Salidroside, the key active component of Rhodiola, possesses anti-hypoxia and anti-fatigue properties, making it valuable in medicine, health products, and cosmetics. This study focuses on efficiently synthesizing salidroside using a dual-enzyme cascade system with sucrose synthase AtSUS3 and glycosyltransferase AtUGT85A1 from Arabidopsis thaliana. This system facilitates the regeneration of UDPG and the conversion of tyrosol into salidroside. First, the two key enzymes AtSUS3 and AtUGT85A1 were expressed and purified with optimized His‑tag positions. Solubility enhancement via tag fusion and linker engineering yielded the fusion proteins TSu and ES85 with desirable activity. Subsequently, semi‑rational design on the poorly thermostable ES85 using the FireProt platform generated an S324R mutant with significantly improved thermostability. Ultimately, a recombinant strain TSR85 co‑expressing both enzymes was constructed. A one‑pot system employing this strain achieved 13.61 g/L salidroside with a conversion rate of 90% within 10 h (space‑time yield 1.36 g/L/h). This study thus integrates solubility enhancement, thermostability engineering, and one‑pot catalysis into a balanced, practical, and industrially relevant platform for salidroside biosynthesis.
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Funding
Key Research and Development Program of China(No. 2022YFA0912204)
National Natural Science Foundation of China(32001064)
Jiangsu Provincial Key Research and Development Program(BE2023685)
RIGHTS & PERMISSIONS
Jiangnan University
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