Dual functions of Lip6 and its regulation of lipid metabolism in the

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Dual functions of Lip6 and its regulation of lipid metabolism in the oleaginous fungus Mucor circinelloides Xinyi Zan, Xin Tang, Linfang Chu, and Yuanda Song J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b06024 • Publication Date (Web): 28 Feb 2018 Downloaded from http://pubs.acs.org on March 1, 2018

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Journal of Agricultural and Food Chemistry

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Dual functions of Lip6 and its regulation of lipid metabolism in the oleaginous

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fungus Mucor circinelloides

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Xinyi Zan1, Xin Tang1, Linfang Chu1, Yuanda Song1,2*

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Technology, Jiangnan University, Wuxi, P.R. China

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State Key Laboratory of Food Science and Technology, School of Food Science and

Colin Ratledge Center for Microbial Lipids, School of Agriculture Engineering and

Food Science, Shandong University of Technology, Zibo, P.R. China

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* Correspondence author: Yuanda Song E-mail: [email protected] (Yuanda Song);

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ABSTRACT

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Although the multiple roles of lipases have been reported in yeasts and

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microalgae, the functions of lipases have not been studied in oleaginous filamentous

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fungi. Lipase Lip6 has been reported in the oleaginous filamentous fungus Mucor

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circinelloides with the consensus lipase motif GXSXG and the typical acyltransferase

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motif of H-(X)4-D. To demonstrate that Lip6 might play dual roles as a lipase and an

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acyltransferase, we performed site-directed mutagenesis in the lipase motif and the

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acyltransferase motif of Lip6. Mutation in the lipase motif increased cell biomass by

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12%-18% and promoted lipid accumulation by 9%-24%, whilst mutation in the

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acyltransferase motif induced lipid degradation. In vitro, purified Lip6 had a slight

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lipase activity but had a stronger phospholipid:DAG acyltransferase activity. Enzyme

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activity assays in vivo and phospholipid synthesis pathway analysis suggested that

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phosphatidyl serine and phosphatidyl ethanolamine can be the supplier of a fatty acyl

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moiety to form TAG in M. circinelloides.

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Key words: Mucor circinelloides; Lipid accumulation; Lipase; Phospholipid;

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Introduction

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Oleaginous microorganisms, including bacteria, yeast, microalgae and fungi, can,

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by definition, accumulate oil to more than 20% of their cell dry weight (CDW)1.

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Lipids in these microorganisms accumulate as discrete droplets that have a neutral

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lipid core surrounded by a phospholipid monolayer2-4. A specific set of proteins are

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associated with or embedded into the phospholipid monolayer where they can

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regulate lipid synthesis and turnover of stored lipids2-5. Proteome analysis of lipid

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droplets (LD) for the oleaginous fungus, Mortierella alpina, revealed that the isolated

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LDs in the fungus were also spherical in structure and had a significantly higher

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proportion of TAG and a corresponding smaller proportion of polar lipids6.

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Lipases and/or hydrolases are the major classes of enzymes found in these

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droplets. They may possibly reside on the outside of the droplet being embedded in a

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phospholipid layer; indeed, such a layer can be identified in the droplets of Mucor6.

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Their intrinsic function is to hydrolyze acylglycerols into glycerol and fatty acids.

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Deletion of lipase genes increased the content of TAG not only in the non-oleaginous

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yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe, but also in the

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oleaginous yeast Yarrowia lipolytica and the oleaginous microalgae Thalassiosira

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pseudonana7-13. In addition to lipolytic activity, some lipases also possess

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phospholipase and/or acyltransferase activities that could contribute to phospholipid

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or triacylglycerol synthesis11-13. These findings suggested that these multifunctional

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lipases may contribute to anabolic as well as catabolic processes. However, the

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molecular mechanism balancing catabolic and anabolic activities of these enzymes 3 ACS Paragon Plus Environment


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needs to be elucidated.

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Lipases belong to the structural super family of α/β-hydrolases14. Their activities

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rely on a catalytic triad formed by Ser, His and Asp residues. Serine is essential for

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the lipolytic activity and usually appears in a highly conserved motif (G/A) XSXG15.

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In addition, multifunctional lipases contain an acyltransferase signature motif

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H-(X)4-D. The motif H-(X)4-D is one of most conserved functional motifs of

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acyltransferase family. Peers’ studies have reported that ICT116, LOR117, CGI5818 and

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At4g2416019 from yeast, mammalian, and plant systems share the highly conserved

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motif H-(X)4-D. Site-directed mutagenesis experiments have demonstrated that the

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histidine residue in this motif is indispensable for acyltransferase activity of Tgl3p8.

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Our previous studies have reported the characterizations of lipases in lipid producing

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fungus Mucor circinelloides20,

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contain the lipase consensus sequence, GXSXG, but also harbor a typical

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acyltransferase motif of H-(X)4-D20. Transcriptional analyses revealed that the Lip6

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gene expression increased significantly during lipid accumulation stage20.

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Over-expression of lipase Lip6 led to a slight increase in cell dry weight and lipid

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accumulation21. Therefore, we propose that Lip6 functions both as an acyltransferase

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and a lipase in M. circinelloides. To further confirm that Lip6 has acyltransferase

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activity, we performed site-directed mutagenesis in the lipase motif GXSXG and the

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acyltransferase motif H-(X)4-D of Lip6, respectively. Then, we investigated the

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effects of over-expression of these variants on cell growth, lipid composition, lipase

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activity and phospholipid:DAG acyltransferase (PDAT) activity in M. circinelloides.

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. Some lipases, especially lipases Lip6, not only


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Our results showed for the first time that the fungus lipase Lip6 played dual roles as a

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lipase and an acyltransferase in M. circinelloides.

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Materials and Methods

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Strains and culture conditions. Mucor circinelloides strains used this study are

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listed in Table 1. 100 µL spore suspension (approx. 107 spores/mL) was inoculated

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into 200 mL K&R medium22 held in a 1 L baffled shake flask. Cultures were

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incubated at 30 oC for 24 h with shaking at 150 rpm and then used at 10% (v/v) to

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inoculate 1 L baffled shake flasks containing 200 mL modified K&R medium (2 g

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diammonium tartrate, 80 g glucose per liter plus essential salts). The cultures were

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incubated for 72 h at 30 oC with shaking at 150 rpm.

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The uridine auxotroph, pleu-MU402, was used as recipient strain in

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transformation experiments to over-express lipase genes of M. circinelloides CBS

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277.4923. This strain could grow at 30oC in YPG media containing 3 g yeast extract /L,

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10 g tryptone /L, and 20 g glucose /L at pH 4.5. The media were supplemented with

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uridine (200 µg/mL) when required.

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Escherichia coli Top 10 was used for all cloning experiments. Escherichia coli BL21 was used for protein expression.

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Site-directed mutagenesis and transformation in M. circinelloides. Plasmids

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pMAT1552 and pMAT1552-Lip6 were obtained as described previously by Zan et

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al21. Plasmids pMAT1552-Lip6S272R and pMAT1552-Lip6H335Q were constructed

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using the Hieff Mut Site-Directed Mutagenesis Kit (Yesen Co., Shanghai, China) with 5 ACS Paragon Plus Environment


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pMAT1552-Lip6 as templates respectively according to the manufacturer’s

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instructions. Primers used to construct these site-directed mutants are listed in

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Supplementary Table 1.

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All mutants were verified by colony PCR and positive mutants were sequenced

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in both directions. These plasmids were introduced into pleu-MU402 protoplasts

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using the transformation method described23. Transformants, containing plasmids

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pMAT1552, pMAT1552-Lip6, pMAT1552-Lip6S272R, and pMAT1552-Lip6H335Q, were

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named as Mc-1552, Mc-Lip6, Mc-6S272R, and Mc-6H335Q, respectively. Mc-1552

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was used as the control strain.

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Construction of plasmids and purification of recombinant Lip6 mutants. To

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obtain

the

purified

Lip6

mutants,

we

further

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pMAT1552-Lip6-His, pMAT1552-Lip6S272R-His, and pMAT1552-Lip6H335Q-His with

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a His-tag using pMAT1552-Lip6, pMAT1552-Lip6S272R, and pMAT1552-Lip6H335Q as

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templates respectively. Primers used to construct these mutants with a His-tag are

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listed in Supplementary Table 2. E. coil BL21 strains transformed with these plasmids

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were cultured in 200 mL LB medium until the OD600 reached approx. 0.6. IPTG at

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0.1 mM was then added to induce protein expression. Cell pellets were collected by

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centrifugation, re-suspended in the lysis buffer [100 mM KH2PO4, 1mM DTT, 1mM

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benzamidine hydrochloride, 20% (v/v) glycerol, pH 8.0] and purified on a HisTrap

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HP column (GE Healthcare, Sweden). Bradford’s method was used to determine the

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protein concentration with BSA as a standard. SDS-PAGE was performed using 12%

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(v) acrylamide gel. The whole purification process was carried out at 4 oC. The 6 ACS Paragon Plus Environment

constructed

plasmids

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purified protein was stored at -80 oC in 40% (v/v) glycerol.

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Analysis of cell dry weight, lipid content and fatty acid composition. Mycelia were

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sampled at 24 h, 48 h and 72 h，collected on a dried, weighed filter paper by filtration

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through a Buchner funnel under reduced pressure. Cells were washed three times with

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distilled water, frozen overnight at -80 oC, and lyophilized. Cell dry weight was

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determined gravimetrically. The lipid was extracted from approx. 20 mg lyophilized

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biomass by using a mixture of chloroform/methanol/4M HCl with a volume ratio of

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2:1:2. The extracted lipid was methylated with 10% (w/w) methanolic HCl at 60 oC

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for 3 h. The resultant fatty acid methyl esters were extracted with n-hexane and were

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analyzed by GC equipped with a 30 m×0.32 mm DB-WAXETR column with 0.25 µm

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film thickness. The program was 120 oC for 3 min, ramp at 200 oC at 5 oC per min,

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ramp to 220 oC at 4 oC per min, and hold 2 min. Pentadecanoic acid (15:0) was used

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as an internal standard before the methylation.

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Fluorescence microscopy. Mycelia were collected, washed three times with PBS

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buffer and then stained by Nile Red (10 mg/mL) for 5 min at room temperature. The

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stained cells were observed with a light microscope (Nikon, Japan).

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Thin-layer chromatography of lipids. Mycelia were collected, washed three times

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with PBS buffer containing 30 g glucose/L and were immersed in 1 mL of PBS buffer

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containing 30 g glucose/L. To destroy the cell walls, mycelia were frozen at -80oC for

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40 min and were thawed at room temperature for 20 min. After the freeze-thaw

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process was repeated, 2 mL of chloroform and 1 mL of methanol were added to

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extract the total lipid. The layer of chloroform was obtained and then dried under N2. 7 ACS Paragon Plus Environment


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The residue was dissolved with 100 µL chloroform and 50 µL was separated by TLC

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silica gel plates (Merck, Germany) using petroleum ether/diethyl ether/acetic acid

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(70:30:1, by vol.)24. To visualize the lipids, the plates were exposed to iodine vapor.

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The lipids were quantified using densitometry and the images were analyzed using the

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software ImageJ 1.50i.

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Lipase ELISA assays and PDAT activity assays. Mycelia were washed by PBS

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buffer (0.01 M and pH 7.4) containing 30 g glucose/L, collected by filtration and

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ground in liquid nitrogen, and then re-suspended in an extraction buffer containing 20%

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(w/v) glycerol, 100 mM KH2PO4/KOH, pH 7.5, 1 mM benzamidine and 1 mM DTT.

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After centrifuging at 10,000×g for 10 min at 4 oC, the supernatant was immediately

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used for enzyme activity measurement. Protein concentrations were determined using

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the method of Bradford with BSA as a standard. Lipase activity unit was determined

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using the microbial lipase ELISA kit (Mlbio, Shanghai, China), and the purified

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Candida albicans lipase was used to generate a standard curve. The in vitro assay of

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PDAT or lipase activity was performed in a 200 µL volume of assay mixture

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containing 50 mM potassium phosphate (pH 7.2), 10 mM MgCl2, the re-suspended

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protein extracts (40 µg protein in total), 250 µM lipid donors phosphatidyl

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ethanolamine (PE), phosphatidyl serine (PS) or phosphatidylcholine (PC), and 250

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µM lipid acceptor (DAG)24, or soybean oil (TAG). Reactions were incubated at 30 oC

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for 1 h, and then the lipids were extracted and analyzed as described above. The

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relative amount of TAG formed or degraded was used to evaluate the PDAT or lipase

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activity. 8 ACS Paragon Plus Environment

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Transcriptional analyses of the major phospholipid synthesis genes by qRT-PCR.

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Quantitative RT-PCR (qRT-PCR) analysis was performed to quantify the

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transcriptional levels of genes. Total RNA was extracted by an RNAiso Plus kit after

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grinding under liquid N2 and was transcribed to cDNA using the Prime ScriptRT

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reagent kit (Takara, Japan) according to the manufacturer’s instructions. Real-Time

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quantitative PCR was performed by standard procedures. Relative quantitative

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analysis was based on the 2-△△Ct method using 18S rRNA of M. circinelloides CBS

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277.49 as a housekeeping gene25. The thermal cycling conditions for the amplification

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reaction were 95 oC 30 s, 58/60 oC 30 s (40 cycles). Three replicates, prepared from

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independent biological samples, were analyzed. The primer sequences used for

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amplification of major phospholipid synthesis genes are listed in Supplementary Table

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3. We applied a fold-change cutoff of >1.5 for up-regulation, and

Dual functions of Lip6 and its regulation of lipid metabolism in the

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