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An ortholog of farA Aspergillus nidulans is implicated in the…
An ortholog of farA Aspergillus nidulans is implicated in the transcriptional activation of genes involved in fatty acid utilization in the yeast Yarrowia lipolyrtica.
Introduction
Yarrowia lipolytica
- able to utilize various hydrophobic substrates such as n-alkanes and fatty acids as carbon sources.
- n-alkanes are converted to fatty acid through sequential terminal oxidation initiates by cytochromes P450
- Fatty acids synthesized endogenously or incorporated from culture media
- are activated to fattyacyl CoA and metabolized by b-oxidation in peroxisome or used
for synthesis of various lipids
- The transcription of ALK1, a pivotal
P450ALK, is induced by n-alkane.
- Regulated by bHLH, transcription factors Yas1p and Yas2p
- , via Alkane Responsive
Elemen1 (ARE1)
- negatively by Yas3p
Oaf1p-Pip2p system
- , Oaf1p and Pip2p, together with the C2H2 zinc finger protein Adr1p:
:- activate the transcription of target genes in
response to fatty acids
- FarA and FarB are encoded in the genomes of wide variety of yeasts
and filamentous fungi
- Ctf1p involved in fatty acid response
- CTF1 alpha, CTF1 beta and Ctf1 engaged in the transcriptional regulation of cutinase genes
Purpose of the study:
- To characterized the ortholog of FarA in Y. lipolytica, Por1p (Primary oleate regulator 1). The results indicate that the Por1p is requited for growth on fatty acids and for the transcriptional activation of genes involved in b-oxidation and peroxisome proliferation
RESULTS
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:star: Two introns of 80 bp
and 90 bp were found in POR1
:star: Por1p has three domains a fungal Zn2Cys6 binuclear cluster domain (amino acids 32 to 71), a fungal specific transcription factor domain (amino acid 327 to 422) and a glutamine-rich (Q-rich) domain (amino acid 661 to 742)
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-
DISCUSSION
- POR1 - Gene code for ortholog of farA
- farA - encodes transcription factor involved in f. acid metabolism & peroximal f(x) in A. nidulans
- Deletion of POR1
~ Severe growth defects on f. acids
~ Extreme defects transcriptional induction of genes involved in fatty acid utilization by oleic acid
~ Por1p regulates genes transcription involved in fatty acid utilization in response to fatty acids in Y. lipolytica
- Deletion mutant of POR1
~ Exhibited weak growth on oleic acid
- In por1 cells
~ Oleic acid regulate slight transcriptional activation of POT1, PAT1, POX1, and PEX5
~ indicate presence of another mechanism for transcriptional activation by oleic acid in Y. lipolytica
- YALI0F13321g, YALI0D17988g, and/or YALI0D10681g
~ exhibited low similarity to Oaf1p and/or Pip2p in their zinc cluster domains
~ no significant defects on oleic acid - less probable orthologs of Oaf1p and/or Pip2p function in fatty acid response in Y. lipolytica
- In F. solani
~ expression of cutinase genes, cut1 and cut2 regulated by CTF1α and CTF1β
CTF1β - participate in the constitutive expression of cut2
CTF1α - activates transcription of cut1 in the presence of cutin monomer
- In A. nidulans
~ Deletion of farA - defects in the induction by both short- and long-chain fatty acids
~ Deletion of farB - reduction in the induction by short-chain fatty acids
- Unidentified functional homolog(s)
~ Plausibly involved in the transcriptional activation in response to fatty acid in Y. lipolytica
- Distinct regulatory mechanisms
~ Involved in the fatty acid response
- In C. albicans
~ Deletion of CTF1 - transcriptional activation of a subset of β-oxidation genes in the presence of oleic acid
Orthologs
:star: Yas1p- Ino4p
:star: Yas2p- Ino2p
:star: Yas3p- Opi1p
- involved in the transcriptional regulation of
phospholipid biosynthetic genes in S. cerevisiae
A variety of yeasts and filamentous fungi have
acquired FarA systems
- for transcriptional regulation of genes involved in fatty acid utilization
- Transcription of POT1, PAT1, POX2, and PEX5
~ Induced by oleic acid and n-decane
- Deletion of POR1
~ Affected transcript levels of the genes on n-decane to varying extent
- In Y. lipolytica
~ n-alkanes oxidized to fatty acids, then metabolized through β-oxidation
:check: In the wildtype cells, transcription of ALK1 is not activated by fatty
:check: PA is consumed for
phosphatidylinositol synthesis in the presence of inositol
:check: Opi1p is released from the ER
membrane and represses the transcription of target genes by binding to Ino2p in the nucleus
:check: Yas3p also localizes to ER in the
:check: presence of n-alkane and in the nucleus in the presence of n-alkane
Materials & Methods
- Y. lipolytica strain CXAU/A1 (wild type)
- carbon
source was added to 0.67% YNB
Solid media:-
- 0.2% fatty acids (adding 1% Triton X100)
- n-Alkanes were supplied by vapor to YNB solid media
- filter paper soaked with n-alkanes was placed in a lid of petri dish.
- The deletion mutant of POR1
- amplified fragments were digested with BamHI and Xbal or EcoRI, cloned into the Xbal-EcoRI sites to obtain pULP1.
- The plasmid, pPOR1, to express POR1 under its native promoter in Y. lipolytica
- pPpro-LacZ, to analyze promoter activity of PAT1
using lacZ as a reporter gene
- Y. lipolytica was transformed by electroporation
- b-Galactosidase activity assay
-b-Galactosidase assay was performed
- RNA ligase-mediated rapid amplification of cDNA end
- RLM-RACE was performed by using the GeneRacer RLM-RACE kit (Invitrogen)
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