Lipase genes in Mucor circinelloides

1 3

J Ind Microbiol Biotechnol (2016) 43:1467–1480DOI 10.1007/s10295-016-1820-0

GENETICS AND MOLECULAR BIOLOGY OF INDUSTRIAL ORGANISMS - ORIGINAL PAPER

Lipase genes in Mucor circinelloides: identification, sub‑cellular location, phylogenetic analysis and expression profiling during growth and lipid accumulation

Xinyi Zan1 · Xin Tang1 · Linfang Chu1 · Lina Zhao1 · Haiqin Chen1,3 · Yong Q. Chen1,3 · Wei Chen1,3 · Yuanda Song1,2

Received: 24 May 2016 / Accepted: 30 July 2016 / Published online: 17 August 2016 © Society for Industrial Microbiology and Biotechnology 2016

lipases also contained a typical acyltransferase motif of H-(X) 4-D, and these lipases may play a dual role in lipid metabolism, catalyzing both lipid hydrolysis and transacyla-tion reactions. The differential expression of all lipase genes were confirmed by quantitative real-time PCR, and the expression profiling were analyzed to predict the possible biological roles of these lipase genes in lipid metabolism in M. circinelloides. We preliminarily hypothesized that lipases may be involved in triacylglycerol degradation, phospho-lipid synthesis and beta-oxidation. Moreover, the results of sub-cellular localization, the presence of signal peptide and transcriptional analyses of lipase genes indicated that four lipase in WJ11 most likely belong to extracellular lipases with a signal peptide. These findings provide a platform for the selection of candidate lipase genes for further detailed functional study.

Introduction

Lipases or triacylglycerol hydrolases (EC 3.1.1.3) are enzymes which hydrolyze ester bonds between fatty acids and glycerol [4]. They hydrolyze triglycerides (TAG) into diglycerides, monoglycerides, fatty aicds and glycerol [15]. Lipases can also catalyze ester synthesis via esterification and transesterification (alcoholysis and acidolysis) and interesterification reactions. Additionally, they can be used in numerous bioconversion reactions such as aminolysis, amide and thioester synthesis and hydrolysis [24]. Due to these diverse properties, lipases are widely used in waste treatment [13], fine chemicals refinery [14], traditional food making [25, 29], citric acid production [17] and phar-maceutical industries [35].

Lipases belong to the structural super family of α/β-hydrolases [10]. Their activities rely on a catalytic triad

Abstract Lipases or triacylglycerol hydrolases are widely spread in nature and are particularly common in the micro-bial world. The filamentous fungus Mucor circinelloides is a potential lipase producer, as it grows well in triacylglyc-erol-contained culture media. So far only one lipase from M. circinelloides has been characterized, while the major-ity of lipases remain unknown in this fungus. In the present study, 47 potential lipase genes in M. circinelloides WJ11 and 30 potential lipase genes in M. circinelloides CBS 277.49 were identified by extensive bioinformatics analysis. An overview of these lipases is presented, including several characteristics, sub-cellular location, phylogenetic analysis and expression profiling of the lipase genes during growth and lipid accumulation. All of these proteins contained the consensus sequence for a classical lipase (GXSXG motif) and were divided into four types including α/β-hydrolase_1, α/β-hydrolase_3, class_3 and GDSL lipase (GDSL) based on gene annotations. Phylogenetic analyses revealed that class_3 family and α/β-hydrolase_3 family were the con-served lipase family in M. circinelloides. Additionally, some

Electronic supplementary material The online version of this article (doi:10.1007/s10295-016-1820-0) contains supplementary material, which is available to authorized users.

* Yuanda Song [email protected]

1 State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, People’s Republic of China

2 Colin Ratledge Center for Microbial Lipids, School of Agriculture Engineering and Food Science, Shandong University of Technology, Zibo, People’s Republic of China

3 Synergistic Innovation Center for Food Safety and Nutrition, Wuxi, People’s Republic of China

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1468 J Ind Microbiol Biotechnol (2016) 43:1467–1480

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formed by Ser, His and Asp residues. Serine is especially essential for lipase activities and usually appears in a highly conserved motif (G/A) XSXG, which is characteris-tic of α/β-hydrolases [16]. The sequence change within the motif (G/A) XSXG is one of the factors to distinguish vari-ous lipolytic families [10]. For example, the true lipolytic enzymes including triacylglycerol lipase usually contain the G (H/F) SQG sequence, while the GDSLG sequence is a conserved characteristic of GDSL family. Recent studies found that some lipases contain not only the lipase motif GXSXG but also an H-(X)4-D acyltransferase motif such as lipases Tgl3p, Tgl4p, and Tgl5p in the yeast Saccharo-myces cerevisiae [24, 28] and ptl1, ptl2 and ptl3 in the fis-sion yeast Schizosaccharomyces pombe [37]. Furthermore, site-directed mutagenesis experiments clearly demon-strated that the histidine residue is indispensable for acyl-transferase activity of Tgl3p [24].

In general, lipases derived from microorganism can be divided into extracellular and intracellular enzymes. Extra-cellular lipases can be excreted into the culture medium such as CdLIP1 in yeast Candida deformans [4] and LIP2, LIP7 and LIP8 in yeast Yarrowia lipolytica [10]. Interest-ingly, CdLIP1, LIP7 and LIP8 are mainly associated to the cell wall to hydrolyze triglycerides, whereas YlLIP2 is the main lipase released in the culture medium at the end of the growth phase [11]. The syntheses and secretion of extracellular lipases cannot be triggered by glucose, glyc-erol or mineral nitrogen compounds (NH4Cl, (NH4)2SO4), but they are highly increased in the presence of hydropho-bic substrates such as fatty acid, methyl esters and oils [8]. Moreover, the expression of extracellular lipase genes such as YlLIP2 also requires the regulation of SOA genes (spe-cific for oleic acid) [7]. In contrast, intracellular lipases are mainly located on the surface of lipid bodies (LBs) and a few of them are located in mitochondria. Distinct sub-cel-lular location could affect the role of intracellular lipases in cell metabolism. For example, lipases Tgl3, Tgl4 and Tgl5 in yeast S. cerevisiae [24, 28] and TGL3 and TGL4 in yeast Y. lipolytica [9] are located in LBs. These enzymes usually participate in the LB-associated lipid metabolism including TAG homeostasis, regulation of fatty acid com-position, and spore formation of cells. However, lipase Tgl2 in yeast S. cerevisiae is located in mitochondria and may be essential for cell survival under stress-inducing conditions [15, 19].

Fungal cells have been considered as important sources of lipase [2, 22]. The filamentous fungus Mucor circinel-loides grows well in triacylglycerol-containing culture media, which indicates that the fungus is a potential extra-cellular lipase producer [2]. Although a few studies have also investigated the potential of immobilized M. circinel-loides whole cells as a catalyst for ethyl esters synthesis, transesterificantion and ethanolysis [1, 3, 5, 23, 30, 31],

only one mycelium-bound lipase has been reported in Mucor javanicus, which has been reclassified as Mucor circinelloide [12]. Previous studies from genetic perspec-tive have revealed that a large number of lipase genes exist in the fungus Mucor circinelloide [33, 36, 40]. Therefore, it is necessary to study the roles and specificity of the remaining lipases in M. circinelloides. Moreover, a lot of enzymes and/or genes related to TAG synthesis such as malic enzyme, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase have been investi-gated to explore the mechanisms of lipid accumulation in M. circinelloides [26, 34, 38, 41]. However, the enzymes involved in TAG degradation such as lipase in this fungus have not been studied at all. We have previously reported several characteristics of the 30 potential lipases in a low lipid-producing strain M. circinelloides CBS 277.49 (15 % w/w lipid, cell dry weight) [40]. Lipase Lip6 or Lip10 from CBS 277.49 may control acyltransferase activity and play a role in fatty acid reconstruction for TAG, while Lip19 or Lip24 was involved in TAG degradation as a TAG lipase. Recently, a new strain M. circinelloides WJ11 isolated in our laboratory is able to produce up to 36 % (w/w) lipid of cell dry weight (CDW) [32]. The differ-ence on lipases genes involved in lipid metabolism might provide an insight for the different lipid content between strain CBS 277.49 and strain WJ11. Thus, in the present study we searched all possible genes encoding lipase in M. circinelloides WJ11 based on its genome database and compared these genes with that of CBS 277.49. The characteristics, sub-cellular location of these lipase genes were analyzed and phylogenetic analysis and expression profiling of the lipase genes during growth and lipid accu-mulation were performed. To our knowledge, this is the first and comprehensive report of the characterizations of lipases in M. circinelloides.

Materials and methods

Identification of lipase genes in M. circinelloides

Based on genomes of M. circinelloides CBS 277.49 and WJ11, related lipase genes were retrieved. Crite-ria for the identification of lipase genes in M. circinel-loides include gene annotations and the presence of con-served domains. The sequence of gene and protein, gene location and size, protein ID, conserved sequence and annotations of lipase genes in CBS 277.49 and WJ11 were acquired from the Joint Genome Institute (JGI) (http://genome.jgi.doe.gov/pages/search-for-genes.jsf?organism=Mucci2) and the National Center for Bio-technology Information (NCBI) (http://www.ncbi.nlm.nih.gov/genome/?term=LGTF00000000), respectively.

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http://genome.jgi.doe.gov/pages/search-for-genes.jsf%3forganism%3dMucci2

http://genome.jgi.doe.gov/pages/search-for-genes.jsf%3forganism%3dMucci2

http://www.ncbi.nlm.nih.gov/genome/?term=LGTF00000000


1469J Ind Microbiol Biotechnol (2016) 43:1467–1480

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The numbers of amino acid and molecular weight were analyzed on the website (http://www.bio-soft.net/sms/prot_mw.html). The protein isoelectric point was analyzed by using isoeletric point calculator (IPC) (http://isoelectric.ovh.org/).

Analysis of sub‑cellular location, signal peptide and transmembrane domains

TargetP1.1 Server (http://www.cbs.dtu.dk/services/Tar-getP/), SignalP 4.1 Server program (http://www.cbs.dtu.dk/services/SignalP/) and TMHMM (http://www.cbs.dtu.dk/service/TMHMM) were used to predict the sub-cellular location, the presence and location of signal peptide and transmembrane spanning region of lipase proteins from M. circinelloides CBS 277.49 and WJ11, respectively.

Phylogenetic and sequence homology analysis of lipase proteins

The phylogenetic tree was constructed based on the full-length sequences of these lipases. A neighbor-joining tree was built using MEGA 6.06, adopting the interior-branch test. Support for the tree obtained was assessed using the bootstrap method with 1000 replicates. Sequence homol-ogy analyses were performed using pairwise sequence alignment.

RNA isolation and transcriptional analyses of lipase genes by qRT‑PCR

100 μL spore suspension (approx. 107 spores/mL) of M. circinelloides CBS 277.49 or WJ11 was inoculated into 150 mL K&R medium [18] held in a 1 L flask equipped with baffles to increase aeration. Cultures were incubated at 30 °C for 24 h with shaking at 150 rpm and then 10 % of the culture were used as the seed culture to inoculate the 2-L fermentor containing 1.5 L modified K&R medium (2 g diammonium tartrate, 80 g glucose per liter plus inor-ganic salts), which was incubated with aeration at 0.5 v/v min−1 and stirring at 700 rpm. pH was maintained at 6.0 by auto-addition of sterilized 4 M KOH or 2 M H2SO4.

Based on the characteristic lipid accumulation of M. circinelloides WJ11 and CBS 277.49, cells at 6, 24 and 72 h were harvested and used for transcriptional analysis of lipase genes. Total RNA was extracted by an RNAiso Plus kit after grinding under liquid N2 and reverse-tran-scribed using the Prime ScriptRT reagent kit (Takara, Japan) according to the manufacturer’s instructions. Real-Time quantitative PCR was performed in BioRad CFX96 (BioRad, CA, USA) using the iTaq™ Universal SYBR® Green Supermix (BioRad, CA, USA). Relative quantifica-tion was based on the 2−△△Ct method using 18S rRNA of

M. circinelloides as a housekeeping gene [21]. The ther-mal cycling conditions for the amplification reaction were as follows: 95 °C 30 s, 51/53/55/58 °C 30 s (40 cycles). Three replicates, prepared from independent biological samples, were analyzed. The primer sequences used for amplification of lipase genes are listed in Tables S1 and S2. We applied a fold-change cutoff of ≥1.5 for up-regulation, and ≤0.5 for down-regulation.

Results and discussion

Identification of lipase genes in M. circinelloides WJ11

Based on the gene annotations and conserved motif sequences, we are surprised to find 47 potential genes of lipase in high lipid-producing strain M. circinelloides WJ11 (Table 1), which is more than the number of poten-tial lipase genes in low lipid-producing strain M. circinel-loides CBS 277.49 (30, Table 2). However, strain WJ11 has similar average lipase gene and protein length, and the average isoelectric point with strain CBS 277.49. All of these proteins contained the consensus sequence for a classical lipase (GXSXG motif) with a classical catalytic triad containing a nucleophile serine. Gene annotations showed that lipases in strain WJ11 are mainly divided into four types including α/β-hydrolase_1, α/β-hydrolase_3, class_3 and GDSL. The sequence variation within the motif (G/A) XSXG may be a factor to distinguish various lipase families. For example, the α/β-hydrolase_1 fam-ily, α/β-hydrolase_3 family and class_3 family in strain WJ11 mainly conserve the GFSQG, GDSAG and GHSLG sequence, respectively, whereas the motif (G/A) XSXG of GDSL family is diverse in strain WJ11. Most lipases from the filamentous fungi including the fungus WJ11 and CBS 277.49 contain the GHSLG motif in class_3 family, while some unusual motifs (GLSVG, GHSFG, GHSYG, and GTSAG) were also observed in class_3 family in strain WJ11. Moreover, 25 lipases in WJ11 not only contain the typical lipase motif (G/A) XSXG, but also the con-sensus sequence motif H-(X) 4-D (a typical of acyltrans-ferase motif), while only 16 similar lipases are observed in strain CBS 277.49. These lipases may play a dual role in lipid metabolism, catalyzing both lipid hydrolysis and transacylation reactions. Similar results have been reported that lipases Tgl3 and Tgl5 in S. cerevisiae not only exhib-ited lipase activity but also catalyzed acylation of lysophos-phatidylethanolamine and lysophosphatidic acid, respec-tively [27, 28]. The microalga phospholipid:diacylglycerol acyltransferase (PDAT) also exerts multiple functions as a typical PDAT, galactolipid:DAG acyltransferase and lipase [39]. We speculate that synthesis of these multi-functional enzymes allows better resource utilization and management

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http://www.bio-soft.net/sms/prot_mw.html

http://www.bio-soft.net/sms/prot_mw.html

http://isoelectric.ovh.org/

http://isoelectric.ovh.org/

http://www.cbs.dtu.dk/services/TargetP/

http://www.cbs.dtu.dk/services/TargetP/

http://www.cbs.dtu.dk/services/SignalP/

http://www.cbs.dtu.dk/services/SignalP/

http://www.cbs.dtu.dk/service/TMHMM

http://www.cbs.dtu.dk/service/TMHMM


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Tabl

e 1

Lip

ase

gene

s an

d pr

otei

ns o

f M

ucor

cir

cine

lloi

des

WJ1

1

Gen

eL

ocat

ion

(Str

and)

Gen

e si

ze

(cD

NA

bps

)A

min

o ac

idSu

buni

t (k

Da)

pIC

onse

rved

seq

uenc

eA

nnot

atio

n

Lip

ase

Acy

ltran

sfer

ase

evm

.mod

el.s

caff

old0

0007

.23

scaf

fold

0000

7:84

648-

8606

1(−

)14

1443

249

.82

6.7

GFS

QG

–α

/β-h

ydro

lase

_ 1

evm

.mod

el.s

caff

old0

0232

.15

scaf

fold

0023

2:45

815-

4755

1(+

)17

3750

658

.47

8G

FSQ

G–

α/β

-hyd

rola

se_

1

evm

.mod

el.s

caff

old0

0091

.18

scaf

fold

0009

1:54

224-

5671

2(−

)24

8863

872

.49

6.6

GA

SSG

HT

EM

IDα

/β-h

ydro

lase

evm

.mod

el.s

caff

old0

0222

.7sc

affo

ld00

222:

2179

0-22

616(+

)82

725

428

.58

5.8

GH

SAG

–α

/β-h

ydro

lase

evm

.mod

el.s

caff

old0

0131

.9sc

affo

ld00

131:

4149

8-42

579(+

)10

8232

236

.72

5.9

GH

SQG

–α

/β-h

ydro

lase

_ 1

evm

.mod

el.s

caff

old0

0012

.36

scaf

fold

0001

2:12

6245

-127

437(−

)11

9332

535

.76

4.9

GD

SAG

–α

/β-h

ydro

lase

_ 3

evm

.mod

el.s

caff

old0

0020

.2sc

affo

ld00

020:

8143

8-84

656(−

)32

1934

137

.04

5.6

GD

SAG

–α

/β-h

ydro

lase

_ 3

evm

.mod

el.s

caff

old0

0034

.45

scaf

fold

0003

4:13

8437

-139

562(+

)11

2632

135

.43

5.5

GD

SAG

–α

/β-h

ydro

lase

_ 3

evm

.mod

el.s

caff

old0

0459

.1sc

affo

ld00

459:

996-

2168

(+)

1173

321

35.5

25.

4G

DSA

G–

α/β

-hyd

rola

se_

3

evm

.mod

el.s

caff

old0

0089

.30

scaf

fold

0008

9:99

882-

1012

25(−

)13

4441

447

.59

6.3

GD

SAG

–α

/β-h

ydro

lase

_ 3

evm

.mod

el.s

caff

old0

0096

.22

scaf

fold

0009

6:75

927-

7756

3(+

)16

3735

139

.34

8G

DSA

G–

α/β

-hyd

rola

se_

3

evm

.mod

el.s

caff

old0

0230

.20

scaf

fold

0023

0:51

490-

5293

5(−

)14

4644

250

.25

5.4

GD

SAG

HN

QK

YD

α/β

-hyd

rola

se_

3

evm

.mod

el.s

caff

old0

0305

.6sc

affo

ld00

305:

1946

0-20

967(+

)15

0840

752

.94

5.9

GD

SAG

HC

SSQ

Dα

/β-h

ydro

lase

_ 3

evm

.mod

el.s

caff

old0

0827

.1sc

affo

ld00

827:

362-

1807

(−)

1446

442

50.2

55.

4G

DSA

GH

NQ

KY

Dα

/β-h

ydro

lase

_ 3

evm

.mod

el.s

caff

old0

0092

.13

scaf

fold

0009

2:49

157-

5064

7(−

)14

9144

449

.95.

3G

DSS

G–

α/β

-hyd

rola

se_

3

evm

.mod

el.s

caff

old0

0017

.8sc

affo

ld00

017:

2765

8-29

156(−

)14

9933

237

.14.

8G

DSA

GH

DL

RPD

α/β

-hyd

rola

se_

3

evm

.mod

el.s

caff

old0

0028

.33

scaf

fold

0002

8:14

4038

-146

158(−

)21

2162

569

.63

7.27

GD

SAG

HG

KM

TD

α/β

-hyd

rola

se_

3

evm

.mod

el.s

caff

old0

0079

.29

scaf

fold

0007

9:92

508-

9383

1(+

)13

2442

347

.77

6.5

GD

SAG

–α

/β-h

ydro

lase

_ 3

evm

.mod

el.s

caff

old0

0080

.18

scaf

fold

0008

0:54

165-

5541

9(−

)12

5537

041

.89

8G

DSA

G–

α/β

-hyd

rola

se_

3

evm

.mod

el.s

caff

old0

0192

.19

scaf

fold

0019

2:35

339-

3673

2(−

)13

9440

245

.24

5.7

GD

SAG

HT

EPL

Dα

/β-h

ydro

lase

_ 3

evm

.mod

el.s

caff

old0

0193

.13

scaf

fold

0019

3:29

937-

3090

5(−

)96

932

236

.67

6.1

GD

SAG

–α

/β-h

ydro

lase

_ 3

evm

.mod

el.s

caff

old0

0233

.1sc

affo

ld00

233:

3978

0-42

953(−

)31

7432

334

.98

5.1

GD

SAG

HSI

PVD

α/β

-hyd

rola

se_

3

evm

.mod

el.s

caff

old0

0001

.31

scaf

fold

0000

1:11

5177

-116

697(−

)15

2138

541

.41

7.9

GH

SLG

HN

VE

DD

clas

s_3

evm

.mod

el.s

caff

old0

0001

.81

scaf

fold

0000

1:26

0964

-262

497(−

)15

3439

543

.46

6G

HSL

GH

LSY

YD

clas

s_3

evm

.mod

el.s

caff

old0

0016

.3sc

affo

ld00

016:

9934

1-10

3768

(+)

4428

554

63.6

28

GH

SLG

HN

ED

YD

clas

s_3

evm

.mod

el.s

caff

old0

0029

.66

scaf

fold

0002

9:19

7369

-199

001(−

)16

3350

555

.89

8G

HSL

G–

clas

s_3

evm

.mod

el.s

caff

old0

0031

.20

scaf

fold

0003

1:69

730-

7233

0(−

)26

0177

185

.98

5.1

GH

SLG

HSE

GSD

clas

s_3

evm

.mod

el.s

caff

old0

0031

.26

scaf

fold

0003

1:92

982-

9572

4(+

)27

4383

394

.54

8.3

GH

SLG

HK

FNPD

clas

s_3

evm

.mod

el.s

caff

old0

0042

.33

scaf

fold

0004

2:11

1358

-114

283(+

)29

2657

764

.07

6.4

GH

SLG

HD

FYK

Dcl

ass_

3

evm

.mod

el.s

caff

old0

0065

.29

scaf

fold

0006

5:92

414-

9439

2(+

)19

7958

566

.58

7.8

GH

SLG

HK

GFW

Dcl

ass_

3

evm

.mod

el.s

caff

old0

0112

.23

scaf

fold

0011

2:65

525-

6823

1(+

)27

0786

696

.56

7.4

GH

SLG

HE

KA

ED

clas

s_3

evm

.mod

el.s

caff

old0

0113

.12

scaf

fold

0011

3:36

624-

3796

4(−

)13

4135

037

.62

7.5

GH

SLG

–cl

ass_

3

evm

.mod

el.s

caff

old0

0427

.3sc

affo

ld00

427:

3864

-538

4(−

)15

2138

541

.41

7.9

GH

SLG

HN

VE

DD

clas

s_3

evm

.mod

el.s

caff

old0

0153

.24

scaf

fold

0015

3:63

604-

6713

0(−

)35

2711

3912

5.23

6.9

GH

SLG

HA

DD

SDcl

ass_

3

evm

.mod

el.s

caff

old0

0024

.66

scaf

fold

0002

4:20

9236

-210

930(+

)16

9556

463

.75

6.2

GH

SLG

HE

VE

ED

clas

s_3

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for maintaining lipid homeostasis necessary for growth and reproduction, as well as for adapting to changing environment.

Analysis of sub‑cellular location, signal peptide and transmembrane domains of lipases in M. circinelloides WJ11

The predicted results of sub-cellular localization and the presence of signal peptide of lipases in strain WJ11 were shown in Table 3. Five lipases in WJ11 (WJ_16, WJ_29, WJ_39, WJ_40 and WJ_43) are most likely belonging to secretary lipases with a signal peptide at the N-terminus based on the TargetP1.1 Server (the location is “S” and RC value >0.8) and SignalP4.1 Server program (the pre-dicted result is “YES”). Our previous study has found that there are three potential extracellular lipases in strain CBS 277.49 (protein ID: 116027, 106404 and 170034) [40]. More genes coding extracellular lipase in WJ11 possibly mean that strain WJ11 has larger potential to utilize extra-cellular oils as carbon source than strain CBS 277.49. Sig-nal peptide sequences in these lipases contain 18, 20 and 28 of amino acids (Table 4), respectively. Interestingly, the amino acid sequence similarity of signal peptide is 96 % (between 170034 in CBS 277.49 and WJ_29 in WJ11) and 79 % (between 106404 in CBS 277.49 and WJ_40 in WJ11). Similar study reported that extracellular lipases CdLIP1 and CdLIP3 [4] contain a putative signal sequence with 18 and 28 of amino acids, respectively, while the N-ter-minal sequence of lipases Lip7p and Lip8p in Y. lipolytica [10] were found to correspond to the 8 aa of the putative signal sequence. These results also indicated that the signal sequence is present as precursor when associated to the cell wall. Moreover, M. circinelloides WJ11 is a potential source of extracellular lipases and would also be interesting models for studying extracellular lipolysis and fat uptake.

The TMHMM program showed that 11 lipases in WJ11 (WJ_2, 5, 11, 16, 25, 26, 30, 37, 38, 44 and 45) were pre-dicted to contain transmembrane spanning regions of 1–4 near their N-terminus (Fig. 1), whereas only two lipases (protein ID: 115761 and 107413) in strain CBS277.49 were predicted to have transmembrane spanning regions [40]. In the yeast S. cerevisiae, Tgl3p and Tgl4p did not contain any transmembrane (TrM) spanning region [27, 28], but lipases TGL3 and TGL4 from Y. lipolytica were predicted to contain one transmembrane spanning region [9]. These differences in TrM structures may play major roles in con-tributing to the separate roles for these enzymes in vivo. In addition, lipases encoded by genes WJ_ 2, WJ_ 44, WJ_ 37 and WJ_ 25 in strain WJ11 have similar position of TrM sequences. Similar finding were observed in the TrM sequences of lipases WJ_11 and 16, WJ_26 and 5, and jgi_CBS 277.49_115761, 107413 and WJ_30.Ta

ble

1 c

ontin

ued

Gen

eL

ocat

ion

(Str

and)

Gen

e si

ze

(cD

NA

bps

)A

min

o ac

idSu

buni

t (k

Da)

pIC

onse

rved

seq

uenc

eA

nnot

atio

n

Lip

ase

Acy

ltran

sfer

ase

evm

.mod

el.s

caff

old0

0103

.29

scaf

fold

0010

3:74

129-

7912

9(−

)50

0112

9114

5.63

7.5

GH

SLG

HN

QL

FDcl

ass_

3

evm

.mod

el.s

caff

old0

0233

.7sc

affo

ld00

233:

2126

9-22

777(−

)15

0942

948

.98

9G

LSH

GH

HH

TQ

Dcl

ass_

3

evm

.mod

el.s

caff

old0

0071

.35

scaf

fold

0007

1:83

350-

8760

8(+

)42

5910

1711

3.74

5.1

GL

SVG

HN

DH

QD

clas

s_3

evm

.mod

el.s

caff

old0

0209

.14

scaf

fold

0020

9:27

134-

2838

3(−

)12

5030

234

.06

5.1

GH

SFG

–cl

ass_

3

evm

.mod

el.s

caff

old0

0209

.18

scaf

fold

0020

9:32

378-

3366

2(+

)12

8533

336

.96

7.6

GH

SYG

–cl

ass_

3

evm

.mod

el.s

caff

old0

0031

.5sc

affo

ld00

031:

1964

00-1

9719

9(−

)80

042

046

.58

5.8

GT

SAG

–cl

ass_

3

evm

.mod

el.s

caff

old0

0170

.9sc

affo

ld00

170:

1950

5-20

753(−

)12

4937

741

.91

4.7

GT

SAG

–cl

ass_

3

evm

.mod

el.s

caff

old0

0029

.42

scaf

fold

0002

9:12

3632

-125

130(+

)14

9938

446

.58

5.8

GA

SIG

HD

LR

IDG

DSL

evm

.mod

el.s

caff

old0

0236

.5sc

affo

ld00

236:

1284

2-14

220(−

)13

7937

341

.68

4.9

GK

SDG

–G

DSL

evm

.mod

el.s

caff

old0

0122

.28

scaf

fold

0012

2:80

179-

8275

2(−

)25

7454

961

.87

5.4

GL

SSG

HD

AL

LD

GD

SL

evm

.mod

el.s

caff

old0

0111

.28

scaf

fold

0011

1:93

208-

9461

7(−

)14

1037

340

.76

5.9

GV

SYG

–G

DSL

evm

.mod

el.s

caff

old0

0079

.34

scaf

fold

0007

9:11

1258

-112

616(−

)13

5937

641

.71

4.9

GY

SKG

HD

LR

MD

GD

SL

Gen

e, L

ocat

ion

(str

and)

, Gen

e Si

ze, A

min

o ac

id, p

I, C

onse

rved

seq

uenc

e an

d A

nnot

atio

n w

ere

deri

ved

from

the

NC

BI

sour

ces

(http

://w

ww

.ncb

i.nlm

.nih

.gov

/gen

ome/

?ter

m=

LG

TF0

0000

000)

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1 3

Tabl

e 2

Lip

ase

gene

s an

d pr

otei

ns o

f M

ucor

cir

cine

lloi

des

CB

S 27

7.49

Gen

e, G

ene,

Loc

atio

n (s

tran

d), G

ene

Size

, Am

ino

acid

, pI,

Con

serv

ed s

eque

nce

and

Ann

otat

ion

wer

e de

rive

d fr

om th

e Jo

int G

enom

e In

stitu

te (

JGI)

dat

abas

e

Gen

eL

ocat

ion

(Str

and)

Gen

e si

ze

(cD

NA

bps

)Pr

otei

n ID

pIA

min

o ai

cdSu

buni

t (k

Da)

Con

serv

ed s

eque

nce

Ann

otat

ion

Lip

ase

Acy

ltran

sfer

ase

Gen

emar

k1.1

1790

_gsc

affo

ld_1

2:55

4802

-556

327(+

)15

2611

6027

4.8

385

42.6

1G

ASV

GH

DL

RV

DL

ipas

e_G

DSL

Gen

emar

k1.1

0741

_gsc

affo

ld_0

9:29

5517

-296

869(−

)13

5311

4978

4.7

369

40.6

4G

YSK

GH

DL

RM

DL

ipas

e_G

DSL

Muc

ci1.

fgen

eshM

C_p

m.3

_#_2

53sc

affo

ld_0

3:19

7704

1-19

7865

9(−

)16

1972

954

6.2

365

39.9

4G

ISY

G–

Lip

ase_

GD

SL

Muc

ci1.

e_gw

1.2.

1562

.1sc

affo

ld_0

2:22

1965

9-22

2097

8(+

)13

2034

010

6.9

388

44.1

7G

FSQ

G–

α/β

-hyd

rola

se_1

e_gw

1.04

.161

5.1

scaf

fold

_04:

7080

41-7

0949

9(+

)14

5914

3450

5.8

432

48.0

6G

FSQ

Gα

/β-h

ydro

lase

_1

Gen

emar

k1.1

1027

_gsc

affo

ld_1

0:85

859-

8674

3(−

)88

511

5264

5.9

295

33.0

9G

DSA

G–

α/β

-hyd

rola

se_3

Muc

ci1.

fgen

eshM

C_p

g.4_

#_98

2sc

affo

ld_0

4:32

0775

4-32

0895

8(−

)12

0581

713

5.4

342

37.1

4G

DSA

G–

α/β

-hyd

rola

se_3

Gen

emar

k1.8

931_

gsc

affo

ld_0

7:13

1119

-132

507(−

)13

8911

3168

5.5

404

45.4

2G

DSA

G–

α/β

-hyd

rola

se_3

fgen

esh1

_pg.

03_#

_541

scaf

fold

_03:

1866

164-

1867

667(+

)15

0416

1426

6.1

445

49.7

6G

DSA

GH

LYL

DD

α/β

-hyd

rola

se_3

Gen

emar

k1.2

47_g

scaf

fold

_01:

7562

08-7

5835

8 (+

)21

5110

4484

7.8

621

68.7

6G

DSA

GH

GK

MT

Dα

/β-h

ydro

lase

_3

fgen

esh1

_pg.

09_#

_300

scaf

fold

_09:

9666

18-9

6805

0(+

)14

3316

7388

6.4

456

51.2

5G

DSA

G–

α/β

-hyd

rola

se_3

estE

xt_G

enem

ark1

.C_0

8061

0sc

affo

ld_0

8:18

4759

5-18

4884

8(−

)12

5418

5587

7.3

371

41.9

4G

DSA

GH

WIV

KD

α/β

-hyd

rola

se_3

estE

xt_G

enem

ark1

.C_0

5106

7sc

affo

ld_0

5:31

7273

5-31

7493

3(−

)21

9918

4709

8.4

625

69.2

8G

HSL

GH

HH

HQ

Dcl

ass_

3

Muc

ci1.

e_gw

1.5.

1135

.1sc

affo

ld_0

5:32

3892

-325

186(−

)12

9539

585

5.4

337

37.0

2G

TSA

G–

clas

s_3

gw1.

06.1

316.

1sc

affo

ld_0

6:21

9347

8-21

9450

2(+

)10

2513

0354

4.7

305

33.8

5G

TSA

G–

clas

s_3

Gen

emar

k1.1

1524

_gsc

affo

ld_1

1:65

9634

-661

823(−

)21

9011

5761

9.1

622

70.5

5G

LSH

GH

HH

SLD

clas

s_3

Gen

emar

k1.3

176_

gsc

affo

ld_0

2:35

3797

9-35

4005

7(+

)20

7910

7413

7.8

593

67.3

9G

HSL

GH

KG

FWD

clas

s_3

Gen

emar

k1.2

167_

gsc

affo

ld_0

2:53

4294

-535

753(+

)14

6010

6404

7.3

345

38.0

9G

HSY

G–

clas

s_3

gw1.

02.4

71.1

scaf

fold

_02:

5288

13-5

2986

5(−

)10

5311

9940

6.1

234

26.2

8G

HSF

GH

TALY

Dcl

ass_

3

fgen

esh1

_kg.

01_#

_278

_#_1

_0sc

affo

ld_0

1:37

6004

1-37

6074

1(−

)70

115

4931

7.2

219

23.1

4G

HSL

G–

clas

s_3

fgen

esh1

_pg.

08_#

_445

scaf

fold

_08:

1489

434-

1492

137(−

)27

0416

6875

7.6

863

95.6

7G

HSL

GH

DK

AE

Dcl

ass_

3

Muc

ci1.

e_gw

1.2.

509.

1sc

affo

ld_0

2:24

1095

7-24

1237

4(−

)14

1835

076

6.9

353

37.9

6G

HSL

GH

LSY

YD

clas

s_3

fgen

esh1

_kg.

03_#

_293

_#_1

528_

1sc

affo

ld_0

3:32

6364

6-32

6534

6(+

)17

0115

5817

7.9

385

41.1

4G

HSL

GH

FSY

YD

clas

s_3

estE

xt_G

enew

ise1

.C_0

2134

4sc

affo

ld_0

2:27

0999

6-27

1179

5(+

)18

0017

0034

7.6

411

45.2

5G

HSL

G–

clas

s_3

Gen

emar

k1.7

497_

gsc

affo

ld_0

5:22

4416

4-22

4630

8(−

)21

4511

1734

6.2

696

77.9

2G

HSL

GH

SKA

WD

clas

s_3

fgen

esh1

_kg.

02_#

_75_

#_50

1_1

scaf

fold

_02:

9247

29-9

2642

8(−

)17

0015

5202

6.2

396

43.4

2G

HSL

G–

clas

s_3

Gen

emar

k1.7

083_

gsc

affo

ld_0

5:10

1832

4-10

2142

7(+

)31

0411

1320

8.4

935

106.

25G

HSL

GH

KFN

PDcl

ass_

3

Gen

emar

k1.1

0208

_gsc

affo

ld_0

8:88

2079

-885

366(−

)32

8811

4445

8.6

949

107.

58G

HSL

GH

NQ

LFD

clas

s_3

Gen

emar

k1.6

316_

gsc

affo

ld_0

4:30

5847

6-30

6137

0(−

)28

9511

0553

5.2

816

90.9

7G

HSL

GH

NQ

LFD

clas

s_3

fgen

esh1

_pg.

09_#

_158

scaf

fold

_09:

4928

42-4

9570

7(−

)28

6616

7246

5.1

840

93.5

8G

HSL

G–

clas

s_3

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1 3

Evolution of lipases in M. circinelloides CBS 277.49 and WJ11

To investigate the evolutionary relationship of lipase genes, a neighbor-joining tree was generated by align-ing protein sequences of lipases in M. circinelloides CBS 277.49 and WJ11, Tgl3p, Tgl4p and Tgl5p in the yeast S. cerevisiae, and TGL3 and TGL4 in the yeast Y. lipolytica. The phylogram was clustered into three major groups (I, II and III), while the group I appeared earlier by 800 million years than group II and III (Fig. 2). The lipases in group I contain nine sequence variation within the motif GXSXG including GHSLG, G (D/T) SAG, GAS (V/I) G, GLS (S/V) G, GFSQG and GKSDG, whereas these sequence variations appear partly in group II or III. Interestingly, 76 % percentage of lipases in class_3 family and 78 % percentage of lipases in α/β-hydrolase_3 fam-ily fall into group I. The class_3 family showed a closer relationship to the α/β-hydrolase_3 family. This result indicates that class_3 family and α/β-hydrolase_3 family are the conserved lipase family in the fungus M. circinel-loides. The sequence GHSLG is a typical conserved motif in the class_3 family as same as the motif GDSAG in the α/β-hydrolase_3 family. Eighteen pairs of lipases between strain WJ11 and CBS 277.49 show closer evolutionary relationships on the phylogram tree, but further homology analyses based on their amino acid sequences reveal that only ten pairs of lipases have higher identify from 60.8 to 95.7 % with the sequence similarity from 63.1 to 98.5 % (Table 5). This result suggests that most of lipases between strain WJ11 and CBS 277.49 have different amino acid sequences, which may result in different functions of these lipases and associated to the different lipid accumulation. The sequence homology analyses between TGL4 and WJ_1, WJ_25, WJ_26 and Tgl3p, and Tgl4p and 167246 were also performed. TGL4 displayed 9.5 % sequence identify to lipase (WJ_1) and 15 % to lipase (WJ_25). The amino acid sequence identity was 3.7 % (between Tgl3p

Table 3 Sub-cellular location and presence and location of signal peptide of lipases in M. circinelloides WJ11

No. Gene Sub-cellular location

Signal peptides

Location RC

WJ_1 evm.model.scaffold00007.23 – 1 No

WJ_2 evm.model.scaffold00232.15 S 2 No










WJ_12 evm.model.scaffold00230.20 M 3 No




WJ_16 evm.model.scaffold00017.8 S 1 Yes






























S secretory pathway, i.e. the sequence contains a signal peptide; M mitochondrion, i.e. the sequence contains a mitochnondrial target-ing peptide; – any other location; RC reliability class, from 1 to 5, which is a measure of the size the difference between the highest and the second highest output scores; 1 diff >0.8; 2 0.8> diff > 0.6; 3 0.6> diff > 0.4; 4 0.4> diff > 0.2; 5 0.2> diff

Table 3 continued

No. Gene Sub-cellular location

Signal peptides

Location RC



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1 3

and lipase WJ_1) and 9.3 % (between Tgl4p and lipase 167246). The lower homology indicated that lipases in M. circinelloides CBS 277.49 and WJ11 could be func-tionally different from lipases in yeast S. cerevisiae and Y. lipolytica.

Transcriptional analysis of lipase genes in M. circinelloides WJ11 during growth and lipid accumulation

In oleaginous fungus M. circinelloides CBS 277.49 and WJ11, the initiation of lipid accumulation during lipid syn-thesis is caused by the exhaustion of nitrogen (N) in the cultural medium. So in this study, to explore the potential relationship between the expression of lipase genes and

lipid accumulation in M. circinelloides, we analyze the changes at the transcriptional level of lipase genes before and after N depletion.

Our previous studies have reported characteristics of the growth and lipid accumulation in oleaginous fungi M. cir-cinelloides WJ11 [32]. Briefly, glucose remained in excess during the entire fermentation process and ammonium was used up at approx. 9 h. Cell dry weight initially increased rapidly up to 9 h and then slowed down after nitrogen exhaustion. The fungus started to accumulate lipid rapidly immediately after nitrogen depletion. From 9 to 48 h, the total fatty acids content increased rapidly and then slow down.

For transcriptional analysis, M. circinelloides cells were collected at 6 h (N rich, i.e., balanced growth stage), 24 h

Table 4 Sequences and location of signal peptide of lipases in M. circinelloides CBS 277.49 and WJ11

No. Protein/gene ID Signal peptides

Sequence Length

170034 MVSTSYLISQGLHLCVAVSCLLIHFTDAA 28/29

106404 MIFRNLIILLLATAAIQCQ 18/19

116027 MRLCKQLVGIMAVYGATAW 18/19

WJ_16 evm.model.scaffold00017.8 MCIIAYLCSVLLIPTVMTA 18/19

WJ_40 evm.model.scaffold00209.18 MIFRNLFILLLVVAATQCQ 18/19

WJ_39 evm.model.scaffold00209.14 MLKTCAQLLLIGAILQPALCN 20/21

WJ_43 evm.model.scaffold00029.42 MRLCTKLVGIIAAYGATAWAL 20/21

WJ_29 evm.model.scaffold00042.33 MVSTAYLISQGLHLCVAVSCLLIHFTDAA 28/29

Fig. 1 The transmembrane spanning region of M. circinelloides CBS 277.49 lipases (115761 and 107413) and WJ11 lipases (WJ_2, 5, 11, 16, 25, 26, 30, 37, 38, 44 and 45) were predicted in TMHMM

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1 3

Fig. 2 Phylogenetic analyses of lipases from M. circinelloides CBS 277.49 and WJ11, Tgl3p, Tgl4p and Tgl5p from yeast S. cerevisiae, and TGL3 and TGL4 from yeast Y. lipolytica

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1 3

(after N depletion, i.e., fast lipid and accumulation stage) and 72 h (after N depletion, i.e., slow lipid accumulation stage), and qRT-PCR was carried out to analyze the tran-scriptional level of lipase genes (Table 6). We applied a fold-change cutoff of ≥1.5 for up-regulation, and ≤0.5 for down-regulation.

The results showed that twenty-one lipase genes in WJ11 were intensely up-regulated at the fast lipid accumu-lation stage (24 h) but slightly up-regulated at the slow lipid accumulation stage (72 h) compared to the balanced growth stage (6 h). The glucose at the fast lipid accumulation stage (24 h) was in excess and enough to provide energy for cell growth, but the question remained why lipase genes were up-regulated at this stage? One possibility might be that fatty acids from TAG degradation induced by lipases may serve primarily for phospholipid synthesis which is vital to maintaining cell growth. Many lipases with acyltransferase activity are prominent contributor to TAG synthesis in the logarithmic growth stage or the exponential growth phase but less crucial under stress conditions or in the stationary phase, such as PDAT in microalga Chlamydomonas rein-hardtii [39] or Tgl3p, Tgl4p and Tgl5p in the yeast S. cer-evisiae [24, 27, 28]. Moreover, Daum et al. demonstrated that the presence of intracellular TAG was important for the acyltransferase activity of these multi-functional lipases [27]. In vitro Tgl3p in S. cerevisiae utilizes acyl-CoAs for efficient acylation of lysophosphatidylethanolamine, indi-cating that fatty acyl activation is required for the phospho-lipid biosynthetic route. Fewer these lipase genes (six) in strain CBS 277.49 possible resulted in the lower content of phospholipid compared with strain WJ11 [40]. Inter-estingly, seven lipase genes in WJ11 were down-regulated at the fast lipid accumulation stage, but were enhanced sharply at the last fermentation stage. These results sug-gested that these lipases were activated under a lower concentration of glucose in the cultural medium, and may be involved in β-oxidation at the later fermentation stage

(72 h). Similarly, Mycobacteriaceae tuberculosis strain lipase gene (F11 TBFG_11087) was predicted to be part of the operon (mtf_TBFG_11089) together with two other genes, TBFG_11089 and TBFG_11088, encoding enoyl-CoA hydrotases, which are involved in β-oxidation of FFA [20]. Fourteen lipase genes in strain WJ11 but five lipase genes in strain CBS 277.49 were down-regulated during the lipid accumulation. The remaining lipase genes in strains WJ11 and CBS 277.49 have no significant changes in the transcriptional level during the entire bioprocess. Similarly, transcriptions of some triacylglycerol lipase genes in the fungus Mortierella alpina were also inhibited during the lipid accumulation [6]. More lipase genes in strain WJ11 were inhibited compared to strain CBS 277.49, which may be associated with high lipid accumulation in WJ11. More-over, this result also supported our previous prediction that lipases (WJ_16, 29, 39 and 40) in WJ11 belong to the fam-ily of secretary lipases except the lipase (WJ_43), because transcriptional levels of extracellular lipase genes should not be triggered by glucose in the culture medium.

Conclusion

In this work we searched all genes encoding lipase in a high lipid-producing strain M. circinelloides WJ11 based on its genome database and compared these genes to a low lipid-producing strain M. circinelloides CBS 277.49, and ana-lyzed several characteristic, sub-cellular location, phyloge-netic analysis and expression profiling of the lipase genes during growth and lipid accumulation. Our results reveal that all of these proteins contain the typical lipase motif of GXSXG and were divided into four types including α/β-hydrolase_1, α/β-hydrolase_3, class_3 and GDSL. Inter-estingly, some lipases also contain a typical acyltransferase motif of H-(X) 4-D, and these lipases may play a dual role in lipid metabolism, catalyzing both lipid hydrolysis and

Table 5 Homology analyses of lipases between strain WJ11 and CBS 277.49 based on their amino acid sequences

WJ11 CBS 277.49 Amino sequence

No. Gene ID Protein ID Identify (%) Similarity (%)

WJ_30 evm.model.scaffold00065.29 107413 92.7 95.4


WJ_35 evm.model.scaffold00024.66 111734 72 76.6




WJ_7 evm.model.scaffold00020.2 81713 93 96.5




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Table 6 The transcriptional levels of lipase genes in strains WJ11 and CBS 277.49 were analyzed by qRT-PCR at 6, 24 and 72 h

Strain No. Gene/protein ID The transcriptional level (h)

6 24 72

WJ11 WJ_1 evm.model.scaffold00007.23 1.00 3.07 ± 0.06a 1.72 ± 0.23b

WJ_2 evm.model.scaffold00232.15 1.00 2.66 ± 0.13a 0.99 ± 0.48b

WJ_3 evm.model.scaffold00091.18 1.00 0.74 ± 0.13b 3.59 ± 0.48a


WJ_6 evm.model.scaffold00012.36 1.00 2.28 ± 0.42b 1.35 ± 0.25b

WJ_7 evm.model.scaffold00020.2 1.00 0.16 ± 0.03a 0.03 ± 0.01a






































CBS 277.49 114978 1.00 0.14 ± 0.05b 0.26 ± 0.14b

72954 1.00 2.25 ± 0.34b 1.01 ± 0.23a

34010 1.00 2.87 ± 0.56b 1.36 ± 0.21b

143450 1.00 0.78 ± 0.08b 0.29 ± 0.11b

115264 1.00 0.56 ± 0.21b 0.44 ± 0.12b

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transacylation reactions. Based on the biological roles of lipases in yeast and the expression profiling of lipase genes in M. circinelloides, we preliminarily hypothesized that some lipases may be involved in TAG degradation, phos-pholipid synthesis and beta-oxidation (Fig. 3). These dif-ferences involved in potential roles of lipase may be asso-ciated with the differential growth and lipid accumulation in these two strains. Moreover, the results of sub-cellular localization, the presence of signal peptide and transcrip-tional analyses of lipase genes indicated that four lipases in WJ11 most likely belong to extracellular lipases, and these extracellular lipases are valued to investigation for pharma-ceutical and chemical applications.

Although bioinformatic analyses and transcriptional analyses are not necessary to identify the intrinsic of lipases in M. circinelloides, this study provides a plat-form for the selection of candidate lipase genes for further detailed functional study. Target gene knockout based on

the expression profile and the determination of enzyme activity could explain the potential roles of lipases in the lipid accumulation.

Acknowledgments This research was supported by the National Natural Science Foundation of China (31271812 and 21276108), the National High Technology Research and Development Program of China (2012AA022105C), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1249), the Program for New Century Excellent Talents (NCET-13-0831).

References

1. Andrade GS, Carvalho AK, Romero CM, Oliveira PC, Castro HF (2014) Mucor circinelloides whole-cells as a biocatalyst for the production of ethyl esters based on babassu oil. Bioprocess Biosyst Eng 37:2539–2548

2. Andrade GSS, Freitas L, Oliveira PC, Castro HF (2012) Screen-ing, immobilization and utilization of whole cell biocatalysts to

A fold-change cutoff of ≥1.5 was applied for up-regulation, and ≤0.5 for down-regulation. Values which do not share common superscripts were significantly different to each othera p value <0.05b p value <0.01

Table 6 continued Strain No. Gene/protein ID The transcriptional level (h)

6 24 72

81713 1.00 1.64 ± 0.23b 0.05 ± 0.03b

185587 1.00 0.23 ± 0.13b 0.14 ± 0.15b

154931 1.00 1.73 ± 0.25b 0.54 ± 0.21b

155817 1.00 1.92 ± 0.41b 0.91 ± 0.21a

155202 1.00 0.10 ± 0.03b 0.15 ± 0.07b

Fig. 3 Potential roles of lipases during cell growth and lipid accumulation in M. circinel-loides. TAG triacylglycerol, DAG diacylglycerol, PA phos-phatidic acid, LPA lysophos-phatidic acid, G-3-P glycerol-3-phosphate, FA fatty acid, FFA free fatty acid

Dow


ic.oup.com/jim



1 3

mediate the ethanolysis of babassu oil. J Mol Catal B Enzym 84:183–188

3. Antczak T (2001) Catalytic properties of fungal lipases from Mucor Circinelloides and Mucor Racemosus. Zeszyty Naukowe. Rozprawy Naukowe/Politechnika Łódzka 298:5–169

4. Bigey F, Bougard TD, Nicaud JM, Moulin G (2003) Identifica-tion of a triacylglycerol lipase gene family in Candida defor-mans: molecular cloning and functional expression. Yeast 20:233–248

5. Carvalho AKF, Faria ELP, Rivaldi JD, Andrade GSS, Oliveira PC (2015) Performance of whole-cells lipase derived from Mucor circinelloides as a catalyst in the ethanolysis of non-edible veg-etable oils under batch and continuous run conditions. Ind Crops Prod 67:287–294

6. Chen H, Hao G, Wang L, Wang H, Gu Z (2014) Identification of a critical determinant that enables efficient fatty acid synthesis in oleaginous fungi. Sci Rep 5:11247. doi:10.1038/srep11247

7. Desfougeres T, Haddouche R, Fudalej F, Neuveglise C, Nicaud JM (2010) SOA genes encode proteins controlling lipase expres-sion in response to triacylglycerol utilization in the yeast Yar-rowia lipolytica. FEMS Yeast Res 10:93–103

8. Destain J, Fickers P, Weekers F, Moreau B, Thonart P (2005) Utilization of methyloleate in production of microbial lipase. Appl Biochem Biotechnol 121–124:269–277

9. Dulermo T, Tréton B, Beopoulos A, Gnankon APK, Haddouche R (2013) Characterization of the two intracellular lipases of Y. lipolytica encoded by TGL3 and TGL4 genes: new insights into the role of intracellular lipases and lipid body organisation. Bio-chim Biophys Acta 1831:1486–1495

10. Fickers P, Marty A, Nicaud JM (2011) The lipases from Yar-rowia lipolytica: genetics, production, regulation, biochemical characterization and biotechnological applications. Biotechnol Adv 29:632–644

11. Fickers P, Nicaud JM, Gaillardin C, Destain J, Thonart P (2004) Carbon and nitrogen sources modulate lipase production in the yeast Yarrowia lipolytica. J Appl Microbiol 96:742–749

12. Garcia-Galan C, Barbosa O, Ortiz C, Torres R, Rodrigues RC (2013) Biotechnological prospects of the lipase from Mucor javanicus. J Mol Catal B Enzym 93:34–43

13. Goncalves C, Lopes M, Ferreira JP, Belo I (2009) Biological treatment of olive mill wastewater by non-conventional yeasts. Bioresour Technol 100:3759–3763

14. Guieysse D, Sandoval G, Faure L, Nicaud JM, Monsan P (2005) New efficient lipase from Yarrowia lipolytica for the resolu-tion of 2-bromo-arylacetic acid esters. Tetrahedron Asymmetry 15(22):3539–3543

15. Ham HJ, Rho HJ, Shin SK, Yoon HJ (2010) The TGL2 gene of Saccharomyces cerevisiae encodes an active acylglycerol lipase located in the mitochondria. J Biol Chem 285:3005–3013

16. Holmes RS, Cox LA, VandeBerg JL (2010) Comparative stud-ies of mammalian acid lipases: evidence for a new gene family in mouse and rat (Lipo). Comp Biochem Physiol Part D Genom Proteom 5:217–226

17. Kamzolova SV, Finogenova TV, Lunina YN, Perevoznikova OA, Minachova LN (2007) Characteristics of the growth on rapeseed oil and synthesis of citric and isocitric acids by Yarrowia lipol-ytica yeasts. Microbiology 76:20–24

18. Kendrick A, Ratledge C (1992) Desaturation of polyunsatu-rated fatty acids in Mucor circinelloides and the involvement of a novel membrane-bound malic enzyme. Eur J Biochem 209:667–673

19. Kurat CF, Natter K, Petschnigg J, Wolinski H, Scheuringer K (2006) Obese yeast: triglyceride lipolysis is functionally con-served from mammals to yeast. J Biol Chem 281:491–500

20. Lazniewski M, Steczkiewicz K, Knizewski L, Wawer I, Ginalski K (2011) Novel transmembrane lipases of alpha/beta hydrolase fold. FEBS Lett 585:870–874

21. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408

22. Pleiss J, Scheib H, Schmid RD (2000) The His gap motif in microbial lipases: a determinant of stereoselectivity toward tria-cylglycerols and analogs. Biochimie 82:1043–1052

23. Purwanto MGM, Maretha MV, Wahyudi M, Goeltom MT (2015) Whole cell hydrolysis of sardine (Sardinella Lemuru) oil waste using Mucor Circinelloides NRRL 1405 immobilized in poly-urethane foam. Procedia Chem 14:256–262

24. Rajakumari S, Daum G (2009) Janus-faced enzymes yeast Tgl3p and Tgl5p catalyze lipase and acyltransferase reactions. Mol Biol Cell 21:501–510

25. Rajendran A, Palanisamy A, Thangavelu V (2009) Lipase cata-lyzed ester synthesis for food processing industries. Braz Arch Biol Technol 52:207–219

26. Rosa Amarilis RF, Adrián G, Santiago TM, Victoriano G (2012) Malic enzyme activity is not the only bottleneck for lipid accu-mulation in the oleaginous fungus Mucor circinelloides. Appl Microbiol Biotechnol 97:3063–3072

27. Schmidt C, Athenstaedt K, Koch B, Ploier B, Daum G (2013) Regulation of the yeast triacylglycerol lipase TGl3p by forma-tion of nonpolar lipids. J Biol Chem 288:19939–19948

28. Sona R, Günther D (2010) Multiple functions as lipase, steryl ester hydrolase, phospholipase, and acyltransferase of Tgl4p from the yeast Saccharomyces cerevisiae. J Biol Chem 285:15769–15776

29. Suzzi G, Lanorte MT, Galgano F, Andrighetto C, Lombardi A (2001) Proteolytic, lipolytic and molecular characterisation of Yarrowia lipolytica isolated from cheese. Int J Food Microbiol 69:69–77

30. Szczesna-Antczak M, Antczak T, Piotrowicz-Wasiak M, Rzyska M, Binkowska N, Bielecki S (2006) Relationships between lipases and lipids in mycelia of two Mucor strains. Enzyme Microbial Technol 39:1214–1222

31. Szczesna-Antczak M, Antczak T, Rzyska M, Modrzejewska Z, Patura J, Kalinowska H, Bielecki S (2004) Stabilization of an intracellular Mucor circinelloides lipase for application in non-aqueous media. J Mol Catal B Enzym 29:163–171

32. Tang X, Chen HQ, Chen YQ, Chen W, Victoriano G (2015) Com-parison of biochemical activities between high and low lipid-producing strains of Mucor circinelloides: an explanation for the high oleaginicity of strain WJ11. PLoS One 10(9):e0137543

33. Tang X, Zhao LN, Chen HQ, Chen YQ, Chen W, Song YD, Ratledge C (2015) Complete genome sequence of a high lipid-producing strain of Mucor circinelloides WJ11 and comparative genome analysis with a low lipid-producing strain CBS 277.49. PLoS One 10(9):e0137543. doi:10.1371/journal.pone.0137543

34. Tang X, Zan X, Zhao L, Chen H, Chen YQ, Chen W, Song Y, Ratledge C (2016) Proteomics analysis of high lipid-producing strain Mucor circinelloides WJ11: an explanation for the mecha-nism of lipid accumulation at the proteomic level. Microb Cell Fact 11:15–35

35. Turki S, Mrabet JG, Marouani A, Thonart P (2010) Preliminary safety assessment of Yarrowia lipolytica extracellular lipase: results of acute and 28-day repeated dose oral toxicity studies in rats. Food Chem Toxicol 48:2393–2400

36. Vongsangnak W, Klanchui A, Tawornsamretkit L, Tatiyabor-wornchai W, Laoteng K, Meechai A (2016) Genome-scale meta-bolic modeling of Mucor circinelloides and comparative analysis with other oleaginous species. Gene 583(2):121–129

Dow


ic.oup.com/jim


http://dx.doi.org/10.1038/srep11247

http://dx.doi.org/10.1371/journal.pone.0137543


1 3

37. Yazawa H, Kumagai H, Uemura H (2012) Characterization of triglyceride lipase genes of fission yeast Schizosaccharomyces pombe. Appl Microbiol Biotechnol 96:981–991

38. Ying Z, Adams IP, Colin R (2007) Malic enzyme: the controlling activity for lipid production? Overexpression of malic enzyme in Mucor circinelloides leads to a 2.5-fold increase in lipid accu-mulation. Microbiology 153:2013–2025

39. Yoon K, Han D, Li Y, Sommerfeld M, Hu Q (2012) Phospho-lipid: diacylglycerol acyltransferase is a multifunctional enzyme involved in membrane lipid turnover and degradation while

synthesizing triacylglycerol in the unicellular green microalga Chlamydomonas reinhardtii. Plant Cell 24:3708–3724

40. Zan XY, Tang X, Zhao LN, Chu LF, Chen HQ, Chen W, Chen YQ, Song YD (2016) Bioinformatical analysis and preliminary study of the role of lipase in lipid metabolism in Mucor circinel-loides. RSC Adv 6:60673–60682

41. Zhao LN, Tang X, Luan X, Chen HQ, Chen YQ, Chen W, Song YD, Ratledge C (2015) Role of pentose phosphate pathway in lipid accumulation of oleaginous fungus Mucor circinelloides. RSC Adv 5:97658–97664

Dow


ic.oup.com/jim


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