Engineering robust Saccharomyces cerevisiae for high-yield sabinene production from lignocellulosic hydrolysate

Microbial synthesis of sabinene from lignocellulosic biomass represents a sustainable and green alternative to traditional production methods. However, achieving high productivity is often hampered by inefficient substrate utilization and significant product toxicity. In this study, Saccharomyces cerevisiae was engineered to efficiently convert xylose and lignocellulosic hydrolysate (LH) to sabinene. Initially, the biosynthetic pathway was established by introducing a heterologous xylose isomerase and a sabinene synthase. We then systematically enhanced sabinene production through combinatorial metabolic engineering, which included optimizing xylose transport, constructing an orthogonal precursor pathway, reinforcing the supply of key precursors (IPP/DMAPP), reducing acetate and xylitol accumulation, dynamically regulating lipid metabolism, and improving key enzyme efficiency as well as optimizing cofactor balance. A critical bottleneck emerged as sabinene accumulation inhibited cell growth. To address this, we employed adaptive laboratory evolution coupled with transcriptomic analysis, which identified FHN1 and VMA3 as novel genetic targets for improving sabinene tolerance. Their coordinated overexpression was shown to restore intracellular pH homeostasis and vacuolar function under sabinene stress, as well as to improve V-ATPase activity, thereby significantly improving cellular robustness. The resulting haploid strain, ZM26, produced 1685.9 mg/L of sabinene. To further enable efficient bioconversion of inhibitory LH, we generated a diploid hybrid (ZM27) by fusing ZM26 with a UV-mutagenized LH-tolerant strain. The final engineered strain ZM27 achieved a highest sabinene titer of 1537.1 mg/L directly from non-detoxified LH. This work not only demonstrates a scalable strategy for sustainable sabinene production but also provides fundamental insights into mechanisms of monoterpene tolerance in yeast.