Combinatorial genome integration of mevalonate pathway genes was performed with the aim of optimizing the metabolic flux for improved production of terpenoids in budding yeast. In the present study, we developed a novel δ-integration platform to achieve multiple genome integrations through modulating the concentration of antibiotics. By exploiting carotenoid biosynthesis as screening module, we successfully created a library of yeast colonies appeared with various intensities of orange color. As proof-of-concept that carotenoid overproducers could serve to boost the titer of other terpenoids, we further tested engineered strains for the production of amorpha-4,11-diene, an important precursor for antimalarial drug. However, we experienced some limitations of the carotenoid-based screening approach as it was only effective in detecting a small range of pathway activity improvement and further increasing mevalonate pathway activity led to a decreased orange color. By far, we were only able to obtain one mutant strain yielded more than 13-fold amorpha-4,11-diene over parental strains, which was approximately 64 mg/L of caryophyllene equivalents. Further qPCR studies confirmed that erg10, erg13, thmg1 and erg12 involved in mevalonate pathway were overexpressed in this mutant strain. We envision the current δ-integration platform would form the basis of a generalized technique for multiple gene integrations in yeast—a method that would be of significant interest to the metabolic engineering community. Biotechnol. Bioeng. 2014;111: 608–617. © 2013 Wiley Periodicals, Inc.