Elucidating steroid alkaloid biosynthesis in Veratrum californicum: production of verazine in Sf9 cells

Abstract

Summary Steroid alkaloids have been shown to elicit a wide range of pharmacological effects that include anticancer and antifungal activities. Understanding the biosynthesis of these molecules is essential to bioengineering for sustainable production. Herein, we investigate the biosynthetic pathway to cyclopamine, a steroid alkaloid that shows promising antineoplastic activities. Supply of cyclopamine is limited, as the current source is solely derived from wild collection of the plant Veratrum californicum. To elucidate the early stages of the pathway to cyclopamine, we interrogated a V. californicum RNA-seq dataset using the cyclopamine accumulation profile as a predefined model for gene expression with the pattern-matching algorithm Haystack. Refactoring candidate genes in Sf9 insect cells led to discovery of four enzymes that catalyze the first six steps in steroid alkaloid biosynthesis to produce verazine, a predicted precursor to cyclopamine. Three of the enzymes are cytochromes P450 while the fourth is a γ-aminobutyrate transaminase; together they produce verazine from cholesterol.

Comprehensive Investigation of Bioactive Steroidal Alkaloids in Veratrum californicum

“…This is consistent with the findings of Keeler and Binns in their original evaluation of alkaloid content by growth stage, and is consistent with the variable teratogenic effect observed in controlled feeding trials [133]. Consistent with prior analysis of Veratrum alkaloid content sampled throughout the growth season, the bar graphs detailed in Figure 4.2 show that aerial alkaloidquantities decrease from early season through late growth phase for the plants[133,143].This was consistent for both locations and for all of the alkaloids quantified. Plants harvested from Elk Meadows retained ~6% of their original alkaloid content from May to September, while the Shindig aerial plants retained closer to ~27% alkaloid content.…”

The Amaryllidaceae alkaloids: biosynthesis and methods for enzyme discovery

“…In this study, a de novo transcriptome assembly of Veratrum californicum is used to find candidates through co-expression analysis. The candidates are expressed in insect cells and the various products are identified by a combination of MS/MS and NMR (Augustin et al, 2015). Existing and new transcriptomic data can be used in combination for the discovery of biosynthetic enzymes.…”

“…This combination of information allows workflows for candidate gene selection based on homology and co-expression to be carried out with a very manageable initial investment and no prior sequence information (Giddings et al, 2011; Yeo et al, 2013; Kilgore et al, 2014). Many transcriptomes have been assembled using second generation sequencing, thereby providing information on a genetic level to previously uncharacterized systems such as Camptotheca acuminata , Catharanthus roseus , Rauvolfia serpentina, Valeriana officinalis , and Veratrum californicum (Góngora-Castillo et al, 2012; Yeo et al, 2013; Augustin et al, 2015). Transcriptomes for the Amaryllidaceae species Narcissus sp.…”

“…In the case of Amaryllidaceae alkaloids, there is a large diversity of specialized synthesis methods that have regularly been reviewed by Zhong Jin and can be used for production of various pathway intermediates (Jin, 2013). As biosynthetic genes are discovered, functional expression of these genes in a heterologous system will facilitate chemo-enzymatic syntheses (Augustin et al, 2015). It is possible that in the future reactions for which the native enzyme is not known could have an alternate enzyme engineered to perform the desired reaction (Arnold, 2015).…”

Evolution of Structural Diversity of Triterpenoids

“…In these plant species, they have had special attention due to their pharmacological properties. For instance, the Liliaceae Veratrun californicum produces the potent anticancer molecule cyclopamine (Augustin et al, 2015). Biochemical and phylogenetic analysis of the enzymes involved in the biosynthesis of Solanum and Veratrum SGAs suggested their convergent origins and their partial recruitment from primary phytosterol metabolism (Augustin et al, 2015;Sonawane et al, 2016).…”