Overexpression of Key Sterol Pathway Enzymes in Two Model Marine Diatoms Alters Sterol Profiles in Phaeodactylum tricornutum

Abstract

Sterols are a class of triterpenoid molecules with diverse functional roles in eukaryotic cells, including intracellular signaling and regulation of cell membrane fluidity. Diatoms are a dominant eukaryotic phytoplankton group that produce a wide diversity of sterol compounds. The enzymes 3-hydroxy-3-methyl glutaryl CoA reductase (HMGR) and squalene epoxidase (SQE) have been reported to be rate-limiting steps in sterol biosynthesis in other model eukaryotes; however, the extent to which these enzymes regulate triterpenoid production in diatoms is not known. To probe the role of these two metabolic nodes in the regulation of sterol metabolic flux in diatoms, we independently over-expressed two versions of the native HMGR and a conventional, heterologous SQE gene in the diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum. Overexpression of these key enzymes resulted in significant differential accumulation of downstream sterol pathway intermediates in P. tricornutum. HMGR-mVenus overexpression resulted in the accumulation of squalene, cycloartenol, and obtusifoliol, while cycloartenol and obtusifoliol accumulated in response to heterologous NoSQE-mVenus overexpression. In addition, accumulation of the end-point sterol 24-methylenecholesta-5,24(24')-dien-3β-ol was observed in all P. tricornutum overexpression lines, and campesterol increased three-fold in P. tricornutum lines expressing NoSQE-mVenus. Minor differences in end-point sterol composition were also found in T. pseudonana, but no accumulation of sterol pathway intermediates was observed. Despite the successful manipulation of pathway intermediates and individual sterols in P. tricornutum, total sterol levels did not change significantly in transformed lines, suggesting the existence of tight pathway regulation to maintain total sterol content.

Keywords: diatoms; metabolic engineering; sterol metabolism; terpenoids.

Figure 1

Upstream reactions and conserved core of sterol biosynthesis pathway in diatoms and genetic targets overexpressed in this study, highlighted in orange. Mevalonate pathway, MVA; 3-hydroxy-3-methylglutaryl-coenzyme A reductase, HMGR; truncated HMGR, tHMGR; alternative squalene epoxidase, AltSQE; squalene epoxidase from N. oceanica, NoSQE; oxidosqualene cyclase, OSC.

Figure 2

Maximum likelihood phylogenetic tree of diatom HMGR proteins and its domains. Numbers at the nodes represent bootstrap support (1000 replicates). HMGR from yeast, mammals and plants were used as outgroups. Arrows represent start of N-terminal truncated version for P. tricornutum and T. pseudonana used in this study.

Figure 3

Confocal microscopy images showing subcellular localization of the mVenus fusion with target proteins in representative transgenic (a) P. tricornutum cells, scale bars correspond to 5 µm, and (b) T. pseudonana cells, scale bars correspond to 10 µm. Wild type (WT) as negative control and control cell lines that only expressed mVenus. 3-hydroxy-3-methylglutaryl-coenzyme A reductase, HMGR-mVenus; truncated HMGR, tHMGR-mVenus; squalene epoxidase from N. oceanica, NoSQE-mVenus. Scale bars correspond to 10 µm.

Figure 4

Sterol levels in P. tricornutum transformants. (a) End-point sterol (b) intermediates accumulation. Identical letters denote no statistically significant differences among groups using the Pairwise Wilcoxon Rank Sum tests (p < 0.05, n = 9).