Functions of PKS Genes in Lipid Synthesis of Schizochytrium sp. by Gene Disruption and Metabolomics Analysis

Abstract

Schizochytrium sp. is a kind of marine microalgae with great potential as promising sustainable source of polyunsaturated fatty acids (PUFAs). Polyketide synthase-like (PKS synthase) is supposed to be one of the main ways to synthesize PUFAs in Schizochytrium sp. In order to study the exact relationship between PKS and PUFA biosynthesis, chain length factor (CLF) and dehydrogenase (DH) were cloned from the PKS gene cluster in Schizochytrium sp., then disrupted by homologous recombination. The results showed that DH- and CLF-disrupted strains had significant decreases (65.85 and 84.24%) in PUFA yield, while the saturated fatty acid (SFA) proportion in lipids was slightly increased. Meanwhile, the disruption of CLF decreased the C-22 PUFA proportion by 57.51% without effect on C-20 PUFA accumulation while DH-disrupted mutant decreased the production of each PUFA. Combined with analysis of protein prediction, it indicated that CLF gene exerted an enormous function on the carbon chain elongation in PUFA synthesis, especially for the final elongation from C-20 to C-22 PUFAs. Metabolomics analysis also suggested that the disruption of both genes resulted in the decrease of PUFAs but increase of SFAs, thus weakening glycolysis and tricarboxylic acid (TCA) cycle pathways. This study offers a broad new vision to research the mechanism of PUFA synthesis in Schizochytrium sp.

The lipid metabolism in thraustochytrids

“…This domain has been functionally characterized in association with its KS neighbour domain [103] and plays an important role in the carbon chain elongation, especially for the elongation from C20 to C22 (EPA to DHA). In thraustochytrids it also controls the final yield of PUFA production [104]. Two types of DH domains are likely required in PUFA synthase to introduce either saturation or cis double bonds on growing acyl chains [105].…”

“…To achieve the final pattern of double bonds observed in DHA (or EPA), not only trans-2,3-cis but also trans-2,2-cis isomerizations are likely to occur at several rounds of elongation during the FA construction process [92]. When such a DH/I domain is disrupted in Schizochytrium sp., the total PUFA yield decreases by more than half, highlighting its key role [104]. A putative scheme showing the sequence of reactions catalysed by the PUFA synthase is shown in Figure 8.…”

“…The transferred genetic material can be either a circular [125] or a linearized plasmid [126], or just the amplified cassette with the flanking regions for the homologous recombination [127]. With these methods, overexpressing [128], knocked-in [129], and disrupted [104] mutants have been obtained. To improve the efficiency of transformation, the cell wall can be weakened by DTT treatments [130], by vortexing cells with zircon beads before resuspension with the amplified DNA construct [127], or by incubation for few hours with a cocktail of various cell-wall degrading enzymes [126].…”

“…Other interesting and promising works indicate that disruption of the CLF domain of the subunit B of the PUFA synthase and disruption of the second DH/I domain of the subunit C (Fig. 7) strongly affect the FA profile and DHA content [104] in Schizochytrium sp., decreasing both the total FA content and the unsaturated/saturated FA ratio. In the same species, disruption of the AT domain of the PUFA synthase subunit B [136] affects the growth rate and the size of the cells.…”

“…Putative scheme illustrating the reactions catalyzed by the PUFA synthase. This scheme relies on previous reports describing the functions of the different domains [103][104][105]. Compared with the reactions catalyzed by the classical FAS I system, the main differences would be at the level of the DH/I and ER steps, allowing or not the formation of a cis-double bond [92,105].…”