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Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides

Authors :
Gerrit Eggink
Jules Beekwilder
Ruud A. Weusthuis
Servé W. M. Kengen
John van der Oost
Marco Dompé
Ioannis Mougiakos
Enrico Orsi
Wilbert Post
HIRI, Helmholtz-Institut für RNA-basierte Infektionsforschung, Josef-Shneider Strasse 2, 97080 Würzburg, Germany.
Source :
Biotechnology for Biofuels, Vol 13, Iss 1, Pp 1-13 (2020), Biotechnology for Biofuels 13 (2020) 1, Biotechnology for biofuels, England, Biotechnology for Biofuels, 13(1)
Publication Year :
2020
Publisher :
BMC, 2020.

Abstract

Background Microbial cell factories are usually engineered and employed for cultivations that combine product synthesis with growth. Such a strategy inevitably invests part of the substrate pool towards the generation of biomass and cellular maintenance. Hence, engineering strains for the formation of a specific product under non-growth conditions would allow to reach higher product yields. In this respect, isoprenoid biosynthesis represents an extensively studied example of growth-coupled synthesis with rather unexplored potential for growth-independent production. Rhodobacter sphaeroides is a model bacterium for isoprenoid biosynthesis, either via the native 2-methyl-d-erythritol 4-phosphate (MEP) pathway or the heterologous mevalonate (MVA) pathway, and for poly-β-hydroxybutyrate (PHB) biosynthesis. Results This study investigates the use of this bacterium for growth-independent production of isoprenoids, with amorpha-4,11-diene as reporter molecule. For this purpose, we employed the recently developed Cas9-based genome editing tool for R. sphaeroides to rapidly construct single and double deletion mutant strains of the MEP and PHB pathways, and we subsequently transformed the strains with the amorphadiene producing plasmid. Furthermore, we employed 13C-metabolic flux ratio analysis to monitor the changes in the isoprenoid metabolic fluxes under different cultivation conditions. We demonstrated that active flux via both isoprenoid pathways while inactivating PHB synthesis maximizes growth-coupled isoprenoid synthesis. On the other hand, the strain that showed the highest growth-independent isoprenoid yield and productivity, combined the plasmid-based heterologous expression of the orthogonal MVA pathway with the inactivation of the native MEP and PHB production pathways. Conclusions Apart from proposing a microbial cell factory for growth-independent isoprenoid synthesis, this work provides novel insights about the interaction of MEP and MVA pathways under different growth conditions.

Details

Language :
English
ISSN :
17546834
Volume :
13
Issue :
1
Database :
OpenAIRE
Journal :
Biotechnology for Biofuels
Accession number :
edsair.doi.dedup.....35f4ebbc44567fa1e0457fa46f683184