Abstract
BackgroundDocosahexaenoic acid (DHA) is an important omega-3 long-chain polyunsaturated fatty acid that has a variety of physiological functions for infant development and human health. Although metabolic engineering was previously demonstrated to be a highly efficient way to rapidly increase lipid production, metabolic engineering has seldom been previously used to increase DHA accumulation in Schizochytrium spp.ResultsHere, a sensitive β-galactosidase reporter system was established to screen for strong promoters in Schizochytrium sp. Four constitutive promoters (EF-1αp, TEF-1p, ccg1p, and ubiquitinp) and one methanol-induced AOX1 promoter were characterized by the reporter system with the promoter activity ccg1p> TEF-1p > AOX1p (induced) > EF-1αp > ubiquitinp. With the strong constitutive promoter ccg1p, Schizochytrium ATP-citrate lyase (ACL) and acetyl-CoA carboxylase (ACC) were overexpressed in Schizochytrium sp. ATCC 20888. The cells were cultivated at 28 °C and 250 rpm for 120 h with glucose as the carbon source. Shake-flask fermentation results showed that the overexpression strains exhibited growth curves and biomass similar to those of the wild-type strain. The lipid contents of the wild-type strain and of the OACL, OACC, and OACL-ACC strains were 53.8, 68.8, 69.8, and 73.0%, respectively, and the lipid yields of the overexpression strains were increased by 21.9, 30.5, and 38.3%, respectively. DHA yields of the wild-type strain and of the corresponding overexpression strains were 4.3, 5.3, 6.1, and 6.4 g/L, i.e., DHA yields of the overexpression strains were increased by 23.3, 41.9, and 48.8%, respectively.ConclusionsAcetyl-CoA and malonyl-CoA are precursors for fatty acid synthesis. ACL catalyzes the conversion of citrate in the cytoplasm into acetyl-CoA, and ACC catalyzes the synthesis of malonyl-CoA from acetyl-CoA. The results demonstrate that overexpression of ACL and ACC enhances lipid accumulation and DHA production in Schizochytrium sp.
Highlights
Docosahexaenoic acid (DHA) is an important omega-3 long-chain polyunsaturated fatty acid that has a variety of physiological functions for infant development and human health
To determine whether the β-galactosidase reporter works in Schizochytrium, we constructed the reporter plasmid pPICZαA-ubiquitinp-lacZ, in which the E. coli lacZ gene was driven by a ubiquitin promoter–terminator system (Fig. 1a). pPICZαA containing lacZ without a ubiquitin promoter was constructed as a control plasmid
ATCC 20888 were selected on glucose–peptone–yeast extract (GPY) plates with zeocin
Summary
Docosahexaenoic acid (DHA) is an important omega-3 long-chain polyunsaturated fatty acid that has a variety of physiological functions for infant development and human health. Most studies of DHA production by Schizochytrium spp. have focused on the adaptive evolution of the strains [9]; on the optimization of medium composition including sources of carbon and nitrogen and the addition of inorganic salts and antioxidants [5, 6, 10, 11]; and on cultivation conditions and cultivation styles [12, 13]. By increasing the number of active ACP domains of PUFA synthase, DHA productivity was increased by 1.8-fold in a recombinant E. coli expressing Schizochytrium PUFA biosynthetic genes [16]. These studies demonstrate that metabolic engineering can increase DHA production by Schizochytrium spp
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