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

• Published in: Nature 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
• Language: English
• 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
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-022-30529-8

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.