Nile red-based lipid fluorometry protocol and its use for statistical optimization of lipids in oleaginous yeasts.
Central composite design
Lipid fluorometry
Nile red
Oleaginous yeasts
Rhodotorula toruloides
Yarrowia lipolytica
Journal
Applied microbiology and biotechnology
ISSN: 1432-0614
Titre abrégé: Appl Microbiol Biotechnol
Pays: Germany
ID NLM: 8406612
Informations de publication
Date de publication:
Dec 2023
Dec 2023
Historique:
received:
15
06
2023
accepted:
07
09
2023
revised:
06
09
2023
medline:
13
11
2023
pubmed:
24
9
2023
entrez:
23
9
2023
Statut:
ppublish
Résumé
As lipogenic yeasts are becoming increasingly harnessed as biofactories of oleochemicals, the availability of efficient protocols for the determination and optimization of lipid titers in these organisms is necessary. In this study, we optimized a quick, reliable, and high-throughput Nile red-based lipid fluorometry protocol adapted for oleaginous yeasts and validated it using different approaches, the most important of which is using gas chromatography coupled to flame ionization detection and mass spectrometry. This protocol was applied in the optimization of the concentrations of ammonium chloride and glycerol for attaining highest lipid titers in Rhodotorula toruloides NRRL Y-6987 and Yarrowia lipolytica W29 using response surface central composite design (CCD). Results of this optimization showed that the optimal concentration of ammonium chloride and glycerol is 4 and 123 g/L achieving a C/N ratio of 57 for R. toruloides, whereas for Y. lipolytica, concentrations are 4 and 139 g/L with a C/N ratio of 61 for Y. lipolytica. Outside the C/N of 33 to 74 and 45 to 75, respectively, for R. toruloides and Y. lipolytica, lipid productions decrease by more than 10%. The developed regression models and response surface plots show the importance of the careful selection of C/N ratio to attain maximal lipid production. KEY POINTS: • Nile red (NR)-based lipid fluorometry is efficient, rapid, cheap, high-throughput. • NR-based lipid fluorometry can be well used for large-scale experiments like DoE. • Optimal molar C/N ratio for maximum lipid production in lipogenic yeasts is ~60.
Identifiants
pubmed: 37741936
doi: 10.1007/s00253-023-12786-9
pii: 10.1007/s00253-023-12786-9
doi:
Substances chimiques
nile red
P476F1L81G
Lipids
0
Glycerol
PDC6A3C0OX
Ammonium Chloride
01Q9PC255D
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
7313-7330Subventions
Organisme : Natural Sciences and Engineering Research Council of Canada
ID : RGPIN-2022-05307
Organisme : Fonds de recherche du Québec - Nature et technologies
ID : 2022-NC-298442
Informations de copyright
© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.
Références
Abdel-Mawgoud AM, Markham KA, Palmer CM, Liu N, Stephanopoulos G, Alper HS (2018) Metabolic engineering in the host Yarrowia lipolytica. Metab Eng 50:192–208. https://doi.org/10.1016/j.ymben.2018.07.016
doi: 10.1016/j.ymben.2018.07.016
pubmed: 30056205
Abdel-Mawgoud AM, Stephanopoulos G (2020) Improving CRISPR/Cas9-mediated genome editing efficiency in Yarrowia lipolytica using direct tRNA-sgRNA fusions. Metab Eng 62:106–115. https://doi.org/10.1016/j.ymben.2020.07.008
doi: 10.1016/j.ymben.2020.07.008
pubmed: 32758536
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(1):3131. https://doi.org/10.1038/ncomms4131
doi: 10.1038/ncomms4131
pubmed: 24445655
Chen W, Zhang C, Song L, Sommerfeld M, Hu Q (2009) A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae. J of Microbiol Methods 77(1):41–47. https://doi.org/10.1016/j.mimet.2009.01.001
doi: 10.1016/j.mimet.2009.01.001
Cirulis JT, Strasser BC, Scott JA, Ross GM (2012) Optimization of staining conditions for microalgae with three lipophilic dyes to reduce precipitation and fluorescence variability. Cytomet Part A 81A(7):618–626. https://doi.org/10.1002/cyto.a.22066
doi: 10.1002/cyto.a.22066
da Silva GP, Mack M, Contiero J (2009) Glycerol: a promising and abundant carbon source for industrial microbiology. Biotechnol Advanc 27(1):30–39. https://doi.org/10.1016/j.biotechadv.2008.07.006
doi: 10.1016/j.biotechadv.2008.07.006
Dobrowolski A, Drzymala K, Rzechonek DA, Mitula P, Mironczuk AM (2019) Lipid production from waste materials in seawater-based medium by the yeast Yarrowia lipolytica. Front in Microbiol 10:9. https://doi.org/10.3389/fmicb.2019.00547
doi: 10.3389/fmicb.2019.00547
Fam TK, Klymchenko AS, Collot M (2018) Recent advances in fluorescent probes for lipid droplets. Mater 11(9):1768
doi: 10.3390/ma11091768
Greenspan P, Fowler SD (1985) Spectrofluorometric studies of the lipid probe, Nile red. J of Lipid Res 26(7):781–789. https://doi.org/10.1016/S0022-2275(20)34307-8
doi: 10.1016/S0022-2275(20)34307-8
Henne WM, Reese ML, Goodman JM (2018) The assembly of lipid droplets and their roles in challenged cells. EMBO J 37(12). https://doi.org/10.15252/embj.201898947
Hildyard JCW, Halestrap AP (2003) Identification of the mitochondrial pyruvate carrier in Saccharomyces cerevisiae. Biochem J 374(3):607–611. https://doi.org/10.1042/bj20030995
doi: 10.1042/bj20030995
pubmed: 12887330
pmcid: 1223651
Isleten-Hosoglu M, Gultepe I, Elibol M (2012) Optimization of carbon and nitrogen sources for biomass and lipid production by Chlorella saccharophila under heterotrophic conditions and development of Nile red fluorescence based method for quantification of its neutral lipid content. Biochem Eng J 61:11–19. https://doi.org/10.1016/j.bej.2011.12.001
doi: 10.1016/j.bej.2011.12.001
Johns AMB, Love J, Aves SJ (2016) Four inducible promoters for controlled gene expression in the oleaginous yeast Rhodotorula toruloides. Front in Microbiol 7(1666). https://doi.org/10.3389/fmicb.2016.01666
Kimura K, Yamaoka M, Kamisaka Y (2004) Rapid estimation of lipids in oleaginous fungi and yeasts using Nile red fluorescence. J of Microbiol Methods 56(3):331–338. https://doi.org/10.1016/j.mimet.2003.10.018
doi: 10.1016/j.mimet.2003.10.018
Kisley L (2019) Chapter 3 - Single molecule spectroscopy at interfaces. In: Johnson CK (ed) Spectroscopy and dynamics of single molecules. Elsevier, pp 117–161
doi: 10.1016/B978-0-12-816463-1.00003-1
Kitcha S, Cheirsilp B (2011) Screening of oleaginous yeasts and optimization for lipid production using crude glycerol as a carbon source. Energy Procedia 9:274–282. https://doi.org/10.1016/j.egypro.2011.09.029
doi: 10.1016/j.egypro.2011.09.029
Kuttiraja M, Douha A, Valéro JR, Tyagi RD (2016) Elucidating the effect of glycerol concentration and C/N ratio on lipid production using Yarrowia lipolytica SKY7. Appl Biochem and Biotechnol 180(8):1586–1600. https://doi.org/10.1007/s12010-016-2189-2
doi: 10.1007/s12010-016-2189-2
Lamprecht A, Benoit J-P (2003) Simple liquid-chromatographic method for Nile red quantification in cell culture in spite of photobleaching. J Chromatogr B 787(2):415–419. https://doi.org/10.1016/S1570-0232(02)00962-5
doi: 10.1016/S1570-0232(02)00962-5
Miranda C, Bettencourt S, Pozdniakova T, Pereira J, Sampaio P, Franco-Duarte R, Pais C (2020) Modified high-throughput Nile red fluorescence assay for the rapid screening of oleaginous yeasts using acetic acid as carbon source. BMC Microbiol 20(1):60. https://doi.org/10.1186/s12866-020-01742-6
doi: 10.1186/s12866-020-01742-6
pubmed: 32169040
pmcid: 7071767
Moreton R (1988) Single cell oil. Longman Scientific Technical, Harlow
Nicaud JM (2012) Yarrowia lipolytica. Yeast 29(10):409–418. https://doi.org/10.1002/yea.2921
doi: 10.1002/yea.2921
pubmed: 23038056
Nielsen J (2014) Synthetic biology for engineering acetyl coenzyme A metabolism in yeast. mBio 5(6):e02153–e02114. https://doi.org/10.1128/mBio.02153-14
doi: 10.1128/mBio.02153-14
pubmed: 25370498
pmcid: 4222110
Orr V, Rehmann L (2015) Improvement of the Nile red fluorescence assay for determination of total lipid content in microalgae independent of chlorophyll content. J of Appl Phycol 27(6):2181–2189. https://doi.org/10.1007/s10811-014-0481-5
doi: 10.1007/s10811-014-0481-5
Papanikolaou S, Aggelis G (2011a) Lipids of oleaginous yeasts. Part I: Biochemistry of single cell oil production. Eur J Lipid Sci Technol 113(8):1031–1051. https://doi.org/10.1002/ejlt.201100014
doi: 10.1002/ejlt.201100014
Papanikolaou S, Aggelis G (2011b) Lipids of oleaginous yeasts. Part II: Technology and potential applications. Eur J Lipid Sci Technol 113(8):1052–1073. https://doi.org/10.1002/ejlt.201100015
doi: 10.1002/ejlt.201100015
Poli JS, Lützhøft H-CH, Karakashev DB, Valente P, Angelidaki I (2014) An environmentally-friendly fluorescent method for quantification of lipid contents in yeast. Bioresour Technol 151:388–391. https://doi.org/10.1016/j.biortech.2013.09.128
doi: 10.1016/j.biortech.2013.09.128
pubmed: 24206637
Pomraning KR, Wei S, Karagiosis SA, Kim Y-M, Dohnalkova AC, Arey BW, Bredeweg EL, Orr G, Metz TO, Baker SE (2015) Comprehensive metabolomic, lipidomic and microscopic profiling of Yarrowia lipolytica during lipid accumulation identifies targets for increased lipogenesis. Plos One 10(4):e0123188. https://doi.org/10.1371/journal.pone.0123188
doi: 10.1371/journal.pone.0123188
pubmed: 25905710
pmcid: 4408067
Ouellet B, Abdel-Mawgoud AM (2023) Strong Expression of Cas9 Under a New 3’-Truncated TEF1α Promoter Enhances CRISPR-Cas9-Mediated Genome Editing in Yarrowia Lipolytica. Yarrowia Lipolytica. https://doi.org/10.2139/ssrn.4499951
doi: 10.2139/ssrn.4499951
Quispe CAG, Coronado CJR, Carvalho JA Jr (2013) Glycerol: production, consumption, prices, characterization and new trends in combustion. Renew Sustain Energy Rev 27:475–493. https://doi.org/10.1016/j.rser.2013.06.017
doi: 10.1016/j.rser.2013.06.017
Ramírez-Castrillón M, Jaramillo-Garcia VP, Lopes Barros H, Pegas Henriques JA, Stefani V, Valente P (2021) Nile red incubation time before reading fluorescence greatly influences the yeast neutral lipids quantification. Front Microbiol 12(445). https://doi.org/10.3389/fmicb.2021.619313
Ratledge C (1997) Microbial Lipids. Biotechnol:133–197
Ratledge C, Wynn JP (2002) The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Advanc Appl Microbiol 51:1–51. https://doi.org/10.1016/s0065-2164(02)51000-5
doi: 10.1016/s0065-2164(02)51000-5
Ray A, Das S, Chattopadhyay N (2019) Aggregation of Nile red in water: prevention through encapsulation in β-cyclodextrin. ACS Omega 4(1):15–24. https://doi.org/10.1021/acsomega.8b02503
doi: 10.1021/acsomega.8b02503
pubmed: 31459307
pmcid: 6649296
Rumin J, Bonnefond H, Saint-Jean B, Rouxel C, Sciandra A, Bernard O, Cadoret J-P, Bougaran G (2015) The use of fluorescent Nile red and BODIPY for lipid measurement in microalgae. Biotechnol Biofuels 8(1):42. https://doi.org/10.1186/s13068-015-0220-4
doi: 10.1186/s13068-015-0220-4
pubmed: 25788982
pmcid: 4364489
Sackett DL, Wolff J (1987) Nile red as a polarity-sensitive fluorescent probe of hydrophobic protein surfaces. Anal Biochem 167(2):228–234. https://doi.org/10.1016/0003-2697(87)90157-6
doi: 10.1016/0003-2697(87)90157-6
pubmed: 3442318
Sitepu IR, Ignatia L, Franz AK, Wong DM, Faulina SA, Tsui M, Kanti A, Boundy-Mills K (2012) An improved high-throughput Nile red fluorescence assay for estimating intracellular lipids in a variety of yeast species. J Microbiol Methods 91(2):321–328. https://doi.org/10.1016/j.mimet.2012.09.001
doi: 10.1016/j.mimet.2012.09.001
pubmed: 22985718
pmcid: 3478415
Tesnière C, Brice C, Blondin B (2015) Responses of Saccharomyces cerevisiae to nitrogen starvation in wine alcoholic fermentation. Appl Microbiol Biotechnol 99(17):7025–7034. https://doi.org/10.1007/s00253-015-6810-z
doi: 10.1007/s00253-015-6810-z
pubmed: 26201494
Van Wychen S, Ramirez K, Laurens LML (2016) Determination of total lipids as fatty acid methyl esters (FAME) by in situ transesterification: laboratory analytical procedure (LAP). Natl Renew Energy Lab: Tech Rep(NREL/TP-5100-60958). https://doi.org/10.2172/1118085
doi: 10.2172/1118085
Zhang HY, Wu C, Wu QY, Dai JB, Song YD (2016) Metabolic flux analysis of lipid biosynthesis in the yeast Yarrowia lipolytica using C-13-labled glucose and gas chromatography-mass spectrometry. Plos One 11(7):14. https://doi.org/10.1371/journal.pone.0159187
doi: 10.1371/journal.pone.0159187