha CK-1, which lacked ergosterol, grew more slowly at 40 and 50°C than its wild-type parent. The mutant grown with *Correspo~i~g a u ~ o r

ergosterol-phosphatidylcholine emulsion at 50°C incorporated ergosterol and the growth rate increased. Another approach to the further understanding of the thermotolerance of this yeast might be the use of mutants with diminished thermotolerance. I n this work, temperature-sensitive mutants of H. p0/ymorpha CK-1 were isolated and compared in lipid composition with the thermotolerant wild type. The results described below confirmed and extended our earlier findings.I) A wild-type strain of H. polymorpha CK-1 (ATCC 64209) used in a previous study 1) was used for isolation of the mutants. The wild type and the mutants described below were maintained on YPD slants (1% yeast extract, 2% peptone, 2% glucose, and 2% agar) at 4°C. One loopful of the wild-type cells from a fresh slant culture was suspended in 10 ml ~f 67 m M phosphate buffer (pH 7.0) in an Lshaped tube. Ethyl methanesulfonate (0.1 ml) was added to the cell suspension and the mixture was incubated on a Monod shake1

456

OHTA, WIJEYARATNE, and I~AYASI,IIDA

at 30°C for 30 to 40 rain. A portion (0.2 ml) of the suspension was then transferred to 8 ml of sterile 5% (w/v) sodium thiosulfate to quench the reaction. A suitable dilution (0.1 ml) of the above suspension was plated on YPD agar. T h e plates were incubated at 30°C for 3 to 4 d to allow colony formation. Colonies were replica-plated onto two YPD plates. One replica was incubated at 40°C and the other at 50°C (the m a x i m u m growth temperature of the wild type) for 3 to 4 d. T h e colonies that grew at 40°C, but not at 50°C, were isolated as temperature-sensitive mutants. One loopful of selected m u t a n t cells from a fresh slant culture was suspended in 5 ml of sterile deionized water. One milliliter of the suspension was transferred to 100 ml of synthetic medium containing 5% (w/v) glucosO) in a 500-ml conical flask plugged with_ cotton wool. Triplicate cultures were incubated statically at the indicated temperatures. Cell growth was monitored by measuring the absorbancy of the culture at 660 rim. T e m p e r a t u r e shift experiments were done for assaying the viability of resting cells of mutants at the restrictive temperature of 50°C. M u t a n t strains were grown at 20°C in synthetic medium as described above. The cells were harvested in the mid-exponential phase of growth and resuspended in 67 m M phosphate buffer ( p H 6.0) supplemented with 2% (w/v) glucose to give a cell concentration of approximately 1 X lO 8 cells/ml. A lO-ml portion of this suspension was transferred into tubes and incubated at

[J. Ferment. Technol.,

T h e viable cell numbers were measured periodically by plating appropriate dilutions of the suspensions on YPD plates and counting colonies after incubation at 30°C for 3 to 4 d. Cells for lipid analysis were grown as described above and harvested in the midexponential phase (2 to 4 d old culture) by eentrifugation. T h e y were then washed three times with deionized water and freezedried. T h e freeze-dried cells were stored at --20°C over silica gel. Lipids were extracted from cells, and the total lipid content and total phospholipids of the extract were measured as previously described.Z~ Sterols were extracted and purified as digitonides by the procedure of Shaw and Jefferies.3) The sterol content was estimated as ergosterol from the extinction at 282 nm by a Shimadzu UV-240 recording spectrophotometer. For fatty acid analysis, freeze-dried cells were directly methanolyzed with methanolic HC1 and the resulting methyl esters of the fatty acids were analyzed by gas-liquid chromatography using methods described earlier. 1) Eleven temperature-sensitive mutants were isolated. Among these, two mutants (TS-1 and TS-7) which difikred in their temperature sensitivity were chosen for comparison with the wild type. Figure 1 shows the growth curves of wild type and mutants TS-1 and TS-7 from 20 to 40°C. Mutant TS-1 showed less growth at all temperatures tested, especially at 40°C, than the wild type and the mutant TS-7. Mutant TS-7 grew nearly as well as the wild type at 20, 30 and 37°C. The o p t i m u m temperatures for growth ok 50°C.

1.2 @tO 1.0 .50.8

o~0.e U3

0.4 0.2

~o

I

I

I

1 234567

I

I

I

S

1 2 3 4 5 6 7 Time (d)

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Fig. 1. Growth curves for wild type (A) and temperature-sensitive mutants TS-1 (B) and TS-7 (C) ofH. polymarpha CK-1 at 20 (O), 30 (Q), 37 (~) and 40°(3 (A).

Temperature-Sensitive Mutants ofH. polymorpha

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T h e m u t a n t cells g r o w n at 20, 30 and 40°C were analyzed for lipid compositions (Table 1). T h e m a j o r changes in lipid composition observed in the wild type with increased growth temperature from 20 to 50°C were a decrease in the phospholipid content of cells and a sharp reduction of the proportion of linolenic ( 1 8 : 3 ) acid in phospholipids and total lipids.1) T o t a l lipid contents o f cells of the mutants on a d r y weight basis were higher t h a n those of the wild type. E Phospholipid contents of cells of the mutants ¢.. T S - I and TS-7 decreased with increases in growth temperature from 20 to 30°C. H o w ever, the phospholipid content o f cells o f the m u t a n t TS-1 r e m a i n e d almost constant and ..Q that of the m u t a n t TS-7 increased slightly 0 with a further increase in growth temperature > from 30 to 40°C. T h e m a j o r sterol in the mutants was ergosterol (data not shown). T h e sterol content of the m u t a n t TS-1 was higher than that o f the wild type at 20, 30 a n d 40°C, but the sterol content of the m u t a n t TS-7 was the same level as that of the wild type. T h e sterol content o f these two Fig. 2. Viability of wild type (©) and temperaturesensitive mutants TS-I (O) and TS-7 (/x) of mutants did not show m u c h change with temperature. T h e molar ratio of sterols to H. polymorpha CK-I after shift to 50°C. Wild type and mutants were grown to mid- phospholipids of the m u t a n t TS-1 increased exponential phase in synthetic medium at 20°C. to 0.50 at 30 and 40°C, w h i c h was comparable The cells were then suspended in 67 mM phos- to the increase in that of the wild type at phate buffer (pH6.0) supplemented with 2% 40°C. T h e m o l a r ratio o f the m u t a n t TS-7 (w/v) glucose and the suspension was incubated grown at 20, 30 and 40°C was between at 50°C. The viable cell count was calculated by 0.31 and 0.34. T h e major fatty acids of the spreading dilutions of cells on YPD agar medium and incubating at 30°C. The results represent mutants T S - I and TS-7 were the same as those of the wild type and were palmitic the average of cell count from three plates. (16 : 0), oleic (18 : 1), linoleic (18 : 2) and linolenic ( 1 8 : 3 ) acids (Table 2). T h e TS-1 a n d TS-7 were 30 and 37°C, respective- decrease of 18 : 3 acid from 14.3 to 10.5% ly. in the m u t a n t TS-1 and from 18.6 to 8.0% T h e viability of resting cells o f the wild type in the m u t a n t TS-7 with increase in temperand the m u t a n t s grown at 20°(3 were com- ature from 20 to 40°(3 was not as significant pared at 50°C (Fig. 2). While 80% of the as noted in the wild type. T h e degree of cells of the wild type remained viable even fatty acid unsaturation (A/tool) was defined after 20 h of incubation at 50°C, the viabilities as (% monoene -k 2 [0/0 diene] -k 3 [ % o f the ceils o f TS-1 and TS-7 after 6 h o f triene])/100. While the A/mol of the mui n c u b a t i o n were 33 and 50%, respectively. tant TS-7 decreased from 1.39 at 20°C to 1.32 at 30 a n d 40°C, there was little variation Further loss o f viability was observed in both mutants after prolonged exposure o f the o f t h e v a l u e (1.26-1.28/mol) o f the m u t a n t TS-1 grown at 20, 30 and 40°C. Similar ceils to 50°C.

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= ~

105 0

5

10 15 20 Time(h)

OHTA,WIJEYARATNE,and I~AYA$HIDA

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[J. Ferment. Technol.,

Table 1. Lipid composition of temperature-sensitive mutants of H. poly~rpha CK-1. a Growth temperature (°C)

Content (mg/100 mg dry weight cells) b of Total Phosnholipid Sterol lipid "(P) (S)

S/P molar ratio

CK-1 (wild type) c 20

3.96

2.84 (71.7%)

0.38 ( 9 . 6 % )

0.26

30 40

3. 80 3.30

2. 68 (70. 5%) 1.90 (57.6%)

0. 41 (10. 8%) 0.53 (16.1%)

0. 30 0.54

20 30

6. 48 5.57

4. 06 (62. 7%) 2.64 (47.4%)

0. 68 (10. 5%) 0.67 (12.0%)

0. 33 0.50

4o

5. 40

2. 53 (46. 9%)

o. 65 (12. 0%)

o. 50

20

5. 08

2. 87 (56. 5%)

0. 45 ( 8. 9%)

0. 31

30 40

4.33 4. 37

2.14 (49.4%) 2.49 (57.0%)

0.37 ( 8 . 5 % ) 0. 42 ( 9. 6%)

0.34 0. 33

TS-1

TS-7

a Cells were grown to the mid-exponential phase in a synthetic medium containing 5% (w/v) glucose as the carbon source at the indicated temperatures, b Results represent average from three experiments. Figures in parentheses indicate the amounts of each lipid as percentages of total lipids. c Data from a previous study, t)

Table 2. Growth temperature (°C)

Fatty acid composition of temperature-sensitive mutants of H. polymorpha CK-I. a Fatty acid composition (%) b /mol~

14:0

16:0

16:1

18:0

18:1

18:2

18:3

Total unsaturated fatty acids

20 30 40

0. 3 1.2 1.3

16.3 18. 0 17.6

2.3 2.4 1.8

3.3 6.3 5.0

24. 9 23. 6 26.6

29. 5 29. 2 41.7

23. 4 19. 3 6.0

80. 1 74. 5 76.1

1.56 1.42 1.30

20 30 40

0. 8 0.9 0. 9

16, 4 17.1 18. 0

4. 8 3.1 2. 6

5. 6 5.7 8. 2

35.7 38.3 27.3

22. 4 19.8 32. 5

14. 3 15.1 10. 5

77.2 76.3 72. 9

1.28 1.26 1.26

20 30 40

0.7 1.4 1.5

17.0 19. 5 18. 5

3.3 2. 7 1.7

8.1 8. 8 7.5

24.9 22.4 19. 3

27.4 28. 3 43. 5

18.6 16. 9 8. 0

74.2 70. 3 72. 5

1.39 1.32 1.32

CK-1 (wild type) d

TS-1

TS-7

a Cells were grown to the told-exponential phase in a synthetic medium containing 5% (w/v) glucose as th~ carbon source at the indicated temperatures. b Fatty acids are denoted by the number of carbon atoms : number of unsaturated linkages. Fatty acid com. position was computed as a percentage of total peak areas and represents an average from two experiments. c The values of umaturation (A/tool) were calculated as (% monoene+2 [% diene] + 3 [% triene])]100. d Data from a previous studyA)

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Temperature-Sensitive Mutants of H. polymorpha

studies of a thermophilic bacillus (Bacillus stearothermophiUus) have shown that a temperaturesensitive mutant of this organism also had an inability to alter its fatty acid composition with temperatureA) I n summary, a temperature-sensitive mutant T S - I of H. polymorpha CK-1 showed little variation of the degree of fatty acid unsaturation (1.26-1.28/mol) and the mutant TS-7 had an almost constant sterol/phospholipid molar ratio (0.31-0.34) at 20, 30 and 40°C, as compared to the wild type showing the decrease of the degree of fatty acid unsaturation from 1.56 at 20°C to 1.30 at 40°C and the increase of sterol/phospholipid molar ratio from 0.26 at 20°C to 0.54 at 40°C. Genetic and biochemical studies on

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the mutation points in the mutants m a y provide useful information about the relationships between their temperature sensitivities and their lipid compositions. References

1) Wijeyaratne, S.C., Ohta, K., Chavanich, S., Mahamontri, V., Nilubol, N., Hayashida, S.: Agric. Biol. Ghem., 50, 827 (1986). 2) Wijeyaratne, S.C., Ohta, K., Hayashida, S.: J. Ferment. Technol., 65, 457 (1987). 3) Shaw, W.H.C., Jefferies, J.P.: Analyst, 78, 514 (195s). 4) Souza, K.A., Kostiw, L.L., Tyson, B.J.: Arch. Microbiol., 97, 89 (1974). (Received September 4, 1987)