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Role of pyruvate carboxylase in accumulation of intracellular lipid of the oleaginous yeast Yarrowia lipolytica ACA-DC 50109

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Abstract

Yarrowia lipolytica ACA-DC 50109 is an oleaginous yeast. In order to know the function of pyruvate carboxylase (PYC) in lipid biosynthesis, the PYC gene cloned from Pichia guilliermondii Pcla22 was overexpressed in the oleaginous yeast. The lipid contents in the wild-type strain ACA-DC 50109 and the transformants P4, P7, and P103 were 30.2 % (w/w) 36.5 % (w/w), 38.2 % (w/w), and 37.9 % (w/w). However, the amount of the secreted citric acids by strains ACA-DC 50109, P4, P77, and P103 were 0.5, 10.1, 11.5, and 9.4 g/L. In order to reduce the amount of the secreted citric acid, the PYC gene and endogenous ACL1 gene encoding ATP citrate lyase (ACL1) were simultaneously overexpressed in the oleaginous yeast. The lipid contents of the transformants PA19, PA56, PA124 were 44.4 % (w/w), 45.3 % (w/w), and 43.7 % (w/w). At the same time, the amount of the secreted citric acid by the transformants PA19, PA56, and PA124 was reduced to 5.4, 6.2, and 6.3 g/L. The PYC and ACL1 activities and their gene transcriptional levels in all the transformants were greatly enhanced compared to those in their wild-type strain ACA-DC 50109. During 10-L fermentation, lipid content in the transformant PA56 was 49.6 % (w/w) and the amount of secreted citric acid was 2.9 g/L. This meant that PYC and ACL1 can play an important role in accumulation of intracellular lipid of the oleaginous yeast Y. lipolytica ACA-DC 50109.

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References

  • Beopoulos A, Nicaud JM, Gaillardin C (2011) An overview of lipid metabolism in yeasts and its impact on biotechnological processes. Appl Microbiol Biotechnol 90:1193–1206

    Article  CAS  PubMed  Google Scholar 

  • Blazeck J, Hill A, Liu L, Knight R, Miller J, Pan A, Otoupal P, Alper HS (2014) Harnessing Yarrowia lipolytica lipogenesis to create a platform for lipid and biofuel production. Nat Commun 5:3131

    Article  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 72:248–253

    Article  CAS  PubMed  Google Scholar 

  • Brown SH, Bashkirova L, Berka R, Chandler T, Doty T, McCall K, McCulloch M, McFarland S, Thompson S, Yaver D, Berry A (2013) Metabolic engineering of Aspergillus oryzae NRRL 3488 for increased production of l-malic acid. Appl Microbiol Biotechnol 97:8903–8912

    Article  CAS  PubMed  Google Scholar 

  • Camp BJ, Farmer L (1967) A rapid spectrophotometric method for the determination of citric acid in blood. Clin Chem 13:501–505

    CAS  PubMed  Google Scholar 

  • Chávez-Cabrera C, Flores-Bustamante ZR, Marsch R, Montes MC, Sánchez S, Cancino-Díaz JC, Flores-Cotera LB (2010) ATP-citrate lyase activity and carotenoid production in batch cultures of Phaffia rhodozyma under nitrogen-limited and nonlimited conditions. Appl Microbiol Biotechnol 85:1953–1960

    Article  PubMed  Google Scholar 

  • Chi ZM, Liu J, Zhang W (2001) Trehalose accumulation from soluble starch by Saccharomycopsis fibuligera sdu. Enzym Microb Technol 28:240–245

    Article  CAS  Google Scholar 

  • Dulermo T, Jean-Marc N (2011) Involvement of the G3P shuttle and β-oxidation pathway in the control of TAG synthesis and lipid accumulation in Yarrowia lipolytica. Metab Eng 13:482–491

    Article  CAS  PubMed  Google Scholar 

  • Folch J, Lees M, Slane-Stanley JA (1957) A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226:497–509

    CAS  PubMed  Google Scholar 

  • Gokarn RR, Eiteman MA, Altman E (1998) Expression of pyruvate carboxylase enhances succinate production in Escherichia coli without affecting glucose uptake. Biotechnol Lett 20:795–798

    Article  CAS  Google Scholar 

  • Guo X, Wang J, Xie X, Xu Q, Zhang C, Chen N (2013) Enhancing the supply of oxaloacetate for l-glutamate production by pyc overexpression in different Corynebacterium glutamicum. Biotechnol Lett 35:943–950

    Article  CAS  PubMed  Google Scholar 

  • Li M, Liu GL, Chi Z, Chi ZM (2010) Single cell oil production from hydrolysate of cassava starch by marine-derived yeast Rhodotorula mucilaginosa TJY15a. Biomass Bioenergy 4:101–107

    Article  Google Scholar 

  • Lietzan AD, Maurice MS (2013) Insights into the carboxyltransferase reaction of pyruvate carboxylase from the structures of bound product and intermediate analogs. Biochem Biophys Res Commun 441:377–382

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Lipmann F, Tuttle LC (1945) A specific micromethod for the determination of acyl phosphates. J Biol Chem 159:21–28

    CAS  Google Scholar 

  • Liu GL, Wang DS, Wang LF, Zhao SF, Chi ZM (2011) Mig1 is involved in mycelial formation and expression of the genes encoding extracellular enzymes in Saccharomycopsis fibuligera A11. Fungal Genet Biol 48:904–913

    Article  CAS  PubMed  Google Scholar 

  • Liu XY, Chi Z, Liu GL, Madzak C, Chi ZM (2013) Both decrease in ACL1 gene expression and increase in ICL1 gene expression in marine-derived yeast Yarrowia lipolytica expressing INU1 gene enhance citric acid production from inulin. Mar Biotechnol 15:26–36

    Article  CAS  PubMed  Google Scholar 

  • Ma Y, Wang GY, Liu GL, Wang ZP, Chi ZM (2013) Overproduction of poly(β-malic acid) (PMA) from glucose by a novel Aureobasidium sp. P6 strain isolated from mangrove system. Appl Microbiol Biotechnol 97:8931–8939

    Article  CAS  PubMed  Google Scholar 

  • Madzak C, Gaillardin C, Beckerich JM (2004) Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica: a review. J Biotechnol 109:63–81

    Article  CAS  PubMed  Google Scholar 

  • Papanikolaou S, Aggelis G (2003) Selective uptake of fatty acids by the yeast Yarrowia lipolytica. Eur J Lipid Sci Technol 105:651–655

    Article  CAS  Google Scholar 

  • Papanikolaou S, Muniglia L, Chevalot I, Aggelis G, Marc I (2002) Yarrowia lipolytica as a potential producer of citric acid from raw glycerol. J Appl Microbiol 92:737–744

    Article  CAS  PubMed  Google Scholar 

  • Ratledge C (2004) Fatty acid biosynthesis in microorganisms being used for single cell oil production. Biochimica 86:807–815

    Article  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Beijing, pp 367–370, Chinese translating ed

    Google Scholar 

  • Spiro RG (1966) Analysis of sugars found in glycoproteins. Methods Enzymol 8:3–26

    Article  CAS  Google Scholar 

  • Tai M, Stephanopoulos G (2013) Engineering the push and pull of lipid biosynthesis in oleaginous yeast Yarrowia lipolytica for biofuel production. Metab Eng 15:1–9

    Article  CAS  PubMed  Google Scholar 

  • Wang F, Yue LX, Wang L, Madzak C, Li J, Wang XH, Chi ZM (2009) Genetic modification of the marine-derived yeast Yarrowia lipolytica with high-protein content using a GPI-anchor-fusion expression system. Biotechnol Prog 25:1297–1303

    Article  CAS  PubMed  Google Scholar 

  • Wang GY, Chi Z, Song B, Wang ZP, Chi ZM (2012) High level lipid production by a novel inulinase-producing yeast Pichia guilliermondii Pcla22. Bioresour Technol 124:77–82

    Article  CAS  PubMed  Google Scholar 

  • Wang ZP, Xu HM, Wang GY, Chi Z, Chi ZM (2013) Disruption of the MIG1 gene enhances lipid biosynthesis in the oleaginous yeast Yarrowia lipolytica ACA-DC 50109. Biochim Biophys Acta 1831:675–682

    Article  CAS  PubMed  Google Scholar 

  • Watanabe H, Hatakeyama N, Sakurai H, Uchimiya H, Sato T (2008) Isolation of industrial strains of Aspergillus oryzae lacking ferrichrysin by disruption of the dffA gene. J Biosci Bioeng 106:488–492

    Article  CAS  PubMed  Google Scholar 

  • Xu G, Liu L, Chen J (2012) Reconstruction of cytosolic fumaric acid biosynthetic pathways in Saccharomyces cerevisiae. Microb Cell Factories 11:24–28

    Article  CAS  Google Scholar 

  • Xuan JM, Fournier P, Gaillardin C (1988) Cloning of the LYS5 gene encoding saccharopine dehydrogenase. Curr Genet 14:15–21

    Article  CAS  Google Scholar 

  • Xue Z, Sharpe PL, Hong SP, Yadav NS, Xie D, Short DR, Damude HG, Rupert RA, Seip JE, Wang J, Pollak DW, Bostick MW, Bosak MD, Macool DJ, Hollerbach DH, Zhang H, Arcilla DM, Bledsoe SA, Croker K, McCord EF, Tyreus BD, Jackson EN, Zhu Q (2013) Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica. Nat Biotechnol 31:734–741

    Article  CAS  PubMed  Google Scholar 

  • Yin X, Madzak C, Du G, Zhou J, Chen J (2012) Enhanced alpha-ketoglutaric acid production in Yarrowia lipolytica WSH-Z06 by regulation of the pyruvate carboxylation pathway. Appl Microbiol Biotechnol 96:1527–1537

    Article  CAS  PubMed  Google Scholar 

  • Zhang F, Wang ZP, Chi Z, Raoufi Z, Abdollahi S, Chi ZM (2013) The changes in Tps1 activity, trehalose content and expression of TPS1 gene in the psychrotolerant yeast Guehomyces pullulans 17–1 grown at different temperatures. Extremophiles 17:241–249

    Article  CAS  PubMed  Google Scholar 

  • Zhao CH, Cui W, Liu XY, Chi ZM, Madzak C (2010) Expression of inulinase gene in the oleaginous yeast Yarrowia lipolytica and single cell oil production from inulin-containing materials. Metab Eng 12:510–551

    Article  CAS  PubMed  Google Scholar 

  • Zhou J, Yin X, Madzak C, Du G, Chen J (2012) Enhanced α-ketoglutarate production in Yarrowia lipolytica WSH-Z06 by alteration of the acetyl-CoA metabolism. J Biotechnol 161:257–264

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This research was supported by Hi-Tech Research and Development Program of China (Grant No. 2013BAB01B05).

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Authors and Affiliations

  1. Unesco Chinese Center of Marine Biotechnology, Ocean University of China, Yushan Road, No. 5, Qingdao, China

    Guang-Yuan Wang, Yan Zhang, Zhe Chi, Guang-Lei Liu, Zhi-Peng Wang & Zhen-Ming Chi

  2. Shandong Provincial Key Laboratory of Applied Mycology, Qingdao Agricultural University, Changcheng Road, No.700, Qingdao, China

    Guang-Yuan Wang

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  1. Guang-Yuan Wang
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  2. Yan Zhang
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  3. Zhe Chi
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  4. Guang-Lei Liu
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Correspondence to Zhen-Ming Chi.

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Wang, GY., Zhang, Y., Chi, Z. et al. Role of pyruvate carboxylase in accumulation of intracellular lipid of the oleaginous yeast Yarrowia lipolytica ACA-DC 50109. Appl Microbiol Biotechnol 99, 1637–1645 (2015). https://doi.org/10.1007/s00253-014-6236-z

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