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POR1 encode the ortholog of farA

farA encode the transcription factor involved in fatty acid metabolism and peroxisomal function in A. nidulans.

-Por1 mutant :

1. fatty acid growth defect(accumulation of harmful metabolites,n-decane) & transcriptional induction of genes involved in utilize by oleic acid was defect.

2. expression of genes involved in b-oxidation and peroxisome proliferation is sufficient to support growth

3.  cause indirect effect on amount of the potential ligand of Yas3p in the ER membrane and resulted in transcriptional activation of ALK1.

cut1 and cut2, is differentially regulated by CTF1a(activates transcription of cut1) and CTF1b(expression cut2).

farA mutant reduce induction by both short- and long-chain fatty acids, while farB mutant by short-chain fatty acids only.

Transcription of POT1, PAT1, POX2, PEX5 induced by oleic acid & n-decane & in por1 mutant.

ALK1 transcription is not activated by fatty acids in the wildtype cells but transcript in por1 mutant

inositol present cause in PA consuming for phosphatidylinositol synthesis, and Opi1p is released from the ER membrane and represses the transcription of target genes by binding to Ino2p in the nucleus.

CGAGCCGA sequence is consistent with the sequence in the palindrome 1 and 2 proposed as a binding site of CTF1a and CTF1b

FarA systems important in yeast and filamentous fungi for transcriptional regulation involved in fatty acid utilization

how fatty acids are sensed and how transcription of target genes is regulated remain unknown in any of those yeasts or fungi.

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Search for transcription factors involved in fatty acid utilization in Y. lipolytica

1.  Genome database for orthologs of Oaf1p of S. cerevisiae and FarA of A. nidulans is searched using BLAST.

2.  YALI0F13321g, YALI0D17988g, and YALI0D10681g (is similar to Oaf1p)in their zinc clustered domain is deleted and  the growth is observed and analysed.

3. The translational product of  YALI0F13321g and YALI0D10681g is similar to  S. cerevisiae Pip2p in Y. lipolytica .

4.  No growth defect was observed on the medium containing oleic acid as a sole carbon source.

5. However, in S.cerevisae, deletion of  OAF1 or PIP2 ,  causes a severe growth defect on oleic acid.

6. The deletion  of YALI0F13321g and YALI0D10681g, or YALI0D10681g and YALI0D17988g  has no effects on the growth on oleic acid.

7. These proves that, Y.lipolytica has a different mechanism compared to S.cerevisae especially in  transcriptional regulation of fatty acid utilization  in which Oaf1p and Pip2p play vital roles in the growth on fatty acids

8. To search for FarA the highest score, 660 bits, was calculated for the YALI0D12628g translational product, which has been proposed that it is an ortholog of  FarA of A. nidulans and Ctf1p of C. albicans in Y. lipolytica .

9.  This gene is designated as POR1 (Primary Oleate Regulator 1).

10.  Por1p has three domains: a fungal Zn2Cys6 binuclear cluster domain ,  fungal specific transcription factor domain  and  a glutamine-rich (Q-rich) domain.  The first and second domains are conserved in orthologs of FarA.  The Q-rich domains were found in Ctf1p of C. albicans  and an ortholog in Neurospora crassa  but not in FarA and FarB in A. nidulans .

Growth analysis of deletion mutant of POR1

1. The mutant por1 grow normally on  glycerol, glucose, and n-hexadecane.

2.  Mutant por1 strain also grew well on n-decane

3. However,  it exhibited severe growth defects on lauric acid and myristic acid.

4.  The mutant por1 growth in oleic acid  was also impaired severely, but less profoundly than that on lauric acid or myristic acid.

5.  These results suggest that Por1p plays critical roles in fatty acid utilization in Y. lipolytica.

Expression of genes involved in b-oxidation and peroxisome proliferation in the Dpor1 strain

1.  The wild-type and mutant por1 strains precultured in glucose-containing medium were transferred to medium containing glycerol, glucose, oleic acid, or n-decane.

2. After 1h incubation, RNAs are extracted.

3.   Supposedly, the transcription of 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.

4. However, induction of these genes by oleic acid was diminished in the mutant por1 strain.

5. Similar goes to transcription of a peroxin gene, PEX5, encoding a receptor for a type-1peroxisomal targeting signal (PTS1),  was induced by oleic acid in the wild-type cells, but lower level in the mutant por1 cells.

6. Deletion of POR1 had no effect on the transcriptional activation of ALK1 encoding a P450ALK involved in terminal hydroxylation of n-alkane by n-decane.

7.  The transcripts of ALK1  is increased in mutant por1 on oleic acid compared to the wild type.

8. POR1 was expressed on all carbon sources in the wild-type strain in contrast with Ctf1p, an ortholog  FarA in C. albicans which its expression is induced in oleic acid.

9.  Reporter (lacZ) activities were increased on oleic acid and n-decane in the wild-type cell.

10.  In contrast, elevation of b-galactosidase activity on oleic acid was not observed in the mutant por1 cells.

11.  These results suggest that Por1p is an essential transcription factor involved in the transcriptional activation in response to fatty acids.

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Growth analysis of deletion mutant of POR1

> mutant's growth(lack of POR 1) was analysed on various carbon sources
1. mutant por1 strain grew
normally on glycerol, glucose, and n-hexadecane and n-decane(sligthly impairment).
2. Mutants exhibited severe growth defects on lauric acid and myristic acid.
3. This results suggest that Por1p plays critical roles in fatty acid
utilization in Y. lipolytica.

Expression of genes involved in b-oxidation and peroxisome
proliferation in the Dpor1 strain

>>> Analysis of the transcript
levels of genes involved in b-oxidation and peroxisome proliferation in the deletion mutant of POR1.
1. Wild-type and mutant por1
strains precultured in glucose-containing medium were transferred
to medium containing glycerol, glucose, oleic acid, or n-decane.
2. POT1, PAT1 & POX2 was activated by oleic acid in the wild-type cells.
POT1-peroxisomal 3-oxoacyl-
CoA thiolase
PAT1-peroxisomal acetoacetyl-CoA thiolase,
POX2-acyl-CoA oxidase

3.The transcript levels of these genes in the mutant por1 strain incubated on n-decane were less than that in the wild-type strain
4. 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
5. POR1 was expressed on all carbon sources in the wild-type strain.
6.

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Introduction

Yarrowia lipolytica can utilize hydrophobic substrates as carbon source.

n-Alkanes converted to fatty acid through sequential terminal oxidation by cyctochromes P450 (P450ALKs)

fatty acids synthesixed are activated to fattyacyl CoA and metabolized by  b-oxidation in peroxisome.

trancription of ALK1 is induced by n-Alkane and positively regulated by hetero-complex composed of 2 helix-loop-helix transc. factor.

Fatty acid utilization regulated by Oaf1 p-Pip2p system, Zn2Cys6 transcription factors Oaf1p and Pip2 with C2H2 zinc finger protein Adr1p.

Oaf1p-Pip2p appears in a subset of yeast species.

FarA and paralog, FarB belong to another Zn2Cys6 that control the transcription of genes involved in the metabolism of fatty acids.

CTF1a and CTF1b, Ctf1 are engaged in regulation of cultinase genes.

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Yeast strains and media

1. Y. lipolytica wild type strain - CXAU/A1 (ura3, ade1::ADE1)

2. Carbon source was added to 0.67% YNB

3. Addition of 2% (w/v) glycerol, 2% (w/v) glucose, 2% (v/v) oleic acid or 2% (v/v) n-decane for liquid media in YNB.

4. Solid media, addition of 1% Triton X- 100 to disperse 0.2% fatty acids.

5. n-Alkanes were supplied by vapour to YNB media.

The deletion mutant of POR1

1. The 5’ and 3’ Untranslated regions (UTRs) of POR1 gene were amplified by using primers 5’-CGTCTAGACACAAGAGACATGGGTCTGC-3’ and 5’-CGGGATCCTATCGTGCGTTGCGTCGTGC-3’, and 5’-CGGGATCCATGAACTCGACGCTCGGTTC-3’ and 5’-CGGAATTCACGTGACGTCGCGATCCATC-3’.

2.  The amplified fragments were digested with BamHI and XbaI or EcoRI

3.  XbaI-EcoRI sites of pBluescript II SK(+) is cloned to obtain pULP1.

4.  Plasmid pDP1 is synthesized by  digestion of pSAT4 with BamHI in fragment ADE1  was inserted into BamHI site of pULP1 .

5.  The deletion cassette was obtained by digestion of pDP1 with XbaI and EcoRV and then introduced into the CXAU1 [3] to obtain Dpor1 strain. Deletion of POR1 was confirmed by Southern blot analysis