Biosynthesis of Triacylglycerol Molecules with Tailored PUFA Profile in

Industrial Microalgae

Yi Xin1,4,5

, Chen Shen1,4,5

, Yiting She1,4

, Hong Chen2, Cong Wang

3, Li Wei

1,4,

Kangsup Yoon2, Danxiang Han

2, Qiang Hu

2, Jian Xu

1,4,*

1Single-Cell Center, CAS Key Laboratory of Biofuels and Shandong Laboratory of

Energy Genetics, Qingdao Institute of BioEnergy and Bioprocess Technology,

Chinese Academy of Sciences, Qingdao, Shandong 266101

2Center for Microalgal Biotechnology and Biofuels, Institute of Hydrobiology,

Chinese Academy of Sciences, Wuhan, Hubei 430072, China

3Core Laboratory, Qingdao Institute of BioEnergy and Bioprocess Technology,

Chinese Academy of Sciences, Qingdao, Shandong 266101

4University of Chinese Academy of Sciences, Beijing 100049, China

5These authors contributed equally to this article.

*Address correspondence to xujian@qibebt.ac.cn

Running title: Producing designer triacylglycerols in industrial microalgae

Key words: microalgae, polyunsaturated fatty acids, diacylglycerol acyltransferase,

Nannochloropsis oceanica

%TA

Gpe

rTot

alLi

pid

0

10

20

30

40

50

60

NoDGAT2B

NoDGAT2E

NoDGAT2F

NoDGAT2G

NoDGAT2H

NoDGAT2I

NoDGAT2J

NoDGAT2K

ScDGA1

EV(pYES2)

%TA

Gpe

rTot

alLi

pid

0

10

20

30

40

50

60

NoDGAT2B

NoDGAT2E

NoDGAT2F

NoDGAT2G

NoDGAT2H

NoDGAT2I

NoDGAT2J

NoDGAT2K

ScDGA1

EV(pYES2)

TAG

*

TAG

*

A

B

Supplemental Figure 1. Complementation of the TAG-deficient phenotype in mutant yeast H1246 by expression of NoDGAT2s. (A) and (B) GC-MS quantification of TAG levels extracted from transformed yeast after the feeding of C18:3 (A) or C20:4 (B). The total amount of TAG was normalized based on that of the total lipids.

ICKHACNYFPVSLYVEISRHVCSYFPITLHVEVWRYFRDYFPIQLVKTVWRYFRDYFPIQLVKTVWKYMRDYFPIRLIKTLWRYFRDYFPISLIINIWKWYCDYFPISLIKTIYQRHPYYAKQDVVFDLWSRFVEYFSVEVVGD

Y

AYVFGYEPHSVLPIG IFYTPFLRHIWTWLGLTAASR

GYSCVLVPGGVQETFH VFLSRRRGFVRIAMEQ PLVPVFCFGQ

AYVFGYEPHSVFPIG VFYTPFLRHIWSWCGLTPATR

GYSCILVPGGVQETFY AFLKARRGFIRIAMQT PLVPVFCFGQ

NYIFGYHPHGIMGLG NFRMPVLREYLMSGGICPVSR

GNAIIIVVGGAAESLS VTLRNRKGFVKLALRH DLVPIYSFGE

NYIFGYHPHGIMGLG NFRMPVLREYLMSGGICPVNR

GNAIIIVVGGAAESLS VTLKNRKGFVKLALRH DLVPTYSFGE

NYIFGYHPHGILCFG NFRLPMFREYLMCGGICPVNR

GNAVVIVIGGAAESLD VMLKKRKGFVKLALKQ DLVPVYSFGE

NYIFAYHPHGIISIG NFKIPFLRDVLMSFGMSSVSK

GESICLVVGGAEESLD ITLKKRKGFIKLALVN SLVPVYSFGE

TYLFGYHPHGIGALG QFHIPLYRDYLLALGISSVSR

NQSICIVVGGARESLL LILNKRKGFIKLAIQT NLVPVFAFGE

KTLMAYHPHGILCCG LFLVPGMSNLLAWFQGGPAGR

GDNIAIIPGGFEEATI VFLKNRKGFLKLALQY KVHPVYTFGE

SAVYAVIPHGTFPFG VLRFPGFGQLIGFAGGVDAGP

GCSVSICPGGIAEMFW AFLQSRKGFIRMAMKH PVIPVYCFGN

PH G P

GG E L R GF A P FG

LPCRQP-MHVVVGKPIEV PTDEEIAKFHGQYVEALRDLFERHKSRVGLPFKNP-MHVVVGRPIEV PTAEEVAEVQREFIASLKNLFERHKARVGVPYSKP-ITTVVGEPITI PTQQDIDLYHTMYMEALVKLFDKHKTKFGVPYSKP-ITTVVGEPITV PTQKDIDLYHAMYMEALVKLFDNHKTKFGVPYCKP-ITTVVGEPITV PTQDVIDMYHAMYIRSLKSLFDNYKTRFGLPVRHK-IVTVVGEPIDI PTDQVIEHYHQIYVEALQNLFDKHKNSCALPFRAP-INVVVGRPIYV PPDDVVNHFHDLYIAELKRLYYENREKYGMPFRSARLTTVVGAPLQL PTVDDVTKYHNAYMAALQALFDKYKGQYAIPYRVP-LLYAVGKPLHL PTPGQIEVAHAEFCRALSDLFDRYKFYYGP VG P P L L

AtDGAT2VfDGAT2

HsDGAT2MmDGAT2

DrDGAT2DdDGAT2ScDGAT2

NoDGAT2JNoDGAT2K

AtDGAT2VfDGAT2

HsDGAT2MmDGAT2

DrDGAT2DdDGAT2ScDGAT2

NoDGAT2JNoDGAT2K

AtDGAT2VfDGAT2

HsDGAT2MmDGAT2

DrDGAT2DdDGAT2ScDGAT2

NoDGAT2JNoDGAT2K

138

153I 205

219II 248

268III

281

296IV 310

325V 329

338VI

385

402VII 409

437VIII

Supplemental Figure 2. Alignment of amino acid sequences among type-2 DGATs derived from animals, higher plants, fungi and microalgae. Eight highly conserved motifs are shown. Accession numbers of protein sequences are shown in Figure 2A. Bold residues indicate the homologies. Grey lines represent putative functional domains.Red arrows indicate NoDGAT2s.

Supplemantal Figure 3. Maps of the NoDGAT2J and 2K overexpression and knockdown vectors. The NoDGAT2 overexpression vectors were based on pXJ004 (A) and pXJ015 (B). Then the NoDGAT2J and 2K were introduced into pXJ427 (C) and pXJ428 (D), respectively. Meanwhile, RNAi expression cassette containing inverted repeat of NoDGAT2J or 2K gene was used for vector construction, forming pXJ440 (E) and pXJ441 (F). The subcellular local-ization of NoDGAT2J and 2K were determined by fusion with fluorescent tag, forming pXJ53-2J (G) and pXJ53-2K(H) respectively. In addition, a known chloroplast stroma marker, employed as a control, was fused with the fluore-scent tag to form pXJ53-stroma (I). Phsp: promoter of hsp70A, Ptub: promoter of β-tublin, Pvcp: promoter of vcp1. ble: zeocin resistance gene, TpsbA: terminator of psbA, TfcpA: terminator of fcpA, Tvcp: terminator of vcp1, Ttub: terminator of β-tublin.

E

TfcpAblePtub

NoDGAT2K Reverse Short

F

TfcpANoDGAT2J Forward LongblePtub

TpsbA TpsbAPhsp Ptubble

C

D

Ptub

TpsbA

ble

XhoI

bleKpnI

EcoRV

SacI

XhoI

HindIII

EcoRV

Phsp

TpsbA

BA

KpnI

AbspTAbspT butPpshP K2TAGDoNelb

NoDGAT2J

NoDGAT2K Forward Long

NoDGAT2J Reverse Short

G

H

IGFPvcpP K2TAGDoN Ttub Ptub ble Tvcp

GFPvcpP J2TAGDoN Ttub Ptub ble Tvcp

GFPvcpP Ttub Ptub ble TvcpStroma

1 000 bp750 bp500 bp

Marker

2 000 bp

A

1 000 bp750 bp500 bp

250 bp

Marker

2Jo12Jo2

EV pXJ427

2Ko12Ko2

EVpXJ428

2Ji1 2Ji2 2Ki1 2Ki2 WT EV

100 bp

B

Supplemental Figure 4. PCR validation of the NoDGAT2 overexpression and knockdown lines of N. oceanica. (A) Validation of the overexpression lines. The products were amplified by the same forward primer (located in Notub promoter) and distinct reverse primers (located in the corresponding NoDGAT2). (B) Validation of the RNAi-based knockdown lines. The products were amplified by the universal primer located in ble.

A B

N- induction time

TAG

prod

uctiv

ity(m

g/L)

0

50

100

150

200

250

0h 24h 48h 72h 96h

* *

N- induction time

TAG

prod

uctiv

ity(m

g/L)

0

50

100

150

200

250

0h 24h 48h 72h 96h** *

EV2Jo12Jo22Ji12Ji2

EV2Ko12Ko22Ki12Ki2

Supplemental Figure 5. TAG productivity of NoDGAT2J (A) or 2K (B) transgenic lines and control at 0 h, 24 h, 48 h, 72 h and 96 h after onset of N-. Data represent mean ± SD (n = 3). An asterisk indicates significance by Student’s t-test (p≤ 0.01).

NoDGAT2A

Tran

scrip

t abu

ndan

ce(N

oDG

AT2

/NoA

ctin

)

0

1

2

0 6 24 48

NoDGAT2C

0

1

2

0 6 24 48

NoDGAT2D

0

1

2

0 6 24 48

NoDGAT2J

0

1

2

0 6 24 48

NoDGAT2K

0

1

2

0 6 24 48 h

A

B

0

1

2

3

3 6 24 484 120

1

2

3

3 6 24 484 120

1

2

3

3 6 24 484 12 0

1

2

3

3 6 24 484 120

1

2

3

3 6 24 484 120 0 0 0 0

N-

Fold

-cha

nge

oftra

nscr

ipt a

bund

ance

N-/N+

hhhh

h h h h h

Supplemental Figure 6. Temporal dynamics of NoDGAT2A/C/D/J/K Transcript Abundance under Various Culture Conditions. (A) Absolute transcript levels (Ct(NoDGAT2)/Ct(NoActin)) of NoDGAT2s under N- conditions. (B) Relative transcript levels of NoDGAT2s between N- and N+ (i.e., N-/N+).

Mus musculus DGAT2 (NP 080660.1)Saccharomyces cerevisiae DGAT2 (NM_001183664.1)

Chlamydomonas reinhardtii DGTT1 (XP_001702848.1)Nannochloropsis oceanica DGAT2C (KX867958)

Nannochloropsis oceanica DGAT2D (KX867959)

Nannochloropsis oceanica DGAT2J (KX867965)

Arabidopsis thaliana DGAT2 (NM 115011.3)

Nannochloropsis oceanica DGAT2K (KX867955)

Chlamydomonas reinhardtii DGTT2 (XP_001694904.1)Nannochloropsis oceanica DGAT2A (KX867956)

77

71

95

94

91

95

41

0.1

Total FA Level

DU-associated FA Level

Single FA level

Supplemental Figure 7. Cladogram of selected highly conserved motifs of DGAT2 from higher plants, fungi, microalgae and animals. Neighbor-joining Method was used for tree construction, with bootstrap values (from 1000 replicates) shown on each node. Cladogram was plotted based on actual branch length. GenBank accession numbers are provided in brackets. The level of specificity in terms of FA CoA preference is highlighted on the right.

1

Table S1. PUFA profiles of the engineered Nannochloropsis oceanica strains. For

each strain, the numeric values are the percentage proportion of each PUFAs among

the FAs in TAG. Bold value indicates a significant increase over EV control.

Gene Strain C18:2 C18:3 C20:4 C20:5 PUFAs

EV control - - 1.55 0.05 0.21 0.40 2.21

Overexpression NoDGAT2A

2Ao1 1.59 0.15 0.57 0.26 2.57

2Ao2 2.07 0.15 0.41 0.26 2.89

NoDGAT2C 2Co1 2.34 0.24 1.13 2.12 5.83

2Co2 2.51 0.23 1.19 2.34 6.27

NoDGAT2D 2Do1 1.32 0.14 0.40 0.09 1.94

2Do2 1.45 0.17 0.43 0.17 2.21

NoDGAT2J 2Jo1 3.66 0.08 0.43 0.93 5.10

2Jo2 3.92 0.07 0.30 1.06 5.35

NoDGAT2K 2Ko1 1.84 0.07 0.33 2.72 4.96

2Ko2 2.04 0.06 0.23 3.12 5.45

Knockdown NoDGAT2A

2Ai1 1.78 0.05 0.41 0.99 3.24

2Ai2 1.40 0.03 0.14 0.28 1.84

NoDGAT2C 2Ci1 0.55 0.13 0.19 0.12 0.99

2Ci2 0.45 0.09 0.23 0.17 0.94

NoDGAT2D 2Di1 0.21 0.11 0.19 0.01 0.53

2Di2 0.60 0.14 0.21 0.01 0.96

NoDGAT2J 2Ji1 0.61 0.07 0.25 0.80 1.73

2Ji2 0.45 0.06 0.22 0.74 1.47

NoDGAT2K 2Ki1 0.97 0.05 0.19 0.12 1.34

2Ki2 0.85 0.05 0.17 0.17 1.24

2

Table S2. Nucleotide sequences of the primers used in this study 1

Gene Name Forward primer (5' to 3') Reverse primer (5' to 3')

Oligonucleotide primers used for cloning of NoDGAT2s and DGA1 and constructing of pYES2 vectors

NoDGAT2B GGTACCACATAATGACGCAGGTC GAATTCTCACTTAATAAGCAGCTTCTTG

NoDGAT2E GGTACCACATAATGGTTCGGCCCGAAG GAATTCTCACGACTTCGGACAGTCCCAAAT

NoDGAT2F GGTACCACATAATGGGTCTATTTGGCAG GAATTCCTAAAAGAAATTCAACGTCCGAT

NoDGAT2G GGATCCACATAATGCTATTGCAGGG GAATTCTTACAACAGGACCAGCCTATGAT

NoDGAT2H GGTACCACATAATGGCCACAACCTCGTCG GAATTCCTACCACAACTCCAACTTCGCCCCCT

NoDGAT2I GGTACCACATAATGTCCTCCTTCTTGC GAATTCCTAATGGCTATTATTCTTACCGC

NoDGAT2J GGTACCACATAATGGCTCACCTCTT GAATTCTCAAGAGATCGCAACGAAC

NoDGAT2K AAGCTTACATAATGTTGCTGGCGTCGT GAATTCTCAGACGATGCGAAGCGTC

DGA1 GGATCCACATAATGTCAGGAACATTCA

ATGATATAAG

TGCGGCCGCTTACCCAACTATCTTCAATTCTG

CATC

Oligonucleotide primers used for constructing of NoDGAT2 overexpressing vectors

NoDGAT2J CCGCTCGAGATGGCTCACCTCTTC GGGGAATTCTCAAGAGATCGCAACG

NoDGAT2K CCGCTCGAGATGTTGCTGGCGT GGGGAATTCTCAGACGATGCGAAGC

Oligonucleotide primers used for preparation of NoDGAT2 overexpressing cassettes

- TCTCGTAAACCCTGTCCCACTC AGGGTAGTGGCGATGGTG

Oligonucleotide primers used for PCR identification of NoDGAT2 overexpression lines

3

NoDGAT2J CACACTATCCCACACGCCTACAAAC CACTGCGTTTTCGTCCACCTTC

NoDGAT2K CACACTATCCCACACGCCTAC GAGTCAGAACAACACACAAAACAAG

Oligonucleotide primers used for construction of NoDGAT2 RNAi vectors

NoDGAT2J long CGGAATTCGCCGCTGCTTATCTTTATCG GCTCTAGAGGAGACGGTGTCTTGTCCTC

NoDGAT2J short CGGAATTCGCCGCTGCTTATCTTTATCG GCTCTAGACATCCTCCAGTGTTCCTGCT

NoDGAT2K long CGGAATTCGCTTCGGCCAACTTATAGGC GCTCTAGAACCCAAGGCGTCTTAGCC

NoDGAT2K short CGGAATTCGCTTCGGCCAACTTATAGGC GCTCTAGAAAGCAGCCCTCCTTTGGATA

Notub promoter ATGCGAGCTCACTGCGCATGGATTGACC

GA

AGCTCCATGGTGCTTCACAAAAAAGACA

GCTTCTTGAT

Oligonucleotide primers used for PCR identification of NoDGAT2 RNAi lines

- CCAAGTTGACCAGTGCCGTTC TCAGTCCTGCTCCTCGGC

Oligonucleotide primers used for qRT-PCR

NoDGAT2J CTATGACTTCGTTTTCTAAAGGCAC CCGTGCTTGACGAGGTAGATG

NoDGAT2K CTCTACCTACACGACGGTGGGACGC GGGACCGAGGGAGACCACGCC

NoActin GACGGCACCAAGGTCAAAAT ACGACGTGGAAGAGGAGGAA

1

Supplemental Online Methods 1

2

Strains, Medium, and Growth Conditions 3

Nannochloropsis oceanica strain IMET1 was cultivated in liquid modified f/2 4

medium containing sterilized seawater (salinity1.5%, w/v) at 25°C under light-dark 5

cycles of 12 h:12 h at an exposure intensity of 50 µmol m-2

s-1

, with the initial OD750 6

value as 1.0 (Dong et al., 2013, Li et al., 2014, Jia et al., 2015). For the experiment of 7

nitrogen depletion induced TAG-synthesis, mid-logarithmic phase algal cells (OD750 8

value of 2.6) were collected and washed three times with axenic seawater. Then equal 9

numbers of cells were re-inoculated in either nitrogen replete medium (N-replete 10

condition, or N+) or nitrogen-deprived medium (N depleted-condition, or N-) to the 11

same OD750 with 50 µmol m-2

s-1

light intensity. 12

13

Phylogenetic Analysis of DGAT2s 14

The encoded protein sequences of known or putative DGAT2 genes were aligned with 15

the MUSCLE version 3.8.31 (Edgar, 2004) and further adjusted manually using 16

BioEdit version 7.0.5.3(Hall, 1999) before all phylogenetic analysis. The optimal 17

substitution model of amino acid substitution was selected using the program 18

ModelGenerator version 0.84(Keane et al., 2006). DGAT2 sequences from other 19

model organisms [including green algae (Chlamydomonas reinhardtii), red algae 20

(Cyanidioschyzon merolae), higher plant (Arabidopsis thaliana), fungi 21

(Saccharomyces cerevisiae) and bacteria (Mycobacterium tuberculosis)] were also 22

included in this tree. DGAT2 from Mycobacterium tuberculosis was used as the 23

outgroup. 24

The curated alignment was then used to construct a phylogenetic tree using the 25

neighbor-joining (NJ) method in MEGA4.1 (Tamura et al., 2007), with the tree tested 26

by bootstrapping with 1000 replicates. The tree was drawn to scale, with branch 27

lengths in units identical to those of the evolutionary distances used to infer the 28

phylogenetic tree. The evolutionary distances were computed using the Poisson 29

correction method and are in the units of the number of amino acid substitutions per 30

2

site. All positions containing alignment gaps and missing data were eliminated only in 31

pairwise sequence comparisons (Pairwise deletion option). 32

33

RNA Isolation and cDNA Synthesis 34

Total N. oceanica RNA under the above conditions was extracted using Trizol 35

reagents (Invitrogen). For mRNA-Seq, the poly(A)-containing mRNA molecules were 36

purified using Sera-mag Magnetic Oligo(dT) Beads (Thermo Scientific) and were 37

fragmented into 200- to 300-bp fragments by incubation in RNA fragmentation 38

reagent (Ambion) according to manufacturer’s instructions. The fragmented mRNA 39

was then purified from the fragmentation buffer using Agencourt RNAClean beads 40

(Beckman Coulter). The purified, fragmented mRNA was subjected to the 41

PrimeScript RT reagent kit with gDNA Eraser (Takara) for cDNA synthesis. 42

43

Gene Cloning and Plasmid Construction 44

N. oceanica DNA was synthesized and used as a template for PCR. All primers used 45

are listed in Table S2. PCR products were then sequenced and manually curated to 46

obtain the full-length NoDGAT2B, 2E, 2F, 2G, 2H, 2I, 2J and 2K protein-coding 47

sequences. Then the protein structure (i.e., distributions of high conserved motifs) was 48

verified by alignment and comparison among NoDGAT2 sequences in 49

Nannochlororpsis and other model organisms. 50

For expression vector construction in yeast, the amplified PCR products were 51

digested with KpnI and EcoRI for NoDGAT2B, 2E, 2F, 2H, 2I and 2J, and with 52

BamHI and EcoRI for NoDGAT2G, and with HindIII and EcoRI for NoDGAT2K. The 53

products were then subcloned into pYES2 vector (Invitrogen) to form pXJ402, 54

pXJ405, pXJ406, pXJ407, pXJ408, pXJ409, pXJ410 and pXJ411, for expression in 55

the yeast Saccharomyces cerevisiae (below for more details). As a positive control in 56

yeast expression assays, the yeast DGA1 gene encoding DGAT2 was cloned, in a 57

manner similar to NoDGAT2s, to form pXJ412. 58

To construct the backbone for the N. oceanica IMET1 overexpression vectors, 59

the IMET1 endogenous promoters (of β-tublin and hsp70A) and psbA terminator were 60

3

amplified using genomic DNA with sequence-specific primers (Table S2) and 61

assembled in pBluescript SK vector (Stratagene). The selected promoter and 62

terminator regions were cloned into pSKB vector at KpnI-XhoI and BamHI-SacI sites, 63

respectively. The ble gene was codon-optimized based on the codon frequency in 64

IMET1 (Wei et al., 2013, Wang et al., 2014) and was subcloned into pBluescript SK 65

between XhoI and EcoV sites. The resulted vectors were named as pXJ004 and 66

pXJ015 (Figure S3A and B). 67

To further construct the vectors for overexpression in N. oceanica IMET1, 68

NoDGAT2J and 2K cDNA were amplified from pXJ410 and pXJ411 by PCR using 69

gene specific primers (Table S2), respectively. NoDGAT2s were subcloned into 70

pXJ004 vector to substitute ble gene (into XhoI and EcoRV sites). Then the expressing 71

cassettes of Ptub-NoDGATs-TpsbA were amplified and subcloned into the HindIII, 72

SacII or SacI sites of pXJ015 to form pXJ427 or pXJ428, which contains NoDGAT2J 73

or 2K respectively (Figure S3C and D). 74

To construct vector for NoDGAT2J or 2K RNAi knockdown, a 219 bp or 145 bp 75

small fragment (corresponding to the NoDGAT2J or 2K nucleotide sequence 53-271 76

bp or 181-384 bp) and a 427 bp or 280 bp long fragment (corresponding to the 77

NoDGAT2J or 2K gene sequence 53-479 bp or 181-595 bp) were amplified from the 78

N. oceanica IMET1 cDNA, respectively, with the primers NoDGAT2J_fw, or 79

NoDGAT2K_fw (containing a EcoRI site) and NoDGAT2J_rv1 or NoDGAT2K_rv1 80

(containing a XbaI site), and NoDGAT2J_fw or NoDGAT2K_fw and 81

NoDGAT2J_rv2 or NoDGAT2K_rv2 (containing a XbaI site) (Table S2). The 82

fragments were digested with EcoRI and XbaI and joined with XbaI sites. The joint 83

fragments with the inverted sequences were ligated to the EcoRI site of the linearized 84

phir-PtGUS vector to create phir-Pt-NoDGAT2J or phir-Pt-NoDGAT2K plasmid, 85

respectively. The promoter region of β-tublin of N. oceanica IMET1 was amplified 86

from genomic DNA using the primers Notub_fw (containing a SacI site) and 87

Notub_rv (containing a NcoI site; Table S2), then was digested with SacI and NcoI 88

and ligated in the phir-Pt-NoDGAT2J or phir-Pt-NoDGAT2K plasmid replacing the 89

Phaeodactylum tricornutum fcpB promoter to form pXJ440 (with NoDGAT2J 90

4

fragments; Figure S3E) or pXJ441 (with NoDGAT2K fragments; Figure S3F). 91

92

Yeast Strains and Cell Culture 93

S. cerevisiae strain H1246 (relevant genotype: MATαare1-Δ::HIS3are2-Δ::LEU2 94

dga1-Δ::KanMX4lro1-Δ::TRP1ADE2) containing knockouts of DGA1, LRO1, 95

ARE1and ARE2 (Sandager et al., 2002) was kindly provided by S. Stymne 96

(Scandinavian Biotechnology Research, Alnarp, Sweden). It is a neutral 97

lipid-deficient quadruple knockout mutant for DiacylGlycerol Acyltransferase1 98

(DGA1), Lecithin cholesterol acyl transferase Related Open reading frame1 (LRO1), 99

Acyl-coenzyme A: cholesterol acyl transferase-Related Enzyme2 (ARE1) and 100

Acyl-coenzyme A: cholesterol acyl transferase-Related Enzyme2 (ARE2). Yeast cells 101

were maintained on YPD plates (1% yeast extract [w/v], 2% peptone [w/v], and 2% 102

glucose [w/v]) solidified with 2% agar (w/v). Cells were transformed using the 103

lithium acetate procedure (Gietz and Woods, 1994), and transformants were selected 104

by growth on synthetic glucose medium (2% glucose [w/v] and 0.67% yeast nitrogen 105

base without amino acids [w/v]) containing appropriate auxotrophic supplements 106

(Clontech). Single yeast colonies were inoculated into liquid synthetic glucose 107

medium and cultured overnight at 30°C and 150 rpm in an orbital shaker. The OD600 108

of the culture was determined, an appropriate volume of cell culture was harvested by 109

centrifugation, and cells were resuspended in synthetic galactose medium (2% 110

galactose [w/v], 1% raffinose [w/v], 0.67% yeast nitrogen base without amino acids 111

[w/v], and appropriate auxotrophic supplements) at an OD600 of 0.4. To determine 112

poly-unsaturated fatty acid (PUFA) substrate preferences among NoDGAT2J and 2K, 113

supplementation of synthetic galactose cultures with linoleic acid (C18:2), linolenic 114

acid (C18:3), arachidonic acid (C20:4) or eicosapentaenoic acid (C20:5) was carried 115

out with 90 μM of the appropriate FA in the presence of 0.1g/L BSA. Following 116

expression, the yeast cells at late stationary phase of growth were used for extraction 117

of total lipids for further analysis. 118

119

Yeast Microsome Preparation and Non-Radiolabeled DGAT In Vitro Assay 120

5

Yeast transformants carrying NoDGAT2s were collected after growing in liquid 121

galactose medium lacking uracil for 15 h at 30°C. The cell pellets were resuspended 122

in cell lysis buffer [containing 5% glycerol, 20 mM Tris-HCl (pH 8.0), 0.3 M 123

ammonium sulfate, 10 mM MgCl2, 1 mM EDTA, 1 mM DTT, 1× EDTA-free protease 124

inhibitor cocktail set X (Calbiochem), 1 mM PMSF] to an OD600 of approximately 125

100 and lysed by passing twice through a French pressure cell (Spectronics 126

Instruments) at an internal pressure of 15,000 PSI. Cell debris was removed from the 127

suspension by centrifugation at 10,000 × g for 10 min at 4°C and the supernatant was 128

centrifuged further at 100,000 × g for 1 h at 4°C. The resulting microsomal membrane 129

pellets were resuspended in microsomal storage buffer (50 mM Tris-HCl, pH 7.5, 10% 130

glycerol) to give a protein concentration of 10 µg µL-1

for immediate use or stored at 131

-80 °C. 132

The DGAT in vitro assay was performed in a 200 uL of assay mixture containing 133

50 mM potassium phosphate (pH 7.5), 10 mM MgCl2, 40 µg of microsomal 134

membrane protein, 250 µM acyl-CoA, and 250 µM DAG (delivered from a 50 mM 135

stock in ethanol), according to our published procedure (Liu et al., 2016). Reactions 136

were incubated at 30°C for 1 h and the lipids were extracted with 137

chloroform/methanol (2:1, v/v) by vigorous vortex. Microsome fraction alone and 138

microsome fraction with DAG were used as controls and the background levels of 139

TAG were subtracted from the data for DGAT activity analysis. The acyl CoAs tested 140

included myristoyl-CoA (C14:0 CoA), palmitoyl-CoA (C16:0 CoA), 141

hexadecenoyl-CoA (C16:1 CoA), stearoyl-CoA (C18:0 CoA), oleoyl-CoA (C18:1 142

CoA), linoleoyl-CoA (C18:2 CoA), γ-linolenoyl-CoA (C18:3n6 CoA), 143

eicosatetraenoyl-CoA (C20:4 CoA) and eicosapentaenoyl-CoA (C20:5 CoA). The 144

C18:1n9/C16:0-DAG was used as the acyl receptor. The other lipid standards were 145

purchased from Avanti Polar Lipids. 146

147

Nuclear Transformation of N. oceanica 148

Nuclear transformation was performed using the linearized overexpressing vector 149

6

construct and the high-voltage (11,000 V/cm) electroporation method (Wang et al., 150

2016). The transformant with empty pXJ015 vector was used as control. Colonies 151

were picked into 50 ml flask and cultivated in 20 ml liquid modified f/2 medium 152

prepared with sterilized seawater (salinity1.5%, w/v) at 25°C under light-dark cycles 153

of 12 h:12 h at an exposure intensity of 50 µmol m-2

s-1

. Mid-logarithmic phase algal 154

cells (OD750 of 2.6) were collected for validation of successful transformants via PCR 155

amplification of the introduced promoter and NoDGAT2s on the overexpression 156

vector for overexpression lines, and PCR amplification of the ble gene for the 157

knockdown lines (Table S2; Figure S4). Positive strains were then cultured for 158

further validation of target-gene expression and subsequent phenotyping. 159

160

Quantitative Real-Time PCR (qRT-PCR) 161

The qRT-PCR analysis was carried out in optical 96-well plates using the CFX96 162

Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA). The qRT-PCR 163

primer pairs were designed for NoDGAT2s and the housekeeping gene NoACT (actin). 164

Each reaction contained 5 μL of iTaq™ Universal SYBR® Green Supermix 165

(Bio-Rad), 20 ng cDNA, and 280 nM of each gene-specific primer pair to a final 166

volume of 10 μL. Further serial dilutions of the cDNAs were prepared, and qRT-PCR 167

was performed with each primer pair to generate a standard curve and to estimate 168

PCR efficiency. PCR cycling conditions consisted of an initial polymerase activation 169

step at 95 °C for 30 s followed by 40 cycles at 95

°C for 5 s and 60

°C for 30 s, and a 170

final melting step at 65-95 °C. Results were analyzed using the formula 171

2Ct(NoAct)

/2Ct(NoDGAT2)

, using the relative expression value of the housekeeping gene 172

NoACT as the calibrator. The experiment was performed twice, with three biological 173

replicates and four technical replicates for each sample. Primer sequences used in 174

qRT-PCR were listed in Table S2. 175

176

Lipid Isolation and Quantification 177

Total lipids were extracted from dried samples using chloroform:methanol (2:1 [v/v]) 178

with 100 mM internal control tri13:0 TAG (Sigma) and separated on a silica TLC 179

7

plate using a mixture of solvents consisting of petroleum ether, ethyl ether and acetic 180

acid (70:30:1, by volume). To quantify the amount of TAG accumulated in yeasts 181

expressing the NoDGAT2 constructs, TAG bands were scraped from the TLC plate. 182

Fatty acid methyl esters (FAMEs) were prepared by acid-catalyzed transmethylation 183

of the TAG bands and then analyzed by GC-MS as previously described (Zhang et al., 184

2003). Mixed analytical standard of FAMEs and pentadecane were used as external 185

and internal standard, respectively. The amounts of TAGs and the profiles of 186

TAG-associated FAs were calculated based on the results derived from GC-MS. 187

188

REFERENCES 189

190

Dong H.P., Williams E., Wang D.Z., Xie Z.X., Hsia R.C., Jenck A., et al, Place 191

A.R. (2013). Responses of Nannochloropsis oceanica IMET1 to long-term nitrogen 192

starvation and recovery. Plant Physiol. 162: 1110-1126. 193

194

Edgar R.C. (2004). MUSCLE: multiple sequence alignment with high accuracy and 195

high throughput. Nucleic. Acids Res. 32: 1792-1797. 196

197

Gietz R.D. and Woods R.A. (1994). High efficiency transformation with lithium 198

acetate. Molecular genetics of yeast: a practical approach. Academic Press: 121-134. 199

200

Hall T.A. (1999). BioEdit: a user-friendly biological sequence alignment editor and 201

analysis program for Windows 95/98/NT. Nucl. Acids. Symp. Ser.: 95-98. 202

203

Jia J., Han D.X., Gerken H.G., Li Y.T., Sommerfeld M., Hu Q. and Xu J. (2015). 204

Molecular mechanisms for photosynthetic carbon partitioning into storage neutral 205

lipids in Nannochloropsis oceanica under nitrogen-depletion conditions. Algal Res. 7: 206

66-77. 207

208

Keane T.M., Creevey C.J., Pentony M.M., Naughton T.J. and McInerney J.O. 209

(2006). Assessment of methods for amino acid matrix selection and their use on 210

empirical data shows that ad hoc assumptions for choice of matrix are not justified. 211

BMC Evol. Biol. 6: 1-17. 212

213

Li J., Han D.X., Wang D.M., Ning K., Jia J., Wei L., et al, Xu J. (2014). 214

Choreography of transcriptomes and lipidomes of Nannochloropsis reveals the 215

mechanisms of oil synthesis in microalgae. Plant Cell. 26: 1645-1665. 216

217

Liu J., Lee Y.Y., Mao X.M. and Li Y.T. (2016). A simple and reproducible 218

8

non-radiolabeled in vitro assay for recombinant acyltransferases involved in 219

triacylglycerol biosynthesis. J. Appl. Phycol. 29: 323-333. 220

221

Sandager L., Gustavsson M.H., Stahl U., Dahlqvist A., Wiberg E., Banas A., et al, 222

Stymne S. (2002). Storage lipid synthesis is non-essential in yeast. J. Biol. Chem. 277: 223

6478-6482. 224

225

Tamura K., Dudley J., Nei M. and Kumar S. (2007). MEGA4: Molecular 226

Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24: 227

1596-1599. 228

229

Wang D.M., Ning K., Li J., Hu J.Q., Han D.X., Wang H., et al, Xu J. (2014). 230

Nannochloropsis genomes reveal evolution of microalgal oleaginous traits. PLoS 231

Genet. 10: e1004094. 232

233

Wang Q.T., Lu Y.D., Xin Y., Wei L., Huang S. and Xu J. (2016). Genome editing 234

of model oleaginous microalgae Nannochloropsis spp. by CRISPR/Cas9. Plant J. 88: 235

1071-1081. 236

237

Wei L., Xin Y., Wang D.M., Jing X.Y., Zhou Q., Su X.Q., et al, Xu J. (2013). 238

Nannochloropsis plastid and mitochondrial phylogenomes reveal organelle 239

diversification mechanism and intragenus phylotyping strategy in microalgae. BMC 240

Genomics. 14: 1-17. 241

242

Zhang Q., Chieu H.K., Low C.P., Zhang S.C., Heng C.K. and Yang H.Y. (2003). 243

Schizosaccharomyces pombe cells deficient in triacylglycerols synthesis undergo 244

apoptosis upon entry into the stationary phase. J. Biol. Chem. 278: 47145-47155. 245

246

247