Δ12 fatty acid desaturase gene from Geotrichum candidum in cheese: molecular cloning and functional characterization

Abstract

Soft cheese with white rind lacks essential fatty acids ( EFA s), and as a result its long‐term consumption may lead to various kinds of cardiovascular and cerebrovascular diseases, such as hyperlipidemia, hypertension, and atherosclerosis. Geotrichum candidum is a dimorphic yeast that plays an important role in the ripening of mold cheese. A gene coding for Δ12 fatty acid desaturase, a critical bifunctional enzyme desaturating oleic acid ( OA ) and linoleic acid ( LA ) to produce LA and α‐linolenic acid ( ALA ), respectively, was isolated from G. candidum , and then cloned and heterologously expressed in Saccharomyces cerevisiae . This gene, named Gc FADS 12 , had an open reading frame of 1257 bp and codes for a protein of 419 amino acids with a predicted molecular mass of 47.5 kDa. Characterization showed that Gc FADS 12 had the ability to convert OA to LA and LA to ALA , and the conversion rates for OA and LA were 20.40 ± 0.66% and 6.40 ± 0.57%, respectively. We also found that the protein product of Gc FADS 12 catalyzes the conversion of the intermediate product ( LA ) to ALA by addition of OA as the sole substrate. The catalytic activity of Gc FADS 12 on OA and LA was unaffected by fatty acid concentrations. Kinetic analysis revealed that Gc FADS 12 had stronger affinity for the OA than for the LA substrate. This study offers a solid basis for improving the production of EFA s by G. candidum in cheese.

Both Determinants of Allosteric and Active Sites Responsible for Catalytic Activity of Delta 12 Fatty Acid Desaturase by Domain Swapping

“…This is a structural analogue of other fatty acids such as 12,13-cyclopropenoid fatty acid, which might also inhibit the activity of FADS12 and prevent the conversion of OA to LA. If so, this could help to explain the low FADS12 activity in G. candidum (Luo et al, 2019) and provided a new clue to study the catalytic mechanism of fatty acid desaturase.…”

“…However, studies on fatty acid desaturase in dominant strains of cheese have not yet been undertaken. Previously, our laboratory identified the FADS12 gene in a Geotrichum candidum ( G. candidum ) genome sequence submitted to the GenBank database (LOCUS: CCBN010000001) by Casaregola, S. The function of this gene has been verified by the addition of substrates in vitro , and, although the conversion rate of the enzyme encoded by this gene has been determined, its ability to transform OA and LA substrates was found to be low (Luo et al, 2019).…”

Linoleic acid enrichment of cheese by okara flour and Geotrichum candidum overexpressing Δ12 fatty acid desaturase

“…Cheese is rich in nutritional components and the lipids account for 20–35% of dry weight. Cheese lipids mainly come from raw milk (30.0–50.0 g kg −1 fat) and are composed of saturated fatty acids (SFAs, about 60–70%) and unsaturated fatty acids (UFAs, about 30–40%) 1‐4 . SFAs and UFAs are essential for human health and should be consumed in a balanced ratio of 1:2–1:3 5 .…”

“…candidum . This enzyme has the ability to convert oleic acid (OA) into linoleic acid (LA), and the conversion rate for OA reached 20.4 ± 0.7%, providing an operable strain for improving UFA profile in cheese 4 . However, this conversion cannot meet the demand for a balanced proportion of SFAs and UFAs in Geotrichum ‐ripened cheese.…”

“…Cheese lipids mainly come from raw milk (30.0-50.0 g kg −1 fat) and are composed of saturated fatty acids (SFAs, about 60-70%) and unsaturated fatty acids (UFAs, about 30-40%). [1][2][3][4] SFAs and UFAs are essential for human health and should be consumed in a balanced ratio of 1:2-1:3. 5 However, the imbalance between the intake of SFAs and UFAs in cheese raises health concerns and increases the risk of cardiovascular and coronary heart diseases.…”