Lipid distribution pattern and transcriptomic insights revealed

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Omics Technologies Applied to Agriculture and Food

Lipid distribution pattern and transcriptomic insights revealed the potential mechanism of docosahexaenoic acid traffics in Schizochytrium sp. A-2 Xiu-Hong Yue, Wen-Chao Chen, Zhi-Ming Wang, Peng-Yang Liu, XiangYu Li, Chu-Bin Lin, Shu-Huan Lu, Feng-Hong Huang, and Xia Wan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03536 • Publication Date (Web): 05 Aug 2019 Downloaded from pubs.acs.org on August 6, 2019

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Journal of Agricultural and Food Chemistry

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Lipid distribution pattern and transcriptomic insights revealed the potential

2

mechanism of docosahexaenoic acid traffics in Schizochytrium sp. A-2

3 4

Xiu-Hong Yuea, 1, Wen-Chao Chena, b, c, d, 1, Zhi-Ming Wange, Peng-Yang Liua, Xiang-

5

Yu Lie, Chu-Bin Lina, Shu-Huan Lue, Feng-Hong Huanga, b, c, d, Xia Wan*, a, b, c, d

6 7

aOil

8

430062, P.R. China

9

bKey

Crops Research Institute of the Chinese Academy of Agricultural Sciences, Wuhan

Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of

10

Agriculture, Wuhan 430062, P.R. China

11

cOil

12

Laboratory, Wuhan 430062, P.R. China

13

dHubei

14

eCABIO

Crops and Lipids Process Technology National & Local Joint Engineering

Key Laboratory of Lipid Chemistry and Nutrition, Wuhan 430062, P.R. China Bioitech (Wuhan) Co., Ltd, Wuhan 430223, P.R. China

15 16

Xiu-Hong Yue, [email protected]

17

Wen-Chao Chen, [email protected];

18

Zhi-Ming Wang, [email protected];

19

Peng-Yang Liu, [email protected];

20

Xiang-Yu Li, [email protected];

21

Chu-Bin Lin, [email protected];

22

Shu-Huan Lu, [email protected];

ACS Paragon Plus Environment


23

Feng-Hong Huang, [email protected];

24 25

Corresponding author: Xia Wan, [email protected].

26

1Xiu-Hong

Yue and Wen-Chao Chen are co-first authors.


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Abstract

28

Schizochytrium sp. A-2 is a heterotrophic marine fungus used for commercial

29

production of docosahexaenoic acid (DHA). However, the pattern of DHA distribution

30

and how DHA is channeled into phospholipid (PL) and triacylglycerol (TAG) are

31

unknown. In this study, we systematically analyzed the DHA distribution in TAG and

32

PL during the cell growth. The migration of DHA from PL to TAG was presumed

33

during the fermentation cycle. DHA and docosapentaenoic acid (DPAn6) were

34

accumulated in both TAG and phosphatidylcholine (PC), whereas eicosapentaenoic

35

acid (EPA) was mainly deposited in PC. RNA-seq revealed malic enzyme may provide

36

lipogenic NADPH. In addition, long chain acyl-CoA synthase (LACS) and acyl-

37

CoA:lysophosphatidylcholine acyltransferase (LPCAT-1) may participate in DHA

38

accumulation in PL. No phosphatidylcholine:diacylglycerol cholinephosphotransferase

39

(PDCT)

40

phospholipid:diacylglycerol acyltransferase (PDAT) mediated acyl-CoA independent

41

TAG synthesis pathway and phospholipase C (PLC) may contribute to the channeling

42

of DHA from PC to TAG.

was

identified

from

the

genome

sequence.

In

contrast,

43 44

Keywords

45

Docosahexaenoic acid, Schizochytrium, triacylglycerol, phospholipid, migration.



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Introduction

47

Docosahexaenoic acid (DHA, 22:6n-3) is one of omega-3 very long chain

48

polyunsaturated fatty acids (ω3-VLCPUFAs), and plays an important role in human

49

cognitive, neurological and visual developments.1-2 Sufficient daily intake of DHA is

50

recommended by U.S. National Institutes of Health for both adults and children

51

(https://ods.od.nih.gov/factsheets/Omega3FattyAcids-HealthProfessional/#h4).

52

addition to fish oil-derived DHA, microorganisms, such as Schizochytrium sp. and

53

Crypthecodinium cohnii are currently used for commercial production of DHA

54

(https://www.fda.gov/media/117920/download). Of them, Schizochytrium sp., an

55

unicellular non-photosynthetic eukaryote in kingdom Stramenopila, can produce up to

56

55% oils of the cell dry weight and DHA compromises as much as 50% or above of the

57

total fatty acids.3-4

58

DHA is synthesized via two distinct biosynthetic pathways in microorganisms. The

59

aerobic pathway involving several desaturases and elongases is mainly found in animals

60

and eukaryotic microbes.5 The anaerobic biosynthetic pathway mediated by polyketide

61

synthase (PKS) is active mainly in marine bacteria and eukaryotic microorganisms.5-6

62

Although both aerobic and anaerobic pathways exist in DHA-producing

63

Thraustochytrium and Schizochytrium, PKS is identified as the only active pathway for

64

de novo DHA synthesis in these species.7-9

65

Production of DHA in Schizochytrium depends on not only DHA synthesis, but also

66

DHA transportation and accumulation. Although de novo synthesis of DHA has been

67

well documented, information on the route for DHA migration and the molecular


In

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mechanism for DHA migration are unclear in Schizochytrium. It has been demonstrated

69

that DHA is released from PKS pathway in the form of free fatty acid (FFA) in

70

Schizochytrium sp.,10 suggesting enzymes responsible for the following acyl activation

71

and channeling into phospholipid (PL) or triacylglycerol (TAG) are active in

72

Schizochytrium. Notably, the in vivo biochemical results show that the released DHA

73

from PKS pathway is first incorporated into PL but not directly into TAG in

74

Thraustochytrium sp. 26185, a strain closely related to Schizochytrium.7 TAG

75

constitutes the majority of lipids and DHA is highly enriched in TAG during lipid

76

accumulation in both Schizochytrium and Thraustochytrium.5, 11 The route for DHA

77

migration in Schizochytrium might be similar to that in Thraustochytrium. However,

78

there is no direct evidence is reported. Thus it is interesting to investigate the

79

distribution and migration of DHA as well as the key enzymes responsible for these

80

processes in Schizochytrium.

81

In this study, we first systematically analyzed the distribution of DHA in different lipids

82

including neutral lipids and PL in Schizochytrium sp. A-2. We then identified the

83

differentially expressed genes involved in DHA synthesis, modification and migration

84

during fermentation cycle based on the transcriptomic analysis. Those genes that were

85

upregulated at the stage of TAG accumulation were further analyzed.

86 87

Materials and method

88

Strains, media, and growth conditions



89

The strain, Schizochytrium sp. A-2, was obtained from China Center for Type Culture

90

Collection (CCTCC No: M2012494). This strain is stored in 20% (v/v) glycerol at

91

−80°C. The initial culture medium contained glucose (40 g/L), yeast extract (6 g/L),

92

sea salts (20 g/L). The fermentation culture medium contained glucose (50 g/L), yeast

93

extract (9 g/L), sodium glutamate (30 g/L), which were dissolved in artificial sea water.

94

The pH of the medium was adjusted to 6.8 before being autoclaved at 121°C for 20

95

min. Cells were cultured in initial culture medium for two generations and then 1.5 mL

96

of culture were transferred to 30 mL fermentation medium in 250 mL flasks at 28°C

97

with shaking at 220 rpm. No extra glucose was added into the medium during the

98

fermentation. For time course experiment, cells were collected by centrifugation at

99

5000 g for 10 min at different time points. The collected cells were used for RNA

100

extraction or immediately stored at −80°C for lipid extraction.

101 102

Glucose Content Measurements

103

Fresh cells were collected by centrifugation at 5000 g for 10 min at different time points.

104

The supernatant from an amount of 1 mL of culture was used to determine glucose

105

content by the 3, 5-dinitrosalicylic acid (DNS) method.12

106 107

Analysis of fatty acid profile from TAGs and TFAs

108

Collected cells were freeze-dried and weighted. Total lipids were extracted from two

109

milligram of the freeze-dried cells according to our previous method.13 Before

110

extraction, FFA-C21:0 or TAG-63:0 (TAG-tri-21:0) was added as an internal standard


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for the quantification of total fatty acids (TFA) or TAG. Fatty acid methyl esters

112

(FAMEs) of TFA (equivalent to 2 mg dry weight cells) were generated by incubating

113

extracted lipids in 0.75 mL of 5% sulfuric acid in methanol at 90°C for 90 min. The

114

resulting FAMEs were analyzed using a 7890A gas chromatography (GC, Agilent

115

technologies, USA) equipped with a flame ionization detector (FID) and an HP-FFAP

116

column (30 m ×250 μm i.d. × 0.25 μm thickness).

117

To analyze the fatty acid profiles in TAG or PL, the extracted lipids were first separated

118

by thin-layer chromatography (TLC) using a solvent system (hexane: diethyl ester:

119

acetic

120

(chloroform:methanol:acetic acid:water = 90:15:10:3, v/v) for PL. TAG-18:1/18:1/18:1,

121

FFA-18:1 and diacylglycerol (DAG)-18:1/18:1 standards (NuChek, USA) were used to

122

confirm the authentic TAG, FFA and DAG bands from samples, respectively. To

123

visualize the lipid bands, the silica plate was sprayed with 0.01% primuline dissolved

124

in 80% acetone. The bands corresponding to TAG were recovered from TLC plates,

125

methylated to FAMEs and analyzed by GC as described above.

acid

=

70:30:1,

v/v)

for

neutral

lipids,

or

a

solvent

system

126 127

Analysis of lipid profile

128

Structure of TAG and PL was further analyzed by liquid chromatography-mass

129

spectrometer (LC-MS) system including 2777C UPLC system (Waters, UK) and Xevo

130

G2-XS QTOF (Waters, UK). An ACQUITY UPLC CSH C18 column (10 cm x 2.1

131

mm, 1.7 μm, Waters, UK) was used for the chromatographic separation. The injection

132

volume was 10 μL and the flow rate was 0.4 mL/min. The mobile phase consisted of



133

solvent A (ACN/H2O, 60/40, v/v+0.1% formate acid and 10 mM ammonium formate)

134

and solvent B (IPA/ ACN, 90/10, v/v+0.1% formate acid and 10 mM ammonium

135

formate). Gradient elution conditions were set as follows: 40-43% phase B from 0 to 2

136

min; 50-54% phase B from 2.1 to 7 min; 70-99% phase B from 7.1 to 13 min; 40%

137

phase B from 13.1 to 15 min. The Q-TOF was used to detect metabolites eluted from

138

the column in both positive and negative ion modes. The MS data were acquired in

139

Centroid MSE mode and the scanned range was from 50 to 2000 Da. The survey scan

140

time was 0.2 s. All precursors were fragmented at energies ranging from 19 eV to 45

141

eV for MS/MS, and the scan time was 0.2 s. The LE signal was collected every 3 s to

142

calibrate the mass accuracy during the data collection. In addition, a QC sample (Pool

143

of all samples) was run after every 10 samples for evaluating the stability of the LC-

144

MS during the whole acquisition.

145

Lipid identification was based on LipidMaps. In this study, we selected the positive

146

(ESI+) scanning data and whose secondary identification scores were not equal to zero

147

for analysis, TAG and phosphatidylcholine (PC) species were identified and relatively

148

quantified. The differentially abundant lipid molecular species were determined by fold

149

change ≥1.2 or ≤0.8 and q-value < 0.05.

150 151

Transcriptomic analysis

152

Schizochytrium sp. A-2 was cultured at 28°C with shaking at 220 rpm. Cells were

153

collected after 8 h, 24 h, or 48 h and immediately used for RNA extraction. Total RNA

154

extraction was performed with RiboPure™ RNA Purification Kit (Ambion, USA) and


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quantified using an Agilent 2100 bioanalyzer. The mRNA was enriched using Ribo-

156

Zero Gold rRNA Removal Kit (Illumina, USA). Then, the mRNAs were fragmented

157

and enriched, followed by the synthesis of cDNA. Afterwards, the cDNA fragments

158

were purified and ligated with sequencing adapters for the PCR amplification as

159

templates. The constructed library was analyzed quantitatively and qualitatively using

160

Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System and was

161

sequenced on HiSeq 2000 platform (Illumina, USA).

162

The clean reads were obtained by removing adaptors from raw data, then they were

163

matched to the reference gene sequences using Bowtie2.14 The expression levels of the

164

genes and transcripts were calculated using RSEM.15 Fragment Per Kilobase per

165

Million mapped reads (FPKM) was used to quantify gene expression level. The

166

differentially expressed genes (DEGs) between different samples were identified by

167

DEGseq method.16 The false discovery rate (FDR) was corrected using Benjamini’s

168

and Hochberg’s two strategies. The genes with absolute value of the fold change ≥ 2

169

and a FDR of ≤ 0.001 were defined as the significantly differentially expressed genes.

170

The unigenes of each sample were subjected to homology searches against seven

171

functional database annotations as follows: Non-redundant protein sequence database

172

(NR), Nucleic acid sequence database (NT), Swiss-Prot; Kyoto Encyclopedia of Genes

173

and Genomes (KEGG), Eukaryotic Orthologous Groups (KOG), Protein Families

174

(Pfam) and Gene Ontology (GO). The protein names were identified by BLASTP with

175

accession number of the top hit.

176



177

Verification of representative genes transcription by Quantitative Real-Time

178

Reverse Transcriptional PCR (qRT-PCR)

179

The same samples used for RNA-seq were analyzed by qRT-PCR. cDNA was

180

synthesized by using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher,

181

USA) and used for Real-Time PCR analysis. Seven genes (PfsA 、 PfsB 、 PfsC 、

182

FAS2 、 LACS 、 FACS1 、 TE) were chosen based on the result of RNA-seq. The

183

relative expression values were calculated according to the 2−△△Ct method and the

184

constitutively expressed gene β -actin was used as internal standard for data

185

normalization. Primers were listed in Table S1.

186 187

Results and discussion

188

Analysis of DHA content in TFAs in Schizochytrium sp. A-2 during the cell growth

189

We first carried out a time course experiment to determine the growth curve and to

190

detect the DHA distribution in TFA. As the culturing time increased from 0 to 72 h, the

191

proportion of VLCPUFAs including arachidonic acid (ARA), eicosapentaenoic acid

192

(EPA, 20:5n3), omega-6 docosapentaenoic acid (DPAn6, 22:5n6) and DHA, increased

193

from 67.2% to 83.7%. In contrast, the percentage of saturated fatty acids (SFAs), such

194

as C14:0, C15:0, C16:0, generated from the fatty acid synthase (FAS) pathway,

195

decreased as the cells grew (Table 1). DHA was the major fatty acid of VLCPUFA,

196

while C16:0 constituted the most abundant component of SFAs. The ratio of DHA to

197

DPAn6 was reduced as the percentage of DPAn6 was increased from 13.8% to 22.8%

198

(Table 1). Monounsaturated fatty acids (MUFAs) including C16:1 and C18:1 were


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199

detected in trace amounts. After cells were cultured for 48 h, glucose was exhausted,

200

while the total biomass and the lipid titer reached their maximal levels of 32.7 g/L and

201

6.5 g/L (Figure 1A and 1B). The highest DHA titer of 3.5 g/L was also achieved at this

202

time point (Figure 1B). Although the percentage of DHA in TFAs continued to increase

203

to as high as 53.8% after 48 h, the titers of TFAs and DHA all dropped mainly due to

204

the decreased total biomass (Table 1 and Figure 1B). Besides, the decreased total

205

biomass at 60 and 72 h (Figure 1A) might partly due to a decrease of the lipid content

206

(Figure 1B).

207 208

The distribution of DHA in TAG during the cell growth

209

In Schizochytrium sp. 20888, DHA is synthesized and released as FFA via the anaerobic

210

PKS pathway,10 then incorporated into TAG as storage lipid or into PL as membrane

211

lipid. However, when and how the DHA is channeled into TAG or PL is still unknown

212

in Schizochytrium. Therefore, we first investigated the distribution of DHA in TAG

213

during the cell growth. TLC results showed that TAG was the major component of

214

neutral lipids regardless of culturing time (Figure 2A). The band corresponding to DAG

215

was also clearly shown in TLC plate, whereas only trace amount of FFA was observed

216

(Figure 2A). This suggested FFAs as the intermediates were efficiently utilized for the

217

synthesis of lipids in strain A-2 during the fermentation. We also noticed that the DHA

218

level in TAG reached 35.8% as early as 8 h (Figure 2B). At this time point, the cells

219

started to enter the log phase and the biomass was only 0.3 g/L. This indicated that

220

DHA was channeled into TAG directly or indirectly in a very efficient way. In the



221

following 36 h, the proportion of DHA in TAG was kept at a stable level around 42%.

222

Then an increase of DHA percentage was observed at 42 h when glucose was almost

223

exhausted. This might be explained by that other fatty acids were consumed and thus

224

the percentage of DHA increased under carbon deprivation conditions.

225

The pattern of DPAn6 increasing was similar to that of DHA (Table 1 and Figure 2B).

226

Our results and previous report

227

DHA. In fact, both DPAn6 and DHA were synthesized via the same PKS pathway.6

228

The same mechanism for synthesis and migration might apply to DPAn6 and DHA.

229

The existence of abundant DPAn6 was not preferable to commercial production of

230

DHA. Thus, improvement of the percentage of DHA may be achieved by conversion

231

of DPAn6 into DHA. However, overexpression of an ω-3 desaturase from Saprolegnia

232

diclina only slightly increased the conversion of DPAn6 to DHA.18 Screening of more

233

specific or more active C22-specific ω-3 desaturase may be needed in future.19 In

234

addition, previous report showed that supplementation of propionic acid in the medium

235

increased the ratio of DHA/DPAn6 slightly, but the concentrations of DHA and DPAn6

236

were all decreased.20 In contrast to the synchronous increase of DHA and DPAn6, the

237

percentage of EPA decreased in TAG although it increased in TFAs during the cell

238

growth (Table 1 and Figure 2B). The migration route of EPA will be discussed in the

239

section on PL.

240

We also noticed that the percentage of fatty acids synthesized by the FASI system in

241

TAG were higher than that in TFAs. Especially, MUFAs C16:1 and C18:1 comprised

242

up to 16.7% of fatty acids in TAG at 8 h (Figure 2B), in contrast to trace amount of

17

suggested that DPAn6 might be co-migrated with


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them found in TFAs (Table 1). Among those fatty acids synthesized by FASI, most of

244

them were reduced in TAG as the cells grew. However, C16:0 was an exception, which

245

was first increased from 19.6% to 25.0% in TAG at 30 h but followed by a decrease to

246

23% at 48 h (Figure 2B). PKS and FAS pathway competed for the substrate acetyl-CoA

247

generated from glycolysis, and C16:0 was the major fatty acid synthesized via FAS

248

pathway. The change of C16:0 level might be explained by that acetyl-CoA was more

249

utilized by PKS over FAS pathway when glucose was limited for glycolysis.

250

The TAG profile was further investigated by using LC-MS. In total, 74 species of TAGs

251

were identified from Schizochytrium sp. A-2 (Figure 3). Among them, TAG-66:18

252

(TAG-22:6/22:6/22:6),

253

22:6/22:6/22:5n6) and TAG-60:11 (TAG-22:6/22:5n6/16:0) were the most abundant

254

species irrespective to the stages of cell growth (Figure 3A). This was consistent with

255

our previous GC results that DHA, DPAn6 and C16:0 were the major three fatty acids

256

in the TAG fraction. The percentage of TAG-66:18 and TAG-66:17 was increased

257

throughout the fermentation and both had an obvious increase at 48 h (Figure 3B).

258

However, TAG-60:12 containing C16:0 increased at the first 24 h and then decreased

259

at 48 h, which was matching to the increasing pattern of C16:0 in TAG (Figure 3B). In

260

addition to the above four abundant TAGs, other DHA-containing TAGs including but

261

not limited to TAG-14:0/22:6/22:6, TAG-16:0/22:6/16:0, TAG-15:0/22:6/15:0, TAG-

262

20:5/22:6/22:6, TAG-14:0/22:5/22:6 were also identified (Figure 3B). We also noticed

263

that the TAGs with the VLCPUFA generated from PKS pathway increased as the cells

264

grew, whereas the TAGs with the fatty acids generated from FAS pathway did not

TAG-60:12

(TAG-22:6/16:0/22:6),


TAG-66:17

(TAG-


265

change obviously throughout the cell growth (Figure 3C). This again demonstrated that

266

DHA as well as other VLCPUFAs synthesized via PKS pathway were the major fatty

267

acids incorporated in TAG after cells entering the log phase. However, in

268

Schizochytrium sp. SR21, TAG-16:0/22:6/16:0 was reported as the most abundant

269

DHA-containing TAG molecule,17 while all identified TAGs contained at least one

270

SFA in Thraustochytrium sp. 26185.21 The existing of abundant TAG-22:6/22:6/22:6

271

may attribute to the very high levels of DHA in strain A-2.

272 273

The distribution of DHA in PL during the cell growth

274

We next investigated the distribution of DHA in PL. The amount of total PL from

275

different time points remained stable at the levels around 20% of the total lipids (Figure

276

4A). PL as membrane lipids are crucial to the cell functions, and the content of PL is

277

strictly regulated by cells to maintain the membrane functions. TLC results clearly

278

showed that PC was the most abundant component among PL during the cell growth

279

(Figure 4B). Thus, PC profile was further analyzed by LC-MS. Twenty-two species of

280

PC were identified, with PC-42:11 (PC-20:5/22:6) and PC-44:12 (PC-22:6/22:6) as the

281

two major species, which contributed to half amount of the total PC (Figure 4C). This

282

demonstrated that both DHA and EPA were dominant in PC. Compared to the TAG

283

profile, we concluded that DHA was accumulated in both TAG and PC, while EPA was

284

mainly deposited in PC. This was also consistent with our above result that the

285

proportion of EPA slightly increased in TFAs but decreased in TAG.

286

Although the amount of total PL was stable, DHA distributed in PC dropped obviously


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as the cells grew, especially, during the lipid accumulation stage (24-48 h). The

288

percentage of DHA in PC dropped from 69.2% to 59.1% (Figure 4D). Combined with

289

the results from TFA and TAG profiles, DHA might be trafficked from PL to TAG,

290

especially during the stage of lipid accumulation in Schizochytrium sp. A-2. In most

291

oleaginous microbes, acyl-CoA will be directly incorporated into glycerol backbone to

292

form TAG via Kennedy pathway.22 However, in Thraustochytrium, DHA is first and

293

largely accumulated in PLs, meanwhile SFAs are more efficiently incorporated into

294

TAG.7 This suggests the existence of an efficient mechanism for DHA migration from

295

PL to TAG although uncharacterized in both Thraustochytrium and Schizochytrium.

296

In addition to neutral lipids and phospholipids, glycolipids are the third major lipid

297

existing in Schizochytrium (Figure 4A). Glycolipids were found to comprise three

298

major classes, monogalactosyldiacyl glycerol (MGDG), digalactosyl diacylglycerol

299

(DGDG) and sulfoquinovosyl diacylglycerol (SQDG) in our strain. However, DHA

300

was not detected in any glycolipid species throughout the cell growth according to LC-

301

MS result (data not shown). Therefore, we did not further analyze the glycolipid profile

302

in this test.

303

In conclusion, the total amount of PL was stable during the cell growth, while the

304

percentage of DHA in PL decreased. On the other hand, both the total amount of TAG

305

and the percentage of DHA in TAG increased during the cell growth. These suggested

306

that DHA might be channeled from PL to TAG via unknown mechanism.

307 308

Analysis of differentially expressed genes involved in the synthesis of DHA in



309

Schizochytrium

310

RNA-seq was used to analyze the differentially expressed genes (DEGs) related to

311

DHA synthesis and migration during the growth of Schizochytrium. Although the

312

genome sequence of strain A-2 was obtained previously (unpublished data), the

313

sequence quality was not good enough. Therefore, we carried out de novo assembly of

314

data from RNA-seq. A total of 17,140 unigenes were obtained, and 13,095 coding

315

sequences were predicted with 58% of average GC content according to online database

316

(Table S2). Among them, 759 genes were hypothesized to participate in carbohydrate

317

metabolism and 298 genes were related to lipid-related metabolism (Figure S1).

318

Normalized FPKM were calculated as described in Materials and Method section for

319

each annotated gene. From an overview of the metabolic changes that occurred during

320

different cell growth phases, a total of 1557 (24 h vs. 8 h) or 4028 (48 h vs. 24 h) genes

321

with obvious different expression levels (log2 value≥1 and p value≤0.001) were

322

identified (Figure S2). These DEGs were annotated against KEGG pathway

323

(https://www.genome.jp/kegg/pathway.html), and the majority of DEGs was classified

324

into the category of metabolism (Figure S3). To verify the transcriptomic result, seven

325

genes associated with fatty acid biosynthesis or modification were further analyzed by

326

qRT-PCR. The results confirmed that the data from transcriptomic sequencing and

327

qRT-PCR were consistent and reliable (Figure S4).

328

Both acetyl-CoA as substrate and NADPH as reduced power are essential for DHA

329

synthesis and accumulation. Thus, we first investigated DEGs involved in central

330

carbon metabolism including glycolysis, pentose phosphate pathway (PPP) and


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331

tricarboxylic acid cycle (TCA cycle), which are mainly responsible for the generation

332

of acetyl-CoA and NADPH (Figure 5). Citrate synthase (CS) is responsible for the

333

condensation of acetyl-CoA and oxaloacetate to generate citrate. The high expression

334

of CS gene was observed throughout the fermentation cycle in this strain (Table 2) and

335

also in reported strain Schizochytrium sp. HX-308.23 This indicated the important roles

336

of CS involved in the TCA cycle for cellular respiration. In addition, the observed

337

upregulation of CS and the low expression levels of isocitrate dehydrogenase (ICDH)

338

encoding gene (Table 2 and Figure 5) might lead to the accumulation of citrate in

339

mitochondrial and the subsequent transportation of citrate into the cytosol. Then

340

cytosolic citrate would be split into acetyl-CoA by the action of ATP:citrate lyase.

341

Two genes encoding malic enzymes (ME1 and ME2) that involved in generating

342

NADPH were all upregulated during the fermentation cycle, but only ME2 was highly

343

expressed (Table 2 and Figure 5). In addition, the gene for malate dehydrogenase (MDH)

344

which catalyzed the formation of malate was expressed at very high levels during

345

fermentation (Table 2), suggesting sufficient malate was produced and it was probably

346

catalyzed by ME1/2 subsequently for the generation of NADPH. In contrast, the genes

347

for

348

phosphogluconate dehydrogenase (6GPD) involved in PPP for generation of NADPH

349

were first upregulated at 24 h by 2.9- and 1.3-fold, then downregulated at 48 h by 2.9

350

and 1.7-fold (Figure 5). Such decrease at the lipid accumulation stage might suggest

351

G6PDH and 6GPD were not responsible for the generation of lipogenic NADPH. Taken

352

together, ME1 and ME2, especially ME2, but not G6PDH/6GPD probably provided

the

enzymes

glucose-6-phosphate

dehydrogenase


(G6PDH)

and

6-


353

lipogenic NDAPH in Schizochytrium sp. A-2. It was reported that all genes participated

354

in PPP were down-regulated when glycerol was used as the carbon resource and the

355

DHA proportion was elevated in Schizochytrium sp. S056.24 Although the authors did

356

not make a conclusion in the discussion section, it clearly showed that PPP was not the

357

major pathway for providing lipogenic NDAPH. Similar conclusions were conducted

358

in oleaginous fungi Mucor circinelloides and Mortierella alpina.25-26 However, in the

359

case of oleaginous Yarrowia lipolytica, G6PDH and 6GPD involved in PPP instead of

360

ME were demonstrated to contribute to the generation of lipogenic NADPH.27

361

Next, DEGs involved in the fatty acid synthetic pathways were analyzed. Acetyl-CoA

362

carboxylase (ACC) and malonyl-CoA:ACP transacylase (MAT) catalyze the formation

363

of malonyl-ACP, which is the substrate for two competing pathways, FAS and PKS

364

pathways in Schizochytrium. ACC encoding gene was highly expressed during the

365

fermentation process, whereas MAT1 and MAT2 genes did not (Table 3 and Figure 5),

366

suggesting MAT activity might be a limiting factor to the formation of sufficient

367

malonyl-ACP under the tested conditions. Overexpression of a MAT encoding gene in

368

Schizochytrium sp. MYA1381 resulted in the increased yields of total lipids and DHA,28

369

confirming that the increase of MAT activity was an effective strategy for improvement

370

of lipid and DHA production. PKS pathway is the only route for the synthesis of DHA

371

in Thraustochytrium and Schizochytrium.7-8 As expected, three genes encoding for PfsA,

372

PfsB and PfsC involved in PKS were all highly expressed and upregulated during

373

fermentation (Table 3). The genes for multi-domain FAS2 protein involved in FAS

374

pathway was slightly downregulated at cell growth stage (24 h) and then obviously


Page 18 of 41

Page 19 of 41


375

upregulated at lipid accumulation stage (48 h) (Table 3). However, the percentage of

376

all FAS-generated fatty acids generally dropped throughout the fermentation regardless

377

of their distributions in TFA or TAG (Table 1 and Figure 2B). This might be explained

378

by that the absolute concentration of SFAs was increased although the percentage of

379

SFAs decreased (Table 1). The other possible explanation is that the very low

380

expression levels of the gene encoding acyl-ACP thioesterase (TE) (Table 3 and Figure

381

5), which might restrict the release of FFAs from acyl-ACP pool.

382

Although the desaturase/elongase pathway is demonstrated to be inactive and irrelevant

383

to DHA formation,7-8,

384

Schizochytrium sp. A-2 (Table 3). Like the genomic analysis result from

385

Thraustochytrium sp. 26185,29 no Δ9 desaturase sequence was detected in

386

Schizochytrium sp. A-2. We predicted that other desaturase(s) might possess Δ9

387

desaturase activity and catalyze the double bond formation at delta-9 position of C16:0

388

or C18:0 since both C16:1 and C18:1 were found in this strain (Table 1). We also

389

noticed that the gene encoding for a putative Δ12 desaturase was highly upregulated at

390

lipid accumulation stage with FPKM value of 628.4 (Table 3). Such observation was

391

also applied to a Δ12 desaturase gene from Schizochytrium sp. HX-308.23 However, the

392

occurrence of C18:2 was negligible in Schizochytrium. It is reported that Δ12 desaturase

393

activity is also missing in Schizochytrium sp. ATCC 20888.9 Here, the BLAST result

394

showed that Δ12 desaturase from the strain A-2 had a long Q-rich linker at the N-

395

terminus (Table S3), which was absent in other active Δ12 desaturases. Based on above

396

information, we predicted that the Δ12 desaturase might be inactive in the strain A-2

29

various desaturases and elongases were found in



397

although the expression levels were high during the cell growth. The function of this

398

predicted Δ12 desaturase gene awaited further investigation.

399 400

Analysis of differentially expressed genes involved in DHA migration

401

DHA is released from PKS pathway in the form of FFA,10 and subsequently activated

402

by the action of acyl-CoA synthase (ACS) to form DHA-CoA, which is later channeled

403

into PL followed by TAG.7, 21 However, the molecular mechanism on how DHA is

404

channeled into PL or TAG pool is undetermined. In this test, DHA was also presumed

405

to be channeled from PL to TAG, especially during the lipid accumulation stage,

406

according to the above analysis of lipid profile in Schizochytrium sp. A-2. Therefore,

407

the catalytic activity and substrate specificity of long chain acyl-CoA synthase (LACS)

408

were crucial for DHA migration. There were 10 acyl-CoA synthase (ACS) encoding

409

genes uncovered in strain A-2, three of them were predicted to encode for long-chain

410

fatty acid specific enzymes, designating as LACS, LACS6 and LACS7. Only LACS

411

gene was highly upregulated at 48 h with FPKM elevated from 63.3 at 24 h to 259.5 at

412

48 h, whereas the expression levels of LACS6 and LACS7 were not obviously changed

413

and remained at low levels (Table 3). This suggested LACS might participate in the

414

transferring DHA-FFA and other VLCPUFAs into acyl-CoA pool. In addition to LACS,

415

Lysophosphatidylcholine acyltransferase (LPCAT) plays central roles in acyl editing

416

between acyl-CoA and PC pools, which is important to the incorporation of DHA into

417

PC.30 Although LPCAT is demonstrated to possess both acylation and de-acylation

418

abilities, forward acylation of lysophosphatidylcholine (LPC) to form PC by using acyl-


Page 20 of 41

Page 21 of 41


419

CoA is the dominant reaction in vivo.30 Three LPCAT genes were identified and only

420

LPCAT-1 was substantially upregulated (10.3-fold upregulation) at 48 h (Table 3),

421

suggesting LPCAT-1 might selectively catalyze the conversion of DHA between CoA

422

and PC pools. We also noticed that none of the genes involved in Kennedy pathway

423

including genes for glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic

424

acid acyltransferase (LPAT), phosphatidic acid phosphatase (PAP) and diacylglycerol

425

acyltransferase (DGAT) were obviously upregulated at 24 h (Figure 5). DGAT2-3 was

426

the only gene highly expressed during the cell growth (Table 3), which was probably a

427

major contributor to the synthesis of TAG in strain A-2. Although GPAT1-1, GPAT1-

428

2 and LPAT-3 were all upregulated during lipid accumulation stage (Figure 5), their

429

transcriptional levels were low (Table 3). Therefore, the de novo DAG synthesis via

430

Kennedy pathway was unlikely the major contributor for the incorporation of DHA into

431

TAG.

432

There are two possible routes for DHA traffics from PL to TAG. The first route is that

433

DHA

434

Phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) or reverse

435

activity of choline phosphotransferase (CPT), then DHA-DAG is converted to DHA-

436

TAG by DGAT. However, no PDCT was identified in strain A-2 or in

437

Thraustochytrium.29 CPT encoding gene was indeed found although the expression

438

levels did not change during the fermentation (Table 3) and the reverse activity of CPT

439

was never confirmed in vivo in any species. Therefore, both PDCT and CPT may not

440

participate in the DHA migration. Phospholipase C (PLC) is defined as

enters

into

the

DAG

flux

mediated


by

the

action

of


that

cleaves

the

proximal

441

phosphodiesterase

442

glycerophospholipid to produce DAG and phosphorylated headgroups.31 Therefore,

443

PLC may also participate in DHA migration from PL to DAG. Two predicted PLC

444

genes were found and PLC1 was slightly upregulated at 48 h (Figure 5). Three PI-PLC

445

encoding genes were also uncovered (Table 3), but they probably do not contribute to

446

the DHA migration since their exclusive activities on phosphatidylinositol (PI).

447

Alternatively, phospholipid:diacylglycerol acyltransferase (PDAT) may redirect DHA

448

from PL to TAG via acyl-CoA independent pathway. PDAT prefers to catalyze the

449

transferring the sn-2 fatty acid of PC to the sn-3 position of DAG to produce TAG.32

450

PDAT possesses broad substrate activities towards various PLs including PC,

451

phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylserine (PS) and

452

PI.33 The acyltransferase activity of PDAT has been well illustrated in yeast, algae and

453

plant.33-34 In addition to PDAT’s catalytic activity, many of them are described to

454

possess specific activity on transferring unusual fatty acids from PL to TAG.35-36

455

Nevertheless, no PDAT has been reported in DHA-producing strains including

456

Schizochytrium and Thraustochytrium. Here we first uncovered one predicted PDAT

457

encoding sequence. Although the relatively low expression levels during the cell

458

growth was observed, PDAT had an increased expression level at 48 h. Furthermore,

459

TAG-22:6/22:6/22:6 was observed as the most abundant TAG species in our strain A-

460

2, indicating active enzyme(s) may catalyze the channeling of DHA into the sn-3

461

positions in TAGs. All above information suggested that PDAT might be an active

462

enzyme involved in channeling fatty acids from PL to TAG. To further confirm above


phosphodiester

Page 22 of 41

bond

of

Page 23 of 41


463

hypothesis, in vitro investigating enzyme activity and substrate specificity of PDAT,

464

LPCAT-1 and LACS should be carried out in near future.

465 466

Acknowledgment

467

We would like to acknowledge Xue-Rong Zhou from CSIRO Agriculture & Food, for

468

his critical reading of this manuscript.

469 470

Supporting Information

471

Primer sequences for qRT-PCR; the quality index of unigene and coding sequence; the

472

coding sequences of genes listed in Table 2 and 3; the distribution of functional genes

473

based on KEGG database; the statistics of differentially expressed genes; classification

474

of differentially expressed genes based on KEGG pathway; quantification of the

475

expressions levels of PfsA, PfsB, PfsC, FAS2, LACS, FACS1 and TE encoding genes

476

by qRT-PCR.

477 478

Author Contributions

479

XY, WC, SL and XW conceived this project. XY, PL and CL carried out experimental

480

work. XY, ZW, XL, FH and XW analyzed and interpreted the data. XY and XW wrote

481

the paper, all authors assisted in the process.

482 483

Funding

484

This work was financially supported by Chinese Academy of Agricultural Sciences



485

(CAAS-ASTIP-2013-OCRI), “3551” Optics Valley Talent Schema (K159) and

486

Ministry of Science and Technology of the People’s Republic of China

487

(2016YFD0501209).

488 489

Competing Interest

490

The authors declare that they have no competing interests.


Page 24 of 41

Page 25 of 41


491

References

492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533

1.

Weiser, M. J.; Butt, C. M.; Mohajeri, M. H., Docosahexaenoic Acid and Cognition throughout the

Lifespan. Nutrients 2016, 8 (2), 99. 2.

Janssen, C. I.; Kiliaan, A. J., Long-chain polyunsaturated fatty acids (LCPUFA) from genesis to

senescence: the influence of LCPUFA on neural development, aging, and neurodegeneration. Prog Lipid Res 2014, 53, 1-17. 3.

Sun, X. M.; Ren, L. J.; Bi, Z. Q.; Ji, X. J.; Zhao, Q. Y.; Jiang, L.; Huang, H., Development of a cooperative

two-factor adaptive-evolution method to enhance lipid production and prevent lipid peroxidation in Schizochytrium sp. Biotech Biofuels 2018, 11, 65. 4.

Zhao, B.; Li, Y.; Li, C.; Yang, H.; Wang, W., Enhancement of Schizochytrium DHA synthesis by plasma

mutagenesis aided with malonic acid and zeocin screening. Appl Microbiol Biotechnol 2018, 102 (5), 2351-2361. 5.

Wallis, J. G.; Watts, J. L.; Browse, J., Polyunsaturated fatty acid synthesis: what will they think of

next? Trends Biochem Sci 2002, 27 (9), 467. 6.

Metz, J. G.; Roessler, P.; Facciotti, D.; Levering, C.; Dittrich, F.; Lassner, M.; Valentine, R.; Lardizabal,

K.; Domergue, F.; Yamada, A.; Yazawa, K.; Knauf, V.; Browse, J., Production of polyunsaturated fatty acids by polyketide synthases in both prokaryotes and eukaryotes. Science 2001, 293 (5528), 290-3. 7.

Zhao, X.; Qiu, X., Analysis of the biosynthetic process of fatty acids in Thraustochytrium. Biochimie

2018, 144, 108-114. 8.

Meesapyodsuk, D.; Qiu, X., Biosynthetic mechanism of very long chain polyunsaturated fatty acids

in Thraustochytrium sp. 26185. J Lipid Res 2016, 57 (10), 1854-1864. 9.

Lippmeier, J. C.; Crawford, K. S.; Owen, C. B.; Rivas, A. A.; Metz, J. G.; Apt, K. E., Characterization of

both polyunsaturated fatty acid biosynthetic pathways in Schizochytrium sp. Lipids 2009, 44 (7), 62130. 10. Metz, J. G.; Kuner, J.; Rosenzweig, B.; Lippmeier, J. C.; Roessler, P.; Zirkle, R., Biochemical characterization of polyunsaturated fatty acid synthesis in Schizochytrium: release of the products as free fatty acids. Plant physiol Bochem 2009, 47 (6), 472-8. 11. Fan, K. W.; Jiang, Y.; Faan, Y. W.; Chen, F., Lipid characterization of mangrove thraustochytrid-Schizochytrium mangrovei. J Agi Food Chem 2007, 55 (8), 2906-10. 12. Miller, G. L., Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Anall chem 1959, 31 (3), 426-428. 13. Xiao, K.; Yue, X. H.; Chen, W. C.; Zhou, X. R.; Wang, L.; Xu, L.; Huang, F. H.; Wan, X., Metabolic Engineering for Enhanced Medium Chain Omega Hydroxy Fatty Acid Production in Escherichia coli. Front Microbiol 2018, 9, 139. 14. Langmead, B.; Salzberg, S. L., Fast gapped-read alignment with Bowtie 2. Nat Methods 2012, 9 (4), 357-9. 15. Li, B.; Dewey, C., Li B, Dewey CN. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12:323. 2011; Vol. 12, p 323. 16. Cock, P. J. A.; Fields, C. J.; Goto, N.; Heuer, M. L.; Rice, P. M., The Sanger FASTQ file format for sequences with quality scores, and the Solexa/Illumina FASTQ variants. Nucleic Acids Res 2010, 38 (6), 1767-1771. 17. Nakahara, T.; Yokochi, T.; Higashihara, T.; Tanaka, S.; Yaguchi, T.; Honda, D., Production of docosahexaenoic and docosapentaenoic acids by Schizochytrium sp. isolated from Yap Islands. J Am Oil



534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577

Chem Soc 1996, 73 (11), 1421-1426. 18. Ren, L. J.; Zhuang, X. Y.; Chen, S. L.; Ji, X. J.; Huang, H., Introduction of omega-3 Desaturase Obviously Changed the Fatty Acid Profile and Sterol Content of Schizochytrium sp. J Agri Food chem 2015, 63 (44), 9770-6. 19. Shrestha, P.; Zhou, X.-R.; Vibhakaran Pillai, S.; Petrie, J.; de Feyter, R.; Singh, S., Comparison of the Substrate Preferences of ω3 Fatty Acid Desaturases for Long Chain Polyunsaturated Fatty Acids. Int J Mo Sci 2019, 20 (12), 3058. 20. Zhang, K.; Li, H.; Chen, W.; Zhao, M.; Cui, H.; Min, Q.; Wang, H.; Chen, S.; Li, D., Regulation of the Docosapentaenoic Acid/Docosahexaenoic Acid Ratio (DPA/DHA Ratio) in Schizochytrium limacinum B4D1. Appl Biochem Biotech 2017, 182 (1), 67-81. 21. Zhao, X.; Qiu, X., Very Long Chain Polyunsaturated Fatty Acids Accumulated in Triacylglycerol Are Channeled From Phosphatidylcholine in Thraustochytrium. Front Microbiology 2019, 10, 645. 22. Ledesma-Amaro, R.; Nicaud, J. M., Yarrowia lipolytica as a biotechnological chassis to produce usual and unusual fatty acids. Prog Lipid Res 2016, 61, 40-50. 23. Bi, Z. Q.; Ren, L. J.; Hu, X. C.; Sun, X. M.; Zhu, S. Y.; Ji, X. J.; Huang, H., Transcriptome and gene expression analysis of docosahexaenoic acid producer Schizochytrium sp. under different oxygen supply conditions. Biotechnol Biofuels 2018, 11, 249. 24. Chen, W.; Zhou, P.; Zhang, M.; Zhu, Y.; Wang, X.; Luo, X.; Bao, Z.; Yu, L., Transcriptome analysis reveals that up-regulation of the fatty acid synthase gene promotes the accumulation of docosahexaenoic acid in Schizochytrium sp. S056 when glycerol is used. Algal Res 2016, 15, 83-92. 25. Zhang, Y.; Adams, I. P.; Ratledge, C., Malic enzyme: the controlling activity for lipid production? Overexpression of malic enzyme in Mucor circinelloides leads to a 2.5-fold increase in lipid accumulation. Microbiology 2007, 153 (Pt 7), 2013-25. 26. Wynn, J. P.; bin Abdul Hamid, A.; Ratledge, C., The role of malic enzyme in the regulation of lipid accumulation in filamentous fungi. Microbiology 1999, 145 ( Pt 8), 1911-7. 27. Wasylenko, T. M.; Ahn, W. S.; Stephanopoulos, G., The oxidative pentose phosphate pathway is the primary source of NADPH for lipid overproduction from glucose in Yarrowia lipolytica. Metab Eng 2015, 30, 27-39. 28. Li, Z.; Meng, T.; Ling, X.; Li, J.; Zheng, C.; Shi, Y.; Chen, Z.; Li, Z.; Li, Q.; Lu, Y.; He, N., Overexpression of Malonyl-CoA: ACP Transacylase in Schizochytrium sp. to Improve Polyunsaturated Fatty Acid Production. J Agri Food Chem 2018, 66 (21), 5382-5391. 29. Zhao, X.; Dauenpen, M.; Qu, C.; Qiu, X., Genomic Analysis of Genes Involved in the Biosynthesis of Very Long Chain Polyunsaturated Fatty Acids in Thraustochytrium sp. 26185. Lipids 2016, 51 (9), 106575. 30. Lager, I.; Yilmaz, J. L.; Zhou, X. R.; Jasieniecka, K.; Kazachkov, M.; Wang, P.; Zou, J.; Weselake, R.; Smith, M. A.; Bayon, S.; Dyer, J. M.; Shockey, J. M.; Heinz, E.; Green, A.; Banas, A.; Stymne, S., Plant acylCoA:lysophosphatidylcholine acyltransferases (LPCATs) have different specificities in their forward and reverse reactions. J Biol Chem 2013, 288 (52), 36902-14. 31. Lyu, Y.; Ye, L.; Xu, J.; Yang, X.; Chen, W.; Yu, H., Recent research progress with phospholipase C from Bacillus cereus. Biotechnol Lett 2016, 38 (1), 23-31. 32. Stahl, U.; Carlsson, A. S.; Lenman, M.; Dahlqvist, A.; Huang, B.; Banas, W.; Banas, A.; Stymne, S., Cloning and functional characterization of a phospholipid:diacylglycerol acyltransferase from Arabidopsis. Plant physiology 2004, 135 (3), 1324-35. 33. Yoon, K.; Han, D.; Li, Y.; Sommerfeld, M.; Hu, Q., Phospholipid:diacylglycerol acyltransferase is a


Page 26 of 41

Page 27 of 41

578 579 580 581 582 583 584 585 586 587 588 589


multifunctional enzyme involved in membrane lipid turnover and degradation while synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii. Plant cell 2012, 24 (9), 3708-24. 34. Dahlqvist, A.; Stahl, U.; Lenman, M.; Banas, A.; Lee, M.; Sandager, L.; Ronne, H.; Stymne, S., Phospholipid:diacylglycerol acyltransferase: an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants. Proc Nat Acad Sci U S A 2000, 97 (12), 6487-92. 35. van Erp, H.; Bates, P. D.; Burgal, J.; Shockey, J.; Browse, J., Castor phospholipid:diacylglycerol acyltransferase facilitates efficient metabolism of hydroxy fatty acids in transgenic Arabidopsis. Plant physiol 2011, 155 (2), 683-93. 36. Marmon, S.; Sturtevant, D.; Herrfurth, C.; Chapman, K.; Stymne, S.; Feussner, I., Two Acyltransferases Contribute Differently to Linolenic Acid Levels in Seed Oil. Plant physiol 2017, 173 (4), 2081-2095.

590



Figure legends Figure 1 Growth curve (A) and DHA content (B) of Schizochytrium sp. A-2. Cells were cultured in the artificial sea salt medium at 28°C with shaking at 220 rpm for 72 h.

Figure 2 Neutral lipid profile (A) and fatty acid composition of TAG (B) in Schizochytrium sp. A-2 during the cell growth. TAG: Triacylglycerol；FFA: Free fatty acid；DAG: Diacylglycerol.

Figure 3 Analysis of TAG profile in Schizochytrium sp. A-2 during the cell growth. (A) A time-course TAG profile shown in pie charts. TAGs with percentage greater than 1% were shown and TAGs greater than 5% were marked. (B) Heat map of TAG species. (C) Heat map of differential TAG species.

Figure 4 Analysis of total lipid profile (A), PL profile (B), PC profile (C) and DHAenriched PC profile (D) in Schizochytrium sp. A-2 during the cell growth. PL: phospholipid, PC: phosphatidylcholine.

Figure 5 Changes in transcript abundance of genes involved in DHA synthesis, migration and accumulation in Schizochytrium sp. A-2. Cells were cultured in medium at 28°C with shaking at 220 rpm for 8, 24 or 48 h. HXY: Hexokinase; G6PDH: Glucose6-phosphate dehydrogenase; 6GPD: 6-phosphogluconate dehydrogenase; PFK: phosphofructokinase; FBA: Fructose-bisphosphate aldolase; PYK: Pyruvate kinase;


Page 28 of 41

Page 29 of 41


PYC: Pyruvate carboxylase; ME: malic enzyme; MDH: Malate dehydrogenase; ACL: ATP citrate lyase; PDH: Pyruvate dehydrogenase; CS: Citrate synthase; ICDH: Isocitrate dehydrogenase; PPP: pentose pathway; TCA Cycle: tricarboxylic acid cycle; NADPH: Nicotinamide adenine dinucleotide phosphate ； ACC: Acetyl-coA carboxylase; MAT: Malonyl-CoA: ACP transacylase; FAS: type I fatty acid synthase; PfsA: Polyunsaturated fatty acid synthase subunit A; PfsB: Polyunsaturated fatty acid synthase subunit B; PfsC: Polyunsaturated fatty acid synthase subunit C; Ppt: Phosphopantetheinyl transferase ； LACS: long chain Acyl-CoA synthase; ACS: Acyl-CoA synthase; LPCAT: lysophosphatidylcholine acyltransferase ； Glycerol-3-phosphate

acyltransferase;

LPAT:

GPAT:

1-acyl-sn-glycerol-3-phosphate

acyltransferase; PAP: Phosphatidic acid phosphatase; DGAT: Diacylglycerol acyltransferase;

CPT:

Diacylglycerol

cholinephosphotransferase;

PDCT:

Phosphatidylcholine: diacylglycerol cholinephosphotransferase; PDAT: Phospholipid: diacylglycerol acyltransferase; TAG: Triacylglycerol; CDS: CDP-DAG synthase; PSS: Phosphatidylserine ; PS: Phosphatidylserine; PSD: Phosphatidylserine decarboxylase; PE: Phosphatidylethanolamine; PEMT: Phosphatidylethanolamine methyltransferase; LPE:

Lysophosphatidylethanolamine;

LPEAT:

Lyso-Phosphatidylethanolamine

acyltransferase.



Page 30 of 41

Table 1 Fatty acid composition of total fatty acids in Schizochytrium sp. A-2 Time (h) SFAs (%) C14:0 C15:0 C16:0 C17:0 C18:0 MUFAs (%) C16:1 C18:1 PUFAs (%) ARA EPA DPAn6 DHA Others (%) FAs from FAS (%) FAs from PKS (%) FAs from FAS (g/L) FAs from PKS (g/L) DHA/DPAn6 ratio

12

24

36

48

60

72

2.6±0.1 2.1±0.0 21.6±0.7 0.9±0.0 0.4±0.0

2.8±0.3 1.5±0.0 22.3±1.5 0.6±0.0 0.8±0.0

2.6±0.1 1.5±0.0 17.9±0.2 0.4±0.0 0.7±0.0

2.1±0.3 1.1±0.0 14.9±0.7 0.3±0.1 0.6±0.0

1.0±0.1 0.5±0.0 11.2±0.3 0.3±0.0 0.4±0.1

1.2±0.1 0.6±0.0 11.8±0.3 0.1±0.0 0.5±0.0

1.2±0.2 2.2±0.2

0.7±0.0 0.5±0.1

0.5±0.0 0.0±0.0

0.4±0.0 0.1±0.1

0.3±0.1 0.0±0.0

0.3±0.2 0.0±0.0

0.0±0.0 2.7±0.1 13.8±0.1 50.6±0.7 1.9±0.0 31.0±0.9 67.2±0.9 1.1±0.0 2.4±0.0 3.7

0.8±0.0 1.5±0.1 16.8±0.8 49.2±1.1 2.7±0.1 29.2±1.9 68.2±1.9 4.3±0.3 10.0±0.3 2.9

0.7±0.0 1.1±0.0 20.1±0.4 51.8±0.0 2.8±0.0 23.6±0.3 73.6±0.3 6.2±0.1 19.2±0.1 2.6

1.1±0.1 2.0±0.2 21.7±0.2 53.2±0.3 2.6±0.1 19.4±0.9 78.0±0.8 6.3±0.3 25.5±0.3 2.4

2.2±0.1 4.9±0.4 22.7±0.2 53.8±0.3 2.5±0.1 13.8±0.2 83.7±0.1 4.2±0.1 25.7±0.0 2.4

2.2±0.2 4.6±0.3 22.8±0.2 53.4±0.1 2.5±0.1 14.6±0.5 82.9±0.4 3.9±0.1 22.2±0.1 2.3

FA: fatty acid; SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; PUFA: polyunsaturated fatty acid. The data was indicated as mean percentage of total peak area ± SEM. The quantification of each fatty acid from samples was repeated in triplicate (n = 3).


Page 31 of 41


Table 2 Differentially expressed genes involved in the generation of acetyl-CoA and NADPH for DHA synthesis. Pathway

Protein name

PPP

G6PDH 6GPD

EMP

PFK

Transhyd -rogenase cycle

FBA PYK1 PYK2 PYK3 ME1 ME2 MDH

TCA

PDH CS ICDH

Protein description

Gene ID

Accession number of the top hit

Glucose-6-phosphate 1Dehydrogenase 6-phosphogluconate dehydrogenase ATP-dependent 6Phosphofructokinase Fructose-bisphosphate aldolase Pyruvate kinase Pyruvate kinase Pyruvate kinase NADP-dependent malic enzyme NADP-dependent malic enzyme Malate dehydrogenase

Unigene462_All

Pyruvate dehydrogenase acetylTransferring kinase Citrate synthase, mitochondrial Isocitrate dehydrogenase NADP

FPKM

Fold change

8h

24 h

48 h

24 h vs 8 h

48 h vs 24 h

GBG29497.1

61.9

180.0

62.1

2.9

-2.9

CL702.Contig1_All

GBG26375.1

234.3

312.8

186.2

1.3

-1.7

Unigene10716_All

GBG29878.1

58.4

98.8

63.2

1.7

-1.6

Unigene3041_All Unigene10866_All Unigene10631_All Unigene6809_All Unigene10526_All CL668.Contig1_All CL728.Contig2_All

GBG34196.1 GBG25431.1 GBG27442.1 GBG27457.1 GBG30842.1 GBG27767.1 XP_013759779.1

1592.4 351.4 38.0 25.4 21.2 101.3 2053.5

2190.9 366.9 55.4 26.6 38.3 154.0 2065.5

1067.5 417.8 44.6 30.7 46.9 306.3 1915.0

1.4 1.0 1.5 1.0 1.8 1.5 1.0

-2.1 1.1 -1.2 1.2 1.2 2.0 -1.1

Unigene3086_All

GBG34078.1

105.9

103.9

72.3

1.0

-1.4

Unigene1997_All CL651.Contig2_All

GBG28992.1 GBG29242.1

516.2 85.6

527.4 87.7

761.8 93.4

1.0 1.0

1.4 1.1



Page 32 of 41

Table 3 Differentially expressed genes related to the DHA synthesis and migration Category

Protein name

Gene description

Gene ID

Accession number of the top hit

Unigene10771_All

FPKM

Fold change

8h

24 h

48 h

24 h vs 8 h

48 h vs 24 h

AHK10581.1

44.4

49.4

36.4

1.1

-1.4

Unigene8585_All

GBG23800.1

18.9

22.0

13.1

1.2

-1.7

ACC FAS2 TE

Malonyl-CoA acyl carrier protein transacylase Malonyl-CoA acyl carrier protein transacylase Acetyl-CoA carboxylase Type I fatty acid synthase (FAS2) Acyl-protein thioesterase 1

CL470.Contig2_All Unigene9070_All Unigene3159_All

GBG29850.1 ABJ98780.1 GBG25039.1

379.6 112.4 82.4

434.6 90.8 78.0

653.2 359.0 58.7

1.1 -1.2 -1.1

1.5 4.0 -1.3

PKS

PfsA PfsB PfsC PPT

Polyunsaturated fatty acid synthase subunit A Polyunsaturated fatty acid synthase subunit B Polyunsaturated fatty acid synthase subunit C Phosphopantetheinyl transferase

CL953.Contig1_All Unigene3873_All CL891.Contig2_All Unigene1616_All

AF378327.2 AAK72880.2 AF378329.2 AOG21007.1

334.9 126.0 253.8 12.6

514.6 160.3 319.7 13

687.8 182.8 582.9 9.8

1.5 1.3 1.3 1.0

1.3 1.1 1.8 -1.3

Desaturase

Des8 Des6 Des12

Delta-8 fatty acid desaturase Delta-6 fatty acid desaturase Delta-12 fatty acid desaturase

Unigene4018_All Unigene9756_All Unigene1056_All

GBG29703.1 GBG26997.1 GBG24140.1

52.0 10.4 142.5

39.7 13.0 64.3

70.4 2.0 628.4

-1.3 1.2 -2.2

1.8 -6.4 9.8

LACS6 LACS7 LACS ACSBG2 FACS-1 FACS-2 SACS

Long chain acyl-CoA ligase 6 Long chain acyl-CoA synthetase 7 Long chain acyl-CoA synthetase Long chain fatty acid CoA ligase ACSBG2 Fatty acyl-CoA synthase Fatty acyl-CoA synthase Short chain fatty acid CoA ligase

Unigene8245_All Unigene2922_All CL13.Contig4_All Unigene6223_All CL683.Contig1_All CL683.Contig2_All Unigene4066_All

GBG25108.1 EWM20588.1 CBJ33608.1 OLP88930.1 SMF20327.1 SMF20327.1 GBG34799.1

74.9 67.9 93.1 13.7 350.0 12.4 20.6

10.7 64.3 63.3 4.3 340.3 23.0 21.0

24.3 66.2 259.5 58.4 278.3 4.2 32.7

-7.0 -1.1 -1.5 -3.2 1.0 1.9 1.0

2.3 1.0 4.1 13.7 -1.2 -5.5 1.6

FAS

MAT-1 MAT-2

Acyl-CoA activation


Page 33 of 41


TAG Synthesis

ACS1 ACS2

Acyl-CoA synthetase, putative Acyl-CoA synthetase, putative

CL829.Contig3_All Unigene7252_All

GBG32653.1 GBG32654.1

9.3 19.1

16.1 31.2

29.4 44.0

1.7 1.6

1.8 1.4

GPAT3 GPAT1-1

Glycerol-3-phosphate acyltransferase 3 Glycerol-3-phosphate O-acyltransferase 1

Unigene3820_All Unigene249_All

GBG34725.1 GBG25294.1

25.1 17.7

31.3 9.5

30.0 32.2

1.2 -1.9

1.0 3.4

GPAT1-2 GPAT1-3 GPAT1-4

Glycerol-3-phosphate O-acyltransferase 1 Glycerol-3-phosphate acyltransferase 1 Glycerol-3-phosphate O-acyltransferase 1 1-acyl-sn-glycerol-3-phosphate acyltransferase 1 1-acyl-sn-glycerol-3-phosphate acyltransferase 3 1-acyl-sn-glycerol-3-phosphate acyltransferase 1-acyl-sn-glycerol-3-phosphate acyltransferase 1-acyl-sn-glycerol-3-phosphate acyltransferase Phosphatidic acid phosphatase type 2

Unigene3587_All Unigene6915_All Unigene387_All

GBG23921.1 GBG28391.1 GBG31449.1 XP_0042425 91.1

6.8 16.7 28.0

6.9 13.9 28.9

27.3 16.1 21.1

1.0 -1.2 1.0

4.0 1.2 1.4

6.3

6.7

4.1

1.1

-1.6

CL432.Contig2_All

GBG31411.1

127.6

71.9

131.2

-1.8

1.8

Unigene1916_All

GBG26176.1

3.7

4.0

7.0

1.1

1.7

Unigene10540_All

GBG34458.1

2.9

4.6

10.1

1.6

2.2

CL432.Contig1_All

GBG34458.1

0.5

0.4

2.4

-1.4

7.0

Unigene2007_All

RKP14015.1

23.1

22.3

27.3

1.0

1.2

Phospholipid phosphatase 2

Unigene7147_All

GBG34077.1

47.5

20.8

37.3

-2.3

1.8

Phospholipid phosphatase 4

Unigene704_All

GBG25135.1

40.6

16.2

32.6

-2.5

2.0

Phosphatidic acid phosphatase Diacylglycerol O-acyltransferase, partial


CEG50069.1 AXK92625.1

24.3 10.2

22.8 8.5

40.1 15.9

-1.1 -1.2

1.8 1.9

Diacylglycerol O-acyltransferase 2

Unigene150_All

GBG31058.1

12.9

11.5

17.2

-1.1

1.5

LPAT1 LPAT3 LPAT-1 LPAT-2 LPAT-3 PAP2 PAP2like -1 PAP2like -2 PAP DGAT DGAT21

Unigene4319_All



DGAT22 DGAT23 Acyl editing/ alternative TAG synthesis PL synthesis

LPCAT-1 LPCAT-2 LPCAT-3 PDAT CPT


Unigene7108_All

GBG34349.1

8.5

10.7

6.1

1.3

-1.8


Unigene3080_All

GBG31037.1

76.3

79.4

102.6

1.0

1.3

Lysophosphatidylcholine acyltransferase Lysophosphatidylcholine acyltransferase Lysophosphatidylcholine acyltransferase 2 Phospholipid:diacylglycerol acyltransferase Diacylglycerol cholinephosphotransferase

Unigene7315_All Unigene4826_All Unigene2851_All unigene5866 Unigene2860_All

GBG29045.1 GBG34814.1 GBG28807.1 GBG32689.1 GBG31343.1

51.4 12.5 12.7 7.1 56.1

43.5 14.1 13.4 6.2 55.6

449.2 18.8 13.2 12.2 53.4

-1.2 1.1 1.1 -1.1 1.0

10.3 1.3 1.0 2.0 1.0

Unigene3956_All

KZS14163.1

38.5

34.1

15.5

-1.1

2.2

CL726.Contig1_All Unigene8676_All Unigene4956_All Unigene4174_All Unigene591_All

GBG25894.1 OLQ07885.1 GBG25300.1 GBG32895.1 GBG24676.1

19.4 6.8 28.0 56.9 96.1

22.8 6.6 29.0 64.8 112.0

25.1 3.5 25.4 62.4 57.1

1.2 1.0 1.0 1.1 1.2

1.1 -1.9 1.1 1.0 -2.0

CL881.Contig2_All

GBG27905.1

20.8

23.5

36.5

1.1

1.6


GBG27555.1 GBG27091.1

25.2 13.4

30.7 10.1

24.5 4.9

1.2 -1.3

-1.3 -2.1

EPT1 LPEAT1

Phosphatidate cytidylyltransferase, mitochondrial Phosphatidate cytidylyltransferase Phosphatidate cytidylyltransferase Phosphatidylserine synthase 2 Phosphatidylserine decarboxylase proenzyme Phosphatidylserine decarboxylase proenzyme Phosphatidylethanolamine Nmethyltransferase Ethanolaminephosphotransferase 1 Lysophospholipid acyltransferase

LPEAT2

Lysophospholipid acyltransferase

Unigene2605_All

GBG25939.1

12.6

12.5

11.3

1.0

-1.1

PI-PLC-1

Phosphoinositide phospholipase C

CL451.Contig2_All

GBG27438.1

9.9

6.5

6.7

-1.5

1.0

PI-PLC-2


Unigene5763_All

GBG32084.1

12.1

8.5

5.2

-1.4

-1.6

CDS-1 CDS-2 CDS-3 PSS PSD-1 PSD-2 PEMT

Degradation of PL/TAG

Page 34 of 41


Page 35 of 41


PI-PLC-3


Unigene7_All

GBG32084.1

35.5

27.9

37.7

-1.3

1.3

PLC1

Phospholipase C

Unigene10280_All

GBG25581.1

16.0

18.5

37.9

1.2

2.1

PLC2

Phospholipase C

Unigene6098_All

GBG31934.1

24.8

21.9

30.1

-1.1

1.4

PLD

Phospholipase D alpha 1

Unigene3515_All

GBG34460.1

62.5

45.8

47.6

-1.4

1.0

PLD

Phospholipase D

Unigene5719_All

GBG32568.1

11.4

10.4

10.9

-1.1

1.0

GDPD

Glycerophosphodiester phosphodiesterase domain containing protein 1

Unigene5398_All

GBG25696.1

9.7

8.8

4.2

-1.1

-2.1

TAG lipase

Triacylglycerol lipase

Unigene6031_All

PBP21411.1

12.5

21.7

19.0

1.7

-1.1



Figure 1


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Figure 2



Figure 3


Page 38 of 41

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Figure 4



Figure 5


Page 40 of 41

Page 41 of 41


Graphic for Table of Content


Lipid distribution pattern and transcriptomic insights revealed

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