<jats:title>Abstract</jats:title><jats:sec><jats:label/><jats:p>The increasing market demands of β‐carotene as colorant, antioxidant and vitamin precursor, requires novel biotechnological production platforms. <jats:italic>Yarrowia lipolytica</jats:italic>, is an industrial organism unable to naturally synthesize carotenoids but with the ability to produce high amounts of the precursor Acetyl‐CoA. We first found that a lipid overproducer strain was capable of producing more β‐carotene than a wild type after expressing the heterologous pathway. Thereafter, we developed a combinatorial synthetic biology approach base on Golden Gate DNA assembly to screen the optimum promoter‐gene pairs for each transcriptional unit expressed. The best strain reached a production titer of 1.5 g/L and a maximum yield of 0.048 g/g of glucose in flask. β‐carotene production was further increased in controlled conditions using a fed‐batch fermentation. A total production of β‐carotene of 6.5 g/L and 90 mg/g DCW with a concomitant production of 42.6 g/L of lipids was achieved. Such high titers suggest that engineered <jats:italic>Y. lipolytica</jats:italic> is a competitive producer organism of β‐carotene.</jats:sec>