Abstract
BackgroundMicrobial 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.ResultsThis 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.ConclusionsApart 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.
Highlights
Microbial cell factories are usually engineered and employed for cultivations that combine product synthesis with growth
Prevention of PHB formation and its effect on amorphadiene biosynthesis Culturing R. sphaeroides under nitrogen-limited conditions could theoretically result in growth-independent isoprenoid synthesis via the native methyl-d-erythritol 4-phosphate (MEP) and the heterologous MVA pathways
We demonstrated that deletion of the phaB gene prevents PHB biosynthesis [31]
Summary
Microbial cell factories are usually engineered and employed for cultivations that combine product synthesis with growth. 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. Isoprenoids ( known as terpenoids) have great industrial value as ingredients of pharmaceuticals, perfumes, food flavourings and most recently biofuels [1,2,3,4,5,6]. They are formed by the condensation of the five-carbon monomers isopentenyl pyrophosphate (IPP) and its isomer.
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