Up Front Unfolded Protein Response Combined with Early Protein Secretion Pathway Engineering in Yarrowia lipolytica to Attenuate ER Stress Caused by Enzyme Overproduction
Abstract
:1. Introduction
2. Results and Discussion
2.1. Enzyme Overproduction Causes ER Stress and Triggers the Unfolded Protein Response (UPR)
2.2. Engineering Protein Translocation Improves Protein Production in Y. lipolytica
2.3. Engineering Protein-Folding Machinery Improves Protein Production in Y. lipolytica
2.4. The Impact of Engineering Protein Translocation and Folding on R-Protein Production in Y. lipolytica
3. Materials and Methods
3.1. Strains, Reagents and Culture Media
3.2. Plasmid and Strain Construction
3.3. Recombinant Protein Production
3.4. Gene Expression Analysis at Transcriptional Level
3.5. Measurement of Enzyme Activity
3.6. Analysis of Glucose Consumption and Biomass Formation
3.7. Statistical Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Strains | Relevant Genotype | Source of Reference |
---|---|---|
E. coli DH5 | Φ80dlacZΔm15, recA1, endA1, gyrA96, thi-1, hsdR17 (rk−, mk+), supE44, relA1, deoR, Δ(lacZYA-argF) U169 | Invitrogen |
Y. lipolytica Po1f | MatA, leu2-270, ura3-302, xpr2-322 axp1 | [14] |
YlLIP | Po1f; pTEF-LIP2-URA3, LEU2 | This investigation |
Yl4LIP | Po1f; p4UASTEF-LIP2-URA3, LEU2 | This investigation |
Yl8LIP | Po1f; p8UASTEF-LIP2-URA3, LEU2 | This investigation |
Yl16LIP | Po1f; p8UASTEF-LIP2-URA3, p8UASTEF-LIP2-LEU2 | This investigation |
Yl16LIP-Hac1 (Kar2, Sls, Sp14, Sp54, Ss5, Ss6, Ss7, Ss8, Sb or Pd) | Yl16LIP; pTEF-HAC1 (or KAR2, SLS1, SRP14, SRP54, SSA5, SSA6, SSA7, SSA8, SSB1, PDI1) | This investigation |
Yl16LIP-Ssa6/Ssa8 | Yl16LIP; pTEF-SSA6, pTEF-SSA8 | This investigation |
Yl16LIP-Ssa6/Ssb1 | Yl16LIP; pTEF-SSA6, pTEF-SSB1 | This investigation |
Yl16LIP-Ssa6/Sls1 | Yl16LIP; pTEF-SSA6, pTEF-SLS1 | This investigation |
Yl16LIP-Ssa6/Pdi1 | Yl16LIP; pTEF-SSA6, pTEF-PDI1 | This investigation |
Yl16LIP-Ssa8/Ssb1 | Yl16LIP; pTEF-SSA8, pTEF-SSB1 | This investigation |
Yl16LIP-Ssa8/Sls1 | Yl16LIP; pTEF-SSA8, pTEF-SLS1 | This investigation |
Yl16LIP-Ssa8/Pdi1 | Yl16LIP; pTEF-SSA8, pTEF-PDI1 | This investigation |
Yl16LIP-Ssb1/Sls1 | Yl16LIP; pTEF-SSB1, pTEF-SLS1 | This investigation |
Yl16LIP-Ssb1/Pdi1 | Yl16LIP; pTEF-SSB1, pTEF-PDI1 | This investigation |
Yl16LIP-Sls1/Pdi1 | Yl16LIP; pTEF-SLS1, pTEF-PDI1 | This investigation |
Yl16LIP-Ssa6/Ssa8/Ssb1 | Yl16LIP; pTEF-SSA6, pTEF-SSA8, pTEF-SSB1 | This investigation |
Yl16LIP-Ssa6/Ssa8/Sls1 | Yl16LIP; pTEF-SSA6, pTEF-SSA8, pTEF-SLS1 | This investigation |
Yl16LIP-Ssa6/Ssa8/Pdi1 | Yl16LIP; pTEF-SSA6, pTEF-SSA8, pTEF-PDI1 | This investigation |
Yl16LIP-Ssa6/Ssb1/Sls1 | Yl16LIP; pTEF-SSA6, pTEF-SSB1, pTEF-SLS1 | This investigation |
Yl16LIP-Ssa8/Ssb1/Sls1 | Yl16LIP; pTEF-SSA8, pTEF-SSB1, pTEF-SLS1 | This investigation |
Yl16LIP-Ssb1/Sls1/Pdi1 | Yl16LIP; pTEF-SSB1, pTEF-SLS1, pTEF-PDI1 | This investigation |
Yl16LIP-Ssa8/Ssb1/Sls1/Hac1 | Yl16LIP; pTEF-SSA8, pTEF-SSB1, pTEF-SLS1, pTEF-spliced HAC1 | This investigation |
YlGAL | Po1f; pTEF-gal-URA3, LEU2 | This investigation |
Yl4GAL | Po1f; p4UASTEF-gal-URA3, LEU2 | This investigation |
Yl8GAL | Po1f; p8UASTEF-gal-URA3, LEU2 | This investigation |
Yl16GAL | Po1f; p8UASTEF-gal-URA3, p8UASTEF-gal-LEU2 | This investigation |
Yl16GAL-Hac1 (Kar2, Sls, Sp14, Sp54, Ss5, Ss6, Ss7, Ss8, Sb or Pd) | Yl16GAL; pTEF-spliced HAC1 (or KAR2, SLS1, SRP14, SRP54, SSA5, SSA6, SSA7, SSA8, SSB1, PDI1) | This investigation |
Yl16GAL-Ssa5/Ssa6 | Yl16GAL; pTEF-SSA5, pTEF-SSA6 | This investigation |
Yl16GAL-Ssa5/Ssb1 | Yl16GAL; pTEF-SSA5, pTEF-SSB1 | This investigation |
Yl16GAL-Ssa5/Sls1 | Yl16GAL; pTEF-SSA5, pTEF-SLS1 | This investigation |
Yl16GAL-Ssa6/Ssb1 | Yl16GAL; pTEF-SSA6, pTEF-SSB1 | This investigation |
Yl16GAL-Ssa6/Sls1 | Yl16GAL; pTEF-SSA6, pTEF-SLS1 | This investigation |
Yl16GAL-Ssb1/Sls1 | Yl16GAL; pTEF-SSB1, pTEF-SLS1 | This investigation |
Yl16GAL-Ssa5/Ssa6/Ssb1 | Yl16GAL; pTEF-SSA5, pTEF-SSA6, pTEF-SSB1 | This investigation |
Yl16GAL-Ssa5/Ssa6/Sls1 | Yl16GAL; pTEF-SSA5, pTEF-SSA6, pTEF-SLS1 | This investigation |
Yl16GAL-Ssa6/Ssb1/Sls1 | Yl16GAL; pTEF-SSA6, pTEF-SSB1, pTEF-SLS1 | This investigation |
Yl16GAL-Ssa5/Ssa6/Ssb1/Sls1 | Yl16GAL; pTEF-SSA5, pTEF-SSA6, pTEF-SSB1, pTEF-SLS1 | This investigation |
Yl16GAL-Ssa6/Ssb1/Sls1/Hac1 | Yl16GAL; pTEF-SSA6, pTEF-SSB1, pTEF-SLS1, pTEF-spliced HAC1 | This investigation |
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Zhu, X.; Li, M.; Zhu, R.; Xin, Y.; Guo, Z.; Gu, Z.; Zhang, L.; Guo, Z. Up Front Unfolded Protein Response Combined with Early Protein Secretion Pathway Engineering in Yarrowia lipolytica to Attenuate ER Stress Caused by Enzyme Overproduction. Int. J. Mol. Sci. 2023, 24, 16426. https://doi.org/10.3390/ijms242216426
Zhu X, Li M, Zhu R, Xin Y, Guo Z, Gu Z, Zhang L, Guo Z. Up Front Unfolded Protein Response Combined with Early Protein Secretion Pathway Engineering in Yarrowia lipolytica to Attenuate ER Stress Caused by Enzyme Overproduction. International Journal of Molecular Sciences. 2023; 24(22):16426. https://doi.org/10.3390/ijms242216426
Chicago/Turabian StyleZhu, Xingyu, Moying Li, Rui Zhu, Yu Xin, Zitao Guo, Zhenghua Gu, Liang Zhang, and Zhongpeng Guo. 2023. "Up Front Unfolded Protein Response Combined with Early Protein Secretion Pathway Engineering in Yarrowia lipolytica to Attenuate ER Stress Caused by Enzyme Overproduction" International Journal of Molecular Sciences 24, no. 22: 16426. https://doi.org/10.3390/ijms242216426