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

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

doi:10.3389/fceng.2021.790261

Abstract

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 Saccharomyces cerevisiae 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 tHMG1 and IDI1 into multi-copy site Ty2. Subsequently, the ACL gene from Yarrowia lipolytica, 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.

Highlights

Squalene is a natural product found in vegetable oils, fish oils, and human sebum
Most of the promoters with low transcription levels resulted in increased squalene accumulation
To further improve HMGR activity without breaking of balance of redox status, an HMG-CoA reductase NADH-HMGR with high specificity for NADH from S. pomeroyi was introduced into SQ10 to produce strain SQ11

Summary

Introduction

Squalene is a natural product found in vegetable oils, fish oils, and human sebum. It is the main hydrocarbon component of human epidermal fats, accounting for about 10% by weight. Acyclic triterpenoid compound formed from six isoprene units. It is the precursor of many biologically active compounds, including steroids and hopene (Katabami et al, 2015). The main method of squalene production is extraction from plant materials, but the scale is limited by the long growing cycle of the plants (Lou-Bonafonte et al, 2018)

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