An in vivo gene amplification system for high level expression in Saccharomyces cerevisiae

Bottlenecks in metabolic pathways due to insufficient gene expression levels remain a significant problem for industrial bioproduction using microbial cell factories. Increasing gene dosage can overcome these bottlenecks, but current approaches suffer from numerous drawbacks. Here, we describe HapAm...

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Published inNature communications Vol. 13; no. 1; pp. 2895 - 12
Main Authors Peng, Bingyin, Esquirol, Lygie, Lu, Zeyu, Shen, Qianyi, Cheah, Li Chen, Howard, Christopher B., Scott, Colin, Trau, Matt, Dumsday, Geoff, Vickers, Claudia E.
Format Journal Article
LanguageEnglish
Published London Nature Publishing Group UK 24.05.2022
Nature Publishing Group
Nature Portfolio
Subjects
38
38/22
38/35
38/39
38/77
42/44
45/23
45/44
631/1647/1511
631/326/2522
631/61/252/318
82/80
Amplification
Dosage
Factories
Gene Amplification
Gene dosage
Gene expression
Genomes
Haploinsufficiency
Humanities and Social Sciences
In vivo methods and tests
Industrial engineering
Integration
Limonene
Limonene - metabolism
Lycopene
Manufacturing engineering
Metabolic engineering
Metabolic Engineering - methods
Metabolic pathways
Metabolism
Microorganisms
multidisciplinary
Nerolidol
Proteins
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - metabolism
Saccharomyces cerevisiae Proteins - genetics
Saccharomyces cerevisiae Proteins - metabolism
Science
Science (multidisciplinary)
Yeast
Yeasts
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Abstract Bottlenecks in metabolic pathways due to insufficient gene expression levels remain a significant problem for industrial bioproduction using microbial cell factories. Increasing gene dosage can overcome these bottlenecks, but current approaches suffer from numerous drawbacks. Here, we describe HapAmp, a method that uses haploinsufficiency as evolutionary force to drive in vivo gene amplification. HapAmp enables efficient, titratable, and stable integration of heterologous gene copies, delivering up to 47 copies onto the yeast genome. The method is exemplified in metabolic engineering to significantly improve production of the sesquiterpene nerolidol, the monoterpene limonene, and the tetraterpene lycopene. Limonene titre is improved by 20-fold in a single engineering step, delivering ∼1 g L −1 in the flask cultivation. We also show a significant increase in heterologous protein production in yeast. HapAmp is an efficient approach to unlock metabolic bottlenecks rapidly for development of microbial cell factories. Gene dosage-based expression upregulation suffers from instability and random gene integration. Here, the authors report HapAmp, a method that uses haploinsufficiency as evolutionary force to drive in vivo gene amplification, and demonstrate its applications in protein and biochemical production in yeast.
AbstractList Bottlenecks in metabolic pathways due to insufficient gene expression levels remain a significant problem for industrial bioproduction using microbial cell factories. Increasing gene dosage can overcome these bottlenecks, but current approaches suffer from numerous drawbacks. Here, we describe HapAmp, a method that uses haploinsufficiency as evolutionary force to drive in vivo gene amplification. HapAmp enables efficient, titratable, and stable integration of heterologous gene copies, delivering up to 47 copies onto the yeast genome. The method is exemplified in metabolic engineering to significantly improve production of the sesquiterpene nerolidol, the monoterpene limonene, and the tetraterpene lycopene. Limonene titre is improved by 20-fold in a single engineering step, delivering ∼1 g L −1 in the flask cultivation. We also show a significant increase in heterologous protein production in yeast. HapAmp is an efficient approach to unlock metabolic bottlenecks rapidly for development of microbial cell factories. Gene dosage-based expression upregulation suffers from instability and random gene integration. Here, the authors report HapAmp, a method that uses haploinsufficiency as evolutionary force to drive in vivo gene amplification, and demonstrate its applications in protein and biochemical production in yeast.
Bottlenecks in metabolic pathways due to insufficient gene expression levels remain a significant problem for industrial bioproduction using microbial cell factories. Increasing gene dosage can overcome these bottlenecks, but current approaches suffer from numerous drawbacks. Here, we describe HapAmp, a method that uses haploinsufficiency as evolutionary force to drive in vivo gene amplification. HapAmp enables efficient, titratable, and stable integration of heterologous gene copies, delivering up to 47 copies onto the yeast genome. The method is exemplified in metabolic engineering to significantly improve production of the sesquiterpene nerolidol, the monoterpene limonene, and the tetraterpene lycopene. Limonene titre is improved by 20-fold in a single engineering step, delivering ∼1 g L-1 in the flask cultivation. We also show a significant increase in heterologous protein production in yeast. HapAmp is an efficient approach to unlock metabolic bottlenecks rapidly for development of microbial cell factories.Bottlenecks in metabolic pathways due to insufficient gene expression levels remain a significant problem for industrial bioproduction using microbial cell factories. Increasing gene dosage can overcome these bottlenecks, but current approaches suffer from numerous drawbacks. Here, we describe HapAmp, a method that uses haploinsufficiency as evolutionary force to drive in vivo gene amplification. HapAmp enables efficient, titratable, and stable integration of heterologous gene copies, delivering up to 47 copies onto the yeast genome. The method is exemplified in metabolic engineering to significantly improve production of the sesquiterpene nerolidol, the monoterpene limonene, and the tetraterpene lycopene. Limonene titre is improved by 20-fold in a single engineering step, delivering ∼1 g L-1 in the flask cultivation. We also show a significant increase in heterologous protein production in yeast. HapAmp is an efficient approach to unlock metabolic bottlenecks rapidly for development of microbial cell factories.
Bottlenecks in metabolic pathways due to insufficient gene expression levels remain a significant problem for industrial bioproduction using microbial cell factories. Increasing gene dosage can overcome these bottlenecks, but current approaches suffer from numerous drawbacks. Here, we describe HapAmp, a method that uses haploinsufficiency as evolutionary force to drive in vivo gene amplification. HapAmp enables efficient, titratable, and stable integration of heterologous gene copies, delivering up to 47 copies onto the yeast genome. The method is exemplified in metabolic engineering to significantly improve production of the sesquiterpene nerolidol, the monoterpene limonene, and the tetraterpene lycopene. Limonene titre is improved by 20-fold in a single engineering step, delivering ∼1 g L −1 in the flask cultivation. We also show a significant increase in heterologous protein production in yeast. HapAmp is an efficient approach to unlock metabolic bottlenecks rapidly for development of microbial cell factories.
Bottlenecks in metabolic pathways due to insufficient gene expression levels remain a significant problem for industrial bioproduction using microbial cell factories. Increasing gene dosage can overcome these bottlenecks, but current approaches suffer from numerous drawbacks. Here, we describe HapAmp, a method that uses haploinsufficiency as evolutionary force to drive in vivo gene amplification. HapAmp enables efficient, titratable, and stable integration of heterologous gene copies, delivering up to 47 copies onto the yeast genome. The method is exemplified in metabolic engineering to significantly improve production of the sesquiterpene nerolidol, the monoterpene limonene, and the tetraterpene lycopene. Limonene titre is improved by 20-fold in a single engineering step, delivering ∼1 g L−1 in the flask cultivation. We also show a significant increase in heterologous protein production in yeast. HapAmp is an efficient approach to unlock metabolic bottlenecks rapidly for development of microbial cell factories.Gene dosage-based expression upregulation suffers from instability and random gene integration. Here, the authors report HapAmp, a method that uses haploinsufficiency as evolutionary force to drive in vivo gene amplification, and demonstrate its applications in protein and biochemical production in yeast.
Bottlenecks in metabolic pathways due to insufficient gene expression levels remain a significant problem for industrial bioproduction using microbial cell factories. Increasing gene dosage can overcome these bottlenecks, but current approaches suffer from numerous drawbacks. Here, we describe HapAmp, a method that uses haploinsufficiency as evolutionary force to drive in vivo gene amplification. HapAmp enables efficient, titratable, and stable integration of heterologous gene copies, delivering up to 47 copies onto the yeast genome. The method is exemplified in metabolic engineering to significantly improve production of the sesquiterpene nerolidol, the monoterpene limonene, and the tetraterpene lycopene. Limonene titre is improved by 20-fold in a single engineering step, delivering ∼1 g L in the flask cultivation. We also show a significant increase in heterologous protein production in yeast. HapAmp is an efficient approach to unlock metabolic bottlenecks rapidly for development of microbial cell factories.
Gene dosage-based expression upregulation suffers from instability and random gene integration. Here, the authors report HapAmp, a method that uses haploinsufficiency as evolutionary force to drive in vivo gene amplification, and demonstrate its applications in protein and biochemical production in yeast.
ArticleNumber 2895
Author Trau, Matt
Lu, Zeyu
Peng, Bingyin
Dumsday, Geoff
Esquirol, Lygie
Cheah, Li Chen
Shen, Qianyi
Howard, Christopher B.
Vickers, Claudia E.
Scott, Colin
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BackLink https://www.ncbi.nlm.nih.gov/pubmed/35610221$$D View this record in MEDLINE/PubMed
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OpenAccessLink https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9130285/
PMID 35610221
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PublicationDate 2022-05-24
PublicationDateYYYYMMDD 2022-05-24
PublicationDate_xml – month: 05
  year: 2022
  text: 2022-05-24
  day: 24
PublicationDecade 2020
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PublicationTitle Nature communications
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Nature Publishing Group
Nature Portfolio
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SSID ssj0000391844
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Snippet Bottlenecks in metabolic pathways due to insufficient gene expression levels remain a significant problem for industrial bioproduction using microbial cell...
Gene dosage-based expression upregulation suffers from instability and random gene integration. Here, the authors report HapAmp, a method that uses...
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38/22
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45/44
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631/326/2522
631/61/252/318
82/80
Amplification
Dosage
Factories
Gene Amplification
Gene dosage
Gene expression
Genomes
Haploinsufficiency
Humanities and Social Sciences
In vivo methods and tests
Industrial engineering
Integration
Limonene
Limonene - metabolism
Lycopene
Manufacturing engineering
Metabolic engineering
Metabolic Engineering - methods
Metabolic pathways
Metabolism
Microorganisms
multidisciplinary
Nerolidol
Proteins
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - metabolism
Saccharomyces cerevisiae Proteins - genetics
Saccharomyces cerevisiae Proteins - metabolism
Science
Science (multidisciplinary)
Yeast
Yeasts
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Title An in vivo gene amplification system for high level expression in Saccharomyces cerevisiae
URI https://link.springer.com/article/10.1038/s41467-022-30529-8
https://www.ncbi.nlm.nih.gov/pubmed/35610221
https://www.proquest.com/docview/2668568580
https://www.proquest.com/docview/2669502243
https://pubmed.ncbi.nlm.nih.gov/PMC9130285
https://doaj.org/article/e833a815673c4afdbb238b2a86a2e193
Volume 13
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