Evaluation of Acyl Coenzyme A Oxidase (Aox) Isozyme Function in the n -Alkane-Assimilating Yeast Yarrowia lipolytica

Abstract

We have identified five acyl coenzyme A (CoA) oxidase isozymes (Aox1 through Aox5) in the n-alkane-assimilating yeastYarrowia lipolytica, encoded by the POX1through POX5 genes. The physiological function of these oxidases has been investigated by gene disruption. Single, double, triple, and quadruple disruptants were constructed. Global Aox activity was determined as a function of time after induction and of substrate chain length. Single null mutations did not affect growth but affected the chain length preference of acyl-CoA oxidase activity, as evidenced by a chain length specificity for Aox2 and Aox3. Aox2 was shown to be a long-chain acyl-CoA oxidase and Aox3 was found to be active against short-chain fatty acids, whereas Aox5 was active against molecules of all chain lengths. Mutations in Aox4 and Aox5 resulted in an increase in total Aox activity. The growth of mutant strains was analyzed. In the presence of POX1 only, strains did not grow on fatty acids, whereas POX4 alone elicited partial growth, and the growth of the double POX2-POX3-deleted mutant was normal excepted on plates containing oleic acid as the carbon source. The amounts of Aox protein detected by Western blotting paralleled the Aox activity levels, demonstrating the regulation of Aox in cells according to the POX genotype.

Observation of the Yarrowia lipolytica peroxisome—vacuole dynamics by fluorescence microscopy with a single filter set

“…POX1 does not contribute to Aox activity, POX2 and POX3 code for the long-and short-chain specific Aox, respectively, and account for 80% of total Aox activity, POX4 codes for the minor Aox and POX5 codes for a nonspecific chain length Aox that is responsible for the remaining Aox activity. Only the Dpox2, 3, 4, 5 quadruple null mutant has the complete growth defect in oleic acid medium (Wang et al, 1999). No one Aox isoform contains a conserved variant of a C-terminal peroxisomal targeting signal 1 (PTS1) or an N-terminal PTS2, though all five Aox isoforms are peroxisomal in Y. lipolytica.…”

Cellular and metabolic engineering of oleaginous yeastYarrowia lipolyticafor bioconversion of hydrophobic substrates into high‐value products

“…During the -oxidation process, six peroxisomal acyl-CoA oxidases (AOX1-6) encoded by Pox1-6 genes are identified as chainlength-specific for fatty acid degradation [106][107][108][109]. Previous studies demonstrated that the Y. lipolytica strains with Pox1-6 gene mutations impaired fatty acid degrading ability resulting in the increase of high-lipid content in biomass [86,[110][111][112][113]. The next two steps of the -oxidation are catalyzed by the multifunctional enzyme (MFE) [218,219].…”

Expression of Alt a 1 allergen from Alternaria alternata in the yeast Yarrowia lipolytica

“…Because of the large number of genetic markers and molecular tools available, this yeast is considered an efficient heterologous protein production system (Muller et al, 1998;Gasmi et al, 2011;Rao et al, 2011). Several Y. lipolytica promoters have been used for recombinant protein expression (Domínguez et al, 1998;Muller et al, 1998;Wang et al, 1999;Pignède et al, 2000). The copper-inducible bi-directional promoter of YlMTPI and YlMTPII genes has been characterized previously (García, 1993;Domínguez et al, 2003).…”