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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] 16
Shaoqi Li:
[email protected] 17
Md. Ahsanul Kabir Khan:
[email protected] 18
Victoriano Garre:
[email protected] 19
Wanwipa Vongsangnak:
[email protected] 20
Yuanda Song:
[email protected] 21
*Corresponding author
22
Yuanda Song:
[email protected] of Zoology, Faculty of Science, Kasetsart University, Bangkok 10900,
Biomodelling Laboratory for Agricultural Science and Technology
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Wanwipa Vongsangnak:
[email protected] 24
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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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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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
278
microalgae30. However, the remaining citrate in ct- and tct-overexpressing strains was
279
still at high level to be excreted into the growth medium, especially at the end of lipid
280
accumulation phase (Fig. 5). Furthermore, the accumulated citrate in the cytoplasm may
281
lead to polysaccharide biosynthesis by inhibiting glycolysis at level of insufficient
282
enzymatic activity of PFKs, and this may compete with lipid biosynthesis31. Thus, in
283
future, more work should be done to decrease carbon outflow towards metabolic
284
pathways competitive to lipogenesis, which may contribute to a higher lipid 14 ACS Paragon Plus Environment
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accumulation.
286
Despite that overexpression of both transporters can increase lipid production, the
287
increase of lipid production was slightly higher in the tricarboxylate transporter
288
overexpressing strain than in the citrate transporter over-expressing strain. One
289
hypothesis for this phenomenon is that the two transporters carry citrate by different
290
mechanisms. Indeed, tricarboxylate transporter has been isolated from other species,
291
such as rat32 and eel33, and only one binding site was found in its three dimensional
292
structure. Some studies conducted in intact mitochondria or with the purified carrier
293
protein reconstituted in proteoliposomes have provided significant evidence in favor of
294
a uniport transport mechanism for citrate by this transporter: tricarboxylate transporter
295
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
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Page 16 of 34
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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
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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
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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
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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
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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:
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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
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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
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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,
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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
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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Fig. 3 Expressions of ct and tct gene in the transformants Mc-ct, Mc-tct and the control
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Mc-1552. Strains were grown in a 2 L fermenter with 1.5 L modified K & R medium,
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and the mycelium was harvested at 3 h(N rich, i.e., initial growth), 24 h (after N
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depletion, i.e., fast lipid accumulation stage) and 48, 72 h (after N depletion, i.e., slow
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lipid accumulation stage). Total RNA of the strains at different times was extracted and
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the mRNA accumulation was quantified by RT-qPCR. a. The relative expressing level
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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
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amplified by WJ11-ct-F/R primers (striped bars). b. The relative expressing level of tct
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gene located in genome was quantified and amplified by cbs-tct-F/R primers (white and
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black bars) and the over expressed tct gene in the plasmid was quantified and amplified
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by WJ11-tct-F/R primers (striped bars). The values were mean of three independent
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fermentation experiments. Error bars represent the standard error of mean.
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Fig. 4 Cell growth and lipid accumulation of ct-overexpressing and tct-overexpressing
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strains. a. Cell dry weight (CDW), b. Glucose concentration, c. Ammonium
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concentration and d. Lipid content, in Mc-ct (triangle), Mc-tct (square) and control
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strain Mc-1552 (circle) cultures grown in 1.5 L modified K & R medium were
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measured. Samples from the fermenter was taken at the indicated times. The values
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were mean of three biological replicates. Error bars represent the standard error of the
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mean.
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Fig. 5 Extracellular citrate concentration in the cultures of ct-overexpressing (Mc-ct),
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tct-overexpressing (Mc-tct), and control strains (Mc-1552). The strains were grown in a 26 ACS Paragon Plus Environment
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Industrial & Engineering Chemistry Research
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2 L fermenter with 1.5 L modified K & R medium. The values are mean of three
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independent fermentation experiments. Error bars represent the standard error of the
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mean.
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Fig. 6 The cycle of citrate transportation and citrate secretion related to lipid
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accumulation in oleaginous fungus M. circinelloides. mCT: mitochondrial membrane
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citrate transporter, cCT: cytomembrane citrate transporter, TCT: tricarboxylate carrier,
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MT: malate transporter, MAT: monocarboxylic acid transporter (pyruvate transporter),
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CS: citrate synthase, ACL: ATP citrate lyase, FAS: fatty acid synthase, ME: malic
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enzyme, PC: pyruvate carboxylase, PDH: pyruvate dehydrogenase, PPP: pentose
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phosphate pathway [This pathway map is modified from the paper of Zhao et al.
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(2015)15].
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Table of Contents graphic
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27 ACS Paragon Plus Environment
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
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)
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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
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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Page 30 of 34
Page 31 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
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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)
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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
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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Table of contents graphic.
322x150mm (96 x 96 DPI)
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