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

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 vincristine1. 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 and reduction to form vinblastine at an industrial scale2,3. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.

A FAIR-compliant parts catalogue for genome engineering and expression control in Saccharomyces cerevisiae.

The synthetic biology toolkit for baker's yeast, Saccharomyces cerevisiae, includes extensive genome engineering toolkits and parts repositories. However, with the increasing complexity of engineering tasks and versatile applications of this model eukaryote, there is a continued interest to expand and diversify the rational engineering capabilities in this chassis by FAIR (findable, accessible, interoperable, and reproducible) compliance. In this study, we designed and characterised 41 synthetic guide RNA sequences to expand the CRISPR-based genome engineering capabilities for easy and efficient replacement of genomically encoded elements. Moreover, we characterize in high temporal resolution 20 native promoters and 18 terminators using fluorescein and LUDOX CL-X as references for GFP expression and OD600 measurements, respectively. Additionally, all data and reported analysis is provided in a publicly accessible jupyter notebook providing a tool for researchers with low-coding skills to further explore the generated data as well as a template for researchers to write their own scripts. We expect the data, parts, and databases associated with this study to support a FAIR-compliant resource for further advancing the engineering of yeasts.

A synthetic RNA-mediated evolution system in yeast.

Laboratory evolution is a powerful approach to search for genetic adaptations to new or improved phenotypes, yet either relies on labour-intensive human-guided iterative rounds of mutagenesis and selection, or prolonged adaptation regimes based on naturally evolving cell populations. Here we present CRISPR- and RNA-assisted in vivo directed evolution (CRAIDE) of genomic loci using evolving chimeric donor gRNAs continuously delivered from an error-prone T7 RNA polymerase, and directly introduced as RNA repair donors into genomic targets under either Cas9 or dCas9 guidance. We validate CRAIDE by evolving novel functional variants of an auxotrophic marker gene, and by conferring resistance to a toxic amino acid analogue in baker's yeast Saccharomyces cerevisiae with a mutation rate >3,000-fold higher compared to spontaneous native rate, thus enabling the first demonstrations of in vivo delivery and information transfer from long evolving RNA donor templates into genomic context without the use of in vitro supplied and pre-programmed repair donors.

Comparing Ligninolytic Capabilities of Bacterial and Fungal Dye-Decolorizing Peroxidases and Class-II Peroxidase-Catalases.

We aim to clarify the ligninolytic capabilities of dye-decolorizing peroxidases (DyPs) from bacteria and fungi, compared to fungal lignin peroxidase (LiP) and versatile peroxidase (VP). With this purpose, DyPs from Amycolatopsis sp., Thermomonospora curvata, and Auricularia auricula-judae, VP from Pleurotus eryngii, and LiP from Phanerochaete chrysosporium were produced, and their kinetic constants and reduction potentials determined. Sharp differences were found in the oxidation of nonphenolic simple (veratryl alcohol, VA) and dimeric (veratrylglycerol-ß- guaiacyl ether, VGE) lignin model compounds, with LiP showing the highest catalytic efficiencies (around 15 and 200 s-1·mM-1 for VGE and VA, respectively), while the efficiency of the A. auricula-judae DyP was 1-3 orders of magnitude lower, and no activity was detected with the bacterial DyPs. VP and LiP also showed the highest reduction potential (1.28-1.33 V) in the rate-limiting step of the catalytic cycle (i.e., compound-II reduction to resting enzyme), estimated by stopped-flow measurements at the equilibrium, while the T. curvata DyP showed the lowest value (1.23 V). We conclude that, when using realistic enzyme doses, only fungal LiP and VP, and in much lower extent fungal DyP, oxidize nonphenolic aromatics and, therefore, have the capability to act on the main moiety of the native lignin macromolecule.