Alter codon bias of the P. pastoris genome to overcome a bottleneck in codon optimization strategy development and improve protein expression
Copyright © 2024 Elsevier GmbH. All rights reserved..
Apart from its role in translation, codon bias is also an important mechanism to regulate mRNA levels. The traditional frequency-based codon optimization strategy is rather efficient in organisms such as N. crassa, but much less in yeast P. pastoris which is a popular host for heterologous protein expression. This is because that unlike N. crassa, the preferred codons of P. pastoris are actually AU-rich and hence codon optimization for extremely low GC content comes with issues of pre-mature transcriptional termination or low RNA stability in spite of translational advantages. To overcome this bottleneck, we focused on three reporter genes in P. pastoris first and confirmed the great advantage of GC-prone codon optimization on mRNA levels. Then we altered the codon bias profile of P. pastoris by introducing additional rare tRNA gene copies. Prior to that we constructed IPTG-regulated tRNA species to enable chassis cells to switch between different codon bias status. As demonstrated again with reporter genes, protein yield of luc and 0788 was successfully increased by 4-5 folds in chassis cells. In summary, here we provide an alternative codon optimization strategy for genes with unsatisfactory performance under traditional codon frequency-based optimization.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:282 |
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| Enthalten in: |
Microbiological research - 282(2024) vom: 15. März, Seite 127629 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Zhang, Siyu [VerfasserIn] |
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| Links: |
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| Themen: |
9014-25-9 |
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| Anmerkungen: |
Date Completed 18.03.2024 Date Revised 18.03.2024 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/j.micres.2024.127629 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 100 | 1 | |a Zhang, Siyu |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Alter codon bias of the P. pastoris genome to overcome a bottleneck in codon optimization strategy development and improve protein expression |
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| 500 | |a Date Revised 18.03.2024 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Copyright © 2024 Elsevier GmbH. All rights reserved. | ||
| 520 | |a Apart from its role in translation, codon bias is also an important mechanism to regulate mRNA levels. The traditional frequency-based codon optimization strategy is rather efficient in organisms such as N. crassa, but much less in yeast P. pastoris which is a popular host for heterologous protein expression. This is because that unlike N. crassa, the preferred codons of P. pastoris are actually AU-rich and hence codon optimization for extremely low GC content comes with issues of pre-mature transcriptional termination or low RNA stability in spite of translational advantages. To overcome this bottleneck, we focused on three reporter genes in P. pastoris first and confirmed the great advantage of GC-prone codon optimization on mRNA levels. Then we altered the codon bias profile of P. pastoris by introducing additional rare tRNA gene copies. Prior to that we constructed IPTG-regulated tRNA species to enable chassis cells to switch between different codon bias status. As demonstrated again with reporter genes, protein yield of luc and 0788 was successfully increased by 4-5 folds in chassis cells. In summary, here we provide an alternative codon optimization strategy for genes with unsatisfactory performance under traditional codon frequency-based optimization | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Chassis cells | |
| 650 | 4 | |a Codon optimization | |
| 650 | 4 | |a Pichia pastoris | |
| 650 | 4 | |a Protein expression | |
| 650 | 7 | |a Codon |2 NLM | |
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| 650 | 7 | |a RNA, Transfer |2 NLM | |
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| 650 | 7 | |a Recombinant Proteins |2 NLM | |
| 700 | 1 | |a Lin, Ru |e verfasserin |4 aut | |
| 700 | 1 | |a Cui, Luyao |e verfasserin |4 aut | |
| 700 | 1 | |a Jiang, Tianyi |e verfasserin |4 aut | |
| 700 | 1 | |a Shi, Jiacheng |e verfasserin |4 aut | |
| 700 | 1 | |a Lu, Chaoyu |e verfasserin |4 aut | |
| 700 | 1 | |a Li, Pengfei |e verfasserin |4 aut | |
| 700 | 1 | |a Zhou, Mian |e verfasserin |4 aut | |
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