Selectively superior production of docosahexaenoic acid in Schizochytrium sp. through engineering the fatty acid biosynthetic pathways.

BACKGROUND: Schizochytrium sp. is commercially used for production of docosahexaenoic acid (DHA). Schizochytrium sp. utilizes the polyketide synthase complex (PKS) and a single type I fatty acid synthase (FAS) to synthesize polyunsaturated fatty acids and saturated fatty acids, respectively. The acyl carrier protein (ACP) domains of FAS or PKS are used to load acyl groups during fatty acids biosynthesis. Phosphopantetheinyl transferase (PPTase) transfers the pantetheine moiety from Coenzyme A to the conserved serine residue of an inactive ACP domain to produce its active form. RESULTS: In this study, in order to improve production and content of DHA, we decreased the expression of fas, strengthened the expression of the PKS pathway, and enhanced the supply of active ACP in Schizochytrium sp. ATCC20888. Weakening the expression of fas or disruption of orfA both led to growth defect and reduction of lipid yields in the resulting strains WFAS and DPKSA, indicating that both FAS and PKS were indispensable for growth and lipid accumulation. Although WFAS had a higher DHA content in total fatty acids than the wild-type strain (WT), its growth defect and low DHA yield hinders its use for DHA production. Overexpression of the orfAB, orfC, orfC-DH (truncated orfC), or ppt promoted DHA and lipid production, respectively. The yields and contents of DHA were further increased by combined overexpression of these genes. Highest values of DHA yield (7.2 g/L) and DHA content (40.6%) were achieved in a recombinant OPKSABC-PPT, ⁓56.5% and 15.3% higher than the WT values, respectively. CONCLUSIONS: This study demonstrates that genetic engineering of the fatty acid biosynthetic pathways provides a new strategy to enhance DHA production in Schizochytrium.

Synthesis of Poly(ε-caprolactone)-Based Miktoarm Star Copolymers through ROP, SA ATRC, and ATRP.

The synthesis of novel branched/star copolymers which possess unique physical properties is highly desirable. Herein, a novel strategy was demonstrated to synthesize poly(ε-caprolactone) (PCL) based miktoarm star (µ-star) copolymers by combining ring-opening polymerization (ROP), styrenics-assisted atom transfer radical coupling (SA ATRC), and atom transfer radical polymerization (ATRP). From the analyses of gel permeation chromatography (GPC), proton nuclear magnetic resonance (¹H NMR), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), well-defined PCL-µ-PSt (PSt: polystyrene), and PCL-µ-PtBA (PtBA: poly(tert-butyl acrylate) µ-star copolymers were successfully obtained. By using atomic force microscopy (AFM), interestingly, our preliminary examinations of the µ-star copolymers showed a spherical structure with diameters of ca. 250 and 45 nm, respectively. We successfully employed combinations of synthetic techniques including ROP, SA ATRC, and ATRP with high effectiveness to synthesize PCL-based µ-star copolymers.