Optimization of Tabersonine Methoxylation to Increase Vindoline Precursor Synthesis in Yeast Cell Factories.

Pamela Lemos Cruz,Nicolas Papon,Grégory Guirimand,Sébastien Besseau,Nathalie Giglioli-Guivarc’H,Audrey Oudin,Johan-Owen De Craene,Arnaud Lanoue,Natalja Kulagina,Vincent Courdavault,Marc Clastre

doi:10.3390/molecules26123596

Abstract

Plant specialized metabolites are widely used in the pharmaceutical industry, including the monoterpene indole alkaloids (MIAs) vinblastine and vincristine, which both display anticancer activity. Both compounds can be obtained through the chemical condensation of their precursors vindoline and catharanthine extracted from leaves of the Madagascar periwinkle. However, the extensive use of these molecules in chemotherapy increases precursor demand and results in recurrent shortages, explaining why the development of alternative production approaches, such microbial cell factories, is mandatory. In this context, the precursor-directed biosynthesis of vindoline from tabersonine in yeast-expressing heterologous biosynthetic genes is of particular interest but has not reached high production scales to date. To circumvent production bottlenecks, the metabolic flux was channeled towards the MIA of interest by modulating the copy number of the first two genes of the vindoline biosynthetic pathway, namely tabersonine 16-hydroxylase and tabersonine-16-O-methyltransferase. Increasing gene copies resulted in an optimized methoxylation of tabersonine and overcame the competition for tabersonine access with the third enzyme of the pathway, tabersonine 3-oxygenase, which exhibits a high substrate promiscuity. Through this approach, we successfully created a yeast strain that produces the fourth biosynthetic intermediate of vindoline without accumulation of other intermediates or undesired side-products. This optimization will probably pave the way towards the future development of yeast cell factories to produce vindoline at an industrial scale.

Highlights

IntroductionPlant specialized metabolites were first described in the middle 1920s and were initially named secondary metabolites because these compounds were thought to be non-essential for plant development, in contrast to primary metabolites
To optimize the synthesis of vindoline in engineered yeast and reduce the biosynthetic flux towards the undesired vindorosine (Figure 1C), we first investigated the competition between T16H2 and tabersonine 3-oxygenase (T3O) for tabersonine
Using yeast cell factories to produce the precursors of these compounds is one of the promising alternatives

Summary

Introduction

Plant specialized metabolites were first described in the middle 1920s and were initially named secondary metabolites because these compounds were thought to be non-essential for plant development, in contrast to primary metabolites. Biological activities of specialized metabolites explain why they have been associated with medicinal plant properties commonly used in traditional medicine [4,5]. Several of these metabolites, such as the analgesic morphine, the antitussive codeine from Papaver somniferum [6], and the Molecules 2021, 26, 3596 antimalarial artemisinin from Artemisia annua [7], for instance, have been of great importance to modern medicine for decades. Along with other active plant-born compounds, they constitute around 25% of all the drugs currently used in human health [8]

Methods

Results

Conclusion