Lipid Fraction and Intracellular Metabolite Analysis Reveal the

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Lipid fraction and intracellular metabolite analysis reveal the mechanism of arachidonic acid-rich oil accumulation in the aging process of Mortierella alpina Ai-Hui Zhang, Xiao-Jun Ji, Wen-Jia Wu, Lujing Re, Ya-Dong Yu, and He Huang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04521 • Publication Date (Web): 20 Oct 2015 Downloaded from http://pubs.acs.org on October 23, 2015

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Journal of Agricultural and Food Chemistry

Lipid fraction and intracellular metabolite analysis reveal the mechanism of arachidonic acid-rich oil accumulation in the aging process of Mortierella alpina

Ai-Hui Zhang, Xiao-Jun Ji*, Wen-Jia Wu, Lu-Jing Ren, Ya-Dong Yu, He Huang*

State Key Laboratory of Materials-Oriented Chemical Engineering, College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing 211816, People’s Republic of China

*

Corresponding authors: Tel: +86 25 58139942; Fax: +86 25 58139389

E-mail: [email protected] (X.-J. Ji); [email protected] (H. Huang)

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ACS Paragon Plus Environment


1

Abstract

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The mechanism of arachidonic acid (ARA) content increase during aging of Mortierella

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alpina was elucidated. Lipid fraction analysis showed that ARA content increased from

4

46.9% to 66.4% in the triacylglycerol (TAG) molecule, and ARA residue occupation

5

increased in the majority of TAG molecule during the aging process. For the first time,

6

intracellular metabolite analysis was conducted to reveal the pathways closely associated

7

with ARA biosynthesis during aging. The main reason for the increased ARA content was not

8

only at the expense of other fatty acids degradation, but also further ARA biosynthesis during

9

aging. Furthermore, translocation played a vital role in ARA redistribution among the

10

glycerol moiety, mycelium did not die immediately with key pathways activated to maintain a

11

relative stable intracellular environment. This study lays a fundamental for further

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improvement of ARA content in the oil product obtained from M. alpina.

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Keywords: Aging; Arachidonic acid; Intracellular metabolite; Lipid fraction; Mortierella

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alpina

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INTRODUCTION

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Arachidonic acid (ARA; ω-6, 5, 8, 11, 14-cis-eicosatetraenoic acid) is an important

18

polyunsaturated fatty acid for human body. It acts as a precursor to eicosanoid hormones

19

(prostaglandins, leukotrienes and thromboxanes), some of which play an important role in

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combating or preventing a number of human diseases. 1 In addition, ARA is present in human

21

milk but is absent from both cow milk and infant formulas, which frequently substitute

22

human milk. Clinical evidence has revealed that ARA could improve the memory and

23

eyesight in babies because it is involved in the development of neural and retinal functions.2

24

Owing to its unique physiological functions, it has been widely applied in the food industry,

25

cosmetics, medicine, and many other fields.3, 4

26

ARA is traditionally isolated from lipids extracted from the adrenal glands and the livers

27

of animals in addition to egg yolks. However, isolation from these organs is typically costly

28

and insufficient to meet the demand since these organs contain only small amounts of ARA.2,5

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Luckily, microbial oils, which have long been suggested as a viable alternative to plant oils

30

and fats, have be used as an alternative source of ARA. The Zygomycete species, especially

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the Mortierella alpina, are regarded as the most prominent candidates for the production of

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ARA.

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Until now, various efforts have been made to improve the ARA accumulation in M.

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alpina by adjusting the fermentation parameters such as pH, temperature, agitation speed, 6-8

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and using proper multi-stage and fed-batch fermentation strategies.9, 10 However, the relative

36

low ARA content in the final oil product still limits the product quality and thus restricts its

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industrial mass production. In our previous work, it has been demonstrated that aging 3



38

technology, which cultures the cells after regular fermentation in another environment

39

without carbon source, efficiently increased the ARA content in the total fatty acids

40

(TFA).10,11 In fact, this phenomenon has been noticed for a long time. The research related to

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fungal aging started in the early 1950s when Rizet described for the first time that cultures of

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the filamentous fungus Podospora anserina did not grow indefinitely but senesce after a

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specific period of growth. 12 Bajpai et al.13 first reported that the ARA content of M. alpina

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mycelium increased as culture got older, i.e., aging process. Furthermore, Radwan et al.14

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found a phenomenon that many Zygomycete species accumulated higher proportion of ARA

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during the aging process.

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While aging technology could be used to improve the ARA content, the mechanism of

48

how and why the ARA content in the TFA increases significantly during aging has not been

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fully understood. In the present study, we characterized the intracellular metabolic states and

50

pathways closely associated with ARA biosynthesis by using the methods of lipid fraction

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and intracellular metabolite analysis, and revealed the mechanism of ARA accumulation

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MATERIALS AND METHODS

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Microorganism and medium

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M. alpina R807 (CCTCC M 2012118), preserved in the China Centre for Type Culture

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Collection, was used in this study.15 It was maintained on potato-dextrose agar (PDA) slants

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at 4 °C and transferred every 3 months. The PDA medium consisted of (g/L): potatoes 200;

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glucose 25; agar 20. The seed medium contained (g/L): glucose 30; yeast extract 6; NaNO3 3;

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KH2PO4 3; MgSO4·7H2O 0.5. The fermentation medium contained (g/L): glucose 80, yeast 4


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extract 10, KH2PO4 4, NaNO3 3, MgSO4·7H2O 0.6.

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Culture conditions

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A loop of M. alpina mycelia were inoculated on a PDA slant and incubated at 25 °C in

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an electro-thermal incubator. After 5–7 days of incubation, the mycelia were collected and

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used for seed culture. Seed culture was prepared in 500-mL baffled flasks containing 100 mL

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fresh medium. The culture was grown for 18–24 h at 25 °C with shaking at 125 rpm.

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The fermentation cultivation was carried out in 500-mL baffled flasks with 100 mL

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medium.10% (v/v) of the seed culture was used to inoculate the fermentation culture. All the

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cultivation was carried out at 25 °C with the initial pH value at 6.0 and shaking at 125 rpm.

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After regular fermentation, cells were cultured in another environment without carbon source

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for aging process. The fermentation time is 0-156 h and the aging time is 156-240 h.

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Analytical methods

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Dry cell weight, total lipids, and fatty acid profiles analysis

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Glucose concentration was measured by a biosensor with glucose oxidase electrode

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(SBA-40C, Institute of Biology, Shandong Academy of Sciences). Dry cell weight (DCW)

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determination was done by harvesting mycelia via filtration through filter paper, followed by

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washing mycelia three times with distilled water, and drying mycelia at 65 °C for 8 h to

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constant weights. The dry biomass was ground into fine powder for lipid extraction and fatty

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acid determination. Lipids were extracted from 2 g powder in a Soxhlet extractor with 150

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mL chloroform/methanol (2:1, v/v) for 8 h at 75 °C, and the solvent was evaporated and

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recycled.16 Fatty acid profiles were analyzed by GC system (GC-2010, Shimadzu, Japan).

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The GC was equipped with a capillary column (DB-23, 60 m × 0.22 mm) and flame 5



82

ionization detector (FID). Nitrogen was used as the carrier gas. The injector was maintained

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at 250 °C with an inject volume of 1 µL. The column was raised from 100 °C to 196 °C at

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25 °C /min and then increased to 220 °C at 2 °C/min, keeping this temperature for 6 min. The

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FID detector temperature was set at 280 °C.17

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Lipid fraction analysis

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Lipid fraction analysis was performed according to Sun et al.18 and Ren et al.19 with

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some modifications. Briefly, the total lipids (TLs, 2.0 g) obtained from extraction were

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fractionated to neutral lipids (NLs) and polar lipids (PLs) by elution on a silica column,

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initially with petroleum ether/diethyl ether (9:1) and then with methanol. After evaporation of

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the eluate, the amount of each lipid fraction was determined gravimetrically. The TLs was

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fractionated by thin-layer chromatography (TLC) on a silica gel plate (G, 25×50 mm) by

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developing with chloroform/acetone/methanol/acetic acid/water (50:20:10:10:5, v/v). The

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fatty acid profiles of TLs, NLs, and PLs were also detected by GC to get deeper

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understanding of fatty acids distribution in different lipid fractions.

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Intracellular metabolite analysis

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The method of sample preparation for intracellular metabolites, GC-MS analysis, and data

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processing were described in our previous study.20, 21 Briefly, 5 mL culture was dropped into

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liquid nitrogen after washing repeatedly, the frozen sample was grounded to powder quickly

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and then used for extraction. About 0.02 g powder sample was extracted in 1.0 mL 75% (v/v)

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ethanol buffered with 60 mM HEPES (pH 7.5) at 80 °C before melting. The mixture was

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immediately cooled on ice, and then centrifuged at 10,000×g, -10 °C for 2 min. The

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supernatant was directly used for metabolite determination. The precipitate was dried to 6


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constant weight at 60 °C and then weighed to determine the metabolite concentration. To

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correct minor variations during analysis, 10 µL ribitol (0.2 mg/mL in water) was added as

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internal standard to 50 µL extract before being dried under a N2 stream using a sample

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concentrator (MD200-1, Aosheng Company, China). Two-stage chemical derivatization was

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performed on the residue according to Ding et al.22

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Intracellular metabolites were determined by GC-MS after chemical derivatization. 1 µL

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sample was used with split less injection into the GC equipped with a fused silica capillary

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column (30 m × 0.25 mm × 0.25 µm, DB-5MS, Agilent). The column and ion source

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temperatures were set according to Ding et al.23 The mass scan range was 50–600 m/z. Peak

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area value of each metabolites was calculated and normalized on the basis of internal

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standard peak intensity and concentration. Intracellular metabolite level was calculated by the

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ratio of metabolite concentration to the corresponding biomass. The levels of all identified

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metabolites in different sampling protocols were clustered and showed in the heat map using

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Expander 5.0 (EXpression Analyzer and DisplayER).

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RESULTS

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Changes of biomass, lipid accumulation, and fatty acid profile during aging process

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As shown in Fig. 1A, ARA-rich oil production by M. alpina was a long fermentation

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process which lasted for 156 h. Aging period began at the time when 80 g/L glucose was

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exhausted. The biomass and lipid increased to 31.3 g/L and 15.1 g/L respectively at the end of

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fermentation, but decreased to 26.6 g/L and 12.3 g/L during the aging process. The non-lipid

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biomass fraction decreased in early stage of aging period but only slightly changed thereafter,

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while the change of lipid was opposite with increase during early stage and then decrease 7



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thereafter (Fig. 1A). The non-lipid biomass decrease was the main reason for biomass

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decrease in the early stage of aging process. However, degradation of lipid was the main

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reason of biomass decrease in the later stage of the aging process.

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The percentage of different fatty acids over the TFA was shown in Fig. 1B, and the

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concentration of each fatty acid was shown in Fig. 1C. It was noted that the ARA percentage

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increased from 43.9% to 63.0% and ARA concentration increased from 6.40 g/L to 7.75 g/L,

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which was a significant increase, during the aging process. As shown in Table 1, the

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percentages and concentrations of other fatty acids decreased while ARA increased, with the

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degree of fatty acid methyl ester (FAME) unsaturation24 increased from 2.27 to 2.88.

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Furthermore, the total decrease of the other fatty acids (3.67 g/L) channeled into 1.35 g/L

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ARA accounting for 37% of the total decreasement, while the residual decreasement (2.32g/L)

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was due to β-oxidation, realized as the lipid decrease. These results demonstrated that other

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fatty acids acted as the precursors for ARA further biosynthesis during the aging. That is to

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say, the main reason for the ARA percentage increase was not only at the expense of simplex

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other fatty acids degradation, but also further ARA biosynthesis from the other fatty acids

141


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Changes of lipid fractions during aging process

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TLs, extracted from dry mycelium, were fractionated to NLs and PLs by elution on a

144

silica column. As shown in Fig. 2, the majority of lipids were NLs accounting for 80% to

145

90%, which was in agreement with the phenomenon that the change tendency of ARA and

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other fatty acid percentage over the TFA in NLs were similar to that in TLs (Fig. 3). At the

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same time, there was no significant change on the ratio of NLs to PLs. 8


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As shown in Table 2, the ARA percentage over the TFA in TLs increased from 46.4% to

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65.5% while other fatty acids decreased significantly. This change was taken place mainly in

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NLs (Fig. 3A, B). Furthermore, an interesting phenomenon was observed that ARA

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percentage increase in PLs especially in the later aging process (Fig. 3C). To our knowledge,

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PLs were the basic composition of membrane structures of cell and organelle. During the life

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span of an organism, reactive oxygen species can lead to the generation of the highly

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aggressive hydroxyl radical, which can efficiently react with cellular components including

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nucleic acids, lipids, and proteins.25 The majority of membrane structures of cell and

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organelle especially mitochondria would be seriously damaged by hydroxyl radical during

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the aging process. ARA had the physiological function of repairing the membrane impairment,

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2

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the fungal cell to the damage of membrane structures.

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Characterization of the important intracellular metabolites closely associated with ARA

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biosynthesis during the aging process

therefore, ARA percentage increase in PLs was probably due to the self-healing response of

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In order to get deep understanding of the correlations between intracellular metabolism

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and ARA synthesis during the aging process, intracellular metabolites closely associated with

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ARA biosynthesis were analyzed. More than 80 intracellular metabolites were detected

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through GC-MS and 48 of them were identified and analyzed in all samples to give deep

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insight of aging process. As shown in Fig. 4, most of these metabolites were metabolically

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active between 156 h and 180 h, which was the critical period for further ARA biosynthesis

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(Fig. 3A, B, and C). Therefore, these metabolites may contribute to ARA increase during the

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aging process. These metabolites mainly included sugars, amino acids, organic acids, as well 9



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as phosphorylated compounds which were in related to many pathways such as

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Embden-Meyerhof-Parnas (EMP) pathway, tricarboxylic acid (TCA) cycle, etc. (Fig. S1).

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Among the metabolites, the concentration of sucrose was observed to be high when

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glucose was exhausted (Fig. 5C). However, it decreased quickly when the aging period began.

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With

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D-glucose-6-phosphate would enter into the EMP pathway, but their concentration were

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lower in the whole aging period (Fig. 5A). This indicated that, due to the lack of glucose, the

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EMP pathway was not active especially in the later aging period. At the same time,

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α-ketoglutaric acid, succinic acid, citric acid, malic acid changed significantly with the

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cellular metabolism active in the aging period (Fig. 5E). This demonstrated that

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malate/pyruvate and TCA cycles were active in the early aging period (156-180 h). Among

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the amino acids, which were metabolically active in the aging process, pyroglutamic acid was

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found to be more active in the later period of the process. As pyroglutamic acid was reported

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to be a signal for activating glutathione metabolism,26 it might play an important role in

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defending the stress derived from reactive oxygen species (ROS) which were harmful to the

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normal cell metabolism and existed in the later aging mycelium.25

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the

decomposition

Phosphorylated

of

compounds

sucrose,

the

including

derived

D-fructose-6-phosphate

phosphoric

acid,

and

α-glycerophosphate,

187

O-phosphorylethanolamine, D-myo-inositol-phosphate which were involved in PLs synthesis

188

and energy metabolism were metabolically active during aging (Fig 5B, F and G). Among

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these compounds, phosphoric acid, which could take part in phosphorylating ADP to ATP,

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was the most active metabolite, thus could provide energy to maintain the normal function of

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cells. α-Glycerophosphate, O-phosphorylethanolamine, and D-myo-inositol-phosphate were 10


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able to act as precursors for the synthesis of PLs which had many physiological functions

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especially against the abnormal metabolic state.27,28 Therefore, in the aging process of M.

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alpina, the intracellular activated phosphorylated compounds might take an important part in

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energy supply, and cell function to answer the abnormal environmental condition, i.e. nutrient

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deficient.

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DISCUSSION

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The accumulated sucrose delayed the lipid degradation during the aging process

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As indicated above, a considerable amount of sucrose accumulated in the fungal cells

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before glucose depletion, and its concentration decreased quickly in the aging period (Fig.

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5C). In fact, during 144 h-168h, carbon continues synthetizing lipid and therefore leading to

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its increase in this period, there also exists cells decomposition during the aging

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simultaneously. Owing to these reason mentioned above and the existence and degradation

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of sucrose, the non-lipid biomass decreased between 156 h and 180 h (Fig. 1A). Lipid

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concentration decreased quickly when the sucrose exhausted after 192 h. The assimilation of

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sucrose was easier and more quickly than lipid degradation when facing carbon starvation.

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This indicated that in the fungus, M. alpina, some of the absorbed glucose was transformed

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into sucrose whose physiological function was just like the role of glycogen to mammalian

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cell. When sucrose was exhausted, the fatty acids were quickly degraded to provide energy.

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Furthermore, as show in Fig. 1C, the ARA concentration rarely increased during 216-240 h,

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while ARA percentage over TFA increased significantly. This demonstrated that the

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degradation of other fatty acids was the reason why ARA content increased in this stage.

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Thus, the time of 156-204 h was the key period for ARA synthesis in the aging process, and 11



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M. alpina had a physiological ability of storing sucrose in response to aging to delay the

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lipid degradation.

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Translocation may play a vital role in ARA redistribution during the aging process

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It was reported that, in the ARA-producing fungi, 70%-90% of the ARA was present as

218

triacylglycerol (TAG) which was the main component of NLs.29, 30 If the majority of ARA

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was located at one position such as sn-1 of the glycerol moiety, the ARA percentage over the

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TFA would reach a maximum of slightly over 33.3%. In the present study, at the beginning

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of aging process, the ARA percentage over the TFA in NLs and TLs exceeded the value of

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33.3%. This indicated that some TAG molecules must contain more than one ARA residues.

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Furthermore, at the end of the aging process, the ARA percentage over the TFA in NLs and

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TLs exceeded 60%, thus demonstrating that most of the TAG molecules contained more

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than one ARA residues. Myher et al. reported that the ARA concentration in ARA-rich oil

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(ARA content exceed 50% of the TFA) in the sn-1 position was double of that in the sn-3

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position, with an intermediate value in the sn-2 position, while the ARA distribution in

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ARA-poor oil (ARA content not exceed 30% of the TFA) was characterized by nearly equal

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proportions of the acid in all three positions of the TAG.31 These results demonstrate that

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ARA transferred to sn-1 position selectively when ARA content increased greatly. At the

231

same time, the precursor fatty acids of ARA were found predominantly on the sn-2 position

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of the glycerol moiety in TAG.

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played a vital role in fatty acids distribution at least. The distribution would also require

234

trans-location at some stage because the sn-3 position was available for phospholipid which

235

was replaced by ARA in the process of ARA-rich oil biosynthesis.

31

Nevertheless, these demonstrated that the trans-location

12


32

Ouyang et al. also

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revealed that the M. alpina like, ARA producing fungus Myrmecia incise is able to convert

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ARA of membrane lipids into storage TAG by the means of transcriptome analysis.33 These

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indicate that the distribution of ARA in TAG was random in the early stage of TAG

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synthesis (before the aging process), the further biosynthesized ARA in the aging process

240

would be trans-located preferentially to sn-1 position of the initial low ARA content TAG,

241

thus, the ARA percentage over the TFA increased rapidly.

242

Some pathways were activated in response to aging for the cell maintenance

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When the fungal mycelia entered the aging period, the cell growth nearly ceased and this

244

led to the inactivity of many pathways, such as EMP and pentose phosphate pathways which

245

were crucial for the cell to functionalize normally. At this situation, one might be curious

246

about how the ARA was further synthesized, where the precursors were from and how an

247

ARA-synthesis favorable intracellular environment was maintained.

248

It was found that during aging, the intracellular malate and pyruvate were at a relatively

249

higher concentration compared with other organic acids (Fig. 5E). This indicated that the

250

malate/pyruvate cycle, which was the main contributor of NADPH for dehydrogenation of

251

fatty acid to synthesize ARA,

252

the concentration of another organic acid, γ-aminobutyric acid (GABA), was extremely high

253

among the organic acids (Fig. 5D). GABA is indicated to be a key intermediate of the

254

bypath of the TCA cycle, named GABA bypass shunt.35 This bypass shunt could transfer

255

α-ketoglutartate to succinate using GABA and L-glutamic acid as the intermediate (Fig. S1).

256

GABA bypass shunt was generally undetectable under normal conditions.36 It was, therefore,

257

easily to understand that the operation of this shunt pathway was a response to the abnormal

34

was induced to be active in the aging process. Meanwhile,

13



258

metabolic states of starvation in the aging period. The exhaust of glucose led to the

259

inactivity of many pathways, especially TCA cycle, and thus the operation of this shunt

260

pathway was selected to supplement NADH for maintenance. Besides, another function of

261

the GABA pass shunt might be the maintenance of the intracellular redox balance in

262

response to the abnormal metabolic sates of starvation in aging period.37 Thus, the activated

263

pathways of malate/pyruvate cycle and GABA shunt in the aging process, which connected

264

with many other metabolites could not only maintain normal physiological function of cells

265

but also provide NADPH, NADH and intermediate for the further ARA synthesis.

266

As mentioned above, the details of cellular metabolism in the aging process of ARA-rich

267

oil producing M. alpina was discussed for the first time in this study. The main reason for

268

the increased ARA content was not only at the expense of other fatty acids degradation, but

269

also further ARA biosynthesis during aging. Translocation played a vital role in ARA

270

redistribution among the glycerol moiety in the aging process. Furthermore, the mycelia did

271

not die immediately in the aging process with some pathways activated to provide the cell a

272

relative stable environment. The mechanism of fungal aging revealed in this work would lay

273

a foundation for high quality ARA-rich oil production, with high ARA content. The aging

274

technology which was developed to increase the ARA percentage over the TFA in M. alpina

275

could be further improved and applied in other similar polyunsaturated fatty acid-rich oil

276

production.

277

ABBREVIATIONS USED

278

C16:0, palmitic acid; C18:0, stearic acid; C18:1, oleic acid; C18:2, linoleic acid; C18:3,

279

γ-linolenic acid; C20:3, dihomo-γ-linolenic acid; C20:4, ARA; TLs, total lipids; NLs, neutral 14


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lipids; PLs, polar lipids; TFA, total fatty acids; TAG, triacylglycerol; PDA, potato-dextrose

281

agar; FAME, fatty acid methyl ester; γ-aminobutyric acid, GABA.

282

ASSOCIATED CONTENT

283

Supporting Information

284

Proposed metabolic pathways closely associated with ARA biosynthesis in M. alpina during

285

the aging process (Figure S1). This material is available free of charge via the Internet at

286

http://pubs.acs.org.

287

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Funding

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This work was financially supported by the National Science Foundation for Distinguished

393

Young Scholars of China (No. 21225626), the National Natural Science Foundation of China

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(Nos. 21376002, 21476111, 21506096), the Natural Science Foundation of Jiangsu Province

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(No. BK20131405), the National High-Tech R&D Program of China (No. 2014AA021703),

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and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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Notes

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The authors declare no competing financial interest.

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FIGURE CAPTIONS Figure 1. Time course of biomass, lipid, non-lipid biomass, residual glucose, arachidonic acid (ARA), and other fatty acid concentrations in the aging process for ARA-rich oil production by Mortierella alpina. A, the concentration of biomass, lipid, non-lipid biomass, and glucose, B, the percentage of ARA and various other fatty acids over the total fatty acids, C, the concentration of ARA and other fatty acids. The error bars show standard deviations of three independent experiments. Figure 2. Different lipid fraction percentage over the total lipids during the aging process Figure 3. Time course of different fatty acid percentages over the total fatty acids (TFA) in different lipid fractions. A, the arachidonic acid (ARA) and the other fatty acid percentages over the TFA in total lipids, B, the ARA and the other fatty acid percentages over the TFA in neutral lipids, C, the ARA and other fatty acid percentages over the TFA in polar lipids. Figure 4. Clustering of the intracellular metabolites during the aging process. The metabolite levels were corresponded to the square colors. Red and green represent higher and lower level of the respective metabolites. Figure 5. Time course of the important intracellular metabolite concentration of Mortierella alpina during the aging process. A, metabolites related to EMP pathway, B, alcohols, C, sugars, D, the metabolites related to GABA shunt, E, metabolites related to TCA cycle, F and G, phosphorylated compounds. The error bars show standard deviations of three independent experiments.

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Table 1. Comparison of Parameters at Different Stages in Fermentative Production of Arachidonic Acid (ARA)-rich Oil by Mortierella alpina.

a

After fermentation

After aging

Increase a

Biomass (g/L)

31.30 ± 1.04

26.60 ± 0.18

-4.70

Lipid (g/L)

14.60 ± 0.66

12.30 ± 0.12

-2.30

C16:0 (g/L)

2.02 ± 0.15

1.09 ± 0.10

-0.93

C18:0 (g/L)

2.05 ± 0.09

1.13 ± 0.10

-0.92

C18:1 (g/L)

1.92 ± 0.12

0.91± 0.12

-1.01

C18:2 (g/L)

1.06 ± 0.06

0.70 ± 0.31

-0.36

C18:3 (g/L)

0.60 ± 0.05

0.42 ± 0.03

-0.18

C20:3 (g/L)

0.54 ± 0.03

0.27 ± 0.00

-0.27

C20:4 (g/L)

6.40 ± 0.20

7.75 ± 0.28

+1.35

ARA/TFA (%)

43.9

63.0

+19.47

b

2.27

2.88

+0.53

Degree of FAME unsaturation

Increase=the value of aging stage minus the value of fermentation stage in each row. - Represents

decrease while + represents increase. b

Degree of FAME unsaturation=[1.0 (% monene)+2.0 (% diene) +3.0 (% triene) + 4.0 (% tetraenes) + 5.0

(% pentanes) +6.0 (% hexane)].24

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Table 2. Fatty Acid Profiles of Total Lipids and the Individual Lipid Classes Produced by Mortierella alpina at Different Stages After fermentation

a

a

After aging

TLs

NLs

PLs

TLs

C16:0 (%)

12.78

12.83

24.61

8.24

7.92

18.72

C18:0 (%)

14.24

15.56

23.58

9.45

9.53

17.42

C18:1 (%)

12.90

11.95

18.36

7.26

7.10

15.56

C18:2 (%)

7.26

6.40

8.26

4.54

4.26

7.93

C18:3 (%)

3.19

2.99

3.19

2.86

2.69

2.62

C20:3 (%)

3.22

3.38

3.38

2.16

2.15

2.04

C20:4 (%)

46.41

46.89

18.62

65.49

66.35

35.71

2.32

2.31

1.29

2.93

2.95

1.88

Degree of FAME unsaturation

NLs

PLs

Degree of FAME unsaturation=[1.0 (% monene)+2.0 (% diene) +3.0 (% triene) + 4.0 (% tetraenes) + 5.0

(% pentanes) +6.0 (% hexane)].24

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Figure 1

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Figure 2

25



Figure 3

26


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Figure 4

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Figure 5

28


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Graphic for Table of Contents

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Lipid Fraction and Intracellular Metabolite Analysis Reveal the

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