Boosting production of pharmacologically relevant diterpenoids through engineered miltiradiene synthase

By engineering specific amino acid insertions in the enzyme SmKSL1, the scientists achieved a 44% increase in miltiradiene yield alongside enhanced protein solubility and catalytic efficiency.

Diterpenoids are among the most structurally diverse groups of natural products, serving vital functions in plant growth and defense while also providing pharmacological compounds with anticancer, anti-inflammatory, and neuroprotective properties. Within this group, labdane-related diterpenoids (LRDs) represent around 60% of identified diterpenoids, featuring a core bicyclic skeleton that can be enzymatically transformed into abietane, cassane, pimarane, and clerodane structures. Abietane diterpenoids, characterized by their tricyclic carbon skeleton, are especially important due to their broad distribution in higher plants and their derivatives’ medicinal relevance. Miltiradiene, a pivotal abietane intermediate, is the biosynthetic precursor of several clinically promising compounds including tanshinone IIA, which exhibits cardioprotective and anticancer activities, triptolide, a potent immunosuppressant, and carnosic acid, known for neuroprotective effects. Despite its importance, natural miltiradiene synthases display low catalytic efficiency, posing a bottleneck for large-scale production.

A study (DOI:10.1016/j.bidere.2025.100028) published in BioDesign Research on 23 May 2025 by Baolong Jin & Luqi Huang’s team, China Academy of Chinese Medical Sciences, establishes new molecular targets for rational enzyme design and open avenues for industrial-scale biosynthesis of abietane diterpenoids, a diverse class of natural products with therapeutic potential.

In this study, researchers employed a combination of in vitro biochemical assays, homologous enzyme mutagenesis, engineered bacterial expression, and enzymatic property analyses to investigate how amino acid modifications influence the function of diterpene synthases in miltiradiene production. First, site-directed mutagenesis was performed on IrKSL3a from Isodon rubescens, generating 11 insertion variants at the 550th residue to test the role of Tyr and Arg in determining catalytic specificity. All mutants shifted activity from producing isopimaradiene to miltiradiene, though yields varied, with IrKSL3a: E550+KR showing the highest activity, suggesting that basic amino acids enhance enzyme function. Building on this, the team introduced KR and AA double-residue insertions into homologous enzymes (IrKSL6 and SmKSL1), finding that while IrKSL6 mutants partially altered activity, SmKSL1: E550+KR consistently achieved higher titers of miltiradiene. To confirm these findings under physiological conditions, the researchers constructed engineered E. coli strains harboring these mutant genes. In vivo assays showed that SmKSL1: E550+KR produced an average of 19.64 mg/L miltiradiene, representing a 44% increase over wild type, while SmKSL1: E550+AA yielded a modest 13% increase, demonstrating functional consistency with in vitro results. In contrast, IrKSL6: K550+KR reverted to isopimaradiene production in vivo, highlighting the influence of intracellular regulation. Further protein characterization revealed that the SmKSL1: E550+KR mutant not only exhibited 24% higher solubility than the wild type but also displayed enhanced substrate affinity (lower Km) and a 1.26-fold increase in catalytic efficiency (Kcat/Km). These results collectively show that distal residue insertions, particularly the introduction of basic amino acids, can simultaneously improve solubility and catalytic performance. The dual advantages of SmKSL1: E550+KR make it a strong candidate for structure-guided optimization, providing a valuable biocatalyst for high-efficiency biosynthesis of pharmacologically relevant abietane diterpenoids.

By improving the efficiency of miltiradiene production, the engineered SmKSL1 mutant provides a robust biocatalytic tool for heterologous biosynthesis platforms in yeast, bacteria, or plant systems. This advancement holds direct implications for the sustainable, large-scale production of tanshinones, triptolide, and carnosic acid—compounds with demonstrated efficacy in treating cancer, cardiovascular diseases, immune disorders, and neurodegenerative conditions.

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References

DOI

10.1016/j.bidere.2025.100028

Original Source URL

https://doi.org/10.1016/j.bidere.2025.100028

Funding information

This work was supported by Key project at central government level: the ability to establish sustainable use of valuable Chinese Medicine Resources (2060302), National Natural Science Foundation of China (82274054 and 82204573), Scientific and technological innovation project of China Academy of Chinese Medical Science (CI2023E002), Guangdong Provincial Bureau of Traditional Chinese Medicine Research Foundation (20251101).

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