Markus, T.; Soldat, M.; Magdevska, V.; Horvat, J.; Kavscek, M.; Kosec, G.; Fujs, S.; Petrovic, U.

Yarrowia lipolytica is an oleaginous yeast with ever growing popularity in the metabolic engineering circles. It is well known for its ability to accommodate a high carbon flux through acetyl-CoA and is being extensively studied for production of chemicals derived from it. We investigated the effects of modifying the upstream metabolism leading to acetyl-CoA on beta-carotene production, including its titer, yield, and content. We examined the pyruvate and the phosphoketolase bypass, both of which are stoichiometrically favorable for the production of acetyl-CoA and beta-carotene. Additionally, we examined a set of genes involved in the carnitine shuttle. We constructed a set of parental strains derived from the Y. lipolytica YB-392 wild-type strain, each with a different capacity for beta-carotene production, and introduced genes for the metabolic bypasses in each of the constructed parental strains. Subsequently, we subjected these constructed strains to a series of fermentation experiments. We discovered that altering the upstream metabolism in most cases led to a decrease in performance for production of beta-carotene. Most notably, a set of genes used for the pyruvate bypass (YlPDC2, YlALD5, and YlACS1) and the phosphoketolase bypass (LmXPK and CkPTA) resulted in the reduction of more than 30%. Our findings contribute to our understanding of Y. lipolytica\'s metabolic capacity and suggest that production of beta-carotene is most likely not limited solely by the acetyl-CoA supply. We also highlight a complex nature of engineering Y. lipolytica, as most of the results from studies using a different strain background did not align with our findings.