Candida glycerinogenes-Promoted α-Pinene and Squalene Co-production Strategy Based on α-Pinene Stress

α-Pinene is a naturally occurring monoterpene, which is widely used in fragrances, cosmetics, and foods. Due to the high cellular toxicity of α-pinene, this work considered the application of Candida glycerinogenes, an effective industrial strain with high resistance, in α-pinene synthesis. It was found that α-pinene-induced stress resulted in an intracellular accumulation of reactive oxygen species with an increased formation of squalene as a cytoprotective compound. As squalene is a downstream product in the mevalonate (MVA) pathway for α-pinene synthesis, a strategy based on the promotion of α-pinene and squalene co-production under α-pinene stress is proposed. By introducing the α-pinene synthesis pathway and enhancing the MVA pathway, the production of both α-pinene and squalene is increased. We have demonstrated that intracellular synthesis of α-pinene is effective in promoting squalene synthesis. The generation of intercellular reactive oxygen that accompanies α-pinene synthesis promotes squalene synthesis with a resultant cellular protection and upregulation of MVA pathway genes that facilitate α-pinene production. In addition, we have overexpressed phosphatase and introduced NPP as a substrate to synthesize α-pinene, where co-dependent fermentation yielded 208 mg/L squalene and 12.8 mg/L α-pinene. This work establishes a viable strategy to promote terpene-co-dependent fermentation based on stress.