Utilization of C20-polyunsaturated fatty acids by a yeast fatty acid desaturase mutant

Vol. 54, No. 4, 1973

BIOCHEMICAL

UTILIZATION

AND ElOPHYSlCAL

RESEARCH COMML’NICATIONS

OF C2C-POLYUNSATURATED FATTY ACIDS

BY A YEAST FATTY ACID DESATURASE MUTANT M. A. Williams, Dept. Calif.

I). W. Taylor,

J. Tinoco,

M. A. Ojakian

and A. D. Keith

of Nutritional Sciences, University of California, 94720 and Dept. of Biophysics, Pennsylvania State University Park, Pennsylvania 16802

Received

August

27,

Berkeley University,

1973

SUMMARY. A study was made of the utilization of C2G-polyunsaturated fatty acids by the S. cerevisiae fatty acid desaturase mutant olel-1 Arachidonic -1 acid, 8,11,14-eicosatrienoic acid, and 5,8,11,14,17-eicosapentaenoic acid were about equally effective in supporting growth with lactate as the carbon source. The relative proportion of these fatty acids in total cell fatty acids was ca. 50%. 5,8,11-eicosatrienoic acid synthesized from oleate was less effective. Very little growth occurred with 11,14,17-eicosatrienoic acid or with ll,lb-eicosadienoic acid. These results indicate the usefulness of the yeast mutant as a eucaryotic model for study of membrane systems enriched in specific C2C-polyunsaturated fatty acids. INTRODUCTION. rase

The S. cerevisiae

mutant,

can use a variety

9-unsaturated

fatty

by the

wild-type

system

containing

the

one which

"essential

acids

This

membrane has been acid

fatty or glycerol

whether

the

linolenate

animals.

These

include acid

(w?),

(C20:5w3), of both

increases

the

in essential

as

only

growth

medium

or oleate

(09)

normally

acid

acid (5). fatty

in rats

is the

acid

deficiency

acid

is

a major

when grown

acids

which

on

acid

of the

are important

(~20:306),

fed linolenic

C20:3w9

fatty

been made to determine

and 5,8,11-eicosatrienoic PGEl and arachidonic

cis-

eucaryotic

or ~20:4w6),

fatty series

the

synthesized

model

a

mutant

have yet

C20-polyunsaturated

desatu-

(l-3).

acid

no tests

acid

to replace

unsaturated

can be used by this

fatty

acid)

acids

serve

8,11,14-eicosatrienoic

C20 -unsaturated (9,12,15-octadecatrienoic which

by the

a fatty

acids)

can thus

in which

However,

the precursor

is the major

acid

lipids

can use other

(w6),

is

mutant

in animals,

linoleate

eicosapentaenoic

fatty

(5,8,11,14-eicosatetraenoic

(1,3).

mutant

(KD 115),

and palmitoleic

supplied

acid"

glucose

olel-1

of unsaturated

(oleic

yeast.

Arachidonic

C20:3&

mutant

5,8,11,14,17acid

(C20:3m9).

(4).

C20:5w3 acid

Cpo-polyunsaturated in

in

animab

fatty (4).

Vol. 54, No. 4, 1973

If

these

of olel-1, cell

polyunsaturated

quite

lipids

acids

high

(2).

should

functions,

If this

(~20:4w6)

were

so,

cell.

other

MATERIALS olel-1

acid.

nitrogen for

base

onto

YNBD plates.

shaking

the

unsaturated

The 125 ml Erlenmeyer cell

growth

filter).

established

(6).

was 105cells/ml. tionary

phase,

1% Tergitol extracted

acids

the

with

cells

measured

were

arachidonic

as well

as abili-

separated

For lipid then

with

evaporator

by thin-layer

chromatography

1561

and transfer with

vigorous

NP-40)

shaker

a Klett

and the late

log

at 3O'C. tubes,

average

CHC13/CH30H

heptadecanoic

has inoculum

or early

analyses,

sta-

twice the

with

cells

(2v/lv)

(8).

and (66

number

washed

and washed

with

photometer

and cell

chromatography with

yeast

and 1% DL-sodium

(O.D.)

into

and

checked

made of Klett

on YEPD-80,

water.

in a roLary

grown

by centrifugation,

20 hours,

were

1% Tergitol

with

reading

(0.67%

dilution

were

extract

of unsaturated

in a water-bath

had grown

harvested

source

cultures

containing

grown

mutant,

of 1% yeast

had sidearms

Klett

cultures

for

desaturase

2% peptone,

turbidity

distilled

by gas-liquid

cells

K2HP04,

were

were

concentrated

acid

by serial

the

flasks

Inocula

95% ethanol

CHC13 phase

with

the

compared

acids

to growth-supporting

The mutant

extract,

between

and once with

solvents

in the

studies,

by reading

After

and

fatty

on YNBD plates

experiments,

incubation

was monitored

fatty

properties

acids,

consisting

at 10 -3 or 10s4M,

acid

The relationship

been

the

growth

adjusted

fatty

fatty

and agar).

For growth

pH 5.6-6.5,

fatty

in

in these

membrane

and Tween 80 as the

in 25 ml of YEPL (1% yeast

lactate, the

after

enriched

we have

the growth

be present

of unsaturated

was done regularly

and 2% dextrose

revertants

affect

on YEPD-80, agar

plating

then

of cells

respect

The S. cerevisiae

dextrose,

Replica

might

report,

with

to support

lipids.

was maintained

2% each of peptone, fatty

cell

AND METHODS.

study

present

acid,

into

(KD 115),

then

acids

C2C-polyunsaturated

4,7,10,13,16,19-docosahexaenoic ty and incorporation

fatty

be able

by the presence

In the

and five

should

on how they

complication

by the

acids

of these

information

without

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

fatty

levels

provide

synthesized acid

BIOCHEMICAL

were (7),

The lipids and the acid

fatty

as internal

Vol. 54, No. 4, 1973

700

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMbNICAIIONS

2Ot4w6

2084~6

YEPL. 30-C.

I5

25

35

45

55

65

IS

25

35

HOURS

45

!I5

::

65

I, I:II II II ;: 5

3

HOURS

Fig. 1A: Growth of the S. cerevisiae fatty acid desaturase mutant olel-1 (KD 115) on lactate medium supplemented with C20- and C22-polyunsaturated fatty acids at 10v3M. Fig. 1B: Growth of the S. cerevisiae fatty acid desaturase mutant olel-1 (KD 115) on lactate m dium with arachidonic acid or 5,8,11-eicosatrienoic acid (C20:3w9) at 10 -6 M.

standard

(9,lO).

All

fatty

5,8,11-eicosatrienoic of essential

acids

acid

fatty

(99% purity)

preparation

were

which

acid-deficient-rats

from Hormel,

we obtained

by column

from

was 77% pure, with . This preparation 3 component (14%) was tentatively identified

1% oleic

major

as C22:3w9

satrienoic 3%.

acid).

Sodium

nitrogen

Other

lactate

fatty

and Tergitol

base and peptone

were

of arachidonic

A maximum response other

sufficient arachidonic

fatty acid

acid

occurred

C20-unsaturated

fatty acid

components

NP-40 were

from

of olel-1

Sigma.

lipids

on silicic

acid.

The other

(7,10,13-doco-

present

was tested

(5,8,11,14-eicosatetraenoic at 10 -3 M with acids

were

was available,

was as rapid

were

the

at levels Yeast

below

extract,

yeast

from Difco.

The growth

RESULTS AND DISCUSSION. trations

acid

carcass

chromatography

acid-AgNO

except

as with

lactate

also as well oleic

1562

tested

various

acid

or C20:406).

as the carbon

at this -4 as at 10 M.

acid

with

concen-

The

source.

concentration The growth

(g-octadecenoic

acid)

when rate

wit.

or with

Vol. 54, No. 4, 1973

Table

1.

BIOCHEMICAL

Fatty acid composition medium and supplemented

Fatty acid added

12:o

M/l

14:o

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of total cell lipids of cells grown on lactate with C20- and C22 -polyunsaturated fatty acids.

Fatty acid in cell lipids 18:o 20:3w6 20:3ug

16:O

20:4w6

20:5w3

22:6u3

% by weight 20:406

10~~

1.7

2.3

32.9

6.1

-

-

55.5

-

-

20:3o6

IO-~

8.9

9.0

21.8

1.6

53.7

-

-

-

-

20:5W3

1O-3

1.7

3.1

32.8

6.8

-

-

-

53.8

-

22:6,3

IO-~

6.8

5.0

40.2

4.4

-

-

-

-

39.2

20:4u6

10 -4

31.

47.

44.2

7.7

-

-

38.0

-

-

20:3w9

10 -4

32.

92.

54.9

5.9

-

19.2

-

-

-

20:3w6

10 -4

g8.

10.3

30.1

2.3

38.3

-

-

-

-

linoleic

acid

incubation than

(9,12-octadecadienoic

time

glucose

urated

acids

similar

Fig.

before

or glycerol,

fatty

be most

required

acid), the

would

be tested

under

to those

in animal

cells.

responses

(C20:5~3),

4,7,10,13,16,19-docosahexaneoic

enoic

(C20:303)

with

arachidonic

acid.

acid,

growth

C20:2w6,

Growth

and only

fatty

acids

were

the poor

growth

with

the

enter

of olel-1

cell

slight

carbon

source

so that

(~22:6w3),

acid

C20:5w3

(c20:2~6),

was slightly

better

than with

less

~22:6w3.

C20:3w3,

from

acid

source,

rather

than

or to be incorporated

into

1563

cell

from

lipids. with

C20:2w6

comparison

than

that

although

resulted

supplemented

would

acid

There

acids

medium

whieh

in

two fatty

lactate

polyunsat-

ll,lb,l7-eicosatri-

finding

fatty

the

conditions

This

latter

rather

5,8,11,14,17-eicosapentaenoic acid

growth

in the

Lactate,

lipids.

on the

present

phase.

in cell

as an unsaturated the

log

respiratory

to

but was significantly

of these

lity

with

was no increase

entered

and 11,14-eicosadienoic

arachidonic with

cells

was used as the

lA shows the growth

acid

and there

was no high

levels

indicates

that

their

unsuitabi-

an inability

The very

with

poor

or C20:3w3

to growth contrasts

Vol. 54, No. 4,1973

with

the results

two fatty

of Proudlock

acids

used as the that

with

inoculum.

(11).

although

because

cells than

acid

arachidonic

acid

(a reading

cells

listed

tected. retention

times

occurrence

less

of oleate

not

The reiative acids

reversion proporticn

fatty

acids

but

were

after

were

minor

either

rate

remained

of C20:4w6, the relative

components

by tbe were low

the

levei

1564

8,11,14-eicocurves at 10 -3 M

fatty

acids

acids.

The fatty

fatty

acids

fatty

(less

were than

with

either

5%).

inoculum

routinely

de-

acids

or to retention initial

in total

un-

Their by the

which

checked

for

had re-

(1 to 2% revertants).

c20:3~6

ana C~O::LU~ in tctal

proportion

at 10 -3, .

supplied

total

was

incubation).

and palmitoleate

The cultures

or C2O:3md at 10-4M,

fatty

to revertants

supplied

conclusion

with

40 hours

unidentified

Oleate

double

The growth

of the major

include

palmitate.

and palmitoleate

was 50-55%,

units

of

less

or arachidonate

90% or more of the

listed

be attributed

an& the

acid

1 represent

than

~2(3:3w6

was

Growth

This

(~20:4w6).

5,8-

acid

an additional

chain.

C2C -polyunsaturated

on Tween 80 media.

vertants,

either

or to the

was considerably

supplemented

proportions

on the

components

could

been grown

donic

grown

acid

acid

of 600 Klett

or when detected,

detectable,

when the

with

the relative

in Table

Minor

cells

supplemented

1 gives

from

or arachidonic

be factors,

was limited.

of a need for

of cultures

~22~6~3,

of 10e4&l fatty

preparation

an indication

to membrane with

acid

were

may indicate

at C-4 may also

preparation

acid

the growth

growth

A level

these

to glycerol

C2O:5~3

to arachidonic

end of the C2C-fatty

(~20:306)

of cells

Table

fatty

acid,

by comparing

identical

lipids

this

with

inferior

bond

preparation.

of the C20:3wg

the methyl

and yield

fatty

response

with

satrienoic

acids

growth

supplemented

supported

were

the

(C20:3@)

preadapted

with

too much disorder

of the

and double

amount

bond nearer

bond at C-17 gives a cause

good growth

growth

length

the

with

inferior

chain

IB compares

eicosatrienoic

who reported

medium when cells

may be also

the longer

Fig.

(3)

The somewhat double

This

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-et al -*

a glycerol

the additional

lipids

used

BIOCHEMICAL

In the

of either

of C22:dw3 cells fatty

grown acid

was only

cell 38%

on arachiin total

cell

Vol. 54, No. 4, 1973

Table

2.

BIOCHEMICAL

Major grown

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

fatty acid components of phospholipid classes from on arachidonic acid at lo-3M in lactate medium.

Phospholipid Class

Fatty 12:o

14:o

2.0 0.5

4.0 2.6 2.0

cells

acid

16:o

18:o

20:4w6

5.9 3.0 7.5

54.4 48.4 50.0

% by weight Phosphatidylcholine Phosphatidylethanolamine Phosphatidylinositol f Phosphatidylserine

lipids the

was 38%. cells

in less

However,

grown

On

growth

the

did

acid

(C22:3w9)

this

fatty

acid

was the major

Table

2 gives

cells

was

the

fatty

grown

demonstrates

the potential

In studies

evaluation will

decreases

in

activity

possible

be

with

to synthesize

However,

with

c20:4~6,

a eucaryotic

C20:3~9

the

or C20:4~6

poor

growth

fatty

acid

was -ca.

fatty availability

as the

of the yeast may be less

only

will

result

now exists

phosphafractions

proportion finding

mutants

fatty C20:3w9

arachidonate

the

speculation

together

membrane

(4,12). C20:309

properties acid is

stabili-

or ~20:4w6

can be obtained

which

with

Nevertheless,

and palmitoleate

mutant

also

acid.

of C20:3w9,

from

of

of phospho-

fatty

in animals,

in which

1565

since

preparation.

in decreased

oleate

with

of doco-

lipids

as a source

resulting

unsaturated

than

yeast

This

enzymes

until

of the

supplemented suitable

50%.

in the proportion

of the yeast

system

the

The relative

deficiency

cells

acids

in

amount

polyunsaturated

properties

animal

resulted

A minor

M.

of the mutant

of membrane-associated cellular

at 10m4M, which

of phosphatidylcholine,

at 10 -3M .

acid

~20:4w6,

19% in

of the C20:3w9

of a single

increases

-4

(Z-3$)

class

fatty

acid

was only

+ phosphatidylserine

acid

level

of essential

of specific

not

unable

a high

10

composition

usefulness

been made that

ty and altered

at

contaminant acid

of C20:3w9

fatty

detected

each phospholipid

containing

corresponding

acid

also

on arachidonic

in

has often

of this

and phosphatidylinositol

arachidonate

lipids

proportion

arachidonic

satrienoic

from

relative

the preparation

than

tidylethanolamine,

1.0

31.9 41.2 35.8

which

series

(13).

can use C20:3w9 of cells

as a structural

or

having The

can be compared. some evidence

are

that

this

component.

Vol. 54, No. 4, 1973

BIOCHEMICAL

Acknowledgment.

This

work

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

was supported

in part

by NIH Grant

AMl2024.

REFERENCES. 1.

2.

3. 4.

5. 6.

Wisnieski, James, R., L, 38-49 Proudlock, 327-349 Guarnieri, (1970). Pudelkewicz, (1968). Wisnieski,

B. J. and Kiyomoto, R. K., J. Bacterial. 2, 186-195 (1972). Branton, D., Wisnieski, B. and Keith, A., J. Supramol. Struct.

(1972). J. W., Haslam, (1971). M. and Johnson, C., B. J.,

Seufert, Keith,

J. M. and Linnane, R. M., Advances J. and Holman,

A. W., J. Bioenergetics in Lipid

R. T.,

A. D. and Resnick,

Research

J. Nutrition

M. A.,

2,

g, 115-174 94,

J. Bacterial.

138-146 101,

160-165 (1970).

11.

Getz, G. S., Jakovcic, S., Heywood, J., Frank, J. and Rabinowitz, M., Biochim. Biophys. Acta 218, 441-452 (1970). Rouser, G. and Fleischer, S., in Methods in Enzymology lo, 385-392 (1967), R. W. Estabrook and M. E. Pullman, eds., Academic Press, N.Y. Stancliff, R. C., Williams, M. A., Utsumi, K. and Packer, L., Arch. Biochem. Biophys. 131, 629-642 (1969). Skipski, V. P., Peterson, R. F. and Barclay, M., Biochem. J. 90, 374-378 (1964). Eletr. S. and Keith, A. D., Proc. Nat. Acad. Sci. U.S. 69, 1353-1357

12.

Bloj,

13.

Acta Bailey,

7. 8.

9. 10.

(1972). B.,

Morero, R. D., Farias, 311, 67-Q (1973). J. M. and Dunbar, L. M.,

R. N. and Trucco,

R. E.,

Exp. Molec.

g,

1566

Path.

Biochim.

Biophys

142-161 (1973).