Skip to main content
SpringerLink
Log in

One-step utilization of inulin for docosahexaenoic acid (DHA) production by recombinant Aurantiochytrium sp. carrying Kluyveromyces marxianus inulinase

  • Research Paper
  • Published:
Bioprocess and Biosystems Engineering Aims and scope Submit manuscript

Abstract

This study aimed to express an inulinase gene from the yeast Kluyveromyces marxianus (KmINU) in Aurantiochytrium sp. and realized one-step utilization of inulin resource for DHA production without any chemical pretreatment. An expression cassette with a length of 6052 bp for expressing the inulinase gene was constructed by a fast two-step PCR method and then was transferred into the Aurantiochytrium sp. cells. The Aurantiochytrium sp. recombinant T39 was selected with an inulinase activity up to 50.1 U/mL in 72 h. In a 5-l fed-batch fermentation, as high as 148.9 g/L of inulin was directly used within 120 h, and only 1.2 g/L of total sugar was left in the medium at the end of fermentation. The biomass of 51.4 g/L with a lipid content of 69.2% DCW and a DHA yield of 14.9 g/L was obtained.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

DHA:

Docosahexaenoic acid

IOS:

Inulooligosaccharide

SP:

Signal peptide

Pac :

Actin promoter

Pubi :

Ubiquitin promoter

LB:

Luria–Bertani medium

Torfc :

OrfC terminator

KmINU:

The inulinase gene from Kluyveromyces marxianus

DCW:

Dry cell weight

References

  1. Riediger ND, Othman RA, Suh M, Moghadasian MH (2009) A systemic review of the roles of n-3 fatty acids in health and disease. J Am Diet Assoc 109:668–679

    CAS  PubMed  Google Scholar 

  2. Yu XJ, Chen H, Huang CY, Zhu XY, Wang ZP, Wang DS, Liu XY, Sun J, Zheng JY, Li HJ, Wang Z (2019) Transcriptomic mechanism of the phytohormone 6-benzylaminopurine (6-BAP) stimulating lipid and DHA synthesis in Aurantiochytrium sp. J Agric Food Chem 67:5560–5570

    CAS  PubMed  Google Scholar 

  3. Barclay W, Weaver C, Metz J, Hansen J (2010) 4-development of a docosahexaenoic acid production technology using Schizochytrium: historical perspective and update. In: Cohen Z, Ratledge C (eds) Single cell oils, 2nd edn. AOCS Press, Boulder

    Google Scholar 

  4. Taoka Y, Nagano N, Okita Y, Izumida H, Sugimoto S, Hayashi M (2009) Extracellular enzymes produced by marine Eukaryotes, Thraustochytrids. Biosci Biotechnol Biochem 73:180–182

    CAS  PubMed  Google Scholar 

  5. Park WK, Moon M, Shin SE, Cho JM, Suh WI, Chang YK, Lee B (2018) Economical DHA (Docosahexaenoic acid) production from Aurantiochytrium sp. KRS101 using orange peel extract and low cost nitrogen sources. Algal Res 29:71–79

    Google Scholar 

  6. Liang Y, Sarkany N, Cui Y, Yesuf J, Trushenski J, Blackburn JW (2010) Use of sweet sorghum juice for lipid production by Schizochytrium limacinum SR21. Bioresour Technol 101:3623–3627

    CAS  PubMed  Google Scholar 

  7. Juntila DJ, Yoneda K, Suzuki I (2019) Genetic modification of the thraustochytrid Aurantiochytrium sp. 18W–13a for cellobiose utilization by secretory expression of beta-glucosidase from Aspergillus aculeatus. Algal Res 40:101503

    Google Scholar 

  8. Unagul P, Assantachai C, Phadungruengluij S, Suphantharika M, Tanticharoen M, Verduyn C (2007) Coconut water as a medium additive for the production of docosahexaenoic acid (C22:6 n3) by Schizochytrium mangrovei Sk-02. Bioresour Technol 98:281–287

    CAS  PubMed  Google Scholar 

  9. Chi Z, Hu B, Liu Y, Frear C, Wen Z, Chen S (2007) Production of omega-3 polyunsaturated fatty acids from cull potato using an algae culture process. Appl Biochem Biotechnol 137–140:805–815

    PubMed  Google Scholar 

  10. Humhal T, Kastanek P, Jezkova Z, Cadkova A, Kohoutkova J, Branyik T (2017) Use of saline waste water from demineralization of cheese whey for cultivation of Schizochytrium limacinum PA-968 and Japonochytrium marinum AN-4. Bioprocess Biosyst Eng 40:395–402

    CAS  PubMed  Google Scholar 

  11. Blecker C, Fougnies C, Van Herck JC, Chevalier JP, Paquot M (2002) Kinetic study of the acid hydrolysis of various oligofructose samples. J Agric Food Chem 50:1602–1607

    CAS  PubMed  Google Scholar 

  12. Chi ZM, Chi Z, Zhang T, Liu GL, Yue LX (2009) Inulinase-expressing microorganisms and applications of inulinases. Appl Microbiol Biotechnol 82:211–220

    CAS  PubMed  Google Scholar 

  13. Song Y, Oh C, Bae HJ (2017) Simultaneous production of bioethanol and value-added D-psicose from Jerusalem artichoke (Helianthus tuberosus L.) tubers. Bioresour Technol 244:1068–1072

    CAS  PubMed  Google Scholar 

  14. Gunnarsson IB, Karakashev D, Angelidaki I (2014) Succinic acid production by fermentation of Jerusalem artichoke tuber hydrolysate with Actinobacillus succinogenes 130Z. Ind Crop Prod 62:125–129

    CAS  Google Scholar 

  15. Yu XJ, Liu JH, Sun J, Zheng JY, Zhang YJ, Wang Z (2016) Docosahexaenoic acid production from the acidic hydrolysate of Jerusalem artichoke by an efficient sugar-utilizing Aurantiochytrium sp. YLH70. Ind Crop Prod 83:372–378

    CAS  Google Scholar 

  16. Cui W, Wang Q, Zhang F, Zhang SC, Chi ZM, Madzak C (2011) Direct conversion of inulin into single cell protein by the engineered Yarrowia lipolytica carrying inulinase gene. Process Biochem 46:1442–1448

    CAS  Google Scholar 

  17. Shi NC, Mao WA, He XX, Chi Z, Chi ZM, Liu GL (2018) Co-expression of Exo-inulinase and endo-inulinase genes in the oleaginous yeast Yarrowia lipolytica for efficient single cell oil production from inulin. Appl Biochem Biotechnol 185:334–346

    CAS  PubMed  Google Scholar 

  18. Liu XY, Chi Z, Liu GL, Wang F, Madzak C, Chi ZM (2010) Inulin hydrolysis and citric acid production from inulin using the surface-engineered Yarrowia lipolytica displaying inulinase. Metab eng 12:469–476

    CAS  PubMed  Google Scholar 

  19. Yu XJ, Yu Q, Liu YL, Sun J, Zheng J-Y, Wang Z (2015) Utilization of high-fructose corn syrup for biomass production containing high levels of docosahexaenoic acid by a newly isolated Aurantiochytrium sp. YLH70. Appl Biochem Biotechnol 177:1229–1240

    CAS  PubMed  Google Scholar 

  20. Yu XJ, Sun J, Sun YQ, Zheng JY, Wang Z (2016) Metabolomics analysis of phytohormone gibberellin improving lipid and DHA accumulation in Aurantiochytrium sp. Biochem Eng J 112:258–268

    CAS  Google Scholar 

  21. Yu XJ, Guo N, Chi ZM, Gong F, Sheng J, Chi Z (2009) Inulinase overproduction by a mutant of the marine yeast Pichia guilliermondii using surface response methodology and inulin hydrolysis. Biochem Eng J 43:266–271

    CAS  Google Scholar 

  22. Spiro RG (1966) Analysis of sugars found in glycoproteins. Methods in enzymology. Academic Press, Cambridge

    Google Scholar 

  23. Kobayashi T, Sakaguchi K, Matsuda T, Abe E, Hama Y, Hayashi M, Honda D, Okita Y, Sugimoto S, Okino N, Ito M (2011) Increase of eicosapentaenoic acid in Thraustochytrids through Thraustochytrid ubiquitin promoter-driven expression of a fatty acid Delta 5 desaturase gene. Appl Environ Microb 77:3870–3876

    CAS  Google Scholar 

  24. Yan JF, Cheng RB, Lin XZ, You S, Li K, Rong H, Ma Y (2013) Overexpression of acetyl-CoA synthetase increased the biomass and fatty acid proportion in microalga Schizochytrium. Appl Microb Biotechnol 97:1933–1939

    CAS  Google Scholar 

  25. Cui GZ, Ma ZX, Liu YJ, Feng YG, Sun ZJ, Cheng YR, Song XJ, Cui Q (2016) Overexpression of glucose-6-phosphate dehydrogenase enhanced the polyunsaturated fatty acid composition of Aurantiochytrium sp. SD116. Algal Res 19:138–145

    Google Scholar 

  26. Ortega-Berlanga B, Banuelos-Hernandez B, Rosales-Mendoza S (2018) Efficient expression of an Alzheimer's disease vaccine candidate in the microalga Schizochytrium sp. using the algevir system. Mol Biotechnol 60:362–368

    CAS  PubMed  Google Scholar 

  27. Zhang XZ, Zhang YHP (2011) Simple, fast and high-efficiency transformation system for directed evolution of cellulase in Bacillus subtilis. Microb Biotechnol 4:98–105

    PubMed  Google Scholar 

  28. Tong X, Tang Q, Wu Y, Wu J, Chen J (2009) Cloning of the gene encoding alpha-glucosidase from Aspergillus niger and its expression in Pichia pastoris. Wei sheng wu xue bao (Acta microbiologica Sinica) 49:262–268

    CAS  Google Scholar 

  29. Chen XM, Xu XM, Jin ZY, Chen HQ (2013) Expression of an exoinulinase gene from Aspergillus ficuum in Escherichia coli and its characterization. Carbohydr Polym 92:1984–1990

    CAS  PubMed  Google Scholar 

  30. Kim TG, Baek MY, Lee EK, Kwon TH, Yang MS (2008) Expression of human growth hormone in transgenic rice cell suspension culture. Plant Cell Rep 27:885–891

    CAS  PubMed  Google Scholar 

  31. Singh RS, Sooch BS, Puri M (2007) Optimization of medium and process parameters for the production of inulinase from a newly isolated Kluyveromyces marxianus YS-1. Bioresour Technol 98:2518–2525

    CAS  PubMed  Google Scholar 

  32. Wang D, Li FL, Wang SA (2016) Engineering a natural Saccharomyces cerevisiae strain for ethanol production from inulin by consolidated bioprocessing. Biotechnol Biofuels 9:11

    CAS  Google Scholar 

  33. 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 12:510–517

    CAS  PubMed  Google Scholar 

  34. Li YF, Jiang H, Hu Z, Liu GL, Chi ZM, Chi Z (2018) Overexpression of an inulinase gene in an oleaginous yeast, Aureobasidium melanogenum P10, for efficient lipid production from inulin. J Mol Microbiol Biotechnol 28:190–200

    CAS  PubMed  Google Scholar 

  35. Madzak C (2015) Yarrowia lipolytica: recent achievements in heterologous protein expression and pathway engineering. Appl Microb Biotechnol 99:4559–4577

    CAS  Google Scholar 

  36. Leong HY, Su CA, Lee BS, Lan JCW, Law CL, Chang JS, Show PL (2019) Development of Aurantiochytrium limacinum SR21 cultivation using salt-rich waste feedstock for docosahexaenoic acid production and application of natural colourant in food product. Bioresour Technol 271:30–36

    CAS  PubMed  Google Scholar 

  37. Ratledge C (2002) Regulation of lipid accumulation in oleaginous micro-organisms. Biochem Soc Trans 30:1047–1050

    CAS  PubMed  Google Scholar 

  38. Patel A, Matsakas L (2019) A comparative study on de novo and ex novo lipid fermentation by oleaginous yeast using glucose and sonicated waste cooking oil. Ultrason Sonochem 52:364–374

    CAS  PubMed  Google Scholar 

  39. Cescut J, Fillaudeau L, Molina-Jouve C, Uribelarrea JL (2014) Carbon accumulation in Rhodotorula glutinis induced by nitrogen limitation. Biotechnol Biofuels 7:164

    PubMed  PubMed Central  Google Scholar 

  40. Ma C, Wen HQ, Xing DF, Pei XY, Zhu JN, Ren NQ, Liu BF (2017) Molasses wastewater treatment and lipid production at low temperature conditions by a microalgal mutant Scenedesmus sp. Z-4. Biotechnol Biofuels 10:13

    Google Scholar 

  41. Zhang Q, Li YB, Xia LM (2014) An oleaginous endophyte Bacillus subtilis HB1310 isolated from thin-shelled walnut and its utilization of cotton stalk hydrolysate for lipid production. Biotechnol Biofuels 7:13

    Google Scholar 

  42. Wang DS, Yu XJ, Zhu XY, Wang Z, Li HJ, Wang Z-P (2019) Transcriptome mechanism of utilizing corn steep liquor as the sole nitrogen resource for lipid and DHA biosynthesis in marine oleaginous protist Aurantiochytrium sp. Biomolecules 9:695

    CAS  PubMed Central  Google Scholar 

  43. Guo DS, Ji XJ, Ren LJ, Yin FW, Sun XM, Huang H, Zhen G (2018) Development of a multi-stage continuous fermentation strategy for docosahexaenoic acid production by Schizochytrium sp. Bioresour Technol 269:32–39

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This study was financially supported by the Zhejiang Provincial Natural Science Foundation of China (No. LY18C010004).

Author information

Author notes

  1. Xin-Jun Yu and Zhi-Peng Wang contributed equally to this paper.

Authors and Affiliations

  1. Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, No.18, Chaowang Road, Hangzhou, 310014, People’s Republic of China

    Xin-Jun Yu, Miao-Jie Liang & Zhao Wang

  2. Marine Science and Engineering College, Qingdao Agricultural University, Qingdao, 266109, People’s Republic of China

    Zhi-Peng Wang

  3. Jiangsu Key Laboratory for Biomass-Based Energy and Enzyme Technology, Huaiyin Normal University, Huaian, 223300, People’s Republic of China

    Xiao-Yan Liu, Lei Hu & Jun Xia

Authors
  1. Xin-Jun Yu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhi-Peng Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Miao-Jie Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhao Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xiao-Yan Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Lei Hu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jun Xia
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xin-Jun Yu.

Ethics declarations

Conflict of interest

The authors have no financial or personal conflicts of interest to declare.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 135 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yu, XJ., Wang, ZP., Liang, MJ. et al. One-step utilization of inulin for docosahexaenoic acid (DHA) production by recombinant Aurantiochytrium sp. carrying Kluyveromyces marxianus inulinase. Bioprocess Biosyst Eng 43, 1801–1811 (2020). https://doi.org/10.1007/s00449-020-02371-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00449-020-02371-z

Keywords

Search

Navigation