A balanced heterologous biosynthetic pathway in microbes is necessary to ensure high titers of the desired product. Expression of synthetic heterologous metabolic pathways in a host may not be favorable due to the toxicity of non-native metabolic intermediates and the but of expression of genes in the pathway. Thus, optimization of gene expression is required to prevent accumulation of the toxic metabolites and to minimize burden. In this study, we used the BglBrick standard cloning vectors to optimize expression of genes in a heterologous mevalonate-based isoprenoid biosynthetic pathway and examined their impact on production of amorpha-4,11-diene, an intermediate in the biosynthesis of the antimalarial drug artemisinin. Amorpha-4,11-diene titer increased almost three-fold when HMG-CoA reductase and phosphomevalonate kinase were overexpressed relative to the original engineered pathway. In addition, selected-reaction monitoring (SRM) mass spectrometry-based targeted proteomics showed that overexpression of HMG-CoA reductase and phosphomevalonate kinase significantly enhances the titer of amorpha-4,11-diene, and that further increases in titer could be achieved if mevalonate kinase were engineered as well. Published by Elsevier Ltd.