Characterization of two key cytochrome P450 enzymes in <i>Isodon amethystoides</i> and <i>de novo</i> production of 3α,15β-dihydroxy-<i>ent-</i>kaurene in yeast.
This study identifies key enzymes and develops a yeast-based platform for producing a precursor to glaucocalyxin A, achieving 24 mg/L in lab conditions.
Where it sits
this study against the rest of the snap-8 corpusSummary and findings
The study characterizes two cytochrome P450 enzymes, IamCYP71D761 and IamCYP706V18, in Isodon amethystoides that mediate hydroxylation of ent-kaurene. An efficient de novo biosynthesis platform for 3α,15β-dihydroxy-ent-kaurene was developed in Saccharomyces cerevisiae. Production reached 24.0 ± 0.2 mg/L in shake flask conditions.
Abstract
Glaucocalyxin A (GLA), a well-known <i>ent-</i>kaurene diterpenoid, has emerged as a high-value bioactive natural product in pharmaceutical research owing to its potent and broad-spectrum activities. To date, its total synthesis has proven extremely challenging due to poor yield and intricate routes, and the biosynthetic pathway of GLA remains poorly understood. Herein, we identified two enzymes, IamCYP71D761 and IamCYP706V18, in <i>Isodon amethystoides</i>, which specifically mediate the C3α- and C15β-hydroxylation of <i>ent-</i>kaurene with high stereoselectivity and regioselectivity, respectively, generating 3α,15β-dihydroxy-<i>ent-</i>kaurene (<b>3</b>), a key precursor in the biosynthesis of GLA. In addition, we established an efficient <i>de novo</i> biosynthesis platform for <b>3</b> in <i>Saccharomyces cerevisiae</i>. Briefly, high-efficiency isozyme screening, protein engineering, increased acetyl-CoA synthesis, and copy number enhancement were applied to the <i>ent-</i>kaurene biosynthesis module. Subsequently, promoter optimization, competitive pathway knockout, and electron transfer optimization were introduced, resulting in production of <b>3</b> to 24.0 ± 0.2 mg/L in shake flask. In summary, this work highlights the pivotal roles of IamCYP71D761 and IamCYP706V18 in the heterologous biosynthesis of <b>3</b>, offering valuable insights for the further pathway reconstruction and <i>de novo</i> production of GLA, GLB, and other <i>ent-</i>kaurene diterpenoids.
Background
This study addresses the biosynthesis of glaucocalyxin A (GLA), a bioactive natural product with significant pharmaceutical potential. The total synthesis of GLA is challenging due to low yields and complex routes, and its biosynthetic pathway is not well understood. Identifying key enzymes in the biosynthesis of GLA could facilitate more efficient production methods.
Methods
The study identified two cytochrome P450 enzymes, IamCYP71D761 and IamCYP706V18, in Isodon amethystoides that mediate specific hydroxylation reactions in the biosynthesis of ent-kaurene. A de novo biosynthesis platform was established in Saccharomyces cerevisiae, employing techniques such as isozyme screening, protein engineering, and pathway optimization to enhance production.
Results
The primary outcome was the production of 3α,15β-dihydroxy-ent-kaurene at a concentration of 24.0 ± 0.2 mg/L in shake flask conditions. This was achieved through a combination of genetic and metabolic engineering strategies, including promoter optimization and competitive pathway knockout.
Interpretation
The findings provide a significant step forward in understanding and optimizing the biosynthetic pathway for GLA and related diterpenoids. While the production level achieved is promising, it remains to be seen whether these methods can be scaled for industrial applications. The study's approach could inform future efforts to produce other complex natural products.
Key findings
- Identified enzymes IamCYP71D761 and IamCYP706V18 mediate C3α- and C15β-hydroxylation of ent-kaurene.
- 3α,15β-dihydroxy-ent-kaurene produced at 24.0 ± 0.2 mg/L in yeast.
- High-efficiency isozyme screening and protein engineering applied.
- Promoter optimization and competitive pathway knockout used.
- Electron transfer optimization contributed to production efficiency.
Limitations
- Not reported in abstract.