Volume 12, Issue 4, Pages 474-488 (April 2019) Biosynthesis of Triacylglycerol Molecules with a Tailored PUFA Profile in Industrial Microalgae  Yi Xin, Chen Shen, Yiting She, Hong Chen, Cong Wang, Li Wei, Kangsup Yoon, Danxiang Han, Qiang Hu, Jian Xu  Molecular Plant  Volume 12, Issue 4, Pages 474-488 (April 2019) DOI: 10.1016/j.molp.2018.12.007 Copyright © 2018 The Author Terms and Conditions

Figure 1 Ex Vivo and In Vitro Substrate Specificities of NoDGAT2J and 2K for Acyl-CoAs. (A and B) GC–MS quantification of TAG extracted from transformed yeast after feeding of C18:2 (A) or C20:5 (B). The total amount of TAG was normalized based on that of the total lipids. The TAG spots detected by thin-layer chromatography are shown. (C) TAG-associated fatty acid composition in NoDGAT2J-carrying yeast fed with C18:2, and 2K-carrying yeast fed with C20:5. (D and E) TLC analysis of lipids from in vitro enzymatic reactions of NoDGAT2J (D) and NoDGAT2K (E) with various acyl-CoAs. C18:1/C16:0 DAG was used as the acyl acceptor. Experiments were conducted in triplicate, each with one representative TLC shown. (F) Quantification of specific activity of NoDGAT2J and 2K by GC–MS. Ctr1, microsome; Ctr2, microsome plus 250 mM DAG. Values are presented as mean ± SD (n = 3). Different letters above the bars grouped by each acyl-CoA indicate significant differences (p < 0.05) based on one-way analysis of variance and Tukey's honest significant difference test. Molecular Plant 2019 12, 474-488DOI: (10.1016/j.molp.2018.12.007) Copyright © 2018 The Author Terms and Conditions

Figure 2 Phenotypes of NoDGAT2K/2J Overexpression or Knockdown in N. oceanica. (A, B, F, and G) Transcript levels of NoDGAT2K/2J in the overexpression lines (2Jo1/2Jo2 [A] and 2Ko1/2Ko2 [F]) and the knockdown lines (2Ji1/2Ji2 [B] and 2Ki1/2Ki2 [G]) plus an empty vector control (EV) under N+ (0 h) and N− (6 h, 24 h, and 48 h after induction), as measured by qRT–PCR. Transcription level of NoDGAT2s was normalized to that of β-actin, the internal control. (C and H) Growth kinetics of the NoDGAT2J (C) and 2K (H) transgenic lines and EV under both N+ and N− conditions. (D and I) TAG content of the NoDGAT2J (D) and 2K (I) transgenic lines under N+ (0 h) and N− (24 h, 48 h, 72 h, and 96 h after induction). (E and J) FA composition of TAG in the NoDGAT2J (E) and 2K (J) transgenic lines under N− (24 h or 0 h after induction). Molecular Plant 2019 12, 474-488DOI: (10.1016/j.molp.2018.12.007) Copyright © 2018 The Author Terms and Conditions

Figure 3 Functional Partitioning among NoDGAT2K, 2J, 2A, 2C, and 2D in Assembling TAGs from the Various Types of FA CoAs. (A) Distinct yet hierarchical preferences for the FA CoA substrates among NoDGAT2K, 2J, 2A, 2C, and 2D. The enzyme activities, based on in vitro and ex vivo data, are shown as bar plots. Dynamics of FA content are shown by heatmap at the various time points under N−. FA contents are normalized by log2[Tc(FA species, Tm)/Avg(FA species, Tm)] (Tc = content of TAG-associated FA, Tm = time points under N−). Not all intermediates or reactions are displayed. Arrows indicate catalytic steps in the pathway. (B) Correlation of temporal dynamics between relative fold changes of NoDGAT2A, 2C, 2D, 2J, and 2K transcripts and TAG-derived FAs. TAG-derived SFA, PUFA, MUFA, LA, and EPA under N−/N+ in wild-type N. oceanica are shown. The arrows indicate the turning point where a specific NoDGAT2 transcript is upregulated or a class of TAG-derived FAs begins a sharp increase. Values are means ± SD (n = 3). Molecular Plant 2019 12, 474-488DOI: (10.1016/j.molp.2018.12.007) Copyright © 2018 The Author Terms and Conditions

Figure 4 Lineage of NoDGAT2s Based on Their FA CoA Preference. (A) Cladogram of selected DGAT2 protein sequences from higher plants, fungi, microalgae, and animals. The neighbor-joining method was used for tree construction. The cladogram was plotted based on actual branch length. GenBank accession numbers are provided in brackets. Arrows represent NoDGAT2s. (B) FA CoA preference-based functional evolvement of NoDGAT2A/C/D/K/J. Fold change of the TAG-associated FA content is calculated as log10[Tc(NoDGAT2)/Tc(EV Ctr)] (Tc = content of TAG-associated FA) and displayed in the heatmap. The data are from NoDGAT2 overexpression lines. Molecular Plant 2019 12, 474-488DOI: (10.1016/j.molp.2018.12.007) Copyright © 2018 The Author Terms and Conditions

Figure 5 NoDGAT2K and 2J Mediate PUFA-TAG Synthesis in the Chloroplast of N. oceanica. (A) A WT cell that shows baseline plastid autofluorescence in the GFP channel. (B–E) N. oceanica cells that express a GFP without any N-terminal fusion (B), a Signal:GFP fusion protein that targets chloroplast stroma (C), a DGAT2J:GFP fusion protein (D), or a DGAT2K:GFP fusion protein (E). TML, transmission light; PAF, plastid autofluorescence. Scale bar, 2 μm. (F) A mechanistic model of NoDGAT2s-mediated PUFA-TAG synthesis in the chloroplast of N. oceanica. For simplicity, a single TAG sink is shown. Not all intermediates or reactions are displayed. Arrows indicate catalytic steps in the pathway. ACP, acyl carrier protein; DAG, diacylglycerol; DES, desaturase; FAE, fatty acid elongase; FAS, fatty acid synthase; G-3-P, glycerol-3-phosphate; GPAT, glycerol-3-phosphate acyltransferase; LA, lysophosphatidic acid; LPAAT, lysophosphatidic acid acyltransferase; PA, phosphatidic acid; PAP, phosphatidic acid phosphatase; TAG, triacylglycerol. Molecular Plant 2019 12, 474-488DOI: (10.1016/j.molp.2018.12.007) Copyright © 2018 The Author Terms and Conditions

Figure 6 Producing Designer Oils by Modulating the Five DGAT2s in Nannochloropsis spp. (A) Comparison of the in vitro activity of NoDGAT2A/C/D/J/K. Values are presented as mean ± SD (n = 3). DGAT activity is measured based on formed TAG, which was separated by TLC and then quantified by GC–MS. (B) PUFA profiles of TAG in the NoDGAT2 overexpression and knockdown lines under N+. The diameter of the pie chart indicates the productivity of PUFA in biomass (mg g−1 dry weight). (C) TAG-associated EPA/LA values among Nannochloropsis strain bank. The TAG-associated EPA/LA values are evaluated among strains with significantly higher PUFA content than EV. The strains are then grouped into high (EPA/LA ≥ 1), medium (1 > EPA/LA ≥ 0.5), or low EPA/LA strains, as compared with EV. Molecular Plant 2019 12, 474-488DOI: (10.1016/j.molp.2018.12.007) Copyright © 2018 The Author Terms and Conditions