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Heterologous biosynthesis of tetrahydrocannabinolic acid (THCA) in yeast is a biotechnological process in Natural Product Biotechnology that was recently introduced. Based on heterologous genes from Cannabis sativa and Streptomyces spp. cloned into Saccharomyces cerevisiae, the heterologous biosynthesis was fully embedded as a proof of concept. Low titer and insufficient biocatalytic rate of most enzymes require systematic optimization of recombinant catalyst by protein engineering and consequent C-flux improvement of the yeast chassis for sufficient precursor (acetyl-CoA), energy (ATP), and NADH delivery. In this review basic principles of in silico analysis of anabolic pathways towards olivetolic acid (OA) and cannabigerolic acid (CBGA) are elucidated and discussed to identify metabolic bottlenecks. Based on own experimental results, yeasts are discussed as potential platform organisms to be introduced as potential cannabinoid biofactories. Especially feeding strategies and limitations in the committed mevalonate and olivetolic acid pathways are in focus of in silico and experimental studies to validate the scientific and commercial potential as a realistic alternative to the plant Cannabis sativa.Key points• First time critical review of the heterologous process for recombinant THCA/CBDA production and critical review of bottlenecks and limitations for a bioengineered technical process• Integrative approach of protein engineering, systems biotechnology, and biochemistry of yeast physiology and biosynthetic cannabinoid enzymes• Comparison of NphB and CsPT aromatic prenyltransferases as rate-limiting catalytic steps towards cannabinoids in yeast as platform organisms Graphical abstract.

Bioengineering studies and pathway modeling of the heterologous biosynthesis of tetrahydrocannabinolic acid in yeast