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Increased lipid accumulation in Mucor Circinelloides by overexpression of mitochondrial citrate transporter genes Junhuan Yang, Shaoqi Li, Md. Ahsanul Kabir Khan, Victoriano Garre, Wanwipa Vongsangnak, and Yuanda Song Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.8b05564 • Publication Date (Web): 17 Jan 2019 Downloaded from http://pubs.acs.org on January 20, 2019

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Industrial & Engineering Chemistry Research

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Increased lipid accumulation in Mucor circinelloides by overexpression of

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mitochondrial citrate transporter genes

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Junhuan Yang1, Shaoqi Li1, Md. Ahsanul Kabir Khan1, Victoriano Garre2, Wanwipa

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Vongsangnak3, 4*, Yuanda Song1*

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1Colin

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Food Sciences, Shandong University of Technology, Shandong, P. R. China

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2Departmento

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Física Rocasolano, Consejo Superior de Investigaciones Científicas), Facultad de

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Biología, Universidad de Murcia, Murcia, 30100, Spain

Ratledge Center for Microbial Lipids, School of Agriculture Engineering and

de Genética y Microbiología (Unidad Asociada al Instituto de Química

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3Department

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Thailand

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4Computational

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(CBLAST), Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.

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E-mails:

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Junhuan Yang: [email protected]

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Shaoqi Li: [email protected]

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Md. Ahsanul Kabir Khan: [email protected]

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Victoriano Garre: [email protected]

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Wanwipa Vongsangnak: [email protected]

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Yuanda Song: [email protected]

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*Corresponding author

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Yuanda Song: [email protected]

of Zoology, Faculty of Science, Kasetsart University, Bangkok 10900,

Biomodelling Laboratory for Agricultural Science and Technology

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Industrial & Engineering Chemistry Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Wanwipa Vongsangnak: [email protected]

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Abstract

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Background

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Mucor circinelloides has been commonly used as the model microbe to investigate lipid

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production as an oleaginous fungus. Mitochondrial citrate transporter can catalyze the

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translocation of the citrate, accumulated from TCA cycle, across the mitochondrial

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inner membrane. The extra-mitochondrial citrate is then cleaved by ATP-citrate lyase to

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oxaloacetate (OAA) and acetyl-CoA. Acetyl-CoA together with NADPH generated in

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cytosol is used for fatty acid biosynthesis. Thus, citrate transporters provide a link

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between TCA cycle in mitochondria and fatty acid biosynthesis in cytosol. However,

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the role of citrate transporters for lipid accumulation in oleaginous fungi is not clear.

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Two genes coding for citrate transporters, named citrate transporter (ct) and

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tricarboxylate transporter (tct) respectively, were present in the genome of oleaginous

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fungus M. circinelloides WJ11, a high lipid producing strain (36 %, lipid/cell dry

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weight). As the mutant of strain CBS 277.49 (15 %, lipid/cell dry weight) has been

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constructed and its genetic engineering tools are available for gene manipulation, so in

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this work, we investigated the role of citrate transporters in regulating lipid biosynthesis

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by overexpressing the citrate transporters of M. circinelloides WJ11 in CBS 277.49.

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Results

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Our results showed that overexpression of ct and tct led to increased lipid accumulation

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by 44 % (from 13.0 % to 18.8 %, w/w, CDW) and 68 % (from 13.0 % to 21.8 %, w/w， 2 ACS Paragon Plus Environment

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CDW), respectively. Moreover, extracellular citrate concentration in ct-overexpressing

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strains (4.91 mM) and tct-overexpressing (3.25 mM) were significantly decreased by

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20 % and 47 % respectively compared to the control (6.09 mM). Furthermore,

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overexpression of the citrate transporter genes activated the downstream steps in lipid

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biosynthesis, such as ATP citrate lyase (acl gene) and fatty acid synthases (fas1 and

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fas2 genes), indicating a greater flux of carbon went into fatty acid biosynthesis.

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Conclusions

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This is the first report showing that citrate transporters involved in lipid accumulation in

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M. circinelloides. Both citrate transporter and tricarboxylate transporter could transport

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mitochondrial citrate to cytoplasm, which could provide more citrate to be cleaved by

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increased ACL to provide more acetyl-CoA and NADPH for increased FAS to

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synthesize fatty acids, thus, play a vital role in lipid biosynthesis in oleaginous fungus

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M. circinelloides.

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Keywords: Mitochondrial citrate transporter, Mucor circinelloides, Lipid accumulation

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1. Introduction

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Mitochondrial citrate transporter belongs to the mitochondrial carrier family. It provides

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a link between mitochondria and cytosol by catalyzing the translocation of citrate across

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the impermeable barrier of the mitochondrial inner membrane1. During cell growth,

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glucose is hydrolyzed by glycolysis pathway, and the final product, pyruvate, of the

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glycolysis goes into mitochondria, where it is cleaved into acetyl-CoA by pyruvate

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dehydrogenase. Acetyl-CoA then enters into the tricarboxylic acid (TCA) cycle by 3 ACS Paragon Plus Environment


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reacting with oxaloacetate (OAA) and generates citrate. Thus it provides the major

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source of cellular energy ATP by complete oxidation2. Upon an environmental stimulus

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such as nutrient (especially N) limitation, the TCA cycle becomes retarded, and citrate

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is accumulated in the mitochondria. The accumulated citrate is carried into the cytosol

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by citrate transporter, and the cytosolic citrate can be then cleaved to OAA and

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acetyl-CoA by ATP-citrate lyase (ACL). Acetyl-CoA is the essential precursor for fatty

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acid and sterol biosynthesis, whereas OAA is reduced to malate, which is transferred

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back into mitochondria. Alternatively, malate can be converted to pyruvate via malic

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enzyme, and provides cytosolic NADPH and H+ for fatty acid and sterol biosynthesis3-5.

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Citrate exerts a significant function as a key regulator of glycolysis, gluconeogenesis,

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and fatty acid synthesis. Thus, mitochondrial citrate transporter regulating the citrate

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shuttle between the mitochondria and the cytosol, is very important in oleaginous

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microorganisms4.

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Mitochondrial citrate transporter genes have been found in several species, including

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animals6, plants, and yeasts7. This enables us to understand their kinetic parameters8,

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function and regulation. Also, a comparative study of citrate efflux from mitochondria

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to cytosol has showed that the rates of citrate efflux were approximately 2.5-times

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greater in oleaginous than in non-oleaginous yeasts9. Thus, mitochondrial citrate

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transporters may play crucial roles in lipid storage by regulating the amount and rate of

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citrate efflux.

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Previous works have studied the structures, activities and kinetics of citrate transporters

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in yeast10-11, whereas, few studies have reported their functions in oleaginous 4 ACS Paragon Plus Environment

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filamentous fungi12. M. circinelloides has been widely used as the model microbe to

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investigate lipid production as an oleaginous fungus since 1980s13. Recently, a

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comparison analysis for oleaginous fungi Mortierella alpina and M. circinelloides at

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genome-scale level, showed that a putative gene (gene ID: 180302) encoding for the

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C4-dicarboxylate transporter/malic acid transport protein is only present in M.

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circinelloides14. Our preliminary work on malate transporter in M. circinelloides CBS

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277.49 has shown that this transporter regulates the influx of malate into the

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mitochondria and lipid accumulation15. The biochemical and molecular comparisons

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between M. circinelloides WJ11, a high lipid-producing strain with 36 % (w/w) lipid of

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cell dry weight (CDW) and CBS 277.49, a low lipid-producing strain with only 15 %

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(w/w) lipid16, have revealed possible regulation mechanism for lipid biosynthesis in this

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fungus17-19. However, the role of citrate transporters in lipid accumulation and the

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molecular mechanism of citrate transport in M. circinelloides are unclear. Based on

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bioinformatic analyses, we found two genes coding for putative citrate transporters. It

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was hypothesized that both transporters would play an important role in citrate

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transportation from the mitochondria to the cytosol for lipid accumulation. As the

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auxotrophic mutant of strain CBS 277.49 has been constructed and its genetic

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engineering tools are available for gene manipulation, so in this work, we used this

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strain as a model organism to investigate the effect of the citrate transporter on lipid

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accumulation by overexpressing the corresponding genes in M. circinelloides CBS

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277.49 and proposed a model of lipid biosynthesis with a citrate transporter (ct) and a

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tricarboxylate transporter (tct) involved suggesting that they may be essential targets for 5 ACS Paragon Plus Environment


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metabolic engineering aimed at increasing lipid accumulation in M. circinelloides.

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2 Results

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2.1 Identification of genes coding for putative mitochondrial transporter in M.

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circinelloides WJ11

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Gene annotation of M. circinelloides WJ11 surprisingly revealed the presence of 51

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genes coding for potential transporters, which may serve for transporting roles in the

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mitochondria (Fig. 1). According to the annotation in the Uniprot and the Transporter

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Classification Database (TCDB), interestingly, two genes coding for citrate transporter

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were found in the genome of WJ11, one corresponding to a citrate transporter

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(scaffold00129.3), named ct, and the other to a tricarboxylate transporter

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(scaffold00069.38), named tct.

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2.2 Bioinformatic analysis of mitochondrial citrate transporter genes in M.

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circinelloides

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Based on gene sequence and annotation, we compared the properties of citrate

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transporters in WJ11 (Table 1). Bioinformatic analysis of the deduced protein sequence

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of CT and TCT showed that they have different properties. The instability index and

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grand average of hydropathicity (GRAVY) indicated that they are stable and

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hydrophobic. Conserved domain prediction using CDD blast in the Conserved Domain

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Database at NCBI-CDD showed that CT has three Mtc_domains pfam00153 (putative

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mitochondrial carrier protein domains, amino acid residues 10 to 104, 106 to 199, and

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209 to 296) which are found in a wide range of mitochondrial transporters, whereas, 6 ACS Paragon Plus Environment

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TCT only have one Mtc_domain pfam03820 (putative tricarboxylate carrier protein

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domain, amino acid residues 12-321), which has been annotated in Homo sapiens20,

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rat21 and Saccharomyces cerevisiae22.

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Table 1. Properties of mitochondrial citrate transporters in WJ11 Properties

CT

TCT

Amino acid

301

321

Subunit (kDa)

32.12

35.22

PI

9.89

9.5

Instability index

18.29

33.92

GRAVY

0.042

0.089

Domain

pfam00153

pfam03820

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2.3 Generation of ct-overexpressing and tct-overexpressing strains of M.

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circinelloides by genetic engineering

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Involvement of ct and tct in fatty acid accumulation was investigated by generation of

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overexpressing strains for both genes assuming that an increase of citrate transport

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would result in higher lipid accumulation. The plasmid pMAT1552 contained pyrG

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gene as a selectable marker and the strong promoter zrt1 of M. circinelloides was used

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to over express the target gene. The ct and tct gene coding regions were cloned

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downstream of the promoter zrt1 to produce plasmids pMAT1552-ct and

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pMAT1552-tct, respectively (Fig. 2a) (see section of Materials and Methods for details).

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These gene-overexpressing plasmids and the empty plasmid pMAT1552 were

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transformed into MU402, of which uridine auxotrophy can be complemented by the 7 ACS Paragon Plus Environment


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pyrG gene present in the plasmids23. For each overexpressing plasmid, three

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independent transformants were selected, named Mc-ct1, Mc-ct2 and Mc-ct3 for

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pMAT1552-ct, Mc-tct1, Mc-tct2, Mc-tct3 for pMAT1552-tct, and one control

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transformant Mc-1552 for the empty plasmid.

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Presence of the plasmids in the transformants was confirmed by PCR analysis.

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Amplification was carried out using a primer pair (1552-F/R) that amplified ct and tct

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gene together with 600 bp of the backbone plasmid pMAT1552, producing fragments of

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1506-bp and 1796-bp for ct and tct expressing transformants respectively, whereas a

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600-bp fragment could be amplified from the control strain Mc-1552. PCR

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amplification (Fig. 2b, 2c) results proved that the selected transformants carried the

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expected plasmids.

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Notably, ct- and tct-overexpressing stains were grown in complete medium in 2 L

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fermenter with 1.5 L modified K & R medium for 4 days. The lipid contents of Mc-ct1,

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Mc-ct2, Mc-ct3 were not significantly different from each other, and the lipid content

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for Mc-tct1, Mc-tct2, Mc-tct3 were also similar (shown in Table S2), so transformants

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Mc-ct3 and Mc-tct3 for each gene were used for further study.

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2.4 Expression levels of ct and tct genes in the transformants

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The mRNA levels of ct and tct in the transformants Mc-ct3 and Mc-tct3 and the control

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Mc-1552 were analyzed by RT-qPCR at 3, 24, 48 and 72 h of cultivation (Fig. 3). As

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there were two similar ct and tct genes, a native one in the genome and an

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overexpressed one in the plasmid, in the transformants Mc-ct3 and M-tct3, respectively,

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two pairs of primers were designed to distinguish one gene type from the other (Table 8 ACS Paragon Plus Environment

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S1). The cloned ct and tct mRNA were expressed at high levels under the control of

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strong promoter zrt1 in the transformants Mc-ct3 and Mc-tct3, respectively, while the

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native ct and tct expression levels in the transformants, under the control of its own

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promoter, were similar to the control. The highest levels of ct and tct mRNA of the

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transformants were detected at 24 h, but they decreased gradually thereafter, although

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they were maintained at high levels during the whole fermentation. This confirmed that

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those two genes were overexpressed in the corresponding transformed strains.

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2.5 Cell growth and lipid accumulation in ct-overexpressing and tct-overexpressing

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strains

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The cell dry weight (CDW), concentrations of ammonium and glucose in the culture

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medium, and lipid accumulation of ct-overexpressing and tct-overexpressing strains

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during growth were analyzed (Fig 4). In general, all strains showed a similar and typical

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growth profile as the control strain (Fig. 4a). Glucose and ammonium consumption rate

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was similar in these three strains, but they were utilized more rapidly in the control

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strain Mc-1552 than ct- or tct-overexpressing strains (Fig. 4b and 4c). After nitrogen

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was depleted at 12 h, the lipid in three strains started to accumulate rapidly.

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Over-expressing of ct and tct genes had a significant influence on lipid accumulation in

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M. circinelloides. Thus, the lipid content in ct-overexpressing strain was increased 44 %

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compared to Mc-1552 (from 13.0 % in the control to 18.8 % in the transformant), while

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it was increased by 68 % in tct-overexpressing strain compared to Mc-1552 (from 13.0 %

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in the control to 21.8 % in the transformant) (Fig. 4d). The fatty acid profiles of these

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strains revealed thatγ-linolenic acid (GLA, 18:3) contents in total fatty acids (TFAs) of 9 ACS Paragon Plus Environment


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ct-overexpressing strain (21.7 % at 72 h) and tct-overexpressing strain (20.8 % at 72 h)

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were lower than that of Mc-1552 (27.3 % at 72 h)(Table 2). However, the contents of

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GLA in CDW of ct-overexpressing strain (4.1 % at 72 h) and tct-overexpressing (4.5 %

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at 72 h) strain were significantly higher than that of strain Mc-1552 (3.6 % at 72 h) due

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to their higher lipid production.

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Table 2. Fatty acid profiles of ct-overexpressing and tct-overexpressing strains. strains

Fatty acid composition (relative %, w/w)

Mc-1552

Mc-ct

Mc-tct

14:0

2.26±0.18 a

2.46± 0.19 a

2.19± 0.23 a

16:0

17.10±1.18 a

19.74±0.67 a

18.88±1.32 a

16:1

3.20±0.41 a

4.21±0.54 a

3.53±0.75 a

17:0

0.94±0.21 a

0.83±0.25 a

1.05±0.15 a

17:1

0.93±0.24 a

0.75±0.26 a

0.87±0.20 a

18:0

3.93±0.28 a

5.42±1.34 a

4.86±0.32 a

18:1

28.96±0.87 a

29.69±1.34 a

27.90±2.98 a

18:2

15.53±0.79 a

14.69±0.52 a

14.36±1.84 a

18:3

27.35±2.12 a

21.77±1.38 b

20.87±2.62 b

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2.6 Extracellular citrate concentration in ct-overexpressing and tct-overexpressing

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strains

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To investigate the role of citrate transporters in M. circinelloides and understand the

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mechanism of citrate metabolism, the extracellular citrate concentration in the cultures 10 ACS Paragon Plus Environment

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of ct-overexpressing and tct-overexpressing strains grown in K & R medium were

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analyzed. Similar to lipid accumulation, extracellular citrate concentration was initially

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low during the balanced growth phase, and then increased rapidly in all strains after

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nitrogen exhaustion at 12 h (Fig. 5). Interestingly, extracellular citrate concentration in

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ct-overexpressing (4.91 mM) and tct-overexpressing strains (3.25 mM) were

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significantly decreased by 20 % and 47 % respectively compared to the control (6.09

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mM). These results indicated that the intracellular citrate metabolism had been affected

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by the overexpression of ct and tct gene, which might lead to the increased

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accumulation of lipid in the fungus.

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2.7 Expression levels of acl, fas1 and fas2 genes in the transformants

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To ascertain whether the increased lipid accumulation in ct- and tct-over expressing

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strains was associated with the increased expression of the key genes for fatty acid

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biosynthesis in these strains, thus, the mRNA levels of acl, fas1 and fas2 in the

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transformants were assessed. As the crucial reaction for fatty acid biosynthesis in

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oleaginous microorganisms24, the acl mRNA levels were higher in both overexpressing

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strains than that of the control, which indicated that more acetyl-CoA, the substrate for

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fatty acid synthesis, may be provided in the transformations than in control strain. Fas1

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and fas2 can catalyze de novo fatty acid synthesis and regulate the extent of lipid

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accumulation in oleaginous microorganisms18. Compared to fas1 mRNA levels of the

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control, an increase of about 4.5-fold and 3-fold was observed in Mc-ct and Mc-tct

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strains, respectively. Meanwhile, the fas2 mRNA level significantly increased by

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1.94-fold and 4.3-fold in Mc-ct and Mc-tct transformant compared to the control, 11 ACS Paragon Plus Environment


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respectively (Table 3).

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Table 3. Expression of acl, fas1 and fas2 genes in the transformants Mc-ct, Mc-tct and

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Mc-1552. Strains

Relative mRNA level (fold)* acl

fas1

fas2

Mc-1552 0.74±0.01b 0.34±0.03c 0.36±0.01c Mc-ct

1.29±0.17a 1.48±0.07a 0.70±0.03b

Mc-tct

0.80±0.03b 0.98±0.07b 1.57±0.04a

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*Strains were grown in a 2 L fermenter with 1.5 L modified K & R medium, and the mycelium was

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harvested at 36 h. Total RNA of strains at different time was extracted and the mRNA accumulation

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was quantified by RT-qPCR. The values are mean of three independent fermentation experiments.

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Error bars represent the standard error of the mean.

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3. Discussion

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Fatty acid synthesis is an essential metabolic pathway in the cytoplasm of microbial

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cells that can be triggered when nitrogen is exhausted in the culture medium25. N

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depletion lead to the retardation of TCA cycle, which results in citrate accumulation in

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the mitochondria, then the accumulated citrate, can be transported to the cytosol where

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it can be cleaved by ACL to generate acetyl-CoA for fatty acid synthesis26. In addition,

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intracellular citrate can also be secreted into the medium when its cytosolic

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concentration is high. Therefore, mitochondrial citrate transporter connects sugar

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metabolism and lipid biosynthesis27-28. 12 ACS Paragon Plus Environment

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M. circinelloides is a model microbe to study the mechanism of lipid accumulation in

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oleaginous fungus. Although citrate transportation appears to be common in eukaryotes,

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the individual transporters are species-specific27. Recently, the genomes of M. alpina

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and M. circinelloides have been analyzed. Surprisingly, one gene coding for a malate

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transporter was found in M. circinelloides CBS 277.49, whereas it was absent in M.

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alpina genome. In addition, two genes coding for citrate transporters, one citrate

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transporter (ct gene) and one tricarboxylate transporter (tct gene), were discovered in M.

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circinelloides genome, whereas, only one gene coding for tricarboxylate transporter was

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found in M. alpina29. The different set of citrate transporters in oleaginous fungi

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suggests that the ways in which citrate is transported in each fungus could be varied.

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In this work, we have analyzed the role of mitochondrial tricarboxylate transporter and

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citrate transporter in citrate transportation and their contribution to fatty acid

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accumulation in M. circinelloides by overexpressing the ct and tct genes of WJ11,

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which is a high lipid producing strain of M. circinelloides, in CBS 277.49, a low lipid

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producing strain. When any of these genes was over expressed, the lipid productions

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were greatly increased in the corresponding transformants (Fig. 4d), suggesting that

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over expression of these two types of transporters improved citrate efflux from

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mitochondrion to cytosol. Citrate in cytoplasm has a negative feedback on glycolysis by

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inhibiting phosphofructokinase 1 (PFK1), 6-phosphofructo-2-kinase / fructose-2,

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6-biphosphatases (PFK2) and pyruvate kinase (PK)2, that could explain the low

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consumption rates of glucose and nitrogen in both overexpressing strains.

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Simultaneously, extracellular citrate concentrations decreased significantly, which may 13 ACS Paragon Plus Environment


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be caused by increased metabolic activity for fatty acid synthesis, as evidenced by

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significant induced expression of acl, fas1and fas2 (Table 3), the genes for fatty acid

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synthesis. This suggested that either the high efflux of citrate to the cytosol or the high

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concentration of citrate in the cytosol should trigger the activation of an unknown

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mechanism controlling the expression of fatty acid synthesis gene. Thus, although more

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citrate is supposed to be transferred into the cytosol in ct- and tct-overexpressing strains,

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the concentration of extracellular citrate was lower in ct- and tct-overexpressing strains

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than the control, because it was cleaved to acetyl-CoA and OAA by ACL, which could

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be at high levels as a result of the increased expression of acl gene. This may force the

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citrate cycle run faster leading to a greater flux of carbon to acetyl-CoA synthesis.

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Simultaneously, increased acetyl-CoA pool is further converted to fatty acids by

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increased expression of fas genes in overexpressing strains, explaining the low

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extracellular citrate concentration and high lipid accumulation in these transformants.

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This is in agreement with previous findings that when lipid accumulation was

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significantly increased, citric acid production was decreased in yeasts and oleaginous

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microalgae30. However, the remaining citrate in ct- and tct-overexpressing strains was

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still at high level to be excreted into the growth medium, especially at the end of lipid

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accumulation phase (Fig. 5). Furthermore, the accumulated citrate in the cytoplasm may

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lead to polysaccharide biosynthesis by inhibiting glycolysis at level of insufficient

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enzymatic activity of PFKs, and this may compete with lipid biosynthesis31. Thus, in

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future, more work should be done to decrease carbon outflow towards metabolic

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pathways competitive to lipogenesis, which may contribute to a higher lipid 14 ACS Paragon Plus Environment

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accumulation.

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Despite that overexpression of both transporters can increase lipid production, the

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increase of lipid production was slightly higher in the tricarboxylate transporter

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overexpressing strain than in the citrate transporter over-expressing strain. One

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hypothesis for this phenomenon is that the two transporters carry citrate by different

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mechanisms. Indeed, tricarboxylate transporter has been isolated from other species,

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such as rat32 and eel33, and only one binding site was found in its three dimensional

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structure. Some studies conducted in intact mitochondria or with the purified carrier

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protein reconstituted in proteoliposomes have provided significant evidence in favor of

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a uniport transport mechanism for citrate by this transporter: tricarboxylate transporter

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can carry citrate out and malate in through mitochondrion simultaneously. What is more,

296

it can carry citrate out the mitochondrion without exchanging for malate8. However,

297

many works have shown that citrate transporters, with two binding sites, can catalyze an

298

electroneutral exchange of citrate for another tricarboxylate6, even citrate34, a

299

dicarboxylate (L-malate)35, or phosphoenolpyruvate36 across the mitochondrial inner

300

membrane37. Citrate carried by this transporter is driven by a chemical ion gradient

301

generated partially by the oxaloacetate decarboxylase38. Moreover, reconstitution of the

302

citrate transport protein from rat liver mitochondria revealed that without the exchanged

303

substrate for citrate, citrate transportation stopped, which is in contrast with the

304

tricarboxylate transporter39. However, the structures and transportation mechanisms of

305

these two transporters need further investigation.

306

Considering the significant increase of lipid amount in the cell (Fig. 4d) and lower 15 ACS Paragon Plus Environment


Page 16 of 34

307

citrate concentration in the medium (Fig. 5) in ct- and tct-overexpressing strain, we can

308

hypothesize that increased expression of citrate transporter and tricarboxylate

309

transporter could carry out more citrate from the mitochondria matrix to the cytoplasm.

310

Therefore, more citrate could be cleaved by increased ACL to provide more acetyl-CoA

311

and NADPH for increased FAS to synthesize fatty acids. In combination with our

312

previous work, in which the malate transporter in CBS 277.49 was over expressed and

313

lipid production was improved by 70 %, we hypothesized that citrate transporter,

314

tricarboxylate transporter, together with malate transporter consisted the citrate transport

315

system and involved in a novel and unknown mechanism regulating the citrate/malate

316

shuttle in M. circinelloides (Fig. 6).

317

We propose a model of lipid biosynthesis that integrates the data of this work (Fig. 6).

318

In this model, N depletion leads to the retardation of TCA cycle, which results in citrate

319

accumulation in the mitochondria, then the accumulated citrate can be transported to the

320

cytosol by CT and/or TCT, where it can be cleaved by ACL to generate acetyl-CoA and

321

oxaloacetate. Acetyl-CoA is then utilized for fatty acid synthesis26, whereas,

322

oxaloacetate is used to produce malate that is imported into mitochondria by MT alone

323

or CT/TCT for citrate exchanging. Malate in mitochondria can be converted to

324

oxaloacetate, which, together with acetyl-CoA can produce more citrate, thus forms the

325

citrate cycle between mitochondria and cytosol.

326 327

4. Conclusions

328

In this work, a putative citrate transporter gene ct and a putative tricarboxylate 16 ACS Paragon Plus Environment

Page 17 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


329

transporter gene tct have been identified in M. circinelloides WJ11, respectively. Gene

330

overexpression experiments revealed that both transporters play an important role in

331

lipid biosynthesis, probably by transporting mitochondrial citrate to cytoplasm, which

332

could provide more acetyl-CoA and NADPH for fatty acid biosynthesis. Interestingly,

333

overexpression of both transporters induced acl and fas genes expression, suggesting

334

the existence of interlocked and connected regulatory mechanism that links citrate

335

accumulation and lipid biosynthesis. Our work suggested that both transporters play a

336

vital role in lipid accumulation in M. circinelloides.

337 338

5. Materials and Methods

339

5.1 Strains, growth and transformation conditions

340

Escherichia coli Top 10 was used for all cloning experiments.

341

M. circinelloides WJ11 was used as the source of the citrate transporter genes, ct and tct.

342

The uridine and leucine auxotroph MU402, which was derived from CBS 277.4940,

343

was used as recipient strain for ct and tct in transformation experiments. Cultures were

344

grown at 28 °C in YPG or MMC medium. The media were supplemented with uridine

345

(200 μg/mL) when required. The pH was adjusted to 4.5 and 3 for mycelia and colonial

346

growth, respectively. Transformation was carried out as described previously3.

347

Strains Mc-ct (ct-overexpression), Mc-tct (tct-overexpression), and Mc-1552 (control)

348

were initially inoculated into 150 ml K & R medium41 held in 1L flask equipped with

349

baffles to improve aeration and scatter the mycelium. The cultures were incubated in a

350

shaker at 28 ºC and rotated at 150 rpm for 24 h and then inoculated into 1.5 L modified 17 ACS Paragon Plus Environment


Page 18 of 34

351

K & R medium held in 2 L fermenter. Fermenter was controlled at 28 ºC, stirred at 700

352

rpm, aerated at 1.0 v/v min-1, and pH of the culture was maintained at 6.0 by automatic

353

addition of 1 M NaOH or 1 M H2SO4. Culture samples of each strain were collected for

354

analysis at 3, 6, 9, 12, 24, 36, 48, 60, 72, 84, 96 h based on the characteristic of lipid

355

accumulation.

356

5.2 Identification of mitochondrial transporter genes in M. circinelloides

357

Putative mitochondrial transporter genes in WJ11 were identified by gene annotations

358

using different databases, i.e. non-redundant proteins (NR), metabolic pathways

359

(KEGG), NCBI-CDD, protein families (Pfam), and TCDB. Then, the mitochondrial

360

transporter genes associated with citrate were selected and analyzed by bioinformatics.

361

The phylogenetic tree was built by using MEGA 6.0 based on the sequence of

362

transporter proteins which were identified by gene annotations.

363

5.3 Bioinformatic analysis of mitochondrial citrate transporter genes in M.

364

circinelloides

365

For each citrate transporter, the molecular weight, protein isoelectric point, instability

366

index,

367

(web.expasy.org/protparam). The secondary structures of proteins were predicted from

368

the amino acid sequence by CFSSP (www.biogem.org/tool/chou-fasman/index.php) and

369

SOPMA (npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html).

370

TMHMM Server v. 2.0 (www.cbs.dtu.dk/services/TMHMM/) and HMMTOP

371

(www.enzim.hu/hmmtop/index.php) were used to predict the presence, number and

372

location of transmembrane spanning regions of transporter proteins in M. Circinelloides

and

aliphatic

index

were

analyzed

using

protein

analysis

tools


Page 19 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


373

WJ11.

374

5.4 Biochemical analysis of fermentation process

375

Determination of cell dry weight (CDW): biomass, in each culture sample, was

376

harvested by filtration with a dried and weighed filter paper, purged with distilled water

377

for 3 times, frozen for 1 h at -80 ºC, then freeze dried, and weighed gravimetrically.

378

Glucose, citrate and ammonium concentration in the culture media were determined

379

using a glucoseoxidase Perid-test kit (Rongsheng), a citrate kit (Suoqiao Biotec.) and

380

the indophenol test42, respectively. Analysis of cell lipid was carried out by extraction of

381

lipids from 20 mg lyophilized biomass with chloroform/methanol (2:1, v/v), with

382

pentadecanoic acid (15:0) as internal standard, then methylated with 10 %

383

HCl/methanol (w/w). Fatty acid methyl esters were extracted with n-hexane and

384

analyzed by gas chromatography (GC) with a column: DM-FFAP, 30 m×0.32 mm，0.22

385

μm (Dikma Tech Co., Ltd.)42.

386

5.5 Gene over-expressing plasmid construction

387

Plasmid pMAT1552, containing the M. circinelloides pyrG gene, coding for Orotidine

388

5'-phosphate decarboxylase, surrounded up- and down-stream by 1 kb of CarRP

389

sequences, was used to construct the ct-overexpressing and tct-overexpressing plasmids.

390

Ct and tct genes were isolated by PCR amplification from the cDNA of WJ11 with

391

corresponding primers ct-F/R, tct-F/R, (Table S1) which contains 25 bp homologous

392

sequences of both sides of XhoI restriction site in pMAT1552. The PCR fragment was

393

then inserted into plasmid pMAT1552 digested by XhoI to generate plasmids

394

pMAT1552-ct, pMAT1552-tct (Fig. 2a) (One step cloning kit, Takara) 19 ACS Paragon Plus Environment


Page 20 of 34

395

5.6 RNA isolation and transcriptional analysis of gene expressing by RT-qPCR

396

Total RNA was isolated from mycelium with Trizol after grinding under liquid N2and

397

reverse-transcribed using ReverTra Ace qPCR RT Kit (Roche) according to the

398

manufacturer’s instruction. Real-Time quantitative PCR was based on the 2-Ct method

399

using actin gene as a housekeeping gene and performed in LightCycler 96 (Roche)

400

using the SYBR Green Realtime PCR Master Mix according to the manufacturer’s

401

instruction. The amplification reaction cycling conditions were as follows: 95 º C

402

incubation for 600 s, then 95 º C 30 s ， 59 º C 10 s, 72 º C 30 s (45 cycles). The

403

primers used for RT-qPCR are listed in Table S1. Three independent biological

404

replicates were analyzed.

405

5.7 Statistical analysis

406

All data were presented as means ± S.D. from three independent experiments and

407

performed using SPSS 16.0 for Windows, followed by a Student`s t test. Differences

408

were considered statistically significant at P＜0.05.

409 410

Supporting Information.

411

1. Primers (Table S1) used in this study

412

2. The result of transformants lipid content analysis (Tables S2).

413 414

Abbreviations Used

415

TCA:

416

non-redundant proteins, NCBI-CDD: the conserved domain database at NCBI, TCDB:

tricarboxylic

acid,

OAA:

oxaloacetate,

ACL:

ATP-citratelyase,

NR:


Page 21 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


417

the transporter classification database, GLA: γ-linolenic acid , TFAs: total fatty acids ,

418

CDW: determination of cell dry weight, GC: gas chromatography, GRAVY: grand

419

average

420

6-phosphofructo-2-kinase / fructose-2, 6-biphosphatases, PK: pyruvate kinase, mCT:

421

mitochondrial membrane citrate transporter, cCT: cytomembrane citrate transporter,

422

TCT: tricarboxylate carrier, MT: malate transporter, MAT: monocarboxylic acid

423

transporter (pyruvate transporter), CS: citrate synthase, FAS: fatty acid synthase, ME:

424

malic enzyme, PC: pyruvate carboxylase, PDH: pyruvate dehydrogenase, PPP: pentose

425

phosphate pathway.

of

hydropathicity,

PFK1:

phosphofructokinase

1,

PFK2:

426 427

Declarations

428

Author Contributions

429

Junhuan Yang performed the experimental design, computational analysis, manuscript

430

writing, and figures and tables arrangement. Shaoqi Li and Md. Ahsanul Kabir Khan

431

carried out on fermentation testing. Wanwipa Vongsangnak carried out on genome

432

annotation, results interpretation and review of final draft. Victoriano Garre was

433

involved in the experimental design. Yuanda song proposed the project, and involved in

434

data analysis, result interpretation, manuscript writing and review of the final draft.

435

Acknowledgment

436

We would like to thank Computational Biomodelling Laboratory for Agricultural

437

Science and Technology (CBLAST), Faculty of Science, Kasetsart University, Thailand

438

for computational facilities and Yao Zhang for data analysis work supported by the Key 21 ACS Paragon Plus Environment


Page 22 of 34

439

Research and Development project of Shandong Province (2018GSF121013).

440

Funding

441

This work was supported by National Natural Science Foundation of China (Grant Nos.

442

31670064 and 31200989), TaiShan Industrial Experts Programme (tscy 20160101), the

443

Key Research and Development project of Shandong Province (2018GNC110039) and

444

starting grant from Shandong University of Technology.

445

Competing interests

446

The authors declare that they have no competing interests.

447

Availability of data and materials

448

The data supporting the conclusions of this article are included with the article. Strains

449

examined are available from the corresponding author.

450

Consent for publication

451

The authors provide consent for publication.

452

Ethics approval and consent to participate

453

This article does not contain any studies with human participants or animals performed

454

by any of the authors.

455 456

References

457 458 459 460 461 462 463 464

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Gnoni, G. V.; Priore, P.; Geelen, M. J.; Siculella, L., The mitochondrial citrate carrier: metabolic role

and regulation of its activity and expression. IUBMB Life 2009, 61, 987-994. 2.

Iacobazzi, V.; Infantino, V., Citrate--new functions for an old metabolite. Biol Chem 2014, 395,

387-399. 3.

Rodriguez-Frometa, R. A.; Gutierrez, A.; Torres-Martinez, S.; Garre, V., Malic enzyme activity is not

the only bottleneck for lipid accumulation in the oleaginous fungus Mucor circinelloides. Appl Microbiol Biotechnol 2013, 97, 3063-3072. 4.

Ratledge, C., The role of malic enzyme as the provider of NADPH in oleaginous microorganisms: a


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reappraisal and unsolved problems. Biotechnol Lett 2014, 36, 1557-1568. 5.

Vongsangnak, W.; Zhang, Y.; Chen, W.; Ratledge, C.; Song, Y., Annotation and analysis of malic

enzyme genes encoding for multiple isoforms in the fungus Mucor circinelloides CBS 277.49. Biotechnol Lett 2012, 34, 941-947. 6.

Klingenberg, M., Kinetic study of the tricarboxylate carrier in rat liver mitochondria. Eur J Biochem

1972, 26, 587-594. 7.

Xu, Y.; Kakhniashvili, D. A.; Gremse, D. A.; Wood, D. O.; Mayor, J. A.; Walters, D. E.; Kaplan, R. S.,

The yeast mitochondrial citrate transport protein. Probing the roles of cysteines, Arg(181), and Arg(189) in transporter function. J Biol Chem 2000, 275, 7117-7124. 8.

De Palma, A.; Prezioso, G.; Scalera, V., Kinetic evidence for the uniport mechanism hypothesis in

the mitochondrial tricarboxylate transport system. J Bioenerg Biomembr 2005, 37, 279-287. 9.

Evans, C. T.; Scragg, A. H.; Ratledge, C., A comparative study of citrate efflux from mitochondria of

oleaginous and non-oleaginous yeasts. Eur J Biochem 1983, 130, 195-204. 10. Walters, D. E.; Kaplan, R. S., Homology-modeled structure of the yeast mitochondrial citrate transport protein. Biophys J 2004, 87, 907-911. 11. Ma, C.; Remani, S.; Sun, J.; Kotaria, R.; Mayor, J. A.; Walters, D. E.; Kaplan, R. S., Identification of the substrate binding sites within the yeast mitochondrial citrate transport protein. J Biol Chem 2007, 282, 17210-17220. 12. Kirimura, K.; Kobayashi, K.; Ueda, Y.; Hattori, T., Phenotypes of gene disruptants in relation to a putative mitochondrial malate-citrate shuttle protein in citric acid-producing Aspergillus niger. Biosci Biotechnol Biochem 2016, 80, 1737-1746. 13. Ratledge, C.; Wynn, J. P., The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Adv Appl Microbiol 2002, 51, 1-51. 14. Vongsangnak, W.; Klanchui, A.; Tawornsamretkit, I.; Tatiyaborwornchai, W.; Laoteng, K.; Meechai, A., Genome-scale metabolic modeling of Mucor circinelloides and comparative analysis with other oleaginous species. Gene 2016, 583 , 121-129. 15. Zhao, L.; Canovas-Marquez, J. T.; Tang, X.; Chen, H.; Chen, Y. Q.; Chen, W.; Garre, V.; Song, Y.; Ratledge, C., Role of malate transporter in lipid accumulation of oleaginous fungus Mucor circinelloides. Appl Microbiol Biotechnol 2016, 100, 1297-1305. 16. Tang, X.; Zhao, L.; Chen, H.; Chen, Y. Q.; Chen, W.; Song, Y.; Ratledge, C., 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 2015, 10, e0137543. 17. Tang, X.; Chen, H.; Gu, Z.; Zhang, H.; Chen, Y. Q.; Song, Y.; Chen, W., Comparative Proteome Analysis between High Lipid-Producing Strain Mucor circinelloides WJ11 and Low Lipid-Producing Strain CBS 277.49. J Agric Food Chem 2017, 65, 5074-5082. 18. Tang, X.; Chen, H.; Chen, Y. Q.; Chen, W.; Garre, V.; Song, Y.; Ratledge, C., Comparison of Biochemical Activities between High and Low Lipid-Producing Strains of Mucor circinelloides:An Explanation for the High Oleaginicity of Strain WJ11. PLoS One 2015, 10, e0128396. 19. Zhang, L.; Zhang, H.; Song, Y., Identification and Characterization of Diacylglycerol Acyltransferase from Oleaginous Fungus Mucor circinelloides. J Agric Food Chem 2018, 66, 674-681. 20. Miyake, S.; Yamashita, T.; Taniguchi, M.; Tamatani, M.; Sato, K.; Tohyama, M., Identification and characterization of a novel mitochondrial tricarboxylate carrier. Biochem Biophys Res Commun 2002, 295, 463-468. 21. Plouhinec, J. L.; Granier, C.; Le Mentec, C.; Lawson, K. A.; Saberan-Djoneidi, D.; Aghion, J.; Shi, D. L.;



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Collignon, J.; Mazan, S., Identification of the mammalian Not gene via a phylogenomic approach. Gene Expr Patterns 2004, 5, 11-22. 22. Dujon, B.; Albermann, K.; Aldea, M.; Alexandraki, D.; Ansorge, W.; Arino, J.; Benes, V.; Bohn, C.; Bolotin-Fukuhara, M.; Bordonne, R.; Boyer, J.; Camasses, A.; Casamayor, A.; Casas, C.; Cheret, G.; Cziepluch, C.; Daignan-Fornier, B.; Dang, D. V.; de Haan, M.; Delius, H.; Durand, P.; Fairhead, C.; Feldmann, H.; Gaillon, L.; Kleine, K.; et al., The nucleotide sequence of Saccharomyces cerevisiae chromosome XV. Nature 1997, 387, 98-102. 23. Zhao, L.; Tang, X.; Luan, X.; Chen, H.; Chen, Y. Q.; Chen, W.; Song, Y.; Ratledge, C., Role of pentose phosphate pathway in lipid accumulation of oleaginous fungus Mucor circinelloides. RSC Adv. 2015, 5, 97658-97664. 24. Zhao, S.; Torres, A.; Henry, R. A.; Trefely, S.; Wallace, M.; Lee, J. V.; Carrer, A.; Sengupta, A.; Campbell, S. L.; Kuo, Y. M.; Frey, A. J.; Meurs, N.; Viola, J. M.; Blair, I. A.; Weljie, A. M.; Metallo, C. M.; Snyder, N. W.; Andrews, A. J.; Wellen, K. E., ATP-Citrate Lyase Controls a Glucose-to-Acetate Metabolic Switch. Cell Rep 2016, 17, 1037-1052. 25. Zhao, L.; Zhang, H.; Wang, L.; Chen, H.; Chen, Y. Q.; Chen, W.; Song, Y., (13)C-metabolic flux analysis of lipid accumulation in the oleaginous fungus Mucor circinelloides. Bioresour Technol 2015, 197, 23-29. 26. Majd, H.; King, M. S.; Smith, A. C.; Kunji, E. R. S., Pathogenic mutations of the human mitochondrial citrate carrier SLC25A1 lead to impaired citrate export required for lipid, dolichol, ubiquinone and sterol synthesis. Biochim Biophys Acta 2018, 1859, 1-7. 27. Dolce,

V.;

Cappello,

A.

R.;

Capobianco,

L.,

Mitochondrial

tricarboxylate

and

dicarboxylate-tricarboxylate carriers: from animals to plants. IUBMB Life 2014, 66, 462-471. 28. Ferramosca, A.; Zara, V., Dietary fat and hepatic lipogenesis: mitochondrial citrate carrier as a sensor of metabolic changes. Adv Nutr 2014, 5, 217-225. 29. Vongsangnak, W.; Ruenwai, R.; Tang, X.; Hu, X.; Zhang, H.; Shen, B.; Song, Y.; Laoteng, K., Genome-scale analysis of the metabolic networks of oleaginous Zygomycete fungi. Gene 2013, 521, 180-190. 30. Bellou, S.; Triantaphyllidou, I. E.; Mizerakis, P.; Aggelis, G., High lipid accumulation in Yarrowia lipolytica cultivated under double limitation of nitrogen and magnesium. J Biotechnol 2016, 234, 116-126. 31. Dourou, M.; Aggeli, D.; Papanikolaou, S.; Aggelis, G., Critical steps in carbon metabolism affecting lipid accumulation and their regulation in oleaginous microorganisms. Appl Microbiol Biotechnol 2018, 102, 2509-2523. 32. Azzi, A.; Glerum, M.; Koller, R.; Mertens, W.; Spycher, S., The mitochondrial tricarboxylate carrier. J Bioenerg Biomembr 1993, 25, 515-524. 33. Capobianco, L.; Ferramosca, A.; Zara, V., The mitochondrial tricarboxylate carrier of silver eel: dimeric structure and cytosolic exposure of both N- and C-termini. J Protein Chem 2002, 21, 515-521. 34. Ferramosca, A.; Savy, V.; Conte, L.; Colombo, S.; Einerhand, A. W.; Zara, V., Conjugated linoleic acid and hepatic lipogenesis in mouse: role of the mitochondrial citrate carrier. J Lipid Res 2006, 47, 1994-2003. 35. Spagnoletta, A.; De Santis, A.; Tampieri, E.; Baraldi, E.; Bachi, A.; Genchi, G., Identification and kinetic characterization of HtDTC, the mitochondrial dicarboxylate-tricarboxylate carrier of Jerusalem artichoke tubers. J Bioenerg Biomembr 2006, 38, 57-65. 36. Shug, A. L.; Shrago, E., Inhibition of phosphoenolpyruvate transport via the tricarboxylate and adenine nucleotide carrier systems of rat liver mitochondria. Biochem Biophys Res Commun 1973, 53,


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659-665. 37. Carrisi, C.; Madeo, M.; Morciano, P.; Dolce, V.; Cenci, G.; Cappello, A. R.; Mazzeo, G.; Iacopetta, D.; Capobianco, L., Identification of the Drosophila melanogaster mitochondrial citrate carrier: bacterial expression, reconstitution, functional characterization and developmental distribution. J Biochem 2008, 144, 389-392. 38. Kastner, C. N.; Prummer, M.; Sick, B.; Renn, A.; Wild, U. P.; Dimroth, P., The citrate carrier CitS probed by single-molecule fluorescence spectroscopy. Biophys J 2003, 84, 1651-1659. 39. Xu, Y.; Mayor, J. A.; Gremse, D.; Wood, D. O.; Kaplan, R. S., High-yield bacterial expression, purification, and functional reconstitution of the tricarboxylate transport protein from rat liver mitochondria. Biochem Biophys Res Commun 1995, 207, 783-789. 40. Nicolas, F. E.; de Haro, J. P.; Torres-Martinez, S.; Ruiz-Vazquez, R. M., Mutants defective in a Mucor circinelloides dicer-like gene are not compromised in siRNA silencing but display developmental defects. Fungal Genet Biol 2007, 44, 504-516. 41. Kendrick, A.; Ratledge, C., Desaturation of polyunsaturated fatty acids in Mucor circinelloides and the involvement of a novel membrane-bound malic enzyme. Eur J Biochem 1992, 209, 667-673. 42. Chaney, A. L.; Marbach, E. P., Modified reagents for determination of urea and ammonia. Clin Chem 1962, 8, 130-132.

570 571

Figure legends

572

Fig. 1 Phylogenetic tree of mitochondrial transporters. The tree was originated from

573

ClustalW multiple-sequence alignments by using the neighbor-joining method

574

implemented in MEGA6. All 51 transporters of M. circinelloides WJ11 were shown.

575

Bootstrap values for 1000 replicates were reported on each node.

576

Fig. 2 Generation of the transporter expressing strains. a. Structure of plasmids

577

pMAT1552, pMAT1552-ct and pMAT1552-tct. Arrows indicate the positions of the

578

primers 1552-F and 1552-R (Table with primers) used in b and c. b. PCR amplification

579

of control strain Mc-1552 (lane 1), and three transformants with ct overexpressing

580

plasmids (lane 2, Mc-ct1; lane3, Mc-ct2; and lane 4, Mc-ct3). c. PCR amplification of

581

control strain Mc-1552 (lane1), and tct gene overexpressing plasmids (lane 2, Mc-tct1;

582

lane 3, Mc-tct2; and lane 4, Mc-tct3) with the primers 1552-F and 1552-R, shown in a

583

M (DL2000 DNA Marker, Takara). 25 ACS Paragon Plus Environment


Page 26 of 34

584

Fig. 3 Expressions of ct and tct gene in the transformants Mc-ct, Mc-tct and the control

585

Mc-1552. Strains were grown in a 2 L fermenter with 1.5 L modified K & R medium,

586

and the mycelium was harvested at 3 h(N rich, i.e., initial growth), 24 h (after N

587

depletion, i.e., fast lipid accumulation stage) and 48, 72 h (after N depletion, i.e., slow

588

lipid accumulation stage). Total RNA of the strains at different times was extracted and

589

the mRNA accumulation was quantified by RT-qPCR. a. The relative expressing level

590

of ct gene located in the genome was quantified and amplified by cbs-ct-F/R primers

591

(white and black bars) and the over expressed ct gene in the plasmid was quantified and

592

amplified by WJ11-ct-F/R primers (striped bars). b. The relative expressing level of tct

593

gene located in genome was quantified and amplified by cbs-tct-F/R primers (white and

594

black bars) and the over expressed tct gene in the plasmid was quantified and amplified

595

by WJ11-tct-F/R primers (striped bars). The values were mean of three independent

596

fermentation experiments. Error bars represent the standard error of mean.

597

Fig. 4 Cell growth and lipid accumulation of ct-overexpressing and tct-overexpressing

598

strains. a. Cell dry weight (CDW), b. Glucose concentration, c. Ammonium

599

concentration and d. Lipid content, in Mc-ct (triangle), Mc-tct (square) and control

600

strain Mc-1552 (circle) cultures grown in 1.5 L modified K & R medium were

601

measured. Samples from the fermenter was taken at the indicated times. The values

602

were mean of three biological replicates. Error bars represent the standard error of the

603

mean.

604

Fig. 5 Extracellular citrate concentration in the cultures of ct-overexpressing (Mc-ct),

605

tct-overexpressing (Mc-tct), and control strains (Mc-1552). The strains were grown in a 26 ACS Paragon Plus Environment

Page 27 of 34 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


606

2 L fermenter with 1.5 L modified K & R medium. The values are mean of three

607

independent fermentation experiments. Error bars represent the standard error of the

608

mean.

609

Fig. 6 The cycle of citrate transportation and citrate secretion related to lipid

610

accumulation in oleaginous fungus M. circinelloides. mCT: mitochondrial membrane

611

citrate transporter, cCT: cytomembrane citrate transporter, TCT: tricarboxylate carrier,

612

MT: malate transporter, MAT: monocarboxylic acid transporter (pyruvate transporter),

613

CS: citrate synthase, ACL: ATP citrate lyase, FAS: fatty acid synthase, ME: malic

614

enzyme, PC: pyruvate carboxylase, PDH: pyruvate dehydrogenase, PPP: pentose

615

phosphate pathway [This pathway map is modified from the paper of Zhao et al.

616

(2015)15].

617

Table of Contents graphic

618



Fig. 1 Phylogenetic tree of mitochondrial transporters. The tree was originated from ClustalW multiplesequence alignments by using the neighbor-joining method implemented in MEGA6. All 51 transporters of M. circinelloides WJ11 were shown. Bootstrap values for 1000 replicates were reported on each node. 170x170mm (300 x 300 DPI)

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Fig. 2 Generation of the transporter expressing strains. a. Structure of plasmids pMAT1552, pMAT1552-ct and pMAT1552-tct. Arrows indicate the positions of the primers 1552-F and 1552-R (Table with primers) used in b and c. b. PCR amplification of control strain Mc-1552(lane 1), and three transformants with ct overexpressing plasmids (lane 2, Mc-ct1; lane 3, Mc-ct2; and lane 4, Mc-ct3). c. PCR amplification of control strain Mc-1552 (lane1), and tct gene overexpressing plasmids (lane 2, Mc-tct1; lane 3, Mc-tct2; and lane 4, Mc-tct3) with the primers 1552-F and 1552-R, shown in a M (DL2000 DNA Marker, Takara). 170x143mm (300 x 300 DPI)



Fig. 3 Expressions of ct and tct gene in the transformants Mc-ct, Mc-tct and the control Mc-1552. Strains were grown in a 2 L fermenter with 1.5 L modified K & R medium, and the mycelium was harvested at 3h (N rich, i.e., initial growth), 24h (after N depletion, i.e., fast lipid and accumulation stage) and 48,72 h (after N depletion, i.e., slow lipid accumulation stage). Total RNA of the strains at different times was extracted and the mRNA accumulation was quantified by RT-qPCR. a. The relative expressing level of ct gene located in the genome was quantified and amplified by cbs-ct-F/R primers (white and black bars) and the over expressed ct gene in the plasmid was quantified and amplified by WJ11-ct-F/R primers striped bars. b. The relative expressing level of tct gene located in genome was quantified and amplified by cbs-tct-F/R primers (white and black bars) and the over expressed tct gene in the plasmid was quantified and amplified by WJ11-tctF/R primers (striped bars). The values were mean of three independent fermentation experiments. Error bars represent the standard error of mean. 85x129mm (300 x 300 DPI)


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Fig. 4 Cell growth and lipid accumulation of ct-overexpressing and tct-overexpressing strains. a. Cell dry weight (CDW), b. Glucose concentration, c. Ammonium concentration and d. Lipid content, in Mc-ct (triangle), Mc-tct (square) and control strain Mc-1552(circle) cultures grown in 1.5 L modified K & R medium were measured. Samples from the fermenter was taken at the indicated times. The values were mean of three biological replicates. Error bars represent the standard error of the mean.


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Fig. 5 Extracellular citrate concentration in the cultures of ct-overexpressing (Mc-ct), tct-overexpressing (Mc-tct), and control strains (Mc-1552). The strains were grown in a 2 L fermenter with 1.5L modified K & R medium. The values are mean of three independent fermentation experiments. Error bars represent the standard error of the mean. 85x75mm (300 x 300 DPI)



Fig. 6 The cycle of citrate transportation and citrate secretion related to lipid accumulation in oleaginous fungus M. circinelloides. mCT: pyruvate mitochondrial membrane citrate transporter, cCT: pyruvate cytomembrane citrate transporter, TCT: pyruvate tricarboxylate carrier, MT: malate transporter, MAT: monocarboxylic acid transporter (pyruvate transporter), CS: citrate synthase, ACL: ATP citrate lyase, FAS: fatty acid synthase, ME: malic enzyme, PC: pyruvate carboxylase, PDH: pyruvate dehydrogenase, PPP: pentose phosphate pathway [This pathway map is mainly modified from the paper of Zhao et al. (2015) [15]].


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