Metabolic pathway optimization using ribosome binding site variants and combinatorial gene assembly |
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Authors: | Farnaz F Nowroozi Edward E K Baidoo Simon Ermakov Alyssa M Redding-Johanson Tanveer S Batth Christopher J Petzold Jay D Keasling |
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Institution: | 1. Department of Bioengineering, University of California, Berkeley, CA, 94720, USA 2. Joint Bioenergy Institute, 5885 Hollis Avenue, Fourth Floor, Emeryville, CA, 94608, USA 3. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA 4. Department of Molecular Cell Biology, University of California, Berkeley, CA, 94720, USA 5. Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA, 94720, USA
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Abstract: | The genes encoding the mevalonate-based farnesyl pyrophosphate (FPP) biosynthetic pathway were encoded in two operons and expressed in Escherichia coli to increase the production of sesquiterpenes. Inefficient translation of several pathway genes created bottlenecks and led to the accumulation of several pathway intermediates, namely, mevalonate and FPP, and suboptimal production of the sesquiterpene product, amorphadiene. Because of the difficulty in choosing ribosome binding sites (RBSs) to optimize translation efficiency, a combinatorial approach was used to choose the most appropriate RBSs for the genes of the lower half of the mevalonate pathway (mevalonate to amorphadiene). RBSs of various strengths, selected based on their theoretical strengths, were cloned 5′ of the genes encoding mevalonate kinase, phosphomevalonate kinase, mevalonate diphosphate decarboxylase, and amorphadiene synthase. Operons containing one copy of each gene and all combinations of RBSs were constructed and tested for their impact on growth, amorphadiene production, enzyme level, and accumulation of select pathway intermediates. Pathways with one or more inefficiently translated enzymes led to the accumulation of pathway intermediates, slow growth, and low product titers. Choosing the most appropriate RBS combination and carbon source, we were able to reduce the accumulation of toxic metabolic intermediates, improve growth, and improve the production of amorphadiene approximately fivefold. This work demonstrates that balancing flux through a heterologous pathway and maintaining steady growth are key determinants in optimizing isoprenoid production in microbial hosts. |
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