Volume 25, Issue 4 (7-2021)                   IBJ 2021, 25(4): 255-264 | Back to browse issues page

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Zarei N, Ghasemi H, Nayebhashemi M, Zahmatkesh M, Jamalkhah M, Moeinian N, et al . Targeted Deletion of Los1 Homologue Affects the Production of a Recombinant Model Protein in Pichia pastoris. IBJ 2021; 25 (4) :255-264
URL: http://ibj.pasteur.ac.ir/article-1-3460-en.html
Background: The methylotrophic yeast Pichia pastoris is an appealing production host for a variety of recombinant proteins, including biologics. In this sense, various genetic- and non-genetic-based techniques have been implemented to improve the production efficiency of this expression platform. Loss of supression (Los1) encodes a non-essential nuclear tRNA exporter in Saccharomyces cerevisiae, which its deletion extends replicative lifespan. Herein, a los1-deficient strain of P. pastoris was generated and characterized. Methods: A gene disruption cassette was prepared and transformed into an anti-CD22-expressing strain of P. pastoris. A δ los1 mutant was isolated and confirmed. The drug sensitivity of the mutant was also assessed. The growth pattern and the level of anti-CD22 single-chain variable fragment (scFv) expression were compared between the parent and mutant strains. Results: The los1 homologue was found to be a non-essential gene in P. pastoris. Furthermore, the susceptibility of los1 deletion strain to protein synthesis inhibitors was altered. This strain showed an approximately 1.85-fold increase in the extracellular level of anti-CD22 scFv (p < 0.05). The maximum concentrations of total proteins secreted by δ los1 and parent strains were 125 mg/L and 68 mg/L, respectively. Conclusion: The presented data suggest that the targeted disruption of los1 homologue in P. pastoris can result in a higher expression level of our target protein. Findings of this study may improve the current strategies used in optimizing the productivity of recombinant P. pastoris strains.
Type of Study: Full Length/Original Article | Subject: Related Fields

1. Ma Y, Lee CJ, Park JS. Strategies for optimizing the production of proteins and peptides with multiple disulfide bonds. Antibiotics 2020; 9(9): 541. [DOI:10.3390/antibiotics9090541]
2. Ahmad M, Hirz M, Pichler H, Schwab H. Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Applied microbiology and biotechnology 2014; 98(12): 5301-5317. [DOI:10.1007/s00253-014-5732-5]
3. Stovicek V, Holkenbrink C, Borodina I. CRISPR/Cas system for yeast genome engineering: advances and applications. FEMS yeast research 2017; 17(5): fox030. [DOI:10.1093/femsyr/fox030]
4. Werten MW, Eggink G, Stuart MAC, de Wolf FA. Production of protein-based polymers in Pichia pastoris. Biotechnology advances 2019; 37(5): 642-666. [DOI:10.1016/j.biotechadv.2019.03.012]
5. Berlec A, Štrukelj B. Current state and recent advances in biopharmaceutical production in Escherichia coli, yeasts and mammalian cells. Journal of industrial microbiology and biotechnology 2013; 40(3-4): 257-274. [DOI:10.1007/s10295-013-1235-0]
6. Looser V, Bruhlmann B, Bumbak F, Stenger C, Costa M, Camattari A, Fotiadis D, Kovar K. Cultivation strategies to enhance productivity of Pichia pastoris: a review. Biotechnology advances 2015; 33(6): 1177-1193. [DOI:10.1016/j.biotechadv.2015.05.008]
7. Steffen KK, Dillin A. A ribosomal perspective on proteostasis and aging. Cell metabolism 2016; 23(6): 1004-1012. [DOI:10.1016/j.cmet.2016.05.013]
8. Anisimova AS, Alexandrov AI, Makarova NE, Gladyshev VN, Dmitriev SE. Protein synthesis and quality control in aging. Aging (Albany NY) 2018; 10(12): 4269-4288. [DOI:10.18632/aging.101721]
9. Kaeberlein M, Burtner CR, Kennedy BK. Recent developments in yeast aging. PLoS genet 2007; 3(5): e84 [DOI:10.1371/journal.pgen.0030084]
10. Dahiya R, Mohammad T, Alajmi MF, Rehman M, Hasan GM, Hussain A, Hassan M. Insights into the Conserved Regulatory Mechanisms of Human and Yeast Aging. Biomolecules 2020; 10(6): 882. [DOI:10.3390/biom10060882]
11. Kaeberlein M, Kirkland KT, Fields S, Kennedy BK. Genes determining yeast replicative life span in a long-lived genetic background. Mechanisms of ageing and development 2005; 126(4): 491-504. [DOI:10.1016/j.mad.2004.10.007]
12. McCormick MA, Delaney JR, Tsuchiya M, Tsuchiyama S, Shemorry A, Sim S, Chou ACZ, Ahmed U, Carr D, Murakami CJ, Schleit J, Sutphin GL, Wasko BM, Bennett CF, Wang AM, Oslen B, Beyer RP, Bammler TK, Prunkard D, Johnson SC, Pennypacker JK, An E, Anies A, Castanza AS, Choi E, Dang E, Enerio S, Fletcher M, Fox L, Goswami S, Higgins SA, Holmberg MA, Hu D, Hui J, Jelic M, Jeong KS, Johnston E, Kerr EO, Kim J, Kim D, Kirkland K, Klum S, Kotireddy S, Liao E, Lim M, Lin MS, Lo WC, Lockshon D, Miller HA, Moller RM, Muller B, Oakes J, Pak DN, Peng ZJ, Pham KM, Pollard TG, Pradeep P, Pruett D, Rai D, Robison B, Rodriguez AA, Ros B, Sage M, Singh MK, Smith ED, Snead K, Solanky A, Spector BL, Steffen KK, Tchao BN, Ting MK, Wende HV, Wang D, Welton KL, Westman EA, Brem RB, Liu XG, Suh Y, Zhou Z, Kaeberlein M, Kennedy BK. A comprehensive analysis of replicative lifespan in 4,698 single-gene deletion strains uncovers conserved mechanisms of aging. Cell metabolism 2015; 22(5): 895-906. [DOI:10.1016/j.cmet.2015.09.008]
13. Chatterjee K, Nostramo RT, Wan Y, Hopper AK. tRNA dynamics between the nucleus, cytoplasm and mitochondrial surface: location, location, location. Biochimica et biophysica acta (BBA)-gene regulatory mechanisms 2018; 1861(4): 373-386. [DOI:10.1016/j.bbagrm.2017.11.007]
14. Kenyon C. A conserved regulatory system for aging. Cell 2001; 105(2): 165-168. [DOI:10.1016/S0092-8674(01)00306-3]
15. Osiewacz HD. Genes, mitochondria and aging in filamentous fungi. Ageing research reviews 2002; 1(3): 425-442. [DOI:10.1016/S1568-1637(02)00010-7]
16. Sambrook J, Russell DW, Maniatis T. Molecular Cloning. Cold Spring Habour Laboratory Press: New York; 2001.
17. Rodriguez-Tudela J, Barchiesi F, Bille J, Chryssanthou E, Cuenca-Estrella M, Denning D, Donnelly JP, Dupont B, Fegeler W, Moore C, Richaedson M. Method for the determination of minimum inhibitory concentration (MIC) by broth dilution of fermentative yeasts. Clinical microbiology and infection 2003; 9(8): i-viii. [DOI:10.1046/j.1469-0691.2003.00789.x]
18. Macauley-Patrick S, Fazenda ML, McNeil B, Harvey LM. Heterologous protein production using the Pichia pastoris expression system. Yeast 2005; 22(4): 249-270. [DOI:10.1002/yea.1208]
19. Zhang AL, Luo JX, Zhang TY, Pan YW, Tan YH, Fu CY, Tu FZz. Recent advances on the GAP promoter derived expression system of Pichia pastoris. Molecular biology reports 2009; 36(6): 1611-1619. [DOI:10.1007/s11033-008-9359-4]
20. Shojaosadati SA, Varedi Kolaei SM, Babaeipour V, Farnoud AM. Recent advances in high cell density cultivation for production of recombinant protein. Iranian journal of biotechnology 2008; 6(2): 63-84.
21. Sagmeister P, Wechselberger P, Herwig C. Information processing: rate-based investigation of cell physiological changes along design space development. PDA journal of pharmaceutical science and technology 2012; 66(6): 526-541. [DOI:10.5731/pdajpst.2012.00889]
22. Xiao A, Zhou X, Zhou L, Zhang Y. Improvement of cell viability and hirudin production by ascorbic acid in Pichia pastoris fermentation. Applied microbiology and biotechnology 2006; 72(4): 837-844. [DOI:10.1007/s00253-006-0338-1]
23. Pekarsky A, Veiter L, Rajamanickam V, Herwig C, Grünwald-Gruber C, Altmann F, Spadiut O. Production of a recombinant peroxidase in different glyco-engineered Pichia pastoris strains: a morphological and physiological comparison. Microbial cell factories 2018; 17(1): 1-15. [DOI:10.1186/s12934-018-1032-6]
24. Minor RK, Allard JS, Younts CM, Ward TM, de Cabo R. Dietary interventions to extend life span and health span based on calorie restriction. Journals of gerontology series A: biomedical sciences and medical sciences 2010; 65(7): 695-703. [DOI:10.1093/gerona/glq042]
25. Polymenis M, Kennedy BK. Chronological and replicative lifespan in yeast: do they meet in the middle? Cell cycle 2012; 11(19): 3531-3532. [DOI:10.4161/cc.22041]
26. Tavernarakis N. Ageing and the regulation of protein synthesis: a balancing act? Trends in cell biology 2008; 18(5): 228-235. [DOI:10.1016/j.tcb.2008.02.004]
27. Tavernarakis N. Protein synthesis and aging: eIF4E and the soma vs. germline distinction. Cell cycle 2007; 6(10): 1168-1171. [DOI:10.4161/cc.6.10.4230]
28. Sampaio-Marques B, Ludovico P. Linking cellular proteostasis to yeast longevity. FEMS yeast research 2018; 18(5): doi: 10.1093/femsyr/foy043. [DOI:10.1093/femsyr/foy043]
29. Nyström T, Liu B. Protein quality control in time and space-links to cellular aging. FEMS yeast research 2014; 14(1): 40-48. [DOI:10.1111/1567-1364.12095]
30. Gonskikh Y, Polacek N. Alterations of the translation apparatus during aging and stress response. Mechanisms of ageing and development 2017; 168: 30-36 [DOI:10.1016/j.mad.2017.04.003]
31. Saarikangas J, Barral Y. Protein aggregates are associated with replicative aging without compromising protein quality control. Elife 2015; 4: e06197. [DOI:10.7554/eLife.06197]
32. Markina-Iñarrairaegui A, Etxebeste O, Herrero-García E, Araújo-Bazán L, Fernández-Martínez J, Flores JA, Osmani SA, Espeso EA. Nuclear transporters in a multinucleated organism: functional and localization analyses in Aspergillus nidulans. Molecular biology of the cell 2011; 22(20): 3874-3886. [DOI:10.1091/mbc.e11-03-0262]
33. Azizi A, SharifiRad A, Enayati S, Azizi M, Bayat M, Khalaj V. Absence of AfuXpot, the yeast Los1 homologue, limits Aspergillus fumigatus growth under amino acid deprived condition. World journal of microbiology and biotechnology 2020; 36(2): 28 [DOI:10.1007/s11274-020-2805-8]
34. McFarland MR, Keller CD, Childers BM, Adeniyi SA, Corrigall H, Raguin A, Romano MC, Stansfield I. The molecular aetiology of tRNA synthetase depletion: induction of a GCN4 amino acid starvation response despite homeostatic maintenance of charged tRNA levels. Nucleic acids research 2020; 48(6): 3071-3088. [DOI:10.1093/nar/gkaa055]
35. Buchetics M, Dragosits M, Maurer M, Rebnegger C, Porro D, Sauer M, Gasser B, Mattanovich D. Reverse engineering of protein secretion by uncoupling of cell cycle phases from growth. Biotechnology and bioengineering 2011; 108(10): 2403-2412. [DOI:10.1002/bit.23198]
36. Puxbaum V, Gasser B, Mattanovich D. The bud tip is the cellular hot spot of protein secretion in yeasts. Applied microbiology and biotechnology 2016; 100(18): 8159-8168. [DOI:10.1007/s00253-016-7674-6]
37. Dumont FJ, Su Q. Mechanism of action of the immunosuppressant rapamycin. Life sciences 1996; 58(5): 373-395. [DOI:10.1016/0024-3205(95)02233-3]
38. Doi A, Fujimoto A, Sato S, Uno T, Kanda Y, Asami K, Tanaka Y, Kita A, Satoh R, Sugiura R. Chemical genomics approach to identify genes associated with sensitivity to rapamycin in the fission yeast Schizosaccharomyces pombe. Genes cells 2015; 20(4): 292-309. [DOI:10.1111/gtc.12223]
39. McKeehan W, Hardesty B. The mechanism of cycloheximide inhibition of protein synthesis in rabbit reticulocytes. Biochemical and biophysical research communications 1969; 36(4): 625-630. [DOI:10.1016/0006-291X(69)90351-9]
40. Schneider-Poetsch T, Ju J, Eyler DE, Dang Y, Bhat S, Merrick WC, Green R, Shen B, Liu JO. Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin. Nature chemical biology 2010; 6(3): 209-217. [DOI:10.1038/nchembio.304]

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