APPLIED
AND
ENVIRONMENTAL
MICROBIOLOGY,
May
1992,
p.
1677-1682
0099-2240/92/051677-06$02.00/0
Copy igh
©)
1992,
Ame ican
Socie y
o
Mic obiology
O e p oduc ion
o
Th eonine
by
Saccha omyces
ce e isiae
Mu an s
Resis an
o
Hyd oxyno aline
CAYO
RAMOS
AND
ISABEL
L.
CALDERON*
Depa amen o
de
Gene ica,
Facul ad
de
Biologia,
Uni e sidad
de
Se illa,
Apa ado
1095,
E-41080
Se ille,
Spain
Recei ed
20
Decembe
1991/Accep ed
22
Feb ua y
1992
In
his
wo k,
we
isola ed
and
cha ac e ized
mu an s
ha
o e p oduce
h eonine
om
Saccha omyces
ce e isiae.
The
mu an s
we e
selec ed
o
esis ance
o
he
h eonine
analog
a-amino-13-hyd oxyno ale a e
(hyd oxyno aline),
and,
o
hese,
he
ones
able
o
exc e e
h eonine
o
he
medium
we e
chosen.
The
mu an
s ains
p oduce
be ween
15
and
30
imes
mo e
h eonine
han
he
wild
ype
does,
and,
o
a
lesse
deg ee,
hey
also
accumula e
isoleucine.
Gene ic
and
biochemical
s udies
ha e
e ealed
ha
he
h eonine
o e p oduc ion
is,
in
all
cases
s udied,
associa ed
wi h
he
p esence
in
he
s ain
o
a
HOM3
allele
coding
o
a
mu an
aspa a e
kinase
ha
is
o ally
o
pa ially
insensi i e
o
eedback
inhibi ion
by
h eonine.
This
enzyme
seems,
he e o e,
o
be
c ucial
in
he
egula ion
o
h eonine
biosyn hesis
in
S.
ce e isiae.
The
esul s
ob ained
sugges
ha
his
s a egy
could
be
e icien ly
applied
o
he
isola ion
o
h eonine-o e p oducing
s ains
o
yeas s
o he
han
S.
ce e isiae,
e en
hose
used
indus ially.
In
Saccha omyces
ce e isiae,
h eonine
and
me hionine
a e
syn hesized
om
aspa a e
h ough
a
common
me abolic
sequence
ha
leads
o
homose ine
in
h ee
s eps.
Homo-
se ine
is
he
b anching
poin
om
which
he
pa hways
o
me hionine
and
h eonine
di e ge.
The
egula ion
o
his
ou e
akes
place
a
a
numbe
o
s eps,
bo h
a
he
le el
o
enzyme
syn hesis
and
a
he
le el
o
ac i i y
( o
a
e iew,
see
e e ence
8).
Th eonine
seems
o
be
he
p ima y
com-
pound
ha
egula es
he
ca bon
low
in o
and
h ough
he
common
pa hway,
speci ically
a
he
le el
o
aspa a e
kinase
and
homose ine
dehyd ogenase,
he
i s
and
hi d
enzymes
o
his
ou e,
espec i ely.
Mo eo e ,
he
low
in o
each
speci ic
a m
is
con olled
by
end
p oduc
inhibi ion
o
he
co esponding
i s
enzymes:
h eonine
inhibi s
he
ho-
mose ine
kinase,
while
me hionine
inhibi s
he
homose ine
O-ace yl ans e ase.
The
egula ion
o
an
amino
acid
biosyn he ic
ou e
can
be
in es iga ed
by
he
use
o
amino
acid
analogs.
Mu an s
ha
a e
esis an
o
a
oxic
analog
a e
good
candida es
o
be
al e ed
in
hei
abili y
o
egula e
he
syn hesis
o
he
na u al
amino
acid.
In
ac ,
many
analogs
ha e
been
used
o
isola e
amino-acid-o e p oducing
s ains,
bo h
in
p oka yo es
(1)
and
in
euka yo es
(15).
o -Amino-,-hyd oxy ale ic
acid
(hyd oxyno aline)
is
a
h eonine
analog
known
o
inhibi
bac e ial
g ow h
mainly
because
i
mimics
h eonine
and
hinde s
he
ac i i y
o
he
h eonine
eedback-inhibi ed
enzymes
(4).
As
a
conse-
quence,
cells
g owing
in
he
p esence
o
hyd oxyno aline
p oduce
smalle
quan i ies
o
he
amino
acids
syn hesized
h ough
his
pa hway,
which
can
e en
become
limi ing
o
g ow h.
Hyd oxyno aline- esis an
mu an s
ha e
been
iso-
la ed
om
bac e ia
such
as
Eschenchia
coli
and
B e ibac e-
num
and
Co ynebac enum
spp.
(13).
In
hese
mu an s,
one
o
he
h eonine-sensi i e
enzymes
has
become
insensi i e
o
he
eedback
con ol
by
his
amino
acid
and,
as
a
*
Co esponding
au ho .
P esen
add ess:
Depa men
o
Yeas
Gene ics,
Ca lsbe g
Labo a o y,
Copenhagen,
Denma k.
consequence,
he
mu an s
o e p oduce
o
a
ce ain
deg ee
and
e en
exc e e
h eonine
in o
he
medium.
To
ou
knowledge,
hyd oxyno aline
has
ne e
been
es ed
be o e
in
any
yeas
species.
I
he
si ua ion
in
his
o ganism
was
simila
o
ha
in
bac e ia,
one
would
expec
ha ,
in
a
ce ain
hyd oxyno aline- esis an
yeas
s ain,
one
o
mo e
s eps
o
he
pa hway
would
ha e
been
eleased
om
h eonine
inhibi ion.
Some
o
hese
mu an s
could
also
o e p oduce
and
exc e e
h eonine.
The
equency
a
which
each
kind
o
mu an
a ises
and
he
deg ee
o
o e p oduc ion
o
he
amino
acid
would
be
indica i e
o
he
ela i e
impo -
ance
o
ha
s ep
in
he
con ol
o
he
pa hway.
Addi ionally,
hyd oxyno aline
esis ance
could
also
a ise
by
a
de ec
in
he
pe mease
ha
anspo s
i
in o
he
cell.
In
his
wo k,
we
ha e
isola ed
hyd oxyno aline- esis an
mu an s
om
S.
ce e isiae
and
ha e
selec ed
om
among
hem
hose
able
o
exc e e
h eonine
in o
medium,
which
indica es
h eonine
o e p oduc ion.
We
ha e
s udied
he
accumula ion
o
h eonine
and
o he
amino
acids
in
he
mu an
cells
and
cha ac e ized
hei
de ec
bo h
biochemi-
cally
and
gene ically.
MATERIALS
AND
METHODS
S ains.
The
S.
ce e isiae
s ains
used
a e
lis ed in
Table
1.
Enzymes
and
chemicals.
Hyd oxyno aline
was
a
gi
om
Degussa
A.B.M.
(F ank u ,
Ge many);
bo elidin
was
a
kind
gi
om
K.
Po alla;
lac a e
dehyd ogenase
(EC
1.1.1.27),
py u a e
kinase
(EC
2.7.1.40)
om
abbi
muscle,
phosphoenol
py u a e,
o-ph halaldehyde,
and
P-me cap o-
e hanol
we e
om
Sigma
Chemical
Co.
(S .
Louis,
Mo.);
N-me hyl-N'-ni o-N-ni osoguanidine
(NTG)
was
om
Al-
d ich
Co.
(Do se ,
Uni ed
Kingdom);
all
o he
biochemicals
we e
pu chased
om
Sigma.
Ino ganic
compounds
we e
om
Me ck
AG
(Da ms ad ,
Ge many).
Media.
Comple e
(YPD)
medium,
minimal
(SD)
medium,
and
p espo ula ion
and
spo ula ion
media
we e
p epa ed
by
he
me hods
o
She man
e
al.
(20).
Minimal
p oline
(SDP)
medium
is
iden ical
o
SD
medium,
excep
ha
L-p oline
(0.1%)
is
used
ins ead
o
ammonium
sul a e
(0.5%)
as
he
sole
ni ogen
sou ce
(6).
Hyd oxyno aline
oxici y
was
1677
Vol.
58,
No.
5
1678
RAMOS
AND
CALDERON
TABLE
1.
S.
ce e isiae
s ains
used
in
his
wo k
S ain(s)
Geno ype
o
pheno ype
Sou cea
D273-11A
MATox
adel
hisl
CSH
F4
MATa
h 4
LCC
MMY1
MA
To
u a3D52
CyhR
SERI
XCR13-5A
MATa
pl-l
lysl-l
u a4
leu2
DGS
XCR28-4A
MATa
u a3
a g6
hom3
hisl
DGS
AHV1
o
AHV6
MATa
pl-l
lysl-l
u a4
Ieu2
This
wo k
Ah R
Th E
YAHV1
MATa
u a4
HOM3-RI
This
wo k
YAHV2
MATa
HOM3-R2
This
wo k
YAHV3
MATa
HOM3-R3
This
wo k
YAHV5
MATa
u a3
HOM3-R5
This
wo k
YAHV6
MATa
u a3
HOM3-R6
This
wo k
a
Abb e ia ions:
CSH,
Cold
Sp ing
Ha bo
Labo a o y
(Cold
Sp ing
Ha -
bo ,
N.Y.);
LCC,
La
C uz
del
Campo
S.A.
(Se ille,
Spain);
SERI,
Sola
Ene gy
Resea ch
Ins i u e
(Golden,
Colo.);
DGS,
Depa amen o
de
Gene ica
y
Bio ecnia,
Uni e sidad
de
Se illa
(Se ille,
Spain).
es ed
in
SDP
pla es
con aining
10
mM
hyd oxyno aline.
Unde
hese
g ow h
condi ions,
he
MIC
o
he
wild
ype
was
ound
o
be
2.5
mM.
When
necessa y,
minimal
media
we e
supplemen ed
wi h
he
app op ia e
equi emen s
(20).
Mu agenesis.
Ea ly-s a iona y-phase
cells
o
s ain
XCR13-
5A
g own
in
YPD
medium
a
30°C
we e
ea ed
wi h
NTG
as
ecommended
by
Calde on
and
Ce da-Olmedo
(3).
Unde
hese
s anda d
condi ions,
he
su i al
a e
o
his
s ain
was
app oxima ely
40%.
C oss- eeding
es
o
h eonine
exc e ion.
Cells
o
he
Th -
s ain
F4
we e
g own
o
he
s a iona y
phase
in
YPD
medium,
cen i uged,
washed
wice,
and
suspended
in
wa-
e .
A
sample
o
his
cell
suspension,
con aining
abou
106
cells,
was
sp ead
on o
SD
pla es.
The
s ains
o
be
es ed
we e
s eaked
on o
his
cell
lawn
wi h
oo hpicks,
and
he
pla es
we e
incuba ed
a
he
empe a u es
s a ed
o
di e en
pe iods
o
ime.
The
p esence
o
a
halo o
F4
colonies
g owing
a ound
a
s eak
was
conside ed
a
posi i e
esul .
Gene ic
analysis.
Con en ional
p ocedu es
we e
used
o
s ain
ma ing,
diploid
selec ion,
and
e ad
analysis
(20).
Pa ial
pu i ica ion
o
he
aspa a e
kinase.
Aspa a e
ki-
nase
was
pa ially
pu i ied
by
he
me hod
o
Ramos
e
al.
(16).
The
me hod
is
basically
ha
desc ibed
as
ollows.
La e-exponen ial-phase
cells,
g own
a
30°C
in
SD
medium,
we e
ha es ed
and
esuspended
in
AT
bu e
(40
mM
phospha e
[pH
7.2],
0.1
M
KCI,
5
mM
MgCl2,
2
mM
EDTA,
1
mM
di hioe y h i ol
1
mM
L- h eonine
[19])
and
dis up ed
in
a
B aun
homogenize
wi h
0.5-mm-diame e
glass
beads.
Cell
deb is
was
emo ed
by
wo
consecu i e
cen i uga ions
a
4°C,
he
i s
o
20
min
a
8,000
x
g
and
he
second
one
o
2
h
a
105,000
x
g.
P o amine
sul a e
(5
mg/g
[we
weigh ]),
neu alized
o
pH
7
wi h
NaOH,
was
added
o
he
c ude
ex ac ,
and,
a e
1
h
o
s i ing
a
4°C,
he
pelle
was
emo ed
by
cen i uga ion.
Ammonium
sul a e
was
added,
and
he
ac ion
p ecipi a ing
be ween
30
and
45%
sa u a ion
ha
con ained
he
aspa a e
kinase
ac i i y
was
edissol ed
in
BT
bu e
(iden ical
o
AT
bu e
excep
ha
he
phos-
pha e
concen a ion
was
20
mM),
dialyzed ex ensi ely
agains
he
same
bu e ,
and
s o ed
a
-80°C.
Aspa a e
kinase
assay.
Aspa a e
kinase
ac i i y
was
assayed
as
p e iously
epo ed
(16)
by
moni o ing
ADP
p oduc ion
by
use
o
he
py u a e
kinase-lac a e
dehyd oge-
nase
coupled
assay
desc ibed
o
he
E.
coli
homose ine
kinase
(2,
21),
excep
ha
10
mM
ATP
and
4
mM
L-aspa a e
we e
used
as
he
subs a es.
One
uni
o
ac i i y
was
de ined
as
he
amoun
o
pa ially
pu i ied
ex ac
equi ed
o
con-
e
1
,umol
o
ATP
o
ADP
pe
min
a
30°C.
The
s anda d
assay
mix u e
con ained
he
componen s
desc ibed
p e i-
ously
(19),
wi h
mino
modi ica ions.
The
eac ion
was
ini ia ed
by
adding
L-aspa a e
o
he
mix u e.
P o ein
con-
cen a ions
we e
de e mined
by
he
me hod
o
Low y
e
al.
(9).
Amino
acid
de e mina ion.
Amino
acids
we e
analyzed
by
e e se-phase
liquid
ch oma og aphy
by
he
me hod
o
Ma -
inez-Fo ce
and
Beni ez
(10).
The
sys em
consis ed
o
a
Wa e s
high-pe o mance
liquid
ch oma og aph
wi h
a
No apack
C-18
column
(18
by
100
mm),
wo
pumps,
and
a
Wa e s
abso bance
de ec o .
An
IBM
AT
compu e
wi h
a
Baseline
810
ch oma og aphy
wo ks a ion
was
used
as
he
con olle ,
in eg a o ,
and
da a
collec o .
The
g adien
p o-
g am
consis ed
o
wo
sepa a e
sol en
mix u es.
Sol en
A
consis ed
o
20
mM
sodium
phospha e
(pH
7.2),
1.5%
e ahyd o u an,
and
93.5%
dis illed
wa e
(by
olume);
sol en
B
consis ed
o
10
mM
sodium
phospha e
(pH
7.2),
55%
ace oni ile,
and
42.5%
dis illed
wa e
(by
olume).
Amino
acid
samples
we e
p epa ed
as
desc ibed
p e i-
ously
(5).
Samples
(40
ml)
o
a
la e-exponen ial-phase
cul u e
(op ical
densi y
a
660
nm,
app oxima ely
0.7),
we e
cen i-
uged.
The
supe na an
was
eeze-d ied
and
esuspended
in
1
ml
o
deionized
wa e ,
and
he
suspension
was
il e ed
h ough
a
0.45-,um-po e-size
Millipo e
il e
and
used
o
measu e
exc e ed
amino
acids.
To
de e mine
in acellula
amino
acids,
he
pelle
was
esuspended
in
2
ml
o
deionized
wa e
and
boiled
o
15
min.
A
clea
supe na an
was
ob ained
a e
a
b ie
cen i uga ion
and
a
il a ion
as
desc ibed
abo e.
Amino
acids
we e
de i a ized
by
mixing
25
ml
o
each
suspension
wi h
75
ml
o
he
ollowing
mix u e:
0.4
M
sodium
bo a e
(pH
10),
4.5
ml;
54
mg
o
o-ph halaldehyde
pe
ml
o
me hanol,
0.5
ml
(10).
RESULTS
Condi ions
o
hyd oxyno aline
oxici y
es .
P elimina y
expe imen s
showed
ha
hyd oxyno aline
is
oxic
in
media
wi h
a
poo
ni ogen
sou ce
such
as
p oline
bu
no
in
media
wi h
a
good
ni ogen
sou ce
such
as
ammonium
sul a e
(da a
no
shown).
This
is
p obably
due
o
he
ac
ha
his
compound
en e s
he
yeas
cell
mainly
h ough
he
gene al
amino
acid
pe mease
(11),
whose
ac i i y
is
bo h
inhibi ed
and
ep essed
by
ammonium
(6).
Consequen ly,
all
o
he
media
in
which
hyd oxyno aline
oxici y
was
o
be
es ed
ca ied
p oline
as
he
sole
ni ogen
sou ce.
I
was
also
ound
(da a
no
shown)
ha
he
addi ion
o
di e en
amino
acids
such
as
h eonine,
homose ine,
aspa -
agine,
and
ci ulline
o
media
con aining
hyd oxyno aline
coun e ac ed
i s
oxici y.
The
deg ee
o
his
e ec
was
a iable,
p obably
depending
on
he
mechanism
in ol ed
in
each
case.
As
expec ed
om
he
p oposed
hyd oxyno aline
mode
o
ac ion
(4),
h eonine
and
homose ine,
a
h eonine
p ecu so ,
imp o ed
he
s ain
g ow h.
I
is
e y
likely
ha
he
h eonine
o
homose ine
addi ion
bypasses
he
need
o
he
enzymes
ac ing
in
his
biosyn he ic
pa hway,
some
o
which
a e
inhibi ed
by
his
d ug
(16).
Consis en
wi h
his
iew,
i
was
ound
ha ,
e en
hough
me hionine
alone
had
no
e ec ,
he
simul aneous
addi ion
o
h eonine
and
me hi-
onine
comple ely
es o ed
g ow h.
Besides,
hese
and
o he
amino
acids
may
exe
hei
in luence
a
he
anspo
le el.
Ci ulline,
o
ins ance,
may
compe e
wi h
hyd oxyno aline
o
he
gene al
amino
acid
pe mease,
while
o he s
ha
se e
as
good
ni ogen
sou ces
(e.g.,
aspa agine)
may
a ec
he
APPL.
ENVIRON.
MICROBIOL.
THREONINE
OVERPRODUCTION
BY
S.
CEREVISIAE
MUTANTS
1679
0-
*1
la
V
2
V
IC
._
S
E
21
a
o_
0-
0
AHV2
AUV3
AHVS
S ain
FIG.
1.
Quan i ies
o
amino
acids
accumula ed
(inside)
and
exc e ed
o
he
medium
(ou side)
by
di e en
Ah R
Th E
s ains.
Symbols:
_,
h eonine
inside;
Eli,
h eonine
ou side;
,
isoleucine
inside;
W,
me hionine
inside.
gene al
amino
acid
pe mease
ac i i y,
hus
hinde ing
i s
en ance
in o
he
cell.
Isola ion
o
hyd oxyno aline- esis an
(AhVR)
mu an s.
Cells
o
he
wild- ype
s ain
XCR13-5A
we e
pla ed,
ei he
di ec ly
o
a e
ni osoguanidine
ea men ,
on o
SDP
me-
dium
con aining
10
mM
hyd oxyno aline,
and
he
pla es
we e
incuba ed
o
5
days.
Spon aneous
and
NTG-induced
mu an s
a ose
among
he
su i o s
a
equencies
o
1.5
x
10-6
and
5.0
x
10-5,
espec i ely.
In
S.
ce e isiae,
he
exc e ion
o
an
amino
acid
by
a
ce ain
s ain
usually
indica es
o e p oduc ion
o
his
amino
acid.
This
c i e ion
was
applied
o
selec
candida es
o
become
h eonine
o e p oduce s
om
he
Ah R
s ains.
Thus,
all
Ah R
colonies
ob ained
we e
subjec ed
o
he
h eonine
c oss- eeding
es
desc ibed
in
Ma e ials
and
Me hods.
While
60
o
he
128
NTG-induced
Ah R
mu an s
es ed
we e
h eonine
exc e o s
(Th E),
none
o
he
60
spon aneous
Ah R
colonies
we e
Th E.
We
do
no
ha e
a
plausible
explana ion
o
his
di e ence.
The
Ah R
Th E
s ains
(designa ed
by
AHV
ollowed
by
a
numbe )
we e
used
in
u he
s udies.
Dominance
o
ecessi eness
es .
The
dominance
o
eces-
si eness
o
he
alleles
esponsible
o
he
Ah R
and
Th E
pheno ypes
wi h
espec
o
he
wild- ype
alleles
was
in es-
iga ed
by
c ossing
he
mu an
s ains
wi h
s ain
MMY1.
All
o
he
esul ing
diploids
we e
able
o
g ow
on
SDP
medium
con aining
10
mM
hyd oxyno aline,
a e
4
days
o
incuba-
ion
a
30°C,
p o ing
he
dominance
o
he
mu an
alleles.
All
o
hem
we e
also
able
o
c oss- eed
he
Th -
s ain,
i.e.,
o
exc e e
h eonine,
when
incuba ed
o
4
days
a
37°C.
Howe e ,
when
incuba ed
a
22°C,
hey
showed
a
a ie y
o
esponses
in
he
c oss- eeding
es :
no
diploid
exc e ed
be o e
4
days
o
incuba ion,
and
while
some
p esen ed
an
exc e ion
halo
a e
7
days,
o he s
did
no ,
e en
a e
11
days
o
incuba ion.
Fi e
AH-V
s ains
whose
espec i e
AHV/
MMY1
diploids
showed
di e en
c oss- eeding
imes
a
22°C
we e
chosen
o
u he
cha ac e iza ion.
The
selec ed
s ains
we e
AHV1
(c oss- eeding
ime,
7
days),
AHV2
and
AHV3
(c oss- eeding
imes,
9
days),
AHV5
(c oss- eeding
ime,
11
days),
and
AHV6
(no
c oss- eeding
obse ed
a e
11
days
o
incuba ion).
Accumula ion
o
amino
acids
by
he
mu an
s ains.
To
es ablish
whe he
he
Ah R
Th E
s ains
we e,
in
ac ,
h eonine
o e p oduce s,
he
quan i ies
o
his
and
o he
amino
acids
accumula ed
and
exc e ed
in o
he
medium
by
hese
s ains
we e
in es iga ed.
The
esul s
(Fig.
1)
show
ha
all
o
hem
accumula e
15
o
30
imes
mo e
h eonine
and
exc e e
abou
3
imes
mo e
h eonine
han
he
pa en al
s ain.
They
also
con ain
mo e
isoleucine,
an
amino
acid
ha
de i es
om
h eonine,
han
he
con ol
does.
Bo h
in
he
mu an s
and
in
he
pa en al
s ain,
he
accumula ed
isoleu-
cine
seems
o
be
p opo ional
o
ha
o
h eonine
(abou
1:5
o
1:3).
Howe e ,
he
amoun
o me hionine,
whose
biosyn-
he ic
pa hway
sha es
se e al
s eps
wi h
ha
o
h eonine,
is
simila
o
ha
o
he
con ol
in
all
mu an s.
Gene ic
cha ac e iza ion.
The
ollowing
expe imen s
we e
designed
o
de e mine
how
many
genes
a e
esponsible
o
he
Ah R
and
Th E
pheno ypes
and
o
y
o
es ablish
wha
gene(s)
is
esponsible
o
he
h eonine
o e p oduc ion
phe-
no ype.
P e ious
kine ic
s udies
demons a ed
ha
he
as-
pa a e
kinase
plays
a
e y
impo an
ole
in
he
egula ion
o
he
pa hway
a
he
enzyma ic
ac i i y
le el
(16).
Thus,
we
i s
ied
o
p o e
i ,
a
leas
in
some
cases,
h eonine
o e p oduc ion
depends
on
he
p esence
in
he
cell
o
a
mu an
allele
o
he
HOM3
gene,
which
codes
o
his
enzyme.
This
kind
o
es
is
usually
ca ied
ou
by
meio ic
analysis
o
c osses
be ween
he
mu an
s ain
and
a
s ain
ca ying
a
known
allele,
in
his
case,
hom3.
Howe e ,
such
a
c oss
p o ides
only
pa ial
in o ma ion
since
he
Hom3-
spo es
equi e
he
p esence
o
homose ine
in
he
medium.
This
would
in e e e
se e ely
wi h
he
hyd oxyno aline
esis ance
es .
Consequen ly,
only
Hom3+
spo es
a e
sus-
cep ible
o
such
an
analysis.
As
s a ed
abo e,
he
p esence
o
amino
acids
in
he
medium
in e e es
wi h
he
hyd oxyno aline
esis ance
es .
Thus,
i
was
ound
necessa y
o
elimina e
as
many
gene ic
ma ke s
as
possible
om
he
c oss.
AHV/MMY1
diploids
we e
spo ula ed,
and
he
esul ing
e ads
we e
VOL.
58,
1992
1680
RAMOS
AND
CALDERON
TABLE
2.
Gene ic
analysis
o
Ah R
Th E
s ains
No.
o
e adsa
C oss
Ma ke s
PD
NPD
T
YAHV2
x
D273-11A
Th E_HISJ
26
0
0
Ah - HIS1
26
0
0
Ah R-Th E
26
0
0
YAHV5
x
D273-11A
Th E_HISJ
19
0
0
AhVR-HIS1
19
0
0
Ah R
-Th E
19
0
0
YAHV6
x
D273-11A
Th E_HISJ
13
0
1
Ah R
HIS1
13
0
1
Ah R-Th E
14
0 0
a
PD,
pa en al
di ype;
NPD,
nonpa en al
di ype;
T,
e a ype.
TABLE
3.
E ec
o
L- h eonine
and
hyd oxyno aline
on
he
aspa a e
kinase
ac i i y
o
he
Ah R
Th E
s ainsa
Inhibi ion
(%)
by:
S ain
Sp
ac
0m
(mU
mg
o
p o ein-)
20
mM
Hyd oxy
L-Th eonine
Hy oxy-
no aline
XCR13-5A
188
90
91
AHV1
38
0
0
AHV2
45
00
AHV3
135
0
32
AHV5
37 0
0
AHV6
44
50
21
a
Each
alue
ep esen s
he
mean
o
a
leas
wo
expe imen s;
he
s anda d
de ia ion
was,
in
all
cases,
a ound
15%.
DISCUSSION
dissec ed.
Meio ic
descendan s
om
hese
diploids
showing
an
Ah R
Th E
pheno ype
and
ei he
a
U a+
o
U a-
pheno-
ype
we e
chosen
and
called
YAHV
( ollowed
by
he
same
numbe
as
ha
o
hei
pa en s).
When
analyzing
his
c oss,
i
could
al eady
be
s a ed
ha ,
in
all
cases,
he
exc e ion
o
h eonine
seg ega ed
in
a
2+
:2-
ashion,
indica ing
he
monogenic
na u e
o
his
cha ac e .
S ains
YAHV1,
YAHV2,
and
YAHV5
we e
c ossed
wi h
s ain
XCR28-4A
(hom3).
Abou
20
e ads
de i ing
om
each
c oss
we e
dissec ed
and
analyzed.
In
hese
c osses,
he
exc e ion
o
h eonine
also
seg ega ed
in
a
2+
:2-
ashion.
Mo eo e ,
all
o
he
Th E
spo es
we e
Hom3+
and
no
ecombinan
Hom3+
Th NE
( h eonine-nonexc e ing)
spo e
was
obse ed.
This
esul
sugges s
ha
hom3
and
he
h ee
mu a ions
a e
allelic.
The e o e,
hese
mu a ions
ha e
been
designa ed
HOM3R-1,
HOM3R-2,
and
HOM3R-5.
This
conclusion
was
u he
p o en
when
Ah R
Th E
mu an s
we e
c ossed
wi h
a
s ain
ca ying
a
hisl
allele.
The
genes
HIS]
and
HOM3
a e
loca ed
in
he
S.
ce e isiae
map
a
a
dis ance
o
2.5
cen imo gans
(12).
This
c oss
allowed
us
o
ca y
ou
whole- e ad
analysis.
Table
2
p esen s
he
esul s
o
he
gene ic
analysis
o
he
c osses
be ween
he
s ains
YAHV
and
D273-11A.
I
clea ly
shows
he
close
linkage
be ween
he
ma ke s
s udied.
Thus,
we
can
in e
ha
he
o e p oduc ion
o
h eonine
depends
on
he
p esence
in
hese
s ains
o
mu an
alleles
o
he
HOM3
gene.
Aspa a e
kinase
ac i i y
in
he
mu an s.
S udies
ca ied
ou
o
de e mine
he
kine ic
pa ame e s
o
he
aspa a e
kinase
showed
ha
bo h
subs a es,
ATP
and
L-aspa a e,
displayed
no mal
Michaelis-Men en
sa u a ion
kine ics,
wi h
Kms
o
3.6
and
4
mM,
espec i ely
(16).
I
has
also
been
es ablished
ha
h eonine
e y
e ec i ely
inhibi s
he
aspa -
a e
kinase
ac i i y,
3
mM
being
he
concen a ion
equi ed
o
hal -maximal
inhibi ion
(16).
O
o he
amino
acids
es ed,
only
hyd oxyno aline
inhibi s
his
ac i i y
(16).
These
and
o he
s udies
sugges
ha
he
egula ion
o
he
aspa a e
kinase
o
S.
ce e isiae
akes
place
mainly
h ough
eedback
inhibi ion
by
h eonine
(16).
Thus,
an
al e a ion
in
his
enzyme,
such
as
making
possible
he
ac i i y
o
he
enzyme
bu
no
i s
egula ion
by
h eonine,
could
cause
o e p oduc-
ion
o
his
amino
acid.
To
es
whe he
his
was
he
case
wi h
he
Ah R
Th E
mu an s,
hei
aspa a e
kinase
was
pa ially
pu i ied
and
he
e ec s
o
h eonine
and
hyd oxy-
no aline
on
he
ac i i y
we e
assayed.
The
esul s
(Table
3)
show,
in
ac ,
ha
all
mu an s
es ed
ha e
an
al e ed
pa e n
o
inhibi ion
by
bo h
amino
acids.
In
his
a icle,
we
desc ibe
he
isola ion
and
cha ac e iza-
ion
o
S.
ce e isiae
mu an s
esis an
o
he
h eonine
analog
hyd oxyno aline.
Among
hem,
hose
able
o
c oss-
eed
a
h eonine
auxo oph
we e
chosen
as
candida es
o
h eonine
o e p oduc ion.
This
c i e ion
has
p o en
o
be
use ul
since
all
selec ed
s ains
a e,
in
ac ,
h eonine
o e -
p oduce s.
Howe e ,
a
nega i e
esul
in his
biological
es
does
no
necessa ily
mean
ha
he
s ain
p oduces
he
same
amoun
o
h eonine
as,
o
less
han,
he
wild
ype.
I
is
possible
ha
e en
i
a
la ge
amoun
o
h eonine
is
p oduced
pe
cell,
ei he
i
is
no
exc e ed
in o
he
medium
o
he
amoun
p oduced
and
exc e ed
is
no
enough
o
eed
he
es e
s ain.
I
is
also
possible
ha
he
g ow h
a e
o
he
s ain
is
so
low
ha
he
inal
biomass,
and
hus
he
o al
p oduc ion
o
h eonine,
is
small.
I
so,
one
would
expec
a
con inuous
dis ibu ion
o
he
Ah R
mu an s
wi h
espec
o
h eonine
p oduc ion;
only
hose
mu an s
p oducing
and
exc e ing
a
ce ain
le el
o
h eonine
would
be
de ec ed
as
such
in
he
es .
In
ac ,
when
he
c oss- eeding
es
was
pe o med
a
22°C,
he
he e ozygo ic
Ah R
Th
/wild
ype
diploids
beha ed
e y
di e en ly.
Howe e ,
his
beha io
does
no
co ela e
wi h
he
h eonine
p oduc ion
o
he
co esponden
haploid
s ain;
in
ac ,
he
AHV6/MMY1
diploid
is
he
slowes
exc e o ,
despi e
he
ac
ha
AHV6
is
he
bes
h eonine
p oduce .
Mo eo e ,
no
co ela ion
was
ound
be ween
he
amoun
o
h eonine
p oduced
and
he
amoun
exc e ed
by
he
mu an s:
he
amoun
o
accumula ed
h eonine
a ied
be ween
15
and
30
imes
mo e
han
ha
in
he
wild
ype,
while
he
quan i y
o
exc e ed
h eonine
was,
in
all
o
he
mu an s,
only
abou
3
imes
highe
han
ha
in
he
wild
ype
(Fig.
1).
Thus,
we
can
in e
ha
he
a ie y
ound
among
he
he e ozygo ic
diploids
in
he
c oss- eeding
es
is
no
due
o
ue
di e ences
in
exc e ion
bu
is
due
o
di e ences
in
o he
ac o s
such
as
g ow h
a e
o
lysis
o
he
s ain
a
ha
empe a u e.
Since
one
o
he
pa en s
o
he
diploids
is,
in
all
cases,
s ain
MMY1,
such
di e ences
should
e lec
di e ences
be ween
he
AHV
mu an s.
These
mu an s
may
di e
no
only
in
hei
HOM3
allele
bu
also
in
he
gene ic
backg ound
due
o
he
indisc imina e
ac ion
o
he
ni osoguanidine.
S.
ce e isiae
mu an s
able
o
exc e e
amino
acids
ha e
been
isola ed
p e iously
(15).
In
all
cases
s udied,
exc e ion
was
shown
o
be
a
di ec
consequence
o
o e p oduc ion.
Two
genes
speci ically
a ec ing
h eonine
exc e ion
(TEX1
and
TEX2)
ha e
been
desc ibed
(5).
These
genes
a e
linked
nei he
o
each
o he
no
o
HOM3
(5).
Labo a o y
s ains
seem
o
ha e
di e en
combina ions
o
mu an
and
wild- ype
APPL.
ENVIRON.
MICROBIOL.
THREONINE
OVERPRODUCTION
BY
S.
CEREVISIAE
MUTANTS
1681
TEX
alleles.
The
close
ela ionship
be ween
o e p oduc ion
and
exc e ion
ound
in
all
o
he
gene ic
c osses
ca ied
ou
in
his
wo k
sugges s
ha
all
s ains
used
a e
isogenic
wi h
espec
o
he
TEX
alleles,
and,
he e o e,
ha
his
ac o
is
i ele an
o
he
in e p e a ion
o
he
esul s.
All
Ah R
Th E
mu an s
examined
so
a
ha e
an
aspa a e
kinase
ha
is
o ally
o
pa ially
insensi i e
o
eedback
inhibi ion
by
h eonine.
O he
au ho s,
using
di e en
s a -
egies,
ha e
ound
simila
esul s.
Nass
and
Po alla
(14)
isola ed
mu an s
esis an
o
he
mac olide
an ibio ic
bo e-
lidin.
Thei
mu an s
ell
in o
ou
g oups.
Only
one
o
hem,
BOR1,
co esponds
o
s ains
ha
o e p oduce
h eonine;
all
BOR1
s ains
bea
mu a ions
in
he
HOM3
gene.
Thus,
bo elidin
is
no
adequa e
o
ob ain
h eonine-o e p oducing
s ains,
since
mos
o
he
esis an
mu an s
isola ed
do
no
accumula e
his
amino
acid.
As
expec ed,
AHV
mu an s
a e
also
esis an
o
20
,uM
bo elidin
(da a
no
shown),
and,
con e sely,
he
BOR1
mu an s
es ed
(BOR1-1
and
BORI-
III
[see
e e ence
14])
a e
esis an
o
hyd oxyno aline
(da a
no
shown).
Delgado
e
al.
(5)
showed
ha
i
is
possible
o
di ec ly
isola e
h eonine-o e p oducing
s ains
by
e e ing
a
Hom3-
mu an ;
all
o
he
isola ed
s ains
had
a
eedback-
insensi i e
aspa a e
kinase.
Gi en
he
mode
o
ac ion
o
hyd oxyno aline
(4),
one
would
expec
o
ind,
among
he
Ah R
Th E
s ains,
mu an s
in
he
enzymes
suscep ible
o
eedback
inhibi ion
by
h eonine,
namely,
aspa a e
kinase,
homose ine
dehyd ogenase,
and
homose ine
kinase.
How-
e e ,
i
has
been
epo ed
ha
yeas
aspa a e
kinase
is
much
mo e
sensi i e
o
inhibi ion
by
hyd oxyno aline
han
ho-
mose ine
kinase
(16).
The e o e,
i
is
logical
ha
only
mu an s
in
he
aspa a e
kinase
we e
isola ed.
On
he
o he
hand,
no
co ela ion
was
ound
be ween
he
deg ee
o
sensi i i y
o
eedback
inhibi ion
by
h eonine
o
hyd oxy-
no aline
o
he
aspa a e
kinase
and
he
p oduc ion
o
h eonine
(Table
3).
This
esul
could
mean
ha
he
condi-
ions
used
o
measu e
he
inhibi ion
do
no
o ally
e lec
he
"in
i o"
ac i i y
o
he
enzymes.
Table
3
also
shows
ha
he
speci ic
ac i i y
o
he
aspa a e
kinase
in
c ude
ex ac s
o
mu an s
is
smalle
han
ha
o
he
wild
ype.
This
e ec
could
be
due
o
ep ession
by
h eonine
o
he
HOM3
gene,
bu
o he
causes
canno
be
dismissed.
Thus,
i
appea s
ha
he
p esence
in
he
cell
o
a
h eonine-insensi i e
aspa a e
kinase
o e comes
o he
cons ain s
imposed
on
i s
ac i i y,
ha
is,
he
egula ion
akes
place
mainly
a
he
enzyma ic
ac i i y
le el.
AHV
mu an s
accumula e
homose ine
o
abou
he
same
le el
as
BORI
mu an s
do
(18),
i.e.,
abou
10
imes
mo e
han
he
wild
ype
(da a
no
shown).
They
also
o e p oduce
isoleucine
bu
o
a
lesse
deg ee
han
h eonine
does
(Fig.
1).
The
egula ion
o
he
pa hway
leading
om
h eonine
o
isoleucine
elies
mainly
on
he
ILV1
gene
and
i s
p oduc
(L- h eonine
deaminase),
which
ca alyzes
he
con e sion
o
h eonine
in o
o -ke obu y a e
(7).
Isoleucine
o e p oduc ion
in
he
mu an s
is
p obably
due
o
he
induc ion
by
h eonine
o
he
gene
CHAI
coding
o
a
ca abolic
L-se ine
(L- h eo-
nine)
deaminase
(17).
This
enzyme
can
ca y
ou
he
same
eac ion
as
he
anabolic
h eonine
deaminase
bu
is
no
sensi i e
o
eedback
inhibi ion.
The
mu an s
do
no
accumula e
mo e
me hionine
han
he
wild
ype
does,
which
indica es
ha
he
me hionine-speci ic
pa
o
he
ou e
is
s ic ly
con olled,
as
expec ed
om
he
epo ed
s ong
inhibi ion
and
ep ession
o
he
homose ine-
O- ansace ylase
exe ed
by
me hionine
and
S-adenosylme-
hionine
(8).
Con e sely,
and
on
he
basis
o
he
esul s
p esen ed
abo e,
he
las
pa
o
he
h eonine
pa hway
seems
o
play
a
seconda y
ole
in
he
egula ion
o
he
biosyn hesis
o
his
amino
acid.
The
ac s
ha
all
Ah R
Th E
mu an s
isola ed
a e
h eo-
nine
o e p oduce s
and
ha
he
HOM3-R
alleles
a e
domi-
nan
sugges
ha
his
s a egy
could
be
e icien ly
used
o
he
isola ion
o
h eonine-o e p oducing
s ains
o
indus ial
yeas
species.
ACKNOWLEDGMENTS
This
wo k
was
suppo ed
by
he
Spanish
CICYT
(g an
BT87-
0010)
and
by
he
Jun a
de
Andalucia.
We
hank
M.
A.
Delgado
and
E.
Ma in-Rend6n
o
hei
many
help ul
discussions,
K.
Po alla,
Uni e si a
Tubingen
(Tubingen,
Ge many)
o
kindly
supplying
he
bo elidin
and
BOR
mu an s,
M.
J.
Fa an
o
collabo a ing
in
some
expe imen s,
and
I.
Ma inez
o
co ec ing
he
manusc ip .
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