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Heterologous expression and characterization of bacterial 2-C-methyl-d-erythritol-4-phosphate pathway in Saccharomyces cerevisiae

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Abstract

Transfer of a biosynthetic pathway between evolutionary distant organisms can create a metabolic shunt capable of bypassing the native regulation of the host organism, hereby improving the production of secondary metabolite precursor molecules for important natural products. Here, we report the engineering of Escherichia coli genes encoding the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway into the genome of Saccharomyces cerevisiae and the characterization of intermediate metabolites synthesized by the MEP pathway in yeast. Our UPLC-MS analysis of the MEP pathway metabolites from engineered yeast showed that the pathway is active until the synthesis of 2-C-methyl-d-erythritol-2,4-cyclodiphosphate, but appears to lack functionality of the last two steps of the MEP pathway, catalyzed by the [4Fe–4S] iron sulfur cluster proteins encoded by ispG and ispH. In order to functionalize the last two steps of the MEP pathway, we co-expressed the genes for the E. coli iron sulfur cluster (ISC) assembly machinery. By deleting ERG13, thereby incapacitating the mevalonate pathway, in conjunction with labeling experiments with U–13C6 glucose and growth experiments, we found that the ISC assembly machinery was unable to functionalize ispG and ispH. However, we have found that leuC and leuD, encoding the heterodimeric iron–sulfur cluster protein, isopropylmalate isomerase, can complement the S. cerevisiae leu1 auxotrophy. To our knowledge, this is the first time a bacterial iron–sulfur cluster protein has been functionally expressed in the cytosol of S. cerevisiae under aerobic conditions and shows that S. cerevisiae has the capability to functionally express at least some bacterial iron–sulfur cluster proteins in its cytosol.

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Acknowledgments

The authors would like to thank Dr. Hang Zhou, Dr. Jose Avalos, and Dr. Gerald R. Fink for contributing the plasmids and yeast strains used in this study and Dr. Christopher Pirie, Manus Biosynthesis, for critical reading of the manuscript. S.C., P.K.A., and G.S. acknowledge the support by the National Institutes of Health (grant no. 1-R01-GM085323-01A1). S.C., L.R.F., M.L.N., A.E.L., and M.C.K.B. gratefully acknowledge the funding from the Technical University of Denmark. K.Z. and T.H.P. acknowledge the support from Singapore-MIT Alliance (SMA-2).

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  1. Parayil Kumaran Ajikumar

    Present address: Manus Biosynthesis, 790 Memorial Drive, Cambridge, MA, 02139, USA

  2. Michael Lynge Nielsen

    Present address: Novozymes, A/S, Krogshoejvej 36, 2880, Bagsvaerd, Denmark

Authors and Affiliations

  1. Department of Systems Biology, Technical University of Denmark, 2800, Kongens Lyngby, Denmark

    Simon Carlsen, Luca Riccardo Formenti, Michael Lynge Nielsen, Anna Eliasson Lantz & Morten C. Kielland-Brandt

  2. Department of Chemical Engineering, Massachusetts Institute of Technology (MIT), Cambridge, MA, 02139, USA

    Simon Carlsen, Parayil Kumaran Ajikumar, Kang Zhou & Gregory Stephanopoulos

  3. Chemical and Pharmaceutical Engineering Program, Singapore-MIT Alliance, Singapore, 117546, Singapore

    Kang Zhou & Too Heng Phon

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  1. Simon Carlsen
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Correspondence to Gregory Stephanopoulos.

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Carlsen, S., Ajikumar, P.K., Formenti, L.R. et al. Heterologous expression and characterization of bacterial 2-C-methyl-d-erythritol-4-phosphate pathway in Saccharomyces cerevisiae . Appl Microbiol Biotechnol 97, 5753–5769 (2013). https://doi.org/10.1007/s00253-013-4877-y

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