Abstract
γ-Linolenic acid (GLA) is an essential n-6 polyunsaturated fatty acid (PUFA) that has received considerable attention in human and animal feed. GLA is used in many nutritional and medicinal applications, such as the treatment of cancer, inflammatory disorders, and diabetes. Currently, plant seed is the primary dietary source of GLA that is not enough to utilize on an industrial scale. To generate a sustainable novel source of GLA, the gene of delta-6 desaturase, one of the essential enzymes in the GLA production pathway, was isolated from Mucor rouxii DSM1194 and expressed in P. pastoris GS115 by pPICZC vector. The recombinant yeast expressed the GLA up to 19.2% (72 mg/g) of total fatty acids. GLA production of recombinant yeast was studied in a fermenter by oil waste for 5 days, and results detected 6.3 g/l lipid, and 103 mg/g GLA was produced in 72 h. The present study may provide an opportunity to develop an alternative host for manufacturing GLA on an industrial scale.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Availability of data and materials
Non-digital data supporting this study are curated.
References
Akhbariyoon HR, Mirbagheri M, Emtiazi G (2016) Isolation and identification of Alicyclobacillus with high dipicolinic acid and heat resistant proteins from mango juice. Appl Food Biotechnol 3:270–274. https://doi.org/10.22037/afb.v3i4.13429
Christie WW (1993) Preparation of ester derivatives of fatty acids for chromatographic analysis. In: Christie W (ed) Advances in lipid methodology – Two. William. Oily Press, pp 69–111
Cui J, He S, Ji X et al (2016) Identification and characterization of a novel bifunctional Δ12/Δ15-fatty acid desaturase gene from Rhodosporidium kratochvilovae. Biotechnol Lett 38:1155–1164. https://doi.org/10.1007/s10529-016-2090-7
De Gyves EM, Sparks CA, Sayanova O et al (2004) Genetic manipulation of γ-linolenic acid (GLA) synthesis in a commercial variety of evening primrose (Oenothera sp.). Plant Biotechnol J 2:351–357. https://doi.org/10.1111/j.1467-7652.2004.00079.x
Fakas S, Čertik M, Papanikolaou S et al (2008) γ-Linolenic acid production by Cunninghamella echinulata growing on complex organic nitrogen sources. Bioresour Technol 99:5986–5990. https://doi.org/10.1016/j.biortech.2007.10.016
Fakas S, Papanikolaou S, Galiotou-Panayotou M, Komaitis M, Aggelis G (2006) Lipids of cunninghamella echinulata with emphasis to gamma-linolenic acid distribution among lipid classes. Appl Microbiol Biotechnol 73(3):676–83. https://doi.org/10.1007/s00253-006-0506-3
Huang YS, Chaudhary S, Thurmond JM et al (1999) Cloning of Δ12- and Δ-desaturases from Mortierella alpina and recombinant production of γ-linolenic acid in Saccharomyces cerevisiae. Lipids 34:649–659. https://doi.org/10.1007/s11745-999-0410-8
Jangbua P, Laoteng K, Kitsubun P et al (2009) Gamma-linolenic acid production of Mucor rouxii by solid-state fermentation using agricultural by-products. Lett Appl Microbiol 49:91–97. https://doi.org/10.1111/j.1472-765X.2009.02624.x
Kapoor R, Huang Y-S (2006) Gamma linolenic acid: an antiinflammatory omega-6 fatty acid. Curr Pharm Biotechnol 7:531–534. https://doi.org/10.2174/138920106779116874
Kenny FS, Pinder SE, Ellis IO et al (2000) Gamma linolenic acid with tamoxifen as primary therapy in breast cancer. Int J Cancer 85:643–648. https://doi.org/10.1002/(SICI)1097-0215(20000301)85:5%3c643::AID-IJC8%3e3.0.CO;2-Z
Kim D-H, Yoo T-H, Lee SH et al (2012) Gamma linolenic acid exerts anti-inflammatory and anti-fibrotic effects in diabetic nephropathy. Yonsei Med J 53:1165–1175. https://doi.org/10.3349/ymj.2012.53.6.1165
Li P, Anumanthan A, Gao XG et al (2007) Expression of recombinant proteins in Pichia pastoris. Appl Biochem Biotechnol 142:105–124
Lu H, Zhu Y (2015) Screening and molecular identification of overproducing γ-linolenic acid fungi and cloning the delta 6-desaturase gene. Biotechnol Appl Biochem 62:316–322. https://doi.org/10.1002/bab.1281
Lu H, Li JN, Chai YR, Zhang XK (2009) Identification and characterization of a novel Δ6-fatty acid desaturase gene from Rhizopus nigricans. Mol Biol Rep 36:2291–2297. https://doi.org/10.1007/s11033-009-9447-0
Mirbagheri M, Nahvi I, Emamzade R (2015) Reduction of chemical and biological oxygen demands from oil wastes via oleaginous fungi: an attempt to convert food by products to essential fatty acids. Iran J Biotechnol 13:25–30. https://doi.org/10.15171/ijb.1026
Mirbagheri M, Nahvi I, Emamzadeh R et al (2016) Oil wastes management: medium optimization for the production of alpha-linolenic acid in Mucor circinelloides. Int J Environ Sci Technol 13:31–38. https://doi.org/10.1007/S13762-015-0849-3
Mohamadi Nasr M, Mirbagheri M, Nahvi I (2014) Use of a carbohydrate source and oil waste in lipid production by Mucor hiemalis. Iran J Nutr Sci Food Technol 9(2):59–666
Nasr M, Nahvi I, Keyhanfar M, Mirbagheri M (2017) The effect of carbon and nitrogen sources on the fatty acids profile of Mortierella vinacea. Biol J Microorg 5:1–8
Needleman P, Truk J, Jakschik BA et al (1986) Arachidonic acid metabolism. Annu Rev Biochem 55:69–102. https://doi.org/10.1146/annurev.bi.55.070186.000441
Nykiforuk CL, Shewmaker C, Harry I et al (2012) High level accumulation of gamma linolenic acid (C18:3Δ6.9,12 cis) in transgenic safflower (Carthamus tinctorius) seeds. Transgenic Res 21:367–381. https://doi.org/10.1007/s11248-011-9543-5
Opperhuizen A (1982) A laboratory manual. Chemosphere 11:N32–N33. https://doi.org/10.1016/0045-6535(82)90062-5
Pan LX, Yang DF, Shao L et al (2009) Isolation of the oleaginous yeasts from the soil and studies of their lipid-producing capacities. Food Technol Biotechnol 47:215–220
Papanikolaou S, Galiotou-Panayotou M, Fakas S et al (2007) Lipid production by oleaginous Mucorales cultivated on renewable carbon sources. Eur J Lipid Sci Technol 109:1060–1070. https://doi.org/10.1002/EJLT.200700169
Passorn S, Laoteng K, Rachadawong S et al (1999) Heterologous expression of Mucor rouxii Δ12-Desaturase Gene in Saccharomyces cerevisiae. Biochem Biophys Res Commun 265:771. https://doi.org/10.1006/bbrc.1999.1740
Qiao K, Imam Abidi SH, Liu H et al (2015) Engineering lipid overproduction in the oleaginous yeast Yarrowia lipolytica. Metab Eng 29:56–65. https://doi.org/10.1016/j.ymben.2015.02.005
Sakuradani E, Shimizu S (2003) Gene cloning and functional analysis of a second Δ6-fatty acid desaturase from an arachidonic acid-producing Mortierella fungus. Biosci Biotechnol Biochem 67:704–711. https://doi.org/10.1271/bbb.67.704
Sakuradani E, Ando A, Ogawa J, Shimizu S (2009) Improved production of various polyunsaturated fatty acids through filamentous fungus Mortierella alpina breeding. Appl Microbiol Biotechnol 84:1–10
Vrinten P, Wu G, Truksa M, Qiu X (2007) Biotechnology and genetic engineering reviews production of polyunsaturated fatty acids in transgenic plants. Transgenic Plants Biotechnol Genet Eng Rev 24:263–280. https://doi.org/10.1080/02648725.2007.10648103
Wan X, Zhang Y, Wang P et al (2009) Production of gamma-linolenic acid in Pichia pastoris by expression of a delta-6 desaturase gene from Cunninghamella echinulata. J Microbiol Biotechnol 19:1098–1102. https://doi.org/10.4014/jmb.0902.071
Wan X, Zhang Y, Wang P, Jiang M (2011) Molecular cloning and expression analysis of a delta 6-fatty acid desaturase gene from Rhizopus stolonifer strain YF6 which can accumulate high levels of gamma-linolenic acid. J Microbiol 49:151–154. https://doi.org/10.1007/s12275-011-0254-7
Wang D, Li M, Wei D et al (2007) Identification and functional characterization of the delta 6-fatty acid desaturase gene from Thamnidium elegans. J Eukaryot Microbiol 54:110–117. https://doi.org/10.1111/j.1550-7408.2006.00136.x
Wang P, Wan X, Zhang Y, Jiang M (2011) Production of γ-linolenic acid using a novel heterologous expression system in the oleaginous yeast Lipomyces kononenkoae. Biotechnol Lett 33:1993–1998. https://doi.org/10.1007/s10529-011-0651-3
Wei DS, Li MC, Zhang XX et al (2006) A novel Δ12-fatty acid desaturase gene from methylotrophic yeast Pichia pastoris GS115. Acta Biochim Pol 53:753–759. https://doi.org/10.18388/abp.2006_3303
Yamada Y, Makimura K, Mirhendi H et al (2002) Comparison of different methods for extraction of mitochondrial DNA from human pathogenic yeasts. Jpn J Infect Dis 55(4):122–125
Zhang Q, Li MC, Sun Y et al (2005) Heteroexpression of Rhizopus arrhizus delta6-fatty acid desaturase gene in Pichia pastoris Sheng Wu Gong Cheng Xue Bao. Chin J Biotechnol 21:871–877
Zhang Y, Luan X, Zhang H et al (2017) Improved γ-linolenic acid production in Mucor circinelloides by homologous overexpressing of delta-12 and delta-6 desaturases. Microb Cell Fact. https://doi.org/10.1186/s12934-017-0723-8
Zhu Q, Jackson EN (2015) Metabolic engineering of Yarrowia lipolytica for industrial applications. Curr Opin Biotechnol 36:65–72
Acknowledgements
The authors would like to thank Dr. Rahman Emamzadeh, Dr. Maryam Fanaei, Zahra fanaei, Dr. Gholamreza Ghezelbash, and Dr. Hossein Ghanavati for their guidance.
Funding
Not applicable.
Author information
Authors and Affiliations
Contributions
MMSFcarried out the experiment and supervised the project, HA helped supervise the project and wrote the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors have no conflicts of interest to declare that are relevant to the content of this article.
Ethical approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Consent to Participate
The authors consent to participate in this article.
Consent to Publish
All authors agree to publish the article in the journal of Applied biochemistry and biotechnology.
Additional information
Communicated by Erko Stackebrandt.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Mirbagheri Firoozabad, M.S., Akhbariyoon, H. Engineering and fermenter production of fungi GLA in Pichia pastoris GS115 using oil waste. Arch Microbiol 204, 635 (2022). https://doi.org/10.1007/s00203-022-03182-4
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1007/s00203-022-03182-4