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An ortholog of farA of Aspergillus nidulans is implicated in the…
An ortholog of
farA
of
Aspergillus nidulans
is implicated in the transcriptional activation of genes involved in fatty acid utilization in the yeast
Yarrowia lipolytica
RESULTS
3.2 Growth analysis of deletion mutant of POR1
Dpor1 strain
grew normally
on glycerol, glucose, and n-hexadecane, a relatively
longer carbon chain n-alkane
severe growth defects on lauric acid and myristic acid
growth on
oleic acid
was impaired severely but less profoundly than that on lauric acid or myristic acid
growth defects of the Dpor1
strain on fatty acids were complemented by expression of POR1 under its native promoter using the l
ow copy plasmid, pPOR1
Por1p plays critical roles in fatty acid
utilization in Y.
lipolytica
3.3. Expression of genes involved in b-oxidation and peroxisome proliferation in the Dpor1 strain
POT1, PAT1, and, POX2
, encoding a peroxisomal 3-oxoacylCoA thiolase, a peroxisomal acetoacetyl-CoA thiolase, and an acyl-CoA oxidase , respectively, all of which are involved in b-oxidation, was activated by oleic acid in the wild-type cells
transcription of a peroxin gene,
PEX5
, encoding a receptor for a type-1 peroxisomal targeting signal (PTS1) was induced by oleic acid in the wild-type cells, but its level was less in the Dpor1 cells
transcript levels
of these genes in the Dpor1 strain incubated on n-decane were
less than
that in the wild-type strain
deletion of POR1
had no apparent effect on the transcriptional activation of
ALK1
encoding a P450ALK involved in terminal hydroxylation of n-alkane by n-decane
transcripts of
ALK1
were somehow
increased in the Dpor1
cells on oleic acid compared to the wild-type cells
POR1
was expressed on all carbon sources in the wild-type
strain in marked contrast to
Ctf1p
, an ortholog of FarA
in C.
albicans
, whose expression is induced by oleic acid
northern blot analysis
reporter activities
increased
on oleic acid and n-decane
in the wild-type cells
elevation of b-galactosidase activity on oleic acid was not observed in the Dpor1 cells
Slight defect
in the activation of the reporter gene was observed Dpor1 cells incubated on n-decane
Por1p is an essential transcription factor involved in the transcriptional activation in response to fatty acids
3.1 Search for transcription factors involved in fatty acid utilization in
Y. lipolytica
identify transcription factors regulating expression of genes
involved in fatty acid metabolism
Deletion mutants of top three genes, YALI0F13321g, YALI0D17988g, and YALI0D10681g,
Translation products exhibited similarity to Oaf1p in their
zinc cluster domains
Translation products - YALI0F13321g and YALI0D10681g included in top three proteins have similarity to
S. cerevisiae
Pip2p in Y. lipolytica
Single or double deletion mutants genes and analyzed their ability to assimilate fatty acid by observing growth on oleic acid
0.2% fatty acid was dispersed in media by adding 1%
Triton X-100
no growth defect was observed
Deletion of OAF1 or PIP2
causes a
severe growth defect on oleic acid
Simultaneous
deletion of YALI0F13321g and YALI0D10681g
, or YALI0D10681g and YALI0D17988g did
not confer any defects
on the growth on oleic acid
Oaf1p and Pip2p play vital roles in the growth on fatty acids
amino acid sequence of FarA
YALI0D12628g highest score (660 bits) translation product, which had been proposed as an ortholog of FarA of
A.nidulans
and Ctf1p of
C. albicans
in
Y. lipolytica
RLM-RACE analysis using RNA perfrom on
glucose and oleic acid
: size of ORF of POR1 is 2751 bp and encodes a 916-amino acid protein, as predicted by the
Y. lipolytica
genome database
Two introns : 80 bp & 90 bp found in POR1
Por1p has
three domains:
Fungal Zn2Cys6 binuclear cluster domain
Fungal specific transcription factor domain
Glutamine-rich (Q-rich) domain
Found in Ctf1p of
C. albicans
and an ortholog in
Neurospora crassa
, but not in FarA and FarB in
A. nidulans
Deduced amino acid sequence of Por1p has 39, 21, and 28% identity to that of FarA, FarB & Ctf1p
INTRODUCTION
Yeast
Yarrowia lipolytica i
s
able to utilize various hydrophobic substrates
such as n-alkanes and fatty acids
as carbon sources
n-Alkanes
are converted to fatty acids through sequential terminal oxidation initiated by cytochromes P450
POR1
is a
Yarrowia lipolytica
ortholog of farA involved in fatty acid response in
A. nidulans.
Deletion of POR1
caused growth defects on fatty acids.
Δpor1 strain
exhibited defects in the induction of genes
involved in fatty acid utilization.
Por1p
is requited for growth on fatty acids and for the transcriptional activation of genes involved in b-oxidation and peroxisome proliferation
Por1p
acts as an essential transcriptional activator for fatty acid induction in
Y. lipolytica.
METHODS
2.1. Yeast strains and media
Y. lipolytica
strain CXAU/A1 (ura3, ade1::ADE1) was used as a
wild-type strain
Carbon source
was added to 0.67% YNB (Yeast Nitrogen Base without amino acids, Difco)
For
liquid
media: 2% (w/v) glycerol, 2% (w/v) glucose, 2% (v/v) oleic acid or 2% (v/v) n-decane
For
solid media
, 0.2% fatty acids were dispersed by adding 1% Triton X100. n-Alkanes were supplied by vapor to YNB solid media
a piece of filter paper soaked with n-alkanes was placed in a lid of petri dish, which was sealed and kept upside down.
If necessary
, uracil (24 mg/l) was added
The
deletion mutant
of POR1 by amplifying 50 and 30 untranslated regions (UTRs) or POR1 gene by using primers
The amplified fragments were
digested
with
BamHI
and
XbaI or EcoR
cloned
into the XbaI-EcoRI sites of pBluescript II SK(+) to obtain pULP1
The fragment containing
ADE1
, obtained by digestion of
pSAT4
with
BamHI
, was inserted into
BamHI
site of
pULP1
, yielding the plasmid
pDP1
Deletion cassete
was obtained by digestion of pDP1 with XbaI and EcoRV then, introduced into the CXAU1 to obtain
Dpor1 strain.
Deletion of POR1 was confirmed
by Southern blot analysis
2.2. Plasmid construction
The plasmid,
pPOR1
, to
express *
POR1
under its native promoter in
Y. lipolytica*
POR1 gene with its 50- and 30-UTRs was
amplified by using primers
The amplified fragment was
digested
with
Xbal
and
BglII
, and
cloned
into
XbaI-BamHI
sites of
pSUT5
The plasmid,
pPpro-LacZ
, to
analyze promoter activity
of
PAT1
using
lacZ
as a reporter gene
PAT1
promoter was
amplified by using primers
The amplified fragment was
digested
with
XbaI
and
StuI
, and
cloned
into
XbaI-Stul
sites of
pSUT5-LacZ
2.4. B-Galactosidase activity assay
B-Galactosidase assay was performed except
the cells that grown in various carbon sources for 4 h.
2.5. RNA ligase-mediated rapid amplification of cDNA end
RLM-RACE performed using GeneRacer RLM-RACE kit (Invitrogen)
Specific primers for 5'-RACE and 3'-RACE, 5'-GCCTCTGACGTTCGCCGTTGCAGAAT-3' & 5'-
-CTGGTGGTGGGAGGGTCCCGAAGAC-3'
,
2.3. Transformation
Y. lipolytica
was transformed by
electroporation
2.6. Northern blot analysis
Hybridization & detection performed using digoxigenin-labeled DNA probes with PCR DIG probe synthesis kit
DNA probes were amplified using genomic DNA of CXAU1 as a template
DISCUSSION
POR1
gene encoding the ortholog of farA that encodes the transcription factor involved in fatty acid metabolism and peroxisomal function in A.
nidulans
Deletion of POR1 resulted in severe growth defects on fatty acids
profound defects in the transcriptional induction of genes involved in fatty acid utilization by oleic acid
Por1p regulates transcription of genes involved in fatty acid
utilization in response to fatty acids in Y.
lipolytica
weak growth on oleic acid
Dpor1 cells
slight transcriptional activation of
POT1, PAT1,
POX1,
and
PEX5
by oleic acid
indicate the presence of another mechanism for transcriptional activation by oleic acid in Y. lipolytica
mutants of YALI0F13321g, YALI0D17988g, and/or YALI0D10681g that exhibited low similarity to Oaf1p and/or Pip2p in their zinc cluster domains showed no significant defects on oleic acid, it is less probable that orthologs of Oaf1p and/or Pip2p function in fatty acid response in Y. lipolytica
F. solani
expression of cutinase genes, cut1 and cut2
regulated by CTF1a and CTF1b. CTF1b
participate in the constitutive expression of cut2
CTF1a activates transcription of cut1 in the presence of cutin monomer
A.
nidulans
deletion of farA caused defects in the induction by
both short- and long-chain fatty acids
deletion of farB resulted in a reduction in the induction by short-chain fatty acids
unidentified functional homolog(s) may be involved in the transcriptional activation in response to fatty acid in Y. lipolytica
distinct regulatory mechanisms are involved in the fatty acid response
C.
albicans
transcriptional activation of a subset of b-oxidation genes in the presence of oleic acid was observed in the
deletion mutant of CTF1