Enhanced bioproduction of anticancer precursor vindoline by yeast cell factories

Abstract

The pharmaceutical industry faces a growing demand and recurrent shortages in many anticancer plant drugs given their extensive use in human chemotherapy. Efficient alternative strategies of supply of these natural products such as bioproduction by microorganisms are needed to ensure stable and massive manufacturing. Here, we developed and optimized yeast cell factories efficiently converting tabersonine to vindoline, a precursor of the major anticancer alkaloids vinblastine and vincristine. First, fine-tuning of heterologous gene copies restrained side metabolites synthesis towards vindoline production. Tabersonine to vindoline bioconversion was further enhanced through a rational medium optimization (pH, composition) and a sequential feeding strategy. Finally, a vindoline titre of 266 mg l^-1 (88% yield) was reached in an optimized fed-batch bioreactor. This precursor-directed synthesis of vindoline thus paves the way towards future industrial bioproduction through the valorization of abundant tabersonine resources.

Fig. 1

Tailoring yeast cell factories for vindoline bioproduction. A. Vindoline biosynthetic pathway and parallel branch vindorosine pathway. Each colour and number correspond to enzyme product, represented by molecular structure, chromatogram, m/z and retention time: 1 tabersonine, 2 16‐hydroxytabersonine, 3 16‐methoxytabersonine, 4 16‐methoxytabersonine epoxide, 5 16‐methoxy‐2,3‐dihydro‐3‐hydroxytabersonine, 6 desacetoxyvindoline, 7 deacetylvindoline, 8 vindoline and the by‐products 9 tabersonine imine alcohol, 10 2,3‐dihydro‐3‐hydroxytabersonine, 11 desacetoxyvindorosine, 12 deacetylvindorosine and 13 vindorosine. MS/MS fragmentation patterns of compounds are presented in Table S1. Cofactors are indicated for each enzyme. B. Time‐course monitoring of extracellular metabolite content in generated S. cerevisiae strains. The feedings were performed with 125 µM of tabersonine in the initial 200 µl of YPD/strain/time point. The names and the number of gene copies integrated into each strain are written on the top of each graphic. The curves represent the means of peak areas relative to tabersonine. Error bars: standard deviation (n = 3 biological replicates). By‐products are shown in dashed lines.

Fig. 2

Optimization of vindoline production at small and medium scales. A. Effect of pH and addition of fresh YP on small‐scale vindoline production. The tested condition is notified on the top of each graphic. The curves represent the means of peak areas relative to tabersonine. Error bars: standard deviation (n = 3 biological replicates). The feedings were performed with 125 µM of tabersonine in the initial 200 µl of YPD/strain/time point. B. Effect of growth substrate and tabersonine feeding strategies on vindoline production in fed‐batch cultivation. Growth substrates are fed by pulses (F1) or continuously (F2‐F4). Tabersonine is fed by one pulse (F1, F2), seven pulses (F3) or equivalent continuous feeding (F4) (Table S4). Bm, biomass. The replicate of F4 feeding strategy is shown in Fig. S3A.