UC Berkeley

Terpenoids are a broad class of natural products that have applications in medicine as well as commodity chemicals and biofuels. However, many terpenoids are sourced from field grown plants, which suffer from inconsistent yield and composition. Additionally, many terpenoid biosynthetic pathways are poorly understood or completely uncharacterized due to limitations regarding genetic resources and heterologous expression. Recent improvements in synthetic biology such as DNA synthesis, targeted genome editing, and next generation sequencing have enabled accelerated research and development in this field. In this work I will describe my contributions to the characterization of two plant-derived medicinal terpenoid pathways: the prostratin and valerenic acid biosynthetic pathways.

Chapter 1 begins with a discussion of the challenges and successes associated with engineering terpenoid production in yeast as well as other commonly engineered microbes. This chapter covers their applications in medicine, biofuels and other commodity chemicals.

Chapter 2 presents my work in developing transcriptomic resources for identifying P450s and other candidate genes involved in the biosynthesis of prostratin. This work also functionally characterized the Jatropha curcas casbene synthase, as well as ~40 P450s from two Euphorbiaceae species, three of which showed activity on the casbene intermediate. Unfortunately, another group published on the activity of two of these P450s before we could. This study shows the utility of coexpression in the discovery of terpenoid biosynthetic genes.

Chapter 3 develops a high-titer yeast chassis for the production of jolkinol C, an important intermediate in the biosynthesis of prostratin and related compounds. The P450s identified in Chapter 2 were optimized to produce >1 g/L total of triply-oxidized casbanes, and 800mg/L of the target product, jolkinol C. Additionally, this study clarified the role of a short chain alcohol dehydrogenase (ADH) in the pathway; while not necessary for jolkinol C formation, the ADH improved the product formation, suggesting it is important in planta, as this gene is found in the jolkinol C biosynthetic gene cluster of J. curcas.

Chapter 4 elucidates the final unidentified step in the biosynthetic pathway of a sesquiterpenoid drug, valerenic acid. Specifically, the P450 VoCYP71DJ, identified from coexpression analysis with the preceding step valerena-4,7(11)-diene synthase, produces oxidized valerena-4,7(11)-diene. Interestingly, the expression of an alcohol dehydrogenase and aldehyde dehydrogenase with VoCYP71DJ1 were necessary to produce valerenic acid in yeast.

The final chapter summarizes this work, as well as suggesting future efforts to further enable the engineering of terpenoid biosynthesis in microbes.