Identification and Characterization of Diacylglycerol

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Identification and characterization of diacylglycerol acyltransferase from oleaginous fungus Mucor circinelloides Luning Zhang, Huaiyuan Zhang, and Yuanda Song J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04295 • Publication Date (Web): 20 Dec 2017 Downloaded from http://pubs.acs.org on December 21, 2017

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Journal of Agricultural and Food Chemistry

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Identification and characterization of diacylglycerol acyltransferase from

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oleaginous fungus Mucor circinelloides

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Luning Zhanga,b,#, Huaiyuan Zhanga,#, Yuanda Songa,*

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a

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Shandong University of Technology, Zibo, 255049, Shandong, People's Republic of China

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b

Shanghai Lida Polytechnic Institute, shanghai 201609, People’s Republic of China

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#

contributed equally to this work

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*

Colin Ratledge Center for Microbial Lipids, School of Agricultural Engineering and Food Science,

Corresponding author: Yuanda Song, E-mail: [email protected]

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ACS Paragon Plus Environment


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Abstract

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Acyl-CoA:diacylglycerol acyltransferase (DGAT) is an pivotal regulator of triacylglycerol (TAG)

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synthesis. The oleaginous fungus Mucor circinelloides has four putative DGATs, McDGAT1A,

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McDGAT1B, McDGAT2A and McDGAT2B, classified into the DGAT1 and DGAT2 subfamilies,

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respectively. To identify and characterize DGATs in M. circinelloides, these four genes were expressed

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in Saccharomyces cerevisiae H1246 (TAG-deficient quadruple mutant), individually. TAG biosynthesis

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was restored only by the expression of McDGAT2B, and TAG content was significantly higher in the

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mutants with McDGAT2B expression than in a S. cerevisiae mutant with endogenous DGA1

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expression. McDGAT2B prefers saturated fatty acids to monounsaturated fatty acids and has an

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obvious preference for C18:3 (ω-6) according to the results of substrate preference experiments.

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Furthermore, only the mRNA expression pattern of McDGAT2B correlated with TAG biosynthesis

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during a fermentation process. Our experiments strongly indicate that McDGAT2B is crucial for TAG

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accumulation, suggesting that it may be an essential target for metabolic engineering aimed at

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increasing lipid content of M. circinelloides.

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Keywords: Diacylglycerol acyltransferase; Mucor circinelloides; Oleaginous; Triacylglycerol

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1. Introduction

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Triacylglycerols (TAGs) is the major source of oils for nutrition and industrial uses or a promising

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renewable feedstock for biofuel production.1 An alternative to plants and animals as a source of oils,2

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the intracellular lipid content in these oleaginous microorganisms is more than 20% of their dry cell

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weight (DCW).3 The sn-glycerol-3-phosphate (G-3-P) pathway or Kennedy pathway is the overriding

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route to TAG biosynthesis, and in the 1950s it was first described by the team of Professor Eugene

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Kennedy; via this pathway, more than 90% of liver TAGs are produced.4 Acyl-CoA:diacylglycerol

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acyltransferase (DGAT) catalyzes the terminal acylation reaction, which converts diacylglycerol (DAG)

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to TAG. DGAT is also known as an essential regulator of this pathway.5 Four DGAT families have been

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identified: DGAT1, DGAT2, WS/DGAT, and DGAT3.6 DGAT1 and DGAT2 are transmembrane

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proteins, are located in the endoplasmic reticulum and cytosolic lipid droplets, and are widespread

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among plants, animals and microorganisms. No homology was found between DGAT1 and DGAT2.

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WS/DGATs are bifunctional membrane-associated enzymes in bacteria that possess both wax ester

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synthase (WS) and DGAT activities.6, 7 DGAT3 is a soluble DGAT-related enzyme, and it has been

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identified in Arachis hypogaea, A. Thaliana, Glycine and Oryza sativa.6,8

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Many studies have been focused on DGAT function for manipulation of the TAG synthesis pathway to

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improve TAG content and accumulate desired fatty acids with superior industrial or nutritional value in

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several microorganisms. DGAT2 is likely to be the most effective enzyme for improving lipid

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accumulation and has been transferred into various cells to produce and store amount fatty acids.

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Expression of soluble forms of Yarrowia lipolytica DGAT2 in Escherichia coli integrates a large

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number of saturated fatty acids in TAG.9 Overexpression of DGAT2 in Yarrowia lipolytica leads to a

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3-fold increase in lipid accumulation.10 Neutral-lipid content increases by 35%, and more



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eicosapentaenoic acid (EPA) is cooperated when DGAT2 is overexpressed in marine diatom

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Phaeodactylum tricornutum.11

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Oleaginous fungus Mucor circinelloides can synthesize and accumulate large amounts of TAGs and is

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rich in γ-linolenic acid (C18:3, GLA).12, 13 Nonetheless, no information is available about the genes and

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functions of DGAT in M. circinelloides. So all putative DGAT genes was identified and characterized

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in M. circinelloides in this study, and the function of DGATs was explored in TAG synthesis in this

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oleaginous fungus.

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2. Materials and methods

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2.1 Microorganism strains and the culture conditions

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M. circinelloides CBS 277.49 was used in this study. S. cerevisiae mutant strain H1246 (∆dga1 ∆lro1

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∆are1 ∆are2) was employed for functional complementation by M. circinelloides putative DGAT genes,

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which was kindly provided by Professor Stymne from Swedish University of Agricultural Sciences. E.

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coli TOP10 served for plasmid construction.

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M. circinelloides CBS 277.49 was grown in a 2-L fermentor for lipid accumulation analysis with a

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nitrogen-limited medium consisting of (g/L): glucose 80, KH2PO4 7.0, MgSO4·7H2O 1.5, L-ammonium

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tartrate 2.0, Na2HPO4 2.0, yeast extract 1.5, MnSO4·5H2O 0.0001, Co(NO3)2·6H2O 0.0001,

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CaCl2·2H2O 0.1, CuSO4·5H2O 0.0001, , ZnSO4·7H2O 0.001 and FeCl3·6H2O 0.008.14

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For functional expression of the DGAT genes in S. cerevisiae H1246, transformants harboring resulting

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plasmids were cultivated in a synthetic minimal medium (SC-Ura−) according to the protocol

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(Invitrogen) consisting of (g/L): raffinose 20, yeast nitrogen base (with ammonium sulfate) 6.7 and


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appropriate amino acid concentrations at 30°C for 4 days.

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2.2 Construction of S. cerevisiae DGATs expressing strains

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Molecular manipulations were performed by standard protocols. Total RNA of M. circinelloides CBS

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277.49 was isolated by TRIzol Reagent (TaKaRa, Dalian, China) and then was reverse-transcribed into

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cDNA. The DGAT genes of M. circinelloides were amplified using cDNA as a template for PCR with

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specific primers (Table S1), and then cloned into the plasmid pYES2/NT C to construct expression

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vector pYES2-McDGAT1A, pYES2-McDGAT1B, pYES2-McDGAT2A, and pYES2-McDGAT2B,

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separately. The plasmids used in this study are shown in Table 1.

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The DGAT expressing plasmids were transfected into S. cerevisiae H1246 (TAG-deficient) by the

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polyethylene glycol/lithium acetate method.15 The mutant strains harboring empty vector pYES2/NT C

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or pYES2-DGA1 served as the negative or positive control, respectively. Yeast transformants were

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selected on SC-Ura− plates. The recombinant DGAT proteins were expressed under the control of the

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GAL1 promoter according to the pYES2 User manual (Invitrogen). Their optical density at 600 nm

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(OD600) was measured to monitory the growth curve of mutant strains. Linoleic acid (C18:2, Sigma,

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99% purity) and γ-linolenic acid (C18:3, Sigma, 99% purity) were dissolved in Tween 80 and added

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into the medium with final concentration of fatty acid of 1mM to determine the fatty acid preference of

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McDGATs with S. cerevisiae endogenous DGA1 as the control. The yeast cell growth curve was also

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constructed, simultaneously. The cells were harvested by centrifugation (5000 g for 5 min), washed

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three times with double-distilled water, and subjected to total fatty acid (TFA) and TAG analysis.

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2.3 Lipid extraction and fatty acid analysis



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20 mg of lyophilized yeast cells were used to extract the total lipids with the solvent mixture

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CHCl4:CH3OH (2:1, v/v) according to the modified method of Bligh and Dyer.16 Thin layer

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chromatography (TLC) was electively employed for separations of TAGs from total lipids using a

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solvent mixture of hexane:diethyl ether:acetic acid (50:50:1, v/v/v). Individual TAG spots were

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visualized by exposure to iodine vapor, then scraped off the plates and subjected to methyl

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esterification using a sodium hydroxide solution in methanol (0.5 mol/L) at 60oC for 3 hours. TFA

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isolation and fatty acid composition analysis were conducted in terms of fatty acid methyl esters as

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described by Zhang et al.17 using C15:0 as an internal standard. Each sample was analyzed in triplicate.

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The differentiate lipid accumulation and relative amounts of lipid in yeast cells were also determined

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with Nile red method via fluorescence microscope according to the method described previously.18

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2.4 M. circinelloides CBS 277.49 fermentation and its DGATs expression pattern

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For analysis of the relationship between the putative DGAT genes expression pattern and lipid

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accumulation in oleaginous fungus M. circinelloides, fermentation by the cells was implemented in a 2

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L fermentor containing 1.5 L of the nitrogen limited medium (0.5 vvm aeration, pH 6.0, 30°C, 700 rpm

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agitation). Biomass and lipid accumulation were analyzed at various time points. Total RNA was also

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extracted and analyzed by real-time quantitative PCR (RT-qPCR) for expression of the putative DGAT

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genes, McDGAT1A, McDGAT1B, McDGAT2A, and McDGAT2B. The SYBR Green I Master Mix Kit

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(TaKaRa) was used for RT-qPCR, and 18S cDNA served as an internal control. And then gene

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expression analyses were performed by the 2−ΔΔCT Method.19 Each sample was analyzed in triplicate.

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2.5 Statistical analysis


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This analysis of the obtained data was performed with the help of the SPSS 16.0 software for Windows

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(SPSS Inc., Chicago, IL). The mean values and the standard errors were calculated from the data

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obtained from three biological replicates. The difference between the means was evaluated by Student’s

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t test, and those with P < 0.05 were considered statistically significant.

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3. Results

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3.1 Protein sequence features of four putative M. circinelloides DGATs

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According to the annotated genome of oleaginous fungus M. circinelloides, four putative DGAT gene

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sequences, McDGAT1A (gene ID: 167165), McDGAT1B (gene ID: 144132), McDGAT2A (gene ID:

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154800/154801), and McDGAT2B (gene ID: 187167), were retrieved from the published genome

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information

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McDGAT2A and McDGAT2B are 487, 412, 362 and 348 amino acid residues in length, respectively,

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and their protein molecular weights are in the range of 40–60 kDa. The theoretical pI values of

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McDGAT1A, McDGAT1B, McDGAT2A, and McDGAT2B are 8.76, 9.36, 9.30 and 9.19, respectively.

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McDGAT2A and McDGAT2B were classified as unstable according to the instability index (46.49 and

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42.90; Table S2). McDGAT1A and McDGAT1B have more regions containing highly probable

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transmembrane helices than McDGAT2A and McDGAT2B (Table S3).

(http://genome.jgi.doe.gov/Rhoto1/Rhoto1.home.html).

McDGAT1A,

McDGAT1B,

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Four M. circinelloides DGATs and 30 representative DGAT amino acid sequences, covering two major

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groups: DGAT1 and DGAT2, were used to derive the phylogenetic tree (Fig. 1). McDGAT1A and

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McDGAT1B proteins grouped into the DGAT1 clade. McDGAT2A and McDGAT2B clustered within

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the DGAT2 family with strong bootstrap support. Multiple-sequence alignment of M. circinelloides



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DGATs was performed with other DGAT amino acid sequences (Figs. 2 and 3). McDGAT1A and

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McDGAT1B harbor seven motifs with remarkable sequence conservation within the DGAT1 subfamily:

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Motif 1 (GL block), Motif 2 (KSR block), Motif 3 (GL block), Motif 4 (PTR block), Motif 5 (QR

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block), Motif 6 (LWLFFEFDRFYWWNWWNPPFSHP block), and Motif 7 (GL block).20 McDGAT2A

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and McDGAT2B were found to contain six highly conserved motifs that were identified as signature

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motifs within the DGAT2 subfamily, namely: Motif 1 (PH Block), Motif 2 (PR Block), Motif 3 (GGE

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Block), Motif 4 (RGFA Block), Motif 5 (VPFG Block), and Motif 6 (G Block).

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3.2 McDGAT2B restored TAG accumulation in the S. cerevisiae mutant

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To test whether the four putative M. circinelloides DGAT proteins showed the DGAT activity,

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McDGAT1A, McDGAT1B, McDGAT2A, and McDGAT2B were expressed individually in S.

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cerevisiae mutant strain H1246 (TAG-deficient quadruple) to obtain recombinant strains HY1A, HY1B,

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HY2A, and HY2B. In addition, empty vector pYES2/NT C and pYES2-DGA1 harboring endogenous

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DGA1 from S. cerevisiae were transformed into S. cerevisiae H1246 as negative (HY1) and positive

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controls (HY2), respectively (Table 1). Yeast cells were harvested for Nile Red staining and lipid

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isolation after cultivation in flasks for 96 h in the synthetic minimal medium. Nile Red staining

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represents a simple method for monitoring lipid accumulation based on a linear relation between the

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amount of neutral lipids in the cell and fluorescence intensity of the Nile Red dye. The oil bodies were

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visualized only in strains HY2 and HY2B (Fig. 4A). Total lipids were separated by TLC, and the

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results exhibited that TAG biosynthesis was restored in S. cerevisiae strains HY2 and HY2B. The

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strains expressing McDGAT1A, McDGAT1B, or McDGAT2A did not accumulate TAG. The TAG

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content of S. cerevisiae strain HY2B was up to 6.6% of DCW, which is higher than that of mutant


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strain HY2 (3.8%). The trend of TFA content was similar to that of TAG content in the respective

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strains, and TFA biosynthesis in strain HY2B (13.2%) was also more abundant than that in HY2

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(11.6%; Fig. 4B).

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3.3 Substrate preference analysis of M. circinelloides DGAT

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To detect the possible substrate preference of McDGAT2B for unsaturated fatty acid that are naturally

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present in M. circinelloides, S. cerevisiae mutant strains were supplied with C18:2 or C18:3 into the

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synthetic minimal medium. The growth curve (Fig. 5) and lipid biosynthesis (Fig. 4C and 4D) of the

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mutant strains were analyzed. The addition of C18:3 did not change the growth curve of S. cerevisiae

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mutant strains, and final OD600 reached ~14.0 for strains HY2A and HY2B. In contrast, the growth

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curve with the C18:2 supplement significantly differed between DGAT-active strains and DGAT-null

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strains. The yeast cell proliferation in DGAT-null strains was suppressed in the early period of

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cultivation, and the biomass was significantly reduced in the later period of the culture cycle with

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addition of C18:2; final OD600 reached only ~6.0 in DGAT-null strain HY2A; however, it was 12.0 in

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DGAT-active strain HY2B (Fig. 5). These results revealed that DGAT-null strains were sensitive to

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C18:2, which greatly inhibited cell growth, but the DGAT-active strain was not affected by C18:2.

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Intracellular lipid was extracted from lyophilized yeast cells at the end of cultivation and separated by

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TLC. Addition of C18:2 or C18:3 increased the TFA and TAG contents in strain HY2B (Fig. 4C and

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4D), and the amounts of TAG and TFA in strain HY2B with the C18:2 supplement were up to 8.7% and

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15.6%, respectively. The supplement of unsaturated fatty acid also altered the fatty acid profile of

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TAGs (Table 2) in S. cerevisiae mutant strains. Compared with the fatty acid composition of TAGs in



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HY2, McDGAT2B preferred saturated fatty acids (C16:0 and C18:0) to monounsaturated fatty acids

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(C16:1 and C18:1). In these supplementation experiments with fatty acids, C18:2 accumulated more

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than 25% of total fatty acids in S. cerevisiae HY2 (29.64%) and HY2B (26.69%). Supplementation

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with C18:3 obviously effected the fatty acid composition of TAGs. There was low C18:3 content in S.

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cerevisiae HY2 (1.23%), however, the concentration of C18:3 was up to 8.87% in S. cerevisiae HY2B

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(Table 2).

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3.4 The relationship between the DGATs mRNA expression pattern and lipid accumulation in M.

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circinelloides

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To explore the roles of DGATs in the accumulated process of total lipids in M. circinelloides, the

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relationship between the DGATs mRNA expression pattern and lipid biosynthesis was determined by

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mycelia fermentation with the nitrogen-limited medium. During formation, biomass increased

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continuously until the glucose in the medium was exhausted, and final biomass was up to 12 g/L (Fig.

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S1). TFA and TAG contents enhanced sharply when nitrogen was exhausted in the medium. The final

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TFA and TAG contents were ~11% and 9% of DCW, respectively. TFA and TAG production levels

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were up to 1.32 and 1.08 g/L (Fig. 6A). The fatty acid profile of TAGs was analyzed by gas

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chromatography, and the results are shown in Table 3. The concentration of C18:3 in TAGs gradually

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increased up to ~20% of total fatty acid. In addition, the transcription pattern of M. circinelloides

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DGAT genes during this cultivation experiment was analyzed by RT-qPCR (Fig. 6B). The mRNA levels

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of McDGAT2A, McDGAT1A, and McDGAT1B were stable in the early period of fermentation and

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markedly increased in the later period of fermentation. The mRNA expression pattern of these three

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genes did not correlate with the process of lipid accumulation. Nevertheless, the transcription of


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McDGAT2B increased significantly when nitrogen in the medium was exhausted and continued to

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increase for 24-36 h, reached a peak at 48 h, and decreased to a low level at 60 h. The transcriptional

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level of McDGAT2B correlated with the rate of lipid accumulation, which was not the case for

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McDGAT1A, McDGAT1B, and McDGAT2A.

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4. Discussion

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The last step of TAG formation was catalyzed by DGAT from DAG and fatty acyl-CoAs in the

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Kennedy pathway. M. circinelloides has four putative DGAT proteins, McDGAT1A and McDGAT1B

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belong to the DGAT1 family, McDGAT2A and McDGAT2B belong to the DGAT2 family. To confirm

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whether these four putative proteins showed the DGAT activity, their genes were expressed in

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S. cerevisiae H1246 (TAG-deficient quadruple mutant), respectively. As A. thaliana DGAT2 was only

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expressed under active form in H1246 strain when its sequence was optimized,21 the codon bias

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between M. circinelloides and S. cerevisiae was checked and their percent of rare codons was

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extremely low. The results of western blotting suggested that four putative DGAT proteins were

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successfully expressed in S. cerevisiae H1246. And the results of lipid analysis indicated that only

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McDGAT2B completely restored TAG biosynthesis in S. cerevisiae H1246, and the lipid content in S.

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cerevisiae HY2B was higher than that in S. cerevisiae HY2. TAG synthesis function as a crucial

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intracellular buffer for detoxifying excess unsaturated fatty acids was highlighted by Petschnigg et al.22

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Addition of exogenous C18:1 resulted in the dysregulation of lipid synthesis, massive proliferation of

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intracellular membranes, and ultimately cell death in mutant lacking neutral lipids. We observed that

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exogenous unsaturated fatty acid induced toxicity was not detectable in DGAT-active strains HY2B and

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HY2. Mutants lacking neutral lipids displayed delayed growth with C18:2 supplement, however, it was



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not sensitive to exogenous C18:3. Furthermore, we found that the mRNA level of McDGAT2B was

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correlated with the process of TAG accumulation in M. circinelloides, McDGAT2B was up-regulated

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with TAG accumulation, and it may functionally lead to the accumulation of large amounts of TAGs.

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These data suggest that McDGAT2B may participate in lipid biosynthesis and plays an important role

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in TAG accumulation in M. circinelloides.

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Metabolic engineering has been performed to improve lipid accumulation in various species.

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Overexpression of key enzymes of TAG biosynthesis pathway is one of such approaches. A

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relationship between DGAT activity and TAG content in the cells of Streptomyces sp. has been

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reported.23 Arabidopsis DGAT2 overexpression leads to a 3- to 9-fold increase in TAG accumulation24

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and overexpression of DGAT2 in Chlamydomonas results in 2.5-fold TAG upregulation.25 Lipid

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content increased from 12.33% to 18.76% of DCW after overexpression of DGAT2 in Brassica

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napus.26 In addition, a knockout of DGAT decreases TAG synthesis.27, 28 In Yarrowia lipolytica, total

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lipids per g DCW in a DGAT2 mutant were found to decrease to 36% of the wild-type level.27 Deletion

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of LPO1 and DGA1 in Y. lipolytica results in a ~70% loss of TAGs.28 All the above results suggest that

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DGAT may be an important rate-limiting step in TAG biosynthesis.

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A correlation was found between the McDGAT2B mRNA expression level and the TAG content in the

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cells of M. circinelloides in this study. McDGAT2B may play a huge role on TAG accumulation and

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functionally contribute to the accumulation of large amounts of TAGs in M. circinelloides. Further

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research is needed to test whether overexpression of DGAT genes alters lipid accumulation in M.

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circinelloides.


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Supporting information

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Primers (Table S1) used in this study and some results of bioinformatic analysis (Tables S2 and S3) and

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other experiments (Fig. S1).

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Funding

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This work was supported by the National Natural Science Foundation of China (No. 31271812 and

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31670064), the National High Technology Research and Development Program of China (863 Program

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No. 2012AA022105C), Taishan Industry Leading Talent Project, and starting grant from Shandong

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University of Technology.

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productivity in oleaginous Colletotrichum fungus through genetic transformation using the yeast

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CtDGAT2b gene under model-optimized growth condition. PLoS One. 2014, 9(11):e111253.

306 307 308 309

(19) Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2–∆∆ CT method. Methods. 2001, 25:402-408. (20) Cao, H. Structure-function analysis of diacylglycerol acyltransferase sequences from 70 organisms[J]. BMC Res. Notes. 2011, 4: 249.

310

(21) Ayme L, Baud S, Dubreucq B, Joffre F, Chadot T. Function and localization of the Arabidopsis

311

thaliana diacylglycerol acyltransferase DGAT2 expressed in yeast. PLoS One, 2014, 9: e92237.

312

(22) Petschnigg J, Wolinski H, Kolb D, Zellning G, Kurat C.F, Natter K, Kohlwein S.D. Good fat,

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essential cellular requirements for triacylglycerol synthesis to maintain membrane homeostasis in

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yeast. J Biol Chem, 2009, 284: 30981-30993.



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(23) Olukoshi, E.R.; Packter, N.M. Importance of stored triacylglycerols in Streptomyces: possible carbon source for antibiotics. Microbiology. 1994, 140:931-43.

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(24) Bouvier-Nave, P.; Benveniste, P.; Oelkers, P.; Sturley, S.L.; Schaller, H. Expression in yeast and

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tobacco of plant cDNAs encoding acyl CoA:diacylglycerol acyltransferase. Eur. J. Biochem. 2000,

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267:85-96.

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(25) Iwai, M.; Ikeda, K.; Shimojima, M.; Ohta, H. Enhancement of extraplastidic oil synthesis in

321

Chlamydomonas reinhardtii using a type-2 diacylglycerol acyltransferase with a phosphorus

322

starvation-inducible promoter. Plant Biotechnol. J. 2014, 12(6):808-819.

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(26) Ahmad, I.; Sharma, A.K.; Daniell, H.; Kumar, S. Altered lipid composition and enhanced lipid

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production in green microalga by introduction of brassica diacylglycerol acyltransferase 2. Plant

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Biotechnol. J. 2015, 13(4):540-550.

326 327

(27) Zhang, H.; Damude, H.G.; Yadav, N.S. Three diacylglycerol acyltransferases contribute to oil biosynthesis and normal growth in Yarrowia lipolytica. Yeast. 2012, 29(1):25-38.

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(28) Karin, Athenstaedt. YALIOE32769g (DGA1) and YALIOE1679g (LRO1) encode major

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triacylglycerol synthases of the oleaginous yeast Yarrowia lipolytica. Biochimica et Biophysica

330

Acta. 2011, 1811:587-596.

331


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332

Figure legends

333

Fig. 1 Phylogenetic analysis of homologous sequences of McDGAT from plants, fungi, and

334

microorganisms. Multiple sequences were aligned by means of the ClustalX 2.1 Multiple alignment

335

software. The phylogenetic tree was generated in the MEGA 6.06 software. Bootstrap values (shown at

336

the nodes) are expressed in percentages of 1000 replicates. At, Arabidopsis thaliana; Bn, Brassica

337

napus var. napus; Bt, Bos taurus; Ce, Caenorhabditis elegans; Cr, Chlamydomonas reinhardtii; Dm,

338

Drosophila melanogaster; Gm, Glycine max; Hs, Homo sapiens; Mc, Mucor circinelloides; Mm, Mus

339

musculus; Mr, Mortierella ramanniana; Nt, Nicotiana tabacum; Os, Oryza sativa Japonica; Rc,

340

Ricinus communis; Sc, Saccharomyces cerevisiae; Tm, Tropaeolum majus; Vf, Vernicia fordii; Vg,

341

Vernonia galamensis.

342

Fig. 2 Protein sequence alignment of McDGAT1A and McDGAT1B with DGAT1s from four

343

organisms. All protein sequences were obtained from GenBank. At, Arabidopsis thaliana; Ce,

344

Caenorhabditis elegans; Hs, Homo sapiens; Mc, Mucor circinelloides; Mm, Mus musculus.

345

Fig. 3 Protein sequence alignment of McDGAT2A and McDGAT2B with DGAT2s from six organisms.

346

All the protein sequences were obtained from GenBank. At, Arabidopsis thaliana; Ce, Caenorhabditis

347

elegans; Hs, Homo sapiens; Mc, Mucor circinelloides; Mm, Mus musculus; Mr, Mortierella

348

ramanniana; Sc, Saccharomyces cerevisiae.

349

Fig. 4 TFA and TAG synthesis in S. cerevisiae mutants cultivated in the synthetic minimal medium

350

with a fatty-acid supplement to a final concentration of 1.0 mM. Samples were harvested after 96 h of

351

cultivation for Nile Red staining and lipid analysis. A, Lipid bodies where neutral lipids accumulate

352

were visualized in yeast cells with the fluorescent dye Nile Red. BF, Bright-field image; FR, image of

353

Nile Red fluorescence. B, TFA and TAG content of S. cerevisiae mutants without a supplement; C, TFA



354

and TAG content of S. cerevisiae strains HY2 and HY2B with a C18:2 supplement; D, TFA and TAG

355

content of S. cerevisiae strains HY2 and HY2B with a C18:3 supplement.

356

Fig. 5 A growth curve of a S. cerevisiae mutant cultivated in the synthetic minimal medium with a

357

fatty-acid supplement to a final concentration of 1.0 mM (◊, no supplement; ■, added C18:2; ▲, added

358

C18:3). Data are presented as mean ± SD from triple biological replicates.

359

Fig. 6 The lipid accumulation and DGAT gene expression pattern in M. circinelloides. (A) TFA (■) and

360

TAG (▲) content of M. circinelloides cultivated in a fermentor. (B) Expression of DGAT genes in M.

361

circinelloides. Data are presented as mean ± SD from triple biological replicates.


Page 18 of 28

Page 19 of 28


Table 1 Plasmids and microorganism strains used in this study Plasmids pYES2/NT C pYES2-DGA1 pYES2-McDGAT1A pYES2-McDGAT1B pYES2-McDGAT2A pYES2-McDGAT2B

Ampicillin resistance, transformation vector for S. cerevisiae, stored in our lab Ampicillin resistance, S. cerevisiae DGA1 gene cloned into Hind Ш/EcoRI-cut pYES2/NT C for positive control Ampicillin resistance, M. circinelloides putative McDGAT1A gene cloned into Hind Ш/EcoRI-cut pYES2/NT C Ampicillin resistance, M. circinelloides putative McDGAT2A gene cloned into Hind Ш/BamHI-cut pYES2/NT C Ampicillin resistance, M. circinelloides putative McDGAT2A gene cloned into Hind Ш/BamHI-cut pYES2/NT C Ampicillin resistance, M. circinelloides putative McDGAT1A gene cloned into Hind Ш/EcoRI-cut pYES2/NT C

Strains E. coli Top10

E. coli host for DNA manipulations

M. circinelloides CBS 277.49

From our lab

H1246

Saccharomyces cerevisiae H1246 (MATα are1-∆::HIS3 are2-∆::LEU2 dga1-∆::KanMX4 lro1-∆::TRP1 ADE2)

HY1

S. cerevisiae H1246 harboring empty plasmid pYES2/NT C

HY2

S. cerevisiae H1246 harboring plasmid pYES2-DGA1

HY1A

S. cerevisiae H1246 harboring plasmid pYES2-McDGAT1A

HY1B

S. cerevisiae H1246 harboring plasmid pYES2-McDGAT1B

HY2A

S. cerevisiae H1246 harboring plasmid pYES2-McDGAT2A

HY2B

S. cerevisiae H1246 harboring plasmid pYES2-McDGAT2B



Page 20 of 28

Table 2 The fatty-acid profile of TAG in S. cerevisiae H1246 mutant strains harboring each plasmid cultivated with a different fatty-acid supplement Fatty acid composition (%)

Strains

C16:0

C16:1

C18:0

C18:1

C18:2

C18:3

No

HY2

14.43±0.49

39.03±1.07

7.45±0.81

39.09±0.33

-

-

supplement

HY2B

15.07±1.22

38.46±2.12

12.43±0.88

34.05±1.61

-

-

Added

HY2

17.42±1.29

16.82±1.19

12.98±1.73

23.13±1.29

29.64±0.57

-

C18:2

HY2B

19.01±0.92

18.46±1.28

15.55±0.97

20.69±0.91

26.69±1.18

-

Added

HY2

16.73±0.62

36.33±2.12

9.45±1.57

36.09±0.73

-

1.23±0.91

C18:3

HY2B

18.58±1.21

27.02±0.98

16.10±1.15

29.43±0.41

-

8.87±0.50

Data are mean ± SD from triple biological replicates. The hyphen means “not detected.”


Page 21 of 28


Table 3 Fatty acid composition of TAGs in M. circinelloides CBS 277.49 cultivated in a fermentor with the nitrogen-limited medium Fatty acid composition (%)

Culture time (h)

C16:0

C16:1

C18:0

C18:1

C18:2

C18:3

other

8

26.22±0.94

3.15±0.12

6.63±0.19

24.80±0.70

17.63±0.48

17.88±0.60

3.66±0.25

12

24.90±0.83

1.01±0.26

9.71±0.11

20.40±0.94

23.22±0.51

14.38±0.94

6.33±0.26

24

19.67±0.61

2.54±0.38

5.76±0.23

23.30±0.46

20.32±0.27

21.95±0.93

6.43±0.11

48

18.34±0.55

3.67±0.22

5.77±0.16

24.52±0.73

19.87±0.41

21.30±0.81

6.51±0.21

72

18.38±0.58

3.27±0.23

5.13±0.12

25.98±0.64

19.73±0.17

21.58±0.95

5.91±0.18



(Fig. 1)


Page 22 of 28

Page 23 of 28


HsDGAT1 AtDGAT1 CeDGAT1 MmDGAT1 McDGAT1A McDGAT1B

----------------------------MGDR---GSSRRRRTGSRPSSHGGGGPAAAEEEVRDAAAGPDVGAAGDAPAPAPN------KDGDAGVGSG-MAILDSAGVTTVTENGGGEFVDLDRLRRRKSRSDSSNGLLLSGSDNNSPSDDVGAPADVRDRIDSVVNDDAQGTANLAGDNNGGGDNNGGGRGGGEGRGNA ----------------------------MQMR--QQTGRRRRQPSETS----NGSLAS-------SRRSSFAQNGNSSR-------------KSSEMRG-----------------------------MGDRGGAGSSRRRRTGSRVSVQGGSGPKVEEDEVRDAAVSPDLGAGGDAPAPAPAPAHTRDKDGRTSVGDG------------------------MTLPPSITRTSTAPVLVTNDAKSTHPSPPIVNTNTSQLQFKLAKQRPLHFKARLTQFDLSN-----TDSSNGAFRG------------------------------------------------------------------------------------------------------


-------HWELRCHRLQDSLFSSDSGFSN-YRGILNWCVVMLILSN-ARLFLENLIKYGILVDPIQVVSLFLKDPY---SWPAPCLVIAANVFAVAAFQVE DATFTYRPSVPAHRRARESPLSSDAIFKQSHAGLFNLCVVVLIAVN-SRLIIENLMKYGWLIR-----TDFWFSSRSLRDWPLFMCCISLSIFPLAAFTVE -------PCEKVVHTAQDSLFSTSSGWTN-FRGFFNLSILLLVLSN-GRVALENVIKYGILITPLQWISTFVEHHYSIWSWPNLALILCSNIQILSVFGME -------YWDLRCHRLQDSLFSSDSGFSN-YRGILNWCVVMLILSN-ARLFLENLIKYGILVDPIQVVSLFLKDPY---SWPAPCVIIASNIFVVAAFQIE -------FYTLFWIAMGMYVIQSIVRCYEQEGILLSLGFYRLISEDGLALLISDLTMVSMTLFSVLFSKLLMWGVLPYETFGVVIQHTCQALFLFVNIYWT -----------------------------------MDTVLVLGASN-IRLIIENWMKYGLLIG-VPHHSHIPVRDLGLFLLAWLSVPLSLLISLVVEYGMG . * : : :.: . : . : . : .


KRLAVG-----------ALTEQAGLLLHVANLATILCFPAAVVLLVESITPVGSLLALMAHTILFLKLFSYRDVNSWCR-------RARAKAASAGKKASS KLVLQK-----------YISEPVVIFLHIIITMTEVLYPVYVTLRCDSAFLSG-VTLMLLTCIVWLKLVSYAHTSYDIR-----------SLANAADKANP KILERG-----------WLGNGFAAVFYTSLVIAHLTIPVVVTLTHKWKNPLWSVVMMGVYVIEALKFISYGHVNYWARDARRKITELKTQVTDLAKKTCD KRLAVG-----------ALTEQMGLLLHVVNLATIICFPAAVALLVESITPVGSVFALASYSIMFLKLYSYRDVNLWCRQR-----RVKAKAVSTGKKVSG FWRNWP-----------WVQSGFFTMHTIVMMMKTHSYTALNGDLSVKLRRLRQLKAELPKLIAASKEDEHAAAEKIQHMES--------EIAFLEGELVH KLAVAAKDAKPDFLKKLTILERVVAVTHVSHLVFLLTFPSYLAYTQIYHPLVG-SGVIVICLITFLKLTSFALVNHELR------------QAFVQGKSED : . . . * * .. .. : . :


-----------AAAPHTVSYPDNLTYRDLYYFLFAPTLCYELNFPRSPRIRKRFLLRRILEMLFFTQLQVGLIQQWMVPTIQNSMKPFKDMDYSRIIERL ------------------EVSYYVSLKSLAYFMVAPTLCYQPSYPRSACIRKGWVARQFAKLVIFTGFMGFIIEQYINPIVRNSKHPLKG-DLLYAIERV PKQFWDLKDELSMHQMAAQYPANLTLSNIYYFMAAPTLCYEFKFPRLLRIRKHFLIKRTVELIFLSFLIAALVQQWVVPTVRNSMKPLSEMEYSRCLERL -----------AAAQQAVSYPDNLTYRDLYYFIFAPTLCYELNFPRSPRIRKRFLLRRVLEMLFFTQLQVGLIQQWMVPTIQNSMKPFKDMDYSRIIERL G---------------NTRYPDNVTIANFLDYLLVPSLVYWMEYPRTDKIRVWYVFEKTTATLGSFLMLYVTTERWILPKLYDP--------NMSEPRVI ------------FYTEDVAYPNNVNAKNLLYAFFAPTLCYQPSYPRSDKFRKSFFFKRVGELITCLVMMYVLTEQYAKPTLANSIQALEDKKFVTIVERV . :. .: : .*:* * .:** :* :. .: : : ::: * : :. .:


LKLAVPNHLIWLIFFYWLFHSCLNAVAELMQFGDREFYRDWWNSESVTYFWQNWNIPVHKWCIRHFYKPMLR-RGSSKWMARTGVFLASAFFHEYLVSVP LKLSVPNLYVWLCMFYCFFHLWLNILAELLCFGDREFYKDWWNAKSVGDYWRMWNMPVHKWMVRHIYFPCLR-SKIPKTLAIIIAFLVSAVFHELCIAVP LKLAIPNHLIWLLFFYTFFHSFLNLIAELLRFADREFYRDFWNAETIGYFWKSWNIPVHRFAVRHIYSPMMR-NNFSKMSAFFVVFFVSAFFHEYLVSVP LKLAVPNHLIWLIFFYWFFHSCLNAVAELLQFGDREFYRDWWNAESVTYFWQNWNIPVHKWCIRHFYKPMLR-HGSSKWVARTGVFLTSAFFHEYLVSVP LELLFPFMINYLFIFYIIFECILNAFAELSRFADRNFYDDWWNSVTYDEFARKWNKPVHHWLLRHVYAQSIESYKLSKTNATFVTFLLSSIFHELVLIIV LKLSTTAVVIWLLMFYALFHAFLNALAEVLRFGDRTFYLAWWNSGNLATYWRLWNRPVYLFFKRHVYIPAVQ-RGVPPAVCQLLVFLISALLHEVLVGIP *:* . :* :** :*. ** .**: *.** ** :**: . : : ** **: : **.* :. . . .*: *:.:** : :


LRMFRLWAFTGMMAQIPLAWFVG--RFFQGN---YGNAAVWLSLIIG-QPIAVLMYVHDYYVLNYEAPAAEA----CRLFKLWAFLGIMFQVPLVFITNYLQERFGST--VGNMIFWFIFCIFGQPMCVLLYYHDLMNRKGSMS--------LKIFRLWSYYGMMGQIPLSIITD--KVVRGGR--TGNIIVWLSLIVG-QPLAILMYGHDWYILNFGVSAVQNQTVGI LRMFRLWAFTAMMAQVPLAWIVG--RFFQGN---YGNAAVWVTLIIG-QPVAVLMYVHDYYVLNYDAPVGV-----THKIRLYMFFIQMLQLPMIVIGR--MPLFRNRFWLGNSFFWLCMLFG-PPLLGILYCREAFWASWGGPLPPASTAITHSITGFAFWGMLGQIPLIAITHGIEKWRGKGTSLGNTIFWIVFCVVGQPTIALLYYYQWTATHKFDSNGNTV---: : : : : *:*: : ** .*. : . * ::* :

Motif 1

Motif 2

Motif 3

Motif 4

Motif 5

Motif 6

Motif 7

(Fig. 2)



Page 24 of 28

MmDGAT2 HsDGAT2 MrDGAT2A MrDGAT2B McDGAT2B McDGAT2A ScDGAT2 CeDGAT2 AtDGAT2

MKTLIAAYSGVLRGERRAEAARSENKNKGSALSREGSGRWGTGSSILSALQDIFSVTWLNRSKVEKQLQVISVLQWVLSFLVLGVACSVILMYTFCTD--C MKTLIAAYSGVLRGERQAEADRSQRSHGGPALSREGSGRWGTGSSILSALQDLFSVTWLNRSKVEKQLQVISVLQWVLSFLVLGVACSAILMYIFCTD--C -------------------------MASKDQHLQQK-------VKHTLEAIPSPRYAPLR-VPLRRRLQTLAVLLWCSMMSIC----MFIFFFLCSIPVLL -----------------------------MEQVQVT---------ALLDHIPKVHWAPLRGIPLKRRLQTSAIVTWLALLPIC----LIIYLYLFTIP-LL -----------------------------MEEKVS---------QVIPN-VPEVQWAPLTGIPFERRIQMAVVLMWIFLLGNC----LTLFCCSLFLP-FL -------------------------MNSSSETLVAPEPSQTPKEKVSPKPTSQVRWAPIRGIPIERRLQMLAVCTWISMMFML----VSLFFFMATYK-FL -------MSGTFNDIRRRKKEEGSPTAGITERHENK--SLSSIDKREQTLKPQLESCCPLATPFERRLQTLAVAWHTSSFVLF----SIFTLFAISTP-AL -------------------------------MLNY----------QIHKKLTDIKWVNIF-SPWDRQRAYFALVVWFGLIYPFCCLCQVAPFVLFFTG--Q ---------------------------------------------------MGGSREFRAEEHSNQFHSIIAMAIWLGAIHFN---VALVLCSLIFLPPSL : : :


WLIAVLYFTWLAFDWNTPKKG---GRRSQWVRNWAVWRYFRDYFPIQLVKT--HNLLT----------------------------TRN-----------WLIAVLYFTWLVFDWNTPKKG---GRRSQWVRNWAVWRYFRDYFPIQLVKT--HNLLT----------------------------TRN-----------WFPIILYLTWILVWDKAPENG---GRPIRWLRNAAWWKLFAGYFPAHVIKE--ADLDP----------------------------SKN-----------WPILIMYTIWLFF-DKAPENG---GRRISLVRKLPLWKHFANYFPVTLIKE--GDLDP----------------------------KGN-----------WPLHIAYIIYLYR-DQSAENG---GRRSDWFRRLPIWNYYAGYFPAKLVKE--QDLDP----------------------------KKN-----------WPILIAYISFLYV-DKAPESG---GRRFERARHWTLWKYFAAYFPAQLIKE--HDLDP----------------------------KHN-----------WVLAIPYMIYFFF-DRSPATGEVVNRYSLRFRSLPIWKWYCDYFPISLIKT--VNLKPTFTLSKNKRVNEKIYKIRLWPTKYSINLKSNSTIDYRNQECTG WIILGLYAVWYLYDRESPRRG---GYRDNWFRNLSLHKWFAEYFPVKLHKT--AELDP----------------------------NQN-----------SLMVLGLLSLFIFIPIDHRSK--YGRKLARY----ICKHACNYFPVSLYVEDYEAFQP----------------------------NRA-----------. . *** : :. . Motif 2

Motif 1

Motif 3


--YIFGYHPHGIMGLGAFCNFSTEATEVSKKFPGIRPYLATLAGNFRMPVLREYLMSGGICPVNRDTIDYLLSKNGSGNAIIIVVGGAAESLSSMPGK --YIFGYHPHGIMGLGAFCNFSTEATEVSKKFPGIRPYLATLAGNFRMPVLREYLMSGGICPVSRDTIDYLLSKNGSGNAIIIVVGGAAESLSSMPGK --YIFGYHPHGIISMGSFCTFSTNATGFDDLFPGIRPSLLTLTSNFNIPLYRDYLMACGLCSVSKTSCQNILTKGGPGRSIAIVVGGASESLNARPGV --YIMSYHPHGIISMAAFANFATEATGFSEQYPGIVPSLLTLASNFRLPLYRDFMMSLGMCSVSRHSCEAILR-SGPGRSIVIVTGGASESLSARPGT --YVFGSHPHGIISISSFASFATEATGFSQLFPGIIPSLLTLTTNFKFPIYRDIILALGIASVSRHSCEKILS-SGPGRSIVIVIGGASESLNARPGI --YVFGYHPHGIISYGAQLAFATEATGFSEKFPGITPSLLTLNSNFRIPFYRDVIMALGIASVSRRSCENILS-SGPGRSIAIVVGGAAESLNARPGT PTYLFGYHPHGIGALGAFGAFATEGCNYSKIFPGIPISLMTLVTQFHIPLYRDYLLALGISSVSRKNALRTLS---KNQSICIVVGGARESLLSSTNG --YLFGYHPHGILGVGAWSCFGFDACNVKQVFKGIRFNICTLPGNFTAMFRREILLSIGMIESSKESIEHVLNSEEKGRAVVIVVGGAAEALEAHPGK --YVFGYEPHSVLPIG-----VVALCDLTGFMPIPNIKVLASSAIFYTPFLRHIWTWLGLTAASRKNFTSLLD---SGYSCVLVPGGVQETFHMQHDA *::. .**.: . : : * . *. *: .: . * . : :* **. *:: .


NAVTLKNRKGFVKLALRHG-ADLVPTYSFGENEVYKQVIFEEGSWGRWVQKKFQKYIGFAPCIFHGRGLFSSDTWGLVPYSKPITTVVGEPITVPKLNAVTLRNRKGFVKLALRHG-ADLVPIYSFGENEVYKQVIFEEGSWGRWVQKKFQKYIGFAPCIFHGRGLFSSDTWGLVPYSKPITTVVGEPITIPKLMDLVLKRRFGFIKIAVQTG-ASLVPTISFGENELYEQIESNENSKLHRWQKKIQHALGFTMPLFHGRGVFNYD-FGLLPHRHPIYTIVGKPIPVPSIK NDLTLKKRLGFIRLAIRNG-ASLVPIFSFGENDIYEQYDNKKGSLIWRYQKWFQKITGFTVPLAHARGIFNYN-AGFIPFRHPIVTVVGKPIAVPLLA ADLVLKKRLGFIRIAIRHG-ADLVPVFSFGENELYEQVDNSTGSWLWKAQKKMQQALGFTMPLFHARGIFNYN-VGLIPYRHPIVTVVGKPIPVPKMK ADLVLRKRLGFIRLAIKHG-ASLVPVFSFGENEVYDQLDNAKGSKVFMYQKKMQAMLGFTMPLFHARGIFNYD-VGIIPFRHPITTVVGKPIPVPALE TQLILNKRKGFIKLAIQTGNINLVPVFAFGEVDCYNVLSTKKDSVLGKMQLWFKENFGFTIPIFYARGLFNYD-FGLLPFRAPINVVVGRPIYVEKKHTLTLANRKGFVREAVKTG-AHLVPVYAFGENDIYKQIDNPEGSKLRKIQEWGKKKMGISLPLIYGRGYFQMA-LGLLPMSRAVNVVVGAPIQVEKEENVFLSRRRGFVRIAMEQG-SPLVPVFCFGQARVYKWWKPDCD-----LYLKLSRAIRFTPICFWG--VFGSP----LPCRQPMHVVVGKPIEVTKT: * .* **:: *:. * *** .**: *. . . :: . * :* .: .:** ** :


---EHPTQKDIDLYHAMYMEALVKLFDNHKTKFGLPETEVLEVN----EHPTQQDIDLYHTMYMEALVKLFDKHKTKFGLPETEVLEVN-YGQTK--DEIIRELHDSYMHAVQDLYDRYKDIYAKDRVKELEFVEEGETEPSEEQMHQVQAQYIESLQAIYDKYKDIYAKDRIKDMTMIAPGQTEPTQEQLLETQALYIEELESIYNKYKDVYAKDRKQDLRIVSEGQTDPTQEQILAVQQLYIDELFSIYNKYKDVYAKDRKQELCITD--ITNPPDDVVNHFHDLYIAELKRLYYENREKYGVP-DAELKIVG---LDPSKEVIDEIHGVYMEKLAELFEEHKAKFGVSKDTRLVFQ----LKPTDEEIAKFHGQYVEALRDLFERHKSRVGYDLELKIL---. .. : : *: : :: . : . :

Motif 4

Motif 6

Motif 5

(Fig. 3)


Page 25 of 28


(Fig. 4)



(Fig. 5)


Page 26 of 28

Page 27 of 28


(Fig. 6)



Abstract Graphics


Page 28 of 28

Identification and Characterization of Diacylglycerol

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