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Comparative proteome analysis between high lipid-producing strain Mucor circinelloides WJ11 and low lipid-producing strain CBS 277.49 Xin Tang, Haiqin Chen, Zhennan Gu, Hao Zhang, Yong Q. Chen, Yuanda Song, and Wei Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00935 • Publication Date (Web): 30 May 2017 Downloaded from http://pubs.acs.org on June 8, 2017
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Journal of Agricultural and Food Chemistry
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Comparative proteome analysis between high
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lipid-producing strain Mucor circinelloides WJ11 and
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low lipid-producing strain CBS 277.49
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Xin Tang,† Haiqin Chen,*,† Zhennan Gu,† Hao Zhang,† Yong Q. Chen,† Yuanda
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Song,*,†,‡ and Wei Chen†,§
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†
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Technology, Jiangnan University, Wuxi, P.R. China
State Key Laboratory of Food Science and Technology, School of Food Science and
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‡
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Food Science, Shandong University of Technology, Zibo, P.R. China
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§
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and Business University, Beijing, P.R. China
Colin Ratledge Center for Microbial Lipids, School of Agriculture Engineering and
Beijing Innovation Center of Food Nutrition and Human Health, Beijing Technology
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* Correspondence authors: Haiqin Chen ＆ Yuanda Song
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E-mail:
[email protected] (Haiqin Chen) ＆
[email protected] 17
(Yuanda Song)
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Telephone：+86 510 85197239 (Haiqin Chen) +86 510 85197130 (Yuanda Song)
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ABSTRACT: Mucor circinelloides is one of few oleaginous fungi that produce a
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useful oil rich in γ-linolenic acid but it usually only produces < 25% total lipid.
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Nevertheless, we isolated a new strain WJ11 that can produce up to 36% lipid of cell
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dry weight. In this study, we have systematically analyzed the global changes in
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protein levels between the high lipid-producing strain WJ11 and the low
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lipid-producing strain CBS 277.49 (15%, lipid/cell dry weight) at lipid accumulation
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phase through comparative proteome analysis. Proteome analysis demonstrated that
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the branched-chain amino acid and lysine metabolism, glycolytic pathway and
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pentose phosphate pathway in WJ11 were up-regulated, while the activity of
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tricarboxylic acid cycle and branch point enzyme for synthesis of isoprenoids were
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retarded compared with CBS 277.49. The coordinated regulation at proteome level
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indicates more acetyl-CoA and NADPH are provided for fatty acid biosynthesis in
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WJ11 compared with CBS 277.49.
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KEYWORDS: Comparative proteome, Lipid accumulation, Mucor circinelloides,
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Amino acid metabolism, Carbon metabolism
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INTRODUCTION
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Polyunsaturated fatty acids (PUFAs) play crucial structural and functional roles in
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human, and they have been recommended for the prevention or treatment of some
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important diseases.1 Consequently, the lipids rich in PUFAs are demanded in the food
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industry as food additives. Recently, microbial lipids are considered as the major
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sources of nutraceuticals in the form of PUFAs and gain more interest.2
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Among various oleaginous microorganisms, Mucor circinelloides has been
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considered as a crucial model organism for lipid accumulation research due to its
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ability to produce γ-linolenic acid (GLA, 18:3; n-6), which is an important PUFA that
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may have beneficial effects for the treatment of premenstrual tension, atopic
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dermatitis and some other diseases.3 Furthermore, its genome data4 and genetic tools5
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are available. Indeed, M. circinelloides is the first microorganism to be used
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commercially to produce an oil rich in GLA for human consumption. However, the
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commercialized strain only produced 25% of its cell biomass as an extractable oil, and
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the process lasted only 6 years as high GLA-producing plant species appeared in the
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market.6
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Although lots of work has been done to investigate the molecular mechanism of
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lipid accumulation in M. circinelloides7-13, a system-level analysis of the mechanism
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underlying lipid accumulation in this fungus is still lacking. Our previous study
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demonstrated that M. circinelloides WJ11 can produce lipid up to 36% (w/w) of cell
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dry weight (CDW), which is 2.3-fold more than in CBS 277.49 (15% lipid of CDW),
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and the activities of the biochemical pathways related to fatty acid biosynthesis in 3
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these two strains have been analyzed.14 After that, we have also processed the
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comparative genome analysis and
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two strains.4, 15 Even though these previous studies have revealed some insights on the
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distinct lipid accumulation between strains WJ11 and CBS 277.49, the exact
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molecular mechanism related to the various lipid accumulation in the two strains is
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still not very clear.
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C-labeled metabolic flux analysis between these
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Lipid accumulation is a complicated cellular process involving numerous metabolic
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pathways and proteomics is a powerful tool to investigate this complex process.16
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Proteomics can find new functions of gene products since it comprehensively
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represents the gene product, translational rate and even post-translational
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modifications.17 Accordingly, comparative proteome have been explored to enhance
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insights on lipid metabolism in microalgae, yeasts and fungi.16, 18-21
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Identification of proteins up/down-regulated between high lipid-producing strain
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WJ11 and low lipid-producing strain CBS 277.49 is important to study the molecular
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mechanisms of lipid accumulation in M. circinelloides. Therefore, in this study, we
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compared the proteome between these two strains at lipid accumulation phase in order
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to reveal new insights on the molecular basis for the lipid accumulation in M.
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circinelloides.
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MATERIALS AND METHODS
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Microorganism and cultivation. M. circinelloides WJ11 previously was
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isolated in our laboratory from Jiangnan University soil and preserved in China
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Center for Type Culture Collection (CCTCC No. M 2014424).14 M. circinelloides 4
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CBS 277.49 was purchased from Centraalbureau voor Schimmelcultures (CBS).
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Approx. 106 spores was cultivated in 150 mL K & R medium [30 g/L glucose, 3.3 g/L
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diammonium tartrate, 7.0 g/L KH2PO4, 2.0 g/L Na2HPO4, 1.5 g/L MgSO4·7H2O, 1.5
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g/L yeast extract, 0.1 g/L CaCl2·2H2O, 8 mg/L FeC13·6H2O, 1 mg/L ZnSO4·7H2O,
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0.1 mg/L CuSO4·5H2O, 0.1 mg/ L Co(NO3)2·6H2O and 0.1 mg/L MnSO4·5H2O.
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Analytically Pure, Sinopharm Chemical Reagent Co., Ltd, China]7 held in 1 L baffle
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flasks for 24 h at 30 °C with shaking at 150 rpm and then inoculated at 10% (v/v) into
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2 L fermenters (BioFlo 115, New Brunswich Scienfific, USA) containing 1.5 L
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modified K & R medium (80 g/L glucose and 2 g/L diammonium tartrate, others were
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same as K & R medium). Fermenters were maintained at 30 °C with aeration at 0.5
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v/v min-1 and stirred at 700 rpm. Moreover, the pH was controlled at 6.0 through
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auto-addition of 4 M KOH or 2 M H2SO4.
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Measurement of cell dry weight (CDW), glucose/ammonium
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concentration and lipid accumulation. The mycelia of strains WJ11 and CBS
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277.49 were periodically (6 h, balanced growth phase; 24 h, fast lipid accumulation
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phase; 60 h, slow lipid accumulation phase) harvested by filtration with a Buchner
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funnel and washed with distilled water. Then the collected mycelia were frozen at
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-80 °C, freeze-dried and the cell dry weight was measured by weighing method. The
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glucose concentration in the culture medium was determined through the glucose
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oxidase kit (Shanghai Rongsheng Biotech Co., Ltd., China) and ammonium
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concentration was measured by the indophenol test.22 The lipid extraction and
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analysis were measured as described in our previous research.14 5
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Protein extraction, two-dimensional electrophoresis (2-DE) and
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image analysis. The mycelia of strains WJ11 and CBS 277.49 were respectively
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collected at the fast lipid accumulation phase (at 24 h) by filtration, washed with cold
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distilled water and then flash-frozen in liquid nitrogen. For each strain, protein
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extraction was carried out according to a modified method of Liu et al.23 and Chen et
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al.24 2-DE was performed as described previously.21 Six 2-DE gels (three independent
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analytical replicate gels for each strain) were scanned by GE Healthcare Image
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Scanner LabScan. Spot detections, gel matching and group analysis of the gels were
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carried out by the software PDQuest 8.0. Before quantitative analyses, the
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normalizing of spot quantities in all gels was performed through total valid spot
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intensity. The mean quantity of each spot was calculated in these two strains, and the
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spots showing a mean value that changed more than 1.5-fold or less than 0.67-fold
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(p
