De Novo Biosynthesis of Vindoline and Catharanthine in Saccharomyces cerevisiae

Abstract

Vinblastine has been used clinically as one of the most potent therapeutics for the treatment of several types of cancer. However, the traditional plant extraction method suffers from unreliable supply, low abundance, and extremely high cost. Here, we use synthetic biology approach to engineer Saccharomyces cerevisiae for de novo biosynthesis of vindoline and catharanthine, which can be coupled chemically or biologically to vinblastine. On the basis of a platform strain with sufficient supply of precursors and cofactors for biosynthesis, we reconstituted, debottlenecked, and optimized the biosynthetic pathways for the production of vindoline and catharanthine. The vindoline biosynthetic pathway represents one of the most complicated pathways ever reconstituted in microbial cell factories. Using shake flask fermentation, our engineered yeast strains were able to produce catharanthine and vindoline at a titer of 527.1 and 305.1 μg·liter −1 , respectively, without accumulating detectable amount of pathway intermediates. This study establishes a representative example for the production of valuable plant natural products in yeast.

Biosynthesis of kratom opioids

“…MsDCS1 was subcloned from the respective pET30b+ vector into pESC‐His‐dCrSTR within the BamHI/SalI sites. The expression cassette of codon‐optimized CrSGD was subcloned from pESC‐His‐CrSGD vector (Gao et al ., 2022) by the primer set (31/32), and ligated into pESC‐His‐dCrSTR‐MsDCS1 vector within the DraIII site. The PcuT4H open reading frame was synthesized (Twist Biosciences, South San Francisco, CA, USA) and subcloned into pESC‐Leu‐CrCPR vector (Shahsavarani et al ., 2022) within the BamHI/SalI sites.…”

EngineeringY. lipolyticafor the biosynthesis of geraniol

“…Seminal work in engineering MIA focused on strictosidine, the first intermediate of the broad MIA class of compounds (Brown et al, 2015). Other papers have also demonstrated production of monoterpenes and strictosidine with key improvements achieved by pathway compartmentalization, using geraniol and tryptamine feeds, and optimizing P450 and P450 reductase coupling (Misa et al, 2022;Yee et al, 2019) S. cerevisiae was recently engineered to produce vincristine (Gao et al, 2022) and vinblastine (J. . Monoterpene indole alkaloid titers could be enhanced further by eliminating geraniol supply limitations through the combined approaches described in this paper and the use of a more favorable host such as Y. lipolytica.…”

Establishing CRISPRi for Programmable Gene Repression and Genome Evolution in Cupriavidus necator

“…The advent of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated nuclease 9) technology holds significant promise in addressing the aforementioned limitation 13 and has also facilitated de novo synthesis of complex products in microorganisms. 14 Xiong et al first employed CRISPR/Cas9 in C. necator and improved gene knockout efficiency to nearly 100%, 15 demonstrating the feasibility and potential of CRISPR tools. CRISPR interference (CRISPRi), a modification of CRISPR/Cas9, substitutes the Cas9 enzyme with the nuclease deactivated mutant (dCas9).…”

“…The advent of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated nuclease 9) technology holds significant promise in addressing the aforementioned limitation and has also facilitated de novo synthesis of complex products in microorganisms . Xiong et al first employed CRISPR/Cas9 in C.…”