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Functional replacement of isoprenoid pathways in Rhodobacter sphaeroides.

ABSTRACT: Advances in synthetic biology and metabolic engineering have proven the potential of introducing metabolic by-passes within cell factories. These pathways can provide a more efficient alternative to endogenous counterparts due to their insensitivity to host's regulatory mechanisms. In this work, we replaced the endogenous essential 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in the industrially relevant bacterium Rhodobacter sphaeroides by an orthogonal metabolic route. The native 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway was successfully replaced by a heterologous mevalonate (MVA) pathway from a related bacterium. The functional replacement was confirmed by analysis of the reporter molecule amorpha-4,11-diene after cultivation with [4-13 C]glucose. The engineered R. sphaeroides strain relying exclusively on the MVA pathway was completely functional in conditions for sesquiterpene production and, upon increased expression of the MVA enzymes, it reached even higher sesquiterpene yields than the control strain coexpressing both MEP and MVA modules. This work represents an example where substitution of an essential biochemical pathway by an alternative, heterologous pathway leads to enhanced biosynthetic performance.

SUBMITTER: Orsi E

PROVIDER: S-EPMC7264872 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Functional replacement of isoprenoid pathways in Rhodobacter sphaeroides.

Orsi Enrico E Beekwilder Jules J van Gelder Dewi D van Houwelingen Adèle A Eggink Gerrit G Kengen Servé W M SWM Weusthuis Ruud A RA

Microbial biotechnology 20200324 4

Advances in synthetic biology and metabolic engineering have proven the potential of introducing metabolic by-passes within cell factories. These pathways can provide a more efficient alternative to endogenous counterparts due to their insensitivity to host's regulatory mechanisms. In this work, we replaced the endogenous essential 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in the industrially relevant bacterium Rhodobacter sphaeroides by an orthogonal metaboli ...[more]

PMID: 32207882

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Project description: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.

Dataset Information

Functional replacement of isoprenoid pathways in Rhodobacter sphaeroides.

Publications

Functional replacement of isoprenoid pathways in Rhodobacter sphaeroides.

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