Woo, Han Min Murray, Gregory W. Batth, Tanveer S. Prasad, Nilu Adams, Paul D. Keasling, Jay D. Petzold, Christopher J. Lee, Taek Soon 2024-01-20T11:03:10Z 2024-01-20T11:03:10Z 2021-08-31 2013-11-15 0009-2509 https://pubs.kist.re.kr/handle/201004/127439 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. English PERGAMON-ELSEVIER SCIENCE LTD HETEROLOGOUS MEVALONATE PATHWAY HIGH-LEVEL PRODUCTION ESCHERICHIA-COLI SYNTHETIC BIOLOGY KINASE ENZYME RECONSTRUCTION EXPRESSION MOLECULES NETWORKS Application of targeted proteomics and biological parts assembly in E. coli to optimize the biosynthesis of an anti-malarial drug precursor, amorpha-4,11-diene Article 10.1016/j.ces.2013.04.033 1 CHEMICAL ENGINEERING SCIENCE, v.103, pp.21 - 28 CHEMICAL ENGINEERING SCIENCE 103 21 28 scie scopus 000325945800004 2-s2.0-84886100543 Engineering, Chemical Engineering Article HETEROLOGOUS MEVALONATE PATHWAY HIGH-LEVEL PRODUCTION ESCHERICHIA-COLI SYNTHETIC BIOLOGY KINASE ENZYME RECONSTRUCTION EXPRESSION MOLECULES NETWORKS Mevalonate pathway Synthetic biology Metabolic engineering Biocatalysis Biological and biomolecular engineering Molecular biology