Role of β-Oxidation Enzymes in γ-Decalactone Production by the Yeast Yarrowia lipolytica

Abstract

Some microorganisms can transform methyl ricinoleate into ␥-decalactone, a valuable aroma compound, but yields of the bioconversion are low due to (i) incomplete conversion of ricinoleate (C 18 ) to the C 10 precursor of ␥-decalactone, (ii) accumulation of other lactones (3-hydroxy-␥-decalactone and 2-and 3-decen-4-olide), and (iii) ␥-decalactone reconsumption. We evaluated acyl coenzyme A (acyl-CoA) oxidase activity (encoded by the POX1 through POX5 genes) in Yarrowia lipolytica in lactone accumulation and ␥-decalactone reconsumption in POX mutants. Mutants with no acyl-CoA oxidase activity could not reconsume ␥-decalactone, and mutants with a disruption of pox3, which encodes the short-chain acyl-CoA oxidase, reconsumed it more slowly. 3-Hydroxy-␥-decalactone accumulation during transformation of methyl ricinoleate suggests that, in wild-type strains, ␤-oxidation is controlled by 3-hydroxyacyl-CoA dehydrogenase. In mutants with low acyl-CoA oxidase activity, however, the acyl-CoA oxidase controls the ␤-oxidation flux. We also identified mutant strains that produced 26 times more ␥-decalactone than the wild-type parents.

Optimization of Yarrowia lipolytica’s β-oxidation pathway for γ-decalactone production

“…Aox1 exhibits no detectable activity in the conditions we tested, Aox2 and Aox3 are longand short-chain specific, respectively and Aox4 and Aox5 exhibit a weak activity on a wide range of substrates [8]. We have observed in previous works the important role of the acyl-CoA oxidase in the pathway, Aox3 (short-chain specific) being responsible for continuation of oxidation after the C10 level [9] and for lactone reconsumption [10]. Mutants with decreased Aox activity accumulated more DECA as the remaining Aox activity was the rate-limiting step of the pathway [10].…”

“…We have observed in previous works the important role of the acyl-CoA oxidase in the pathway, Aox3 (short-chain specific) being responsible for continuation of oxidation after the C10 level [9] and for lactone reconsumption [10]. Mutants with decreased Aox activity accumulated more DECA as the remaining Aox activity was the rate-limiting step of the pathway [10].…”

“…This esterification occurs competitively to ␤-oxidation and can happen between the four reactions depending on kinetics data. An exit of the acyl-CoA from the ␤-oxidation loop before the oxidation step, yielding [3,10]). The substrate enters ␤-oxidation and, after four oxidation loops gives rise to C10 intermediates: decanoyl-CoA can be hydrolyzed and lactonized to ␥-decalactone (DECA, horizontal arrow) or be oxidized by 1 (acyl-CoA oxidase) to 2-decenoyl-CoA, being itself lactonized to dec-2-en-4-olide (2-DECEN) or hydrated by 2 (2-enoyl-CoA hydratase) to 3-hydroxyacyl-CoA.…”

Decalactone Production by Yarrowia lipolytica under increased O2 Transfer Rates

“…The strictly aerobic yeast, Yarrowia lipolytica, can transform methyl ricinoleate, using peroxisomal b-oxidation (Wache´et al 2001), into c-decalactone, and is used industrially (Barth & Gaillardin 1996).…”

“…The accumulation of these different decalactones in the medium gives an indication of the various activities of the enzymes of the pathway. For example, c-decalactone accumulation increases when acyl-CoA oxidase activity decreases (Wache´et al 2001). However, for the most part, the regulation of this pathway in yeast remains misunderstood.…”