3-Hydroxyisobutyryl-CoA hydrolase involved in isoleucine catabolism regulates triacylglycerol accumulation in Phaeodactylum tricornutum

Abstract

Since methylmalonyl-CoA epimerase appears to be absent in the majority of photosynthetic organisms, including diatoms, (S)-methylmalonyl-CoA, the intermediate of isoleucine (Ile) catabolism, cannot be metabolized to (R)-methylmalonyl-CoA then to succinyl-CoA. In this study, propionyl-CoA carboxylase (PCC) RNAi silenced strains and 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) overexpression strains were constructed to elucidate the Ile degradation pathway and its influence on lipid accumulation in Phaeodactylum tricornutum based on growth, neutral lipid content and metabolite profile analysis. Knockdown of PCC disturbed the metabolism of Ile through propionyl-CoA to methylmalonyl-CoA, as illustrated by much higher Ile content at day 6. However, Ile decreased to comparable levels to the wild-type at day 10. PCC silencing redirected propionyl-CoA to acetyl-CoA via a modified β-oxidation pathway, and transcript levels for some branched-chain amino acid (BCAA) degradation-related genes, especially HIBCH, significantly upregulated in the PCC mutant, which enhanced the BCAA degradations and thus resulted in higher triacylglycerol (TAG) content. Overexpression of HIBCH accelerates Ile degradation and results in a lowered Ile content in the overexpression strains, thus enhancing carbon skeletons to the tricarboxylic acid cycle and giving rise to increasing TAG accumulation. Our study provides a good strategy to obtain high-lipid-yield transgenic diatoms by modifying the propionyl-CoA metabolism.This article is part of the themed issue 'The peculiar carbon metabolism in diatoms'.

Keywords: branched-chain amino acid catabolism; diatom; propionyl-CoA carboxylase; β-oxidation.

Figure 1.

Pathways of BCAA degradation in P. tricornutum. Transcriptionally upregulated genes during TAG accumulation are in red. BCAT, branched-chain amino acid transaminase; BCKDH, branched-chain α-keto acid dehydrogenase; DHLTA, dihydrolipoyllysine-residue (2-methylpropanoyl) transferase; IVD, isovaleryl-CoA dehydrogenase; MCC, methylcrotonyl-CoA carboxylase; MGCHS, methylglutaconyl-CoA hydratase; HCL, hydroxymethylglutaryl-CoA lyase; IPMS, 2-isopropylmalate synthase; IPMDH, isopropylmalate dehydratase; IPMDCase, 3-isopropylmalate dehydrogenase; MCD, 2-methylacyl-CoA dehydrogenase; ECHS, enoyl-CoA hydratase; HAD, 3-hydroxyacyl-CoA dehydrogenase; ACAT, acetyl-CoA C-acyltransferase; ACD, acyl-CoA dehydrogenase; HIBCH, 3-hydroxyisobutyryl-CoA hydrolase; HIBADH, 3-hydroxyisobutyrate dehydrogenase; MMSDH, methylmalonate-semialdehyde dehydrogenase; ALDH, aldehyde dehydrogenase; PCC, propionyl-CoA carboxylase; MCM, methylmalonyl-CoA mutase; MCEE, methylmalonyl-CoA epimerase, VMOAT, valine-3-methyl-2-oxovalerate transaminase.

Figure 2.

Effect of PCC gene silencing on growth and neutral lipid accumulation. (a) Relative mRNA levels of PCC (error bars represent s.e. of triplicate technical replicates of duplicate cultures), (b) growth, (c) accumulation of TAGs detected by thin-layer chromatography (TLC), (d) accumulation of neutral lipid detected by Nile Red assay (fluorescence intensity normalized to cell number) and (e) nitrate utilization of wild-type (WT) and four RNAi silenced lines (pcc1p, pcc1q, pcc2b and pcc2c) grown in f/2 (NaNO₃ concentration was reduced to 500 µM) enriched artificial seawater medium. mRNA levels were performed at day 8. The values above the TLC panel indicate the relative TAGs normalized to the WT at day 6, which was set as 1. Error bars in (b), (d) and (e) represent s.e. of three biological replicates.

Figure 3.

Metabolite contents in wild-type (WT), two HIBCH overexpression lines (hibch-OE1 and hibch-OE3) and one PCC1 RNAi line (pcc1p) grown in f/2 (NaNO₃ concentration was reduced to 500 µM) enriched artificial seawater media at day 2, 6 and 10. Error bars represent s.e. of three biological replicates.

Figure 4.

Transcript levels of genes encoding components involved in BCAA degradation in wild-type (WT, relative to 36 h), PCC RNAi silenced line (pcc1p, relative to WT) and HIBCH overexpression line (hibch-OE3, relative to WT). Transcriptional fold changes from triplicate technical replicates of duplicate cultures (n = 2). BCAT, branched-chain amino acid transaminase; BCKDH, branched-chain α-keto acid dehydrogenase; DHLTA, dihydrolipoyllysine-residue (2-methylpropanoyl) transferase; IVD, isovaleryl-CoA dehydrogenase; MCC, methylcrotonyl-CoA carboxylase; MGCHS, methylglutaconyl-CoA hydratase; HCL, hydroxymethylglutaryl-CoA lyase; MCD, 2-methylacyl-CoA dehydrogenase; ECHS, enoyl-CoA hydratase; HAD, 3-hydroxyacyl-CoA dehydrogenase; ACAT, acetyl-CoA C-acyltransferase; PCC, propionyl-CoA carboxylase; ACD, acyl-CoA dehydrogenase; HIBCH, 3-hydroxyisobutyryl-CoA hydrolase; HIBADH, 3-hydroxyisobutyrate dehydrogenase; MMSDH, methylmalonate-semialdehyde dehydrogenase.

Figure 5.

Fluorescent microscope images of cells transformed with egfp fusions comprised full-length HIBCH. From left to right, panels show microscopical images of transmitted light, chlorophyll autofluorescence, GFP fluorescence, mitochondria stained by MitoTracker Orange and a merged image, scale bar represents 5 μm.

Figure 6.

Effect of HIBCH gene overexpression on growth and neutral lipid accumulation. (a) Relative mRNA levels of HIBCH (error bars represent s.e. of triplicate technical replicates of duplicate cultures), (b) growth, (c) accumulation of TAGs detected by thin-layer chromatography (TLC), (d) accumulation of neutral lipid detected by Nile Red assay (fluorescence intensity normalized to cell number) and (e) nitrate utilization of wild-type (WT) and two overexpression lines (hibch-OE1 and hibch-OE3) grown in f/2 (NaNO₃ concentration was reduced to 500 µM) enriched artificial seawater medium. mRNA levels were performed at day 8. The values above the TLC panel indicate the relative TAGs normalized to the WT at day 6, which was set as 1. Error bars in (b), (d) and (e) represent s.e. of three biological replicates.