Fatty acids are produced by eukaryotes like baker's yeastSaccharomyces cerevisiaemainly using a large multifunctional type I fatty acid synthase (FASI) where seven catalytic steps and a carrier domain are shared between one or two protein subunits. While this system may offer efficiency in catalysis, only a narrow range of fatty acids are produced. Prokaryotes, chloroplasts and mitochondria rely instead on a FAS type II (FASII) where each catalytic step is carried out by a monofunctional enzyme encoded by a separate gene. FASII is more flexible and capable of producing a wider range of fatty acid structures, such as the direct production of unsaturated fatty acids. An efficient FASII in the preferred industrial organismS. cerevisiaecould provide a platform for developing sustainable production of specialized fatty acids. We functionally replaced either yeast FASI genes (FAS1orFAS2) with a FASII consisting of nine genes fromEscherichia coli(acpP,acpSandfab-A, -B, -D, -F, -G, -H, -Z) as well as three fromArabidopsis thaliana(MOD1,FATA1andFATB). The genes were expressed from an autonomously replicating multicopy vector assembled using the Yeast Pathway Kit forin-vivoassembly in yeast. Two rounds of adaptation led to a strain with a maximum growth rate (μmax) of 0.19 h−1without exogenous fatty acids, twice the growth rate previously reported for a comparable strain. Additional copies of theMOD1orfabHgenes resulted in cultures with higher final cell densities and three times higher lipid content compared to the control.