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Systematic overexpression study to find target enzymes enhancing production of terpenes in Synechocystis PCC 6803, using isoprene as a model compound
KTH, School of Biotechnology (BIO). KTH, Centres, Science for Life Laboratory, SciLifeLab. Department of Chemistry – Ångström, Uppsala University, Box 523, SE-751 20 Uppsala, Sweden.
KTH, School of Biotechnology (BIO). KTH, Centres, Science for Life Laboratory, SciLifeLab.
KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO).ORCID iD: 0000-0003-1899-7649
2018 (English)In: Metabolic engineering, ISSN 1096-7176, E-ISSN 1096-7184, Vol. 49, p. 164-177Article in journal (Refereed) Published
Abstract [en]

Of the two natural metabolic pathways for making terpenoids, biotechnological utilization of the mevalonate (MVA) pathway has enabled commercial production of valuable compounds, while the more recently discovered but stoichiometrically more efficient methylerythritol phosphate (MEP) pathway is underdeveloped. We conducted a study on the overexpression of each enzyme in the MEP pathway in the unicellular cyanobacterium Synechocystis sp. PCC 6803, to identify potential targets for increasing flux towards terpenoid production, using isoprene as a reporter molecule. Results showed that the enzymes Ipi, Dxs and IspD had the biggest impact on isoprene production. By combining and creating operons out of those genes, isoprene production was increased 2-fold compared to the base strain. A genome-scale model was used to identify targets upstream of the MEP pathway that could redirect flux towards terpenoids. A total of ten reactions from the Calvin-Benson-Bassham cycle, lower glycolysis and co-factor synthesis pathways were probed for their effect on isoprene synthesis by co-expressing them with the MEP enzymes, resulting in a 60% increase in production from the best strain. Lastly, we studied two isoprene synthases with the highest reported catalytic rates. Only by expressing them together with Dxs and Ipi could we get stable strains that produced 2.8 mg/g isoprene per dry cell weight, a 40-fold improvement compared to the initial strain. 

Place, publisher, year, edition, pages
Academic Press Inc. , 2018. Vol. 49, p. 164-177
Keywords [en]
Carbon flux, Cyanobacteria, Isoprene, MEP pathway, Metabolic engineering, Metabolic modeling, Enzymes, Lipids, Metabolism, Strain, Carbon fluxes, Commercial productions, Cyanobacterium synechocystis, MEP pathways, Reporter molecules, Synechocystis pcc 6803, bacterial enzyme, enzyme Dxs, enzyme Ipi, enzyme IspD, isoprene synthase, methylerythritol 4 phosphate, phosphate, synthetase, terpene derivative, terpenoid, unclassified drug, Agrobacterium tumefaciens, Article, bacterial genome, bacterial growth, bacterial strain, biotechnological production, Botryococcus braunii, Coleus, Coleus forskohlii, controlled study, Deinococcus radiodurans, enzyme activity, Escherichia coli, gene expression level, gene overexpression, gene targeting, glycolysis, heterologous expression, molecular cloning, nonhuman, operon, priority journal, protein analysis, protein expression level, Synechocystis sp. PCC 6803, upregulation
National Category
Biological Sciences
Identifiers
URN: urn:nbn:se:kth:diva-236716DOI: 10.1016/j.ymben.2018.07.004ISI: 000447634700016Scopus ID: 2-s2.0-85051682232OAI: oai:DiVA.org:kth-236716DiVA, id: diva2:1257960
Funder
Swedish Energy Agency, 38334-1Swedish Foundation for Strategic Research , RBP-14-0013
Note

Export Date: 22 October 2018; Article; CODEN: MEENF; Correspondence Address: Lindberg, P.; Department of Chemistry – Ångström, Uppsala University, Box 523, Sweden; email: pia.lindberg@kemi.uu.se; QC 20181106

Available from: 2018-10-23 Created: 2018-10-23 Last updated: 2018-11-06Bibliographically approved

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Shabestary, KiyanHudson, Elton P.

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