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A microbial supply chain for production of anti-cancer drug vinblastine

ABSTRACT: Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine. As MIAs are difficult to chemically synthesize, the world’s supply chain for vinblastine relies on low-yielding extractions of precursors vindoline and catharanthine from the plant Catharanthus roseus, followed by chemical coupling and reduction to form vinblastine. Here, we demonstrate de novo microbial biosynthesis of vindoline and catharanthine from renewable feedstocks such as simple sugar and amino acids using highly engineered yeast. The study showcases the longest biosynthetic pathway refactored into a microbial cell factory to date, including 29 enzymatic steps from the yeast native metabolite geranyl pyrophosphate to catharanthine and vindoline. We made 44 genetic edits to yeast that include expression of 35 heterologous genes from plants as well as deletions, knock-downs, and overexpression of 10 yeast genes or variants thereof to improve the precursor supply. Finally, we demonstrate one-step in vitro vinblastine production using chemical coupling and reduction of vindoline and catharanthine. Not only is the yeast a scalable platform for production of vinblastine, it is also a platform for production of more than 2,000 different natural and new-to-nature MIAs.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Tune Wulff

LAB HEAD: Michael K. Jensen

PROVIDER: PXD025067 | Pride | 2022-08-21

REPOSITORIES: pride

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A microbial supply chain for production of the anti-cancer drug vinblastine.

Zhang Jie J Hansen Lea G LG Gudich Olga O Viehrig Konrad K Lassen Lærke M M LMM Schrübbers Lars L Adhikari Khem B KB Rubaszka Paulina P Carrasquer-Alvarez Elena E Chen Ling L D'Ambrosio Vasil V Lehka Beata B Haidar Ahmad K AK Nallapareddy Saranya S Giannakou Konstantina K Laloux Marcos M Arsovska Dushica D Jørgensen Marcus A K MAK Chan Leanne Jade G LJG Kristensen Mette M Christensen Hanne B HB Sudarsan Suresh S Stander Emily A EA Baidoo Edward E Petzold Christopher J CJ Wulff Tune T O'Connor Sarah E SE Courdavault Vincent V Jensen Michael K MK Keasling Jay D JD

Nature 20220831 7926

Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine<sup>1</sup>. As MIAs are difficult to chemically synthesize, the world's supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling ...[more]

PMID: 36045295

Publication: 1/2

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Project description:Saccharomyces cerevisiae is an established microbial host for the production of non-native compounds. The synthesis of these compounds typically demands energy and competes with growth for carbon and energy substrate. Uncoupling product formation form growth would benefit product yields and decrease formation of by-product biomass. Studying non-growing metabolically-active yeast cultures provides a first step towards developing S. cerevisiae as a non-growing, robust cell factory. Non-growing metabolically-active cultures can be obtained in retentostat, a glucose-limited, continuous bioreactor system in which biomass accumulates while spent medium is constantly removed. Hitherto retentostat cultures of S. cerevisiae have only been reported under anaerobiosis, condition inappropriate for the production of energy-demanding products. The present study, using retentostat cultures, explores the physiology of non-dividing, fully respiring S. cerevisiae, focusing on industrially-relevant features. Following model-aided experimental design, retentostat cultivations were optimized for accelerated but smooth transition of S. cerevisiae from exponential growth to near-zero growth rates. During 20 days in retentostat the biomass concentration increased, leading very slow growth rates (specific growth rates below 0.001 h-1) but high culture viability (over 80% of viable cells). The maintenance requirement (mATP) was estimated at 0.64 mmolATP.gX-1.h-1, which is remarkably ca. 35% lower than the mATP measured in anaerobic retentostat cultures. Transcriptional down-regulation of genes involved in biosynthesis and up-regulation of stress-responsive genes towards near-zero growth rates corresponded well with data from anaerobic retentostats. More striking was the extreme heat-shock tolerance of S. cerevisiae, which exceeded by far previously reported heat shock tolerance of notoriously robust yeast cultures such as stationary phase cultures. Furthermore, while the metabolic fluxes in the retentostats were relatively low as a result of extreme caloric restriction, off-line measurements revealed that S. cerevisiae retained a high catabolic capacity. The high viability and extreme heat-shock tolerance revealed the robustness of S. cerevisiae at near-zero growth in retentostat. In addition, the relatively low maintenance requirements and high metabolic capacity under severe calorie restriction underline the potential of S. cerevisiae as a non-dividing microbial cell factory for the production of energy-intensive compounds. The retentostat is a promising tool to identify the molecular basis of this extreme robustness. The goal of the present study is to investigate the physiology of aerobic fully respiring S. cerevsiae at near-zero growth rates. Fundamental but industrially-relevant questions were addressed thanks to the design, implementation and study of aerobic retentostat cultivations enabling a rapid but smooth transition of S. cerevisiae from exponential growth to near-zero growth rates.

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