AUTHOR=Xia Lu , Lv Yunbin , Liu Song , Yu Shiqin , Zeng Weizhu , Zhou Jingwen 

TITLE=Enhancing Squalene Production in Saccharomyces cerevisiae by Metabolic Engineering and Random Mutagenesis

JOURNAL=Frontiers in Chemical Engineering

VOLUME=3

YEAR=2022

URL=https://www.frontiersin.org/journals/chemical-engineering/articles/10.3389/fceng.2021.790261

DOI=10.3389/fceng.2021.790261

ISSN=2673-2718

ABSTRACT=<p>Squalene is an important polyunsaturated triterpene with wide applications in the food, cosmetics, and pharmaceutical industries. Currently, the main method for squalene production is extraction from oil-producing plants, but the scale is limited. The microbial fermentation with <italic>Saccharomyces cerevisiae</italic> still needs improvement to be economically viable. This study aimed to improve squalene production by metabolic engineering and random mutagenesis. First, the mevalonate (MVA) pathway was enhanced, by integrating <italic>tHMG1</italic> and <italic>IDI1</italic> into multi-copy site <italic>Ty2</italic>. Subsequently, the <italic>ACL</italic> gene from <italic>Yarrowia lipolytica</italic>, encoding citrate lyase was introduced and the β-oxidation pathway was enhanced with multiple copies of key genes. In addition, a high throughput screening strategy based on Nile red staining was established for high squalene-producer screening. After treatment with ARTP mutagenesis, a higher-producing mutant was obtained, with squalene production enhanced by 18.4%. A two-stage fermentation of this mutant in a 5 L bioreactor produced 8.2 g/L of squalene. These findings may facilitate the development of industrial squalene production by fermentation and potentially, other terpenoids.</p>