Role of AMP deaminase during fatty acids accumulation in

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Role of AMP deaminase during fatty acids accumulation in oleaginous fungus Mortierella alpina Lulu Chang, Xin Tang, Hengqian Lu, Hao Zhang, Yong Q. Chen, Haiqin Chen, and Wei Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03603 • Publication Date (Web): 05 Aug 2019 Downloaded from pubs.acs.org on August 7, 2019

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Journal of Agricultural and Food Chemistry

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Role of AMP deaminase during fatty acids accumulation in

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oleaginous fungus Mortierella alpina

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Lulu Chang†,‡, Xin Tang*†,‡, Hengqian Lu†,‡, Hao Zhang†,‡,§,¶, Yong Q. Chen†,‡,§,¶,

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Haiqin Chen*†,‡, Wei Chen†,‡,§,||

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†

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Jiangsu 214122, P. R China

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‡ School

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P. R China

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§

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi,

of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122,

National Engineering Research Center for Functional Food, Jiangnan University,

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Wuxi, Jiangsu 214122, P. R China

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¶

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Research Institute Wuxi Branch, Wuxi, Jiangsu 214122, P. R China

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||

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and Business University (BTBU), Beijing 100048, P.R. China

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* Corresponding author: Xin Tang & Haiqin Chen

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E-mail: [email protected]

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Wuxi Translational Medicine Research Center and Jiangsu Translational Medicine

Beijing Innovation Centre of Food Nutrition and Human Health, Beijing Technology

[email protected] Telephone: 0086-510-85197239

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ACS Paragon Plus Environment


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ABSTRACT: In oleaginous microorganisms, nitrogen limitation activates adenosine

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monophosphate deaminase (AMPD) and promotes lipogenesis via the inhibition of

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isocitrate dehydrogenase (IDH). We found that overexpression of homologous AMPD

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in Mortierella alpina favoured lipid synthesis over cell growth. Total fatty acids content

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in the recombinant strain was 15.0%−34.3% higher than that in the control, even though

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their biomass was similar. During early fermentation stage, intracellular AMP level

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reduced by 40%−60%, together with a 1.9−2.7-fold increase in citrate content

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compared with the control, therefore provided more precursors for fatty acids synthesis.

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Moreover, the decreased AMP level resulted in metabolic reprogramming, reflected by

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blocked TCA cycle and reduction of amino acids, distributing more carbon to lipid

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synthesis pathways. By coupling energy balance with lipogenesis, this study provides

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new insights into cell metabolism under nitrogen-limited conditions and targets to

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regulate fatty acids accumulation in oleaginous microorganisms.

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KEYWORDS: Mortierella alpina, AMPD, Fatty acids accumulation, Energy balance,

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Metabolic reprogramming

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 INTRODUCTION

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Mortierella alpina is a type of oleaginous filamentous fungus with considerable oil-

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producing capacity. Its fatty acids content accounts for more than 50% of dry cell

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weight 1. M. alpina has the enzyme system to synthesise various polyunsaturated fatty

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acids (PUFAs) such as dihomo-gamma-linolenic acid (DGLA), alpha linolenic acid

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(ALA), arachidonic acid (ARA) and eicosapentaenoic acid (EPA) 2-6, making this strain

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a good model microorganism for lipid metabolism research. Several studies have been

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conducted to exploit M. alpina for specific fatty acids production

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high ARA yield (over 50% of total fatty acids content), M. alpina has already been used

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in food additives as well as in infant formula production 10-11.

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The fermentation process in oleaginous microorganisms include three phases, namely

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the cell growth phase, fatty acids accumulation phase and reserve fatty acids turnover

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phase, and the physiological stages are regulated by carbon to nitrogen ratio in culture

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medium

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intracellular carbon is diverted to fatty acids synthesis pathways

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acids accumulation is suggested to be a stress response or an adaptation to nutrient shifts

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14-15.

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Lipid biosynthesis is facilitated by several key enzymes including adenosine

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monophosphate deaminase (AMPD), ATP-citrate lyase (ACL), malic enzymes (MEs)

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and acetyl-CoA carboxylase 1 (ACC1), which together divert intracellular carbon to

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synthesis fatty acids

10, 12-13.

3, 7-9,

and due to its

Under nitrogen-limited conditions, cell growth is inhibited and

10, 16-21.

12.

Therefore, fatty

A metabolic model of lipid accumulation under nitrogen



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limitation has previously been proposed 22. AMPD, activated by nitrogen insufficiency,

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breaks down AMP to inosine monophosphate (IMP) and NH4+. AMP depletion inhibits

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mitochondrial NAD+ dependent isocitrate dehydrogenase (NAD+-IDH) and induces

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lipid accumulation (Figure 1).

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Some attempts have been performed to regulate lipid synthesis by modifying AMPD.

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Indeed, simultaneous overexpression of ME and AMPD in Yarrowia lipolytica

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increases lipogenesis. Moreover, lipogenesis can be further enhanced by co-

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overexpression of diacylglycerol acyltransferase (DGAT) and AMPD, possibly due to

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the overproduction of fatty acid precursors16. In mammals, increased AMPD activity

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activates fatty acids synthase (FAS), ACC1 and ACL, and inhibits oxidation of fatty

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acids by inhibiting the energy decomposition pathway, and thus resulted in hepatic fat

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accumulation 23.

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In this study, we explored the role of homologous MaAMPD on lipid accumulation in

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Mortierella alpina under fermentation conditions. We measured transcriptional level

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and enzyme activity of fatty acids metabolism-related proteins, and carried out

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metabolomics analysis to explore in depth the function of MaAMPD, focusing emphasis

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on energy and nitrogen metabolism pathways. By combining energy balance with

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lipogenesis, we attempted to better understand the global regulatory mechanisms of

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lipogenesis in oleaginous microorganisms under nitrogen-limited conditions.

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 MATERIALS AND METHODS


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Strains and plasmids. The uracil-auxotrophic strain Mortierella alpina CCFM 501

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was the transformation recipient to construct a recombinant strain, and the prototroph

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Mortierella alpina CCFM 505 (uracil+) was the control 24. Agrobacterium tumefaciens

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CCFM 834 was the transfer DNA (T-DNA) donor for Agrobacterium tumefaciens-

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mediated transformations (ATMT) 25. The binary expression vector pBIG2-ura5s-ITs

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was constructed in a previous study, and conserved in Escherichia coli Top 10 strain

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24.

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alpina 24,26.

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The coding sequence of MaAMPD (accession number: MN168296) was obtained using

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the E.C. number (E.C. 3.5.4.6) and homologous alignment in the genome database

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constructed in a previous study

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sequence in this strain, gene blast in whole M. alpina was carried out using M. alpina

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ATCC 32222 local database and used MaAMPD as query. The gene segment was

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obtained from M. alpina ATCC 32222 cDNA and ligated to vector pBIG2-ura5s-ITs to

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construct the recombinant expression vector pBIG2-ura5s-MaAMPD and transformed

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into A. tumefaciens CCFM 834 by electroporation (2.5 kV, 5.0 ms). M. alpina CCFM

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2056 was the MaAMPD overexpression strain. The primers used in these steps are listed

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in Table S1

95 96

The promotor of the vector a modified H4.1 genes from histone H4 isolated from M.

26.

To make sure there was only one AMPD coding

(CCFM refers to Culture Collection of Food Microorganisms of Jiangnan university, School of Food Science and Technology, Research Center of Food Biotechnology).



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Culture conditions of Mortierella alpina. M. alpina was cultured on glucose-yeast

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(GY) agar slant [20 g/L glucose, 10 g/L yeast extract, 2 g/L KNO3, 1 g/L NaH2PO4, 3

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g/L MgSO4•7H2O, and 0.1 g/L uracil (for uracil auxotroph strain)] at 28°C for two

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weeks for mycelium growth then at 4°C for two weeks to produce spore. Spores were

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collected using normal saline and filtered through Miracloth (Calbiochem, Germany),

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then centrifuged at 12,000 g for 25 min at 4°C 22, 25. M. alpina spores were inoculated

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into Broth medium [20 g/L glucose, 5 g/L yeast extract, 10 g/L KNO3, 1 g/L K2HPO4,

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0.25 g/L MgSO4•7H2O, and 0.1 g/L uracil (for uracil auxotroph strain)] at 28°C for 48

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h. The mycelium pellets were dispersed using basic ultra-turrax (IKA, Germany) and

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transferred into new Broth medium (inoculated dose was 1%, v/v). This was repeated

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for three generations, and the mycelium was inoculated and cultured in Broth medium

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(with 50 g/L glucose) or modified Kendrick medium supplemented with 50 g/L glucose

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at 28°C for 240 h to facilitate fermentation

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analyse the physiological properties of M. alpina-MaAMPD in nitrogen-sufficient

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conditions (36 h), nitrogen-limited conditions (96 h) and at final stages of the

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fermentation process (168 h and 240 h).

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Agrobacterium tumefaciens-mediated transformation (ATMT). ATMT was adapted

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from previously described methods

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vector pBIG2-ura5s-MaAMPD was activated in YEP medium [10 g/L yeast extract, 10

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g/L tryptone, 5 g/L NaCl ] with 100 µg/mL kanamycin and 100 µg/mL rifampicin at

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30°C for 36 h away from light, then transferred in antibiotic-free MM medium for about

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36 h at 30°C until OD660 reached 1.5. Next, A. tumefaciens was diluted and cultured in

27.

18-19, 28-29.

Four sampling time points was set to

Specifically, A. tumefaciens with the


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IM liquid medium supplemented with 100 µg/mL acetosyringone (AS) away from light

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at an initial OD660 of 0.2 for about 12 h at 30°C until OD660 reached 0.8. A. tumefaciens

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was gradient-diluted to OD660 of 0.2, 0.4 and 0.6, mixing with M. alpina spore

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suspension (107−8 spores/mL) respectively and spread on IM agar medium covered with

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cellophane membranes. The samples were incubated away from light for 24 h at 16°C

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and then for 24 h at 28°C. Next, the cellophane membranes were moved to uracil-free

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SC agar plates containing 100 µg/mL cefotaxime and 100 µg/mL spectinomycin until

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colonies appeared. Mycelium from fungal colonies was picked and transferred to new

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uracil-free SC agar plates. This step was repeated three times to select stable

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transformants. The preparation methods of transformation medium, including

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synthetic-complete (SC) medium, minimal medium (MM) and induction medium (IM),

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were as described previously 24.

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Extraction of genomic DNA (gDNA) and identification of transformants. Biospin

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Fungus genomic DNA extraction kit (Bioflux, China) was used to extract genomic

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DNA of M. alpina according to instructions. The presence of T-DNA in the genome of

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transformants was identified using universal primers Hispro F1 and TrpC R1 as

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previously described (Table S1)

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and purified with DNA purification kit (Thermo scientific, USA) and sent for

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sequencing.

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RT-qPCR analysis. Total RNA of M. alpina was extracted using Trizol (Thermo

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Scientific, USA) according to the manufacturer’s instructions, and reverse-transcribed

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to generate cDNA using a PrimeScript RT reagent kit (Takara, Japan). The quantitative

30-31.

PCR product was collected with gel extraction



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PCR (qPCR) reaction was performed using a CFX Connect RealTime System (Bio-Rad,

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USA) and SYBR Green PCR Supermix (Bio-Rad, USA) for quantitative analysis

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The housekeeping gene M. alpina 18S rRNA was used as the internal control. The

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primers used are listed in Table S1.

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Biomass analysis, lipid extraction and fatty acid methyl ester (FAME) analysis.

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Fresh biomass was strained through a 200-mesh filter, washed with distilled water to

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remove culture medium, and frozen overnight at -80°C. They were then dried in a

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vacuum freeze-dryer for quantification of the dry cell weight (DCW). All types of lipids

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were hydrolysed with 4 mol/L hydrochloric acid solution, and extracted with methanol

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and chloroform from approximately 50 mg dried sample, then methyl-esterified as

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described previously

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FAME using gas chromatography mass spectrometry analysis (GCMS-QP2010 Ultra.

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Shimadzu, Japan). Pentadecanoic acid (C15:0) was used as an internal standard for

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quantification. The temperature programme was as previously described 30-31.

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Metabolomics analysis. Metabolomics analysis was conducted according to our

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previous study

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through Büchner flask fitted with filter paper (Whatman, England) to remove culture

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medium and quenched in liquid nitrogen. Metabolites were extracted using methanol-

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water (1:1,v/v) solution and dried at 30°C in vacuum concentrator. The vacuum-dried

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samples were resuspended in MeOX-pyridine and MSTFA with 1% TMCS for

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derivatization. The resulting solution was centrifuged and the supernatant was

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transferred into vials for gas chromatography mass spectrometry (GC-MS) analysis.

32.

18-19, 28.

19.

The fatty acids profiles were analysed in the form of

The fresh biomass were washed with normal saline and filtered


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The quantification was normalised to the fresh biomass weight. The peaks exaction,

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retention time adjustment, peak alignment, deconvolution analysis and identification

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were performed by using MSDIAL3.70 equipped with DB_FiehnBinbase-FiehnRI

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database 33. Principal component analysis (PCA) was performed by SIMCA14.1 and

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heat map was drew using TBtools 0.665. The difference of intracellular metabolites

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content between the two strains was shown by fold change and the control group (M.

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alpina prototroph strain) was defined as “1”.

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Preparation of crude protein and enzyme activity analysis. Crude protein

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preparation was conducted as previously described 34-35. The fresh biomass was ground

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in liquid nitrogen. Adding extraction buffer [100 mM KH2PO4 (pH 7.5), containing 20%

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(w/v) glycerol, 1 mM benzamidine HCl and 1 mM DTT] to extract the protein. Protein

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concentration was determined using Bradford assays and bovine serum albumin (BSA)

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was used as a standard. Enzyme activity analysis of ACL (EC 4.1.3.8), NADP+-IDH

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(EC 1.1.1.42), AMPD (EC 3.5.4.6) and NAD+-IDH (EC 1.1.1.41) was conducted

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according to a previous study 34, 36.

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 RESULTS

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Screening and identification of M. alpina-MaAMPD recombinant strain. We

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obtained the coding sequence of MaAMPD through basic local alignment search tool

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(blast), finding that MaAMPD contains 2655 base pairs and corresponds to 884 amino

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acids. Result also showed there was only one AMPD coding sequence in M. alpina

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ATCC 32222. Using Agrobacterium tumefaciens-mediated transformation (ATMT),



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the T-DNA region with MaAMPD and selection marker ura5s successfully inserted into

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genome of the uracil auxotroph strain M. alpina CCFM 501. Eight transformants were

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selected for identification, and the MaAMPD segment was successfully amplified by

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PCR (Figure 2). After cultured in Broth medium (with 50 g/L glucose for fermentation)

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for 168 h, the MaAMPD overexpression strains showed increased lipid accumulation

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(Figure 3). The extent of fatty acids content differed between transformants, possibly

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due to different insertion locations 24, 29, 37. Among them, the transformant No. 5 showed

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the most significant increase in fatty acids content and was chosen for further study.

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Analysis of growth and lipid content of M. alpina-MaAMPD. In Kendrick medium,

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M. alpina-MaAMPD exhibited increased fatty acids content and similar total biomass

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during the entire fermentation process compared with the control (Figure 4). The fatty

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acids content of the recombinant strain increased 34.3%, 15.0%, 24.1% and 17.6%,

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respectively, at the four sampling time points, and the fatty acids composition remained

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unchanged (Table 1). However, at 36 h when nitrogen was sufficient (Figure 5), the

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lipid-free biomass of the recombinant strain was 23% lower than that of the control

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(Figure S1a). In both strains, the total biomass continued to increase during the final

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stages of fermentation (168 h and 240 h). This was mainly due to lipid accumulation

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(Figure S1b). As the fermentation progressed, the lipid-free biomass remained stable

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and the gap between the two strains gradually decreased (Figure S1a). These results

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suggested that homologous MaAMPD overexpression increased carbon flux into fatty

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acids synthesis pathways and inhibited cell growth under both nitrogen-sufficient and

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nitrogen-limited conditions.


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Determination of gene transcription and enzymatic activity. As expected, both the

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transcriptional level and enzymatic activity of MaAMPD were more than 2-fold higher

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in lipid accumulation phase (96 h) compared with the control (Figures 6 and 7).

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Consistent with the increase in fatty acids content, the transcriptional level of acetyl-

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CoA carboxylase 1 (ACC1, the rate-limiting enzyme in fatty acids synthesis) was 20

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times higher in the recombinant strain. ATP-citrate lyase (ACL) consists of two

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subunits in Mortierella alpina (ACL1 and ACL2), although transcriptional level of both

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genes significantly increased, its enzymatic activity was coincident with that in the

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control group (Figure 7).

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activity 22, but such inhibition was not reflected on gene transcriptional level of each

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IDH (Figure 6). It is also unexpected that the activity of mitochondrial NAD+-IDH only

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decreased by 10% in the recombinant strain, while the activity of cytoplasmic NADP+-

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IDH was similar to that in control group (Figure 7).

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Analysis of intracellular metabolites through metabolomics study. To visualize the

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effects of MaAMPD overexpression on cellular metabolism, a metabolomics study was

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carried out to analyse the difference in intracellular metabolites. Emphasis was placed

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on energy and nitrogen metabolism pathways (Figure 8 and 9).

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The AMP level in M. alpina-MaAMPD was 41.4% lower at 36 h (nitrogen sufficient)

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and 61.1% lower at 96 h (nitrogen limited) compared with the control (Figure 8),

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indicating that MaAMPD overexpression reduced intracellular AMP and altered energy

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balance even under nitrogen-sufficient growth conditions. During the late fermentation

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stages, AMP content in the recombinant strain gradually recovered to the same level as

It is reported that AMPD negatively effect NAD+-IDH



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in the control, and was consistent with the trend of MaAMPD activity. Besides, at 36 h

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and 96 h, the citrate level in the recombinant strain was 1.91−2.68 fold higher than that

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in the control, which was contributed to decreased AMP level and thereby increased

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fatty acid content. Alpha-ketoglutarate (α-KG), a decomposition product of isocitrate,

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was lower than the control group. Similar trends were also observed for other TCA

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metabolites, such as succinic acid, fumaric acid and malate. Moreover, a reduction of

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amino acids and metabolite involved in urea cycle was observed in the recombinant

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strain (Figure 9), suggesting that MaAMPD took part in nitrogen metabolism in M.

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alpina. From the heatmap, it was obvious to see the metabolic reprogramming in the

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recombinant strain during nitrogen sufficient conditions (36 h and 96 h), including

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increased fatty acids synthesis, nitrogen substance catabolism and inhibited TCA cycle,

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which was supposed to occur after nitrogen exhaustion.

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

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Nitrogen availability is a key regulator of fatty acids accumulation in oleaginous

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microorganisms, and the increased AMPD activity is one of the inducer of lipogenesis

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10, 12-13, 22.

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regulating fatty acids accumulation. Our results indicate that MaAMPD overexpression

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strain favoured fatty acids synthesis over cell growth. It was obvious to see that M.

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alpina-MaAMPD exhibited higher fatty acids synthesis efficiency (Figure 4c),

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suggesting that the recombinant strain synthesized more fatty acids with less biomass,

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which could also be proved in oleaginous microalgae Phaeodactylum tricornutum 38-40.

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In support of our hypothesis, both M. alpina strains exhibited similar glucose

DISCUSSION

In this study, we aimed to analyse the mechanism of M. alpina AMPD in


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consumption (Figure 5), suggesting that the increased lipids accumulation observed in

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the recombinant strain was the result of a metabolic shift.

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Intracellular factors that affect cell growth include sensitivity and adaptation to the

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environment, distribution of nutrients and aspects of metabolic homeostasis (especially

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energy level) that may affect cell viability

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levels and affects the ATP/AMP ratio and cellular energy balance 22, 36. Consequently,

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AMP-dependent enzymes are affected by decreased AMP level, and the unbalanced

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energy state can drive a stressed phenotype, such as nutrient-starvation. Under such

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energy-starved conditions, protein synthesis and cell growth are typically suppressed to

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minimise energy expenditure 13, 43.

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In this study, as well as in other oleaginous microorganisms, activated AMPD decreases

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the intracellular AMP level and represses TCA cycle, leading to the accumulation of

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intra-mitochondrial citrate. The citrate is then transferred to the cytoplasm in exchange

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with malate

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important precursor for fatty acid synthesis 13-14, 32. Our results indicate that the enzyme

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activity of MaAMPD increased by more than 2-fold during the stationary phase (96 h),

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which is correlated with increased gene transcriptional level of genes related to fatty

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acids biosynthesis (Figure 6). Therefore, more fatty acids are synthesized in the

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recombinant strain, indicating the positive regulatory function of MaAMPD in lipid

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accumulation. However, the NAD+-IDH activity was only slightly repressed (Figure

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7). Given that there are three NAD+-IDH isotypes located in the mitochondria of M.

12.

12, 41-42.

AMPD reduces intracellular AMP

Citrate can be cleaved by cytosolic ACL to form acetyl-CoA, an



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alpina, we are not able to conclude the specific effects of AMPD on the enzymatic

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activity of individual NAD+-IDHs in vivo yet.

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Metabolomics study makes it possible to visualize the effects of AMPD on different

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metabolic pathways. Principal component analysis (PCA) showed that the intracellular

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metabolites were quite different among the four time points (Figure S2). In the control

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group, samples at 36 h (nitrogen sufficient) and 96 h (nitrogen exhausted) were well

277

distinguished, while samples at later stages of fermentation (168 h and 240 h) showed

278

better clustering, indicating that intracellular metabolites tend to be stable when lipid

279

began to accumulate. In M. alpina-MaAMPD strain, however, intracellular metabolites

280

at 36 h and 96 h clustered with that at 168 h and 240 h in the control, which suggested

281

that overexpression of MaAMPD lead to a similar oleaginous phenotype even in cell

282

growth phase of fermentation.

283

In MaAMPD overexpression strain, the TCA cycle was inhibited while citrate

284

accumulated, which was then distributed to fatty acids synthesis pathways instead of

285

energy supply 12.

286

can be converted into oxaloacetate and acetyl-CoA by ACL. Our results show that

287

expression level of two ACL subunits maintained at high levels (Figure 6), and that the

288

enzymatic activity of ACL did not change (Figure 7). Previous studies have shown that

289

ACL activity can be regulated by glucose concentration and acetylation 44-45. Our results

290

indicate that although large amounts of citrate accumulated in M. alpina-MaAMPD, its

291

conversion to acetyl-CoA, and thus fatty acids synthesis, was limited by ACL activity,

292

which might be a rate-limiting step in fatty acids accumulation. Therefore,

Citrate is the most important intermediate in the TCA cycle, and


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simultaneously overexpress AMPD and ACL might be an effective way to increase

294

lipid content in M. alpina.

295

Corroborating cell growth inhibition, we observed reduced amino acids levels in the

296

MaAMPD recombinant strain compared with the control strain, as well as decreased

297

metabolites involved in urea cycle (Figure 9). Such phenomenon was also observed in

298

oleaginous microorganism Rhodosporidium toruloides and Mucor aircinelloides

299

Amino acid is also a source of acetyl-CoA 48. It is reported that lipid accumulation is

300

associated with regulation of amino acid biosynthesis, resulting in redirection of carbon

301

flux during nitrogen limitation from amino acids to lipids 21. The branched-chain amino

302

acids (BCAAs) was also reported as a contributor to refill the acetyl-CoA pool for

303

biofuel production in microalga Dunaliella tertiolecta 48. In this study, the decreased

304

amino acids level in MaAMPD overexpression strain may result from activated amino

305

acids catabolism, therefore led to overproduced acetyl-CoA which was channelled into

306

the fatty acids reservoir (Figure 9). Although no previous studies have reported AMPD

307

is involved in amino acid metabolism under nitrogen stress, our results suggested that

308

MaAMPD affected nitrogen recycling in M. alpina, however, the molecular mechanism

309

needs further investigation.

310

Energy homeostasis plays a pivotal role in the regulation of cell proliferation, gene

311

expression and metabolic balance 49-51. In this study, we found that overexpression of

312

MaAMPD led to metabolite reprogramming mainly reflected in TCA cycle, lipogenesis

313

and nitrogen substance catabolism (Figure 8 and 9). Specifically, during growth phase

314

(36 h), MaAMPD overexpression strain showed oleaginous phenotype that was


46-47.


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supposed to occur after nitrogen exhaustion (Figure 9), which is resulted from the

316

unbalanced energy (AMP) level12.

317

AMP deaminase regulates intracellular AMP level, which in turn regulates the activity

318

of the key nutrient-sensing enzyme AMP-activated protein kinase (AMPK) (or SNF1

319

in yeast) 52-53. In its active form, AMPK reduces fatty acid accumulation by initiating

320

mitochondrial fatty acid β-oxidation and inhibiting the activity of acetyl-CoA

321

carboxylase 1 (ACC1)

322

substrate (AMP). In hepatocytes, the subunit AMPD2 can regulate glucose metabolic

323

homeostasis functions including maintenance of AMPK

324

demonstrated that activated AMPD represses AMPK activity and fatty liver in

325

hibernating animals

326

inhibition 55. These results indicate that AMPD is involved in the regulation of energy

327

balance through opposing the activity of AMPK, and that alterations in energy

328

homeostasis caused by AMPD activation may reprogramme metabolite flux and

329

enhance the accumulation of fatty acids and their precursors. Considering the spectrum

330

of AMPK function, future studies could focus on the relationship between fatty acid

331

accumulation and energy balance.

332

In this study, we revealed the mechanism behind the classical model initially put

333

forward by Ratledge

334

accumulation under nitrogen-limited conditions. Specifically, we showed that

335

homologous overexpression of MaAMPD in M. alpina altered the intracellular energy

336

balance (reflected by AMP level reduction and TCA cycle inhibition) and resulted in

53.

23.

22,

Importantly, AMPD and AMPK compete for the same

54.

Another study has

Moreover, metformin activates AMPK through AMPD

demonstrating how activated AMPD affects fatty acids


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metabolite reprogramming, and provided more citrate as precursors for lipid

338

biosynthesis. In conclusion, by using various methods to examine the effects of AMPD

339

overexpression on lipid accumulation, and coupling lipogenesis with energy balance

340

and nutrient levels, this study provides new insights into lipid metabolism in

341

Mortierealla alpina under nitrogen-limited conditions.

342



343

 Acknowledgment

344

This research was supported by the National Natural Science Foundation of China

345

(31722041, 31530056), the Fundamental Research Funds for the Central Universities

346

(JUSRP51702A), the Natural Science Foundation of Jiangsu Province (BK20160172),

347

the Project funded by China Postdoctoral Science Foundation (2017M611701), the

348

Postdoctoral Science Foundation of Jiangsu Province (1701061C), the National First-

349

class Discipline Program of Food Science and Technology (JUFSTR20180102), the

350

Jiangsu Province “Collaborative Innovation Center for Food Safety and Quality

351

Control”, and the Postgraduate Research & Practice Innovation Program of Jiangnan

352

University (JNKY19_010).

353

 Notes

354

The authors declare no competing ﬁnancial interest.

355

 Supporting Information description

356

Figure S1. Fatty acids yield and lipid-free biomass in M. alpina-MaAMPD

357

(a) Fatty acids yield (g/L); (b) Lipid-free biomass (g/L). Solid black circle referred to

358

M. alpina prototrophic strain (control group) and solid grey square referred to

359

recombinant strain M. alpina-MaAMPD.

360

Figure S2. Principal component analysis (PCA) of the two strains at four time

361

points


Page 18 of 39

Page 19 of 39


362

The PCA score plot showing the discrimination of M. alpina and M. alpina-MaAMPD

363

at different time points. Circle referred to M. alpina and triangle refers to M. alpina-

364

MaAMPD. Different colour referred to different time point.

365

Table S1. Primers used for MaAMPD amplification and RT-qPCR analysis

366

367

 AUTHOR INFORMATION

368

Corresponding Author

369

(Xin Tang) E-mail: [email protected]

370

(Haiqin Chen) E-mail: [email protected]

371

Telephone: 0510-85197239

372

ORCID

373

Xin Tang: 0000-0001-6391-3075

374

Haiqin Chen: 0000-0002-6850-3160



375

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376

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535

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536

537

 Figure Caption

538

Figure 1. Lipid accumulation model under nitrogen limited culture condition

539

AMPD, AMP deaminase; IDH, mitochondrial NAD+-isocitrate dehydrogenase; ACL:

540

ATP: citrate lyase (ACL)；ACC: acetyl CoA carboxylase; FAS: fatty acid synthase.

541

The limited nitrogen activated AMPD, which has the ability to break down AMP to

542

IMP and NH4+, and decreased AMP concentration inhibits the activity of NAD+-

543

dependent isocitrate dehydrogenase (IDH), leading to the accumulation of isocitrate and

544

citrate, which will then be cleaved by cytosolic ACL to acetyl-CoA, another important

545

precursor for fatty acids synthesis. In this way, lipid begins to accumulate in the cell.

546

Figure 2. PCR identification of eight M. alpina-MaAMPD recombinant strains.

547

(a) M, marker; NC, negative control (M. alpina prototrophic strain); 1-8, eight M.

548

alpina-MaAMPD recombinant strains; PC, positive control (plasmid pBIG2-ura5s-

549

MaAMPD).

550

(b) Plasmid pBIG2-ura5s-MaAMPD. The primer Hispro F1 was located on promotor

551

His550 and TrpCR1 was located on terminator TrpCR1. Two fragments were amplified,

552

the ura5s (880 bp) and targeted gene MaAMPD (2895 bp).

553

Figure 3. Fatty acids content of eight M. alpina-MaAMPD recombinant strains.

554

Fatty acids content was measured in form of fatty acid methyl ester (FAME), and

555

calculated as the content of dry cell weight (DCW), and number 1-8 referred to eight


Page 26 of 39

Page 27 of 39


556

transformants of M. alpina-MaAMPD recombinant strain. Different letters on the

557

histogram represented that there were significant differences among data, P＜0.05. For

558

each group, three biological repetitions were used.

559

Figure 4. Biomass and total fatty acids content in M. alpina-MaAMPD

560

（a）Total biomass (g/L); (b) Fatty acids content (DCW%); (c) Fatty acids synthesis

561

efficiency (fatty acids yield / lipid-free biomass). Solid black circle referred to M. alpina

562

prototrophic strain (control group) and solid grey square referred to recombinant strain

563

M. alpina-MaAMPD. For each group, three biological repetitions were used.

564

Figure 5. Residual glucose and ammonium in culture medium

565

Solid line referred to glucose concentration and dot-dashed line referred to NH4+

566

concentration. Black circle referred to M. alpina prototrophic strain (control group),

567

and grey square referred to recombinant strain M. alpina-MaAMPD.

568

Figure 6. Changing fold of gene transcription levels at stationary phase (96 h)

569

Control group referred to M. alpina prototrophic strain (M. alpina CCFM 505); AMPD,

570

AMP deaminase; ACC: acetyl CoA carboxylase; ACL, ATP-citrate lyase, there existed

571

two subunits in M. alpina showing as ACL1 and ACL2; NAD+-IDH, mitochondrial

572

NAD+-isocitrate dehydrogenase, there existed three subunits in M. alpina showing as

573

NAD+-IDH1, NAD+-IDH2 and NAD+-IDH3; NADP+-IDH, cytoplasmic NADP+-

574

isocitrate dehydrogenase, there existed three subunits in M. alpina showing as NADP+-

575

IDH1, NADP+-IDH2 and NADP+-IDH3. Different letters on the histogram represented

576

that there were significant differences among data, P ＜ 0.05. For each group, three

577

biological repetitions were used.



578

Figure 7. Enzymatic activity at stationary phase (96 h)

579

AMPD, AMP deaminase; ACL, ATP-citrate lyase; NAD+-IDH, mitochondrial NAD+-

580

isocitrate dehydrogenase; NADP+-IDH, cytoplasmic NADP+-isocitrate dehydrogenase.

581

Different letters on the histogram represented that there were significant differences

582

among data, P＜0.05. For each group, three biological repetitions were used.

583 584

Figure 8. Networks of central carbon metabolism related to lipid accumulation in

585

M. alpina-MaAMPD

586

The difference of intracellular metabolites content in the two strains was shown by fold

587

change and every control group (black column) was defined as “1”. Different columns

588

in each graph referred to four time points (36 h, 96 h, 168 h and 240 h).

589

Figure 9. Heatmap of the intracellular metabolites in M. alpina-MaAMPD

590

Heatmap was drew to show the content and clustering of intracellular metabolites

591

between the two strains. The darkness of the color indicates the ranking: the darkest red

592

marked the increased value, the darkest green marked the decreased.

593



TOC Graphic


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Table 1. Fatty acids compositions and proportion Fatty acids composition

36 h

96 h

168 h

240 h

M. alpina

M. alpina-MaAMPD

M. alpina

M. alpina-MaAMPD

M. alpina

M. alpina-MaAMPD

M. alpina

M. alpina-MaAMPD

C14:0

1.59±0.05

1.58±0.05

2.39±0.04

2.2±0.03

2.34±0.02

2.36±0.03

1.51±0.07

1.56±0.06

C16:0

15.37±0.22

14.18±0.02

12.95±0.29

12.33±0.21

12.67±0.09

12.79±0.16

14.33±0.24

13.94±0.19

C18:0

11.68±0.08

12.94±0.25

11.28±0.13

11.41±0.16

12.43±0.09

12.36±0.16

13.97±0.23

13.96±0.13

C18:1

13.19±0.12

13.38±0.17

12.16±0.14

11.75±0.34

11.87±0.28

12.13±0.08

14.6±0.16

13.64±0.17

C18:2

5.33±0.21

5.49±0.31

11.81±0.24

11.91±0.34

13.2±0.13

13.2±0.14

16.52±0.4

17.11±0.34

C18:4

7.24±0.44

5.97±0.02

5.55±0.05

5.55±0.04

5.54±0.23

5.36±0.02

4.19±0.06

4.44±0.06

C20:0

0.57±0.02

0.7±0.01

1.15±0.07

1.16±0.04

1.31±0.05

1.35±0.02

0.98±0.05

0.92±0.02

C20:3

6.43±0.08

6.01±0.07

4.42±0.14

4.39±0.13

4.77±0.15

4.65±0.05

2.69±0.06

2.59±0.06

C20:4

29.24±0.66

31.17±0.1

27.59±0.26

28.55±0.29

25.84±0.45

25.53±0.11

24.7±0.7

25.62±0.41

C22:0

1.53±0.25

1.56±0.01

2.18±0.1

2.14±0.11

2.38±0.11

2.45±0.04

1.36±0.03

1.35±0.03

Note: Fatty acids that accounting for more than 1% of total fatty acids were listed



Figure 1. Lipid accumulation model under nitrogen limited culture condition


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Figure 2. PCR identification of eight M. alpina-MaAMPD recombinant strains.



Figure 3. Fatty acids content of eight M. alpina-MaAMPD recombinant strains.


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Figure 4 Growth and total fatty acids content in M. alpina-MaAMPD



Figure 5. Residual glucose and ammonium in culture solution


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Page 35 of 39


Figure 6. Changing fold of gene transcription levels at stationary phase (96 h)



Figure 7. Enzyme activity at stationary phase (96 h)


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Figure 8. Networks of central carbon metabolism related to lipid accumulation in M. alpina-MaAMPD



Figure 9. Heat map of the intracellular metabolites in M. alpina-MaAMPD


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 TOC Graphic


Role of AMP deaminase during fatty acids accumulation in

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