Functional replacement of isoprenoid pathways in <i>Rhodobacter sphaeroides</i>

Orsi, Enrico; Beekwilder, Jules; Gelder, Dewi van; Houwelingen, Adèle van; Eggink, Gerrit; Kengen, S.W.M.; Weusthuis, Ruud A.

doi:10.1111/1751-7915.13562

Cited by 17 publications

(14 citation statements)

References 38 publications

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“…1). This strain relies exclusively on the non-native isoprenoid route (MVA-only) [16]. Also here, a substantial increase in the amorphadiene/biomass ratio was observed during both nitrogen excess and nitrogen-limited conditions (Fig.…”

Section: Prevention Of Phb Formation and Its Effect On Amorphadiene Bsupporting

confidence: 52%

“…Therefore, regulatory control affecting the MEP pathway should not affect the MVA pathway, and vice versa. Moreover, in a recent study, functional replacement of the native MEP with the heterologous MVA pathway was described in the bacterium Rhodobacter sphaeroides [16], a microbial platform organism that is gaining interest for isoprenoid biosynthesis. This organism is able to synthesize intracellular membranes which can accommodate isoprenoids such as carotenoids and bacteriochlorophylls.…”

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confidence: 99%

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Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides

Orsi

Mougiakos

Post

et al. 2020

Biotechnol Biofuels

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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 13 C-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.

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Section: Prevention Of Phb Formation and Its Effect On Amorphadiene Bsupporting

confidence: 52%

mentioning

confidence: 99%

Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides

Orsi

Mougiakos

Post

et al. 2020

Biotechnol Biofuels

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“…Efforts for improving bioproduction have been focusing either on engineering of the endogenous MEP pathway (11)(12)(13), or by co-expressing the heterologous MVA counterpart (10,14,15). Moreover, to limit the effect of unfavorable regulatory control of the endogenous pathway, substitution by an orthogonal isoprenoid pathway has been reported (16).…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, regulatory control affecting the MEP pathway should not affect the MVA pathway, and vice versa. Moreover, in a recent study, functional replacement of the native MEP with the heterologous MVA pathway was described in the bacterium Rhodobacter sphaeroides (16), a microbial platform organism that is gaining interest for isoprenoid biosynthesis. This organism is able to synthesize intracellular membranes which can accommodate isoprenoids such as as carotenoids and bacteriochlorophylls.…”

Section: Introductionmentioning

confidence: 99%

Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides

Orsi

Mougiakos

Post

et al. 2020

Preprint

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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.

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“…1). This strain relies exclusively on the non-native isoprenoid route (MVAonly) (201). Also here, a substantial increase in the amorphadiene/biomass ratio was observed during both nitrogen excess and nitrogen-limited conditions (Fig.…”

Section: Prevention Of Phb Formation and Its Effect On Amorphadiene Bmentioning

confidence: 51%

Racing-red chassis : Advancing Rhodobacter sphaeroides as platform for isoprenoid production

Orsi¹

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Diminishing the turnaround time of DBTL cycles is a crucial aspect for accelerating the development of microbial chassis (31, 32). Cutting edge advances in fields like systems biology (33), machine learning (34), metabolomics (35), automation (31), or genome engineering (36-38) can contribute to accelerate one or more parts of the DBTL cycle. Biofoundries have been realized, in which automated and high-throughput DBTL cycles are integrated for rapidly and efficiently reprogramming cell factories (31, 39). While rapid and standardized DBTL cycles can be performed for model organisms like E. coli or S. cerevisiae, their use for non-model microorganisms is not trivial (15). For example, potentially interesting chassis might lack genetic accessibility, which would render the 'build' phase cumbersome. Also, the metabolic pathways supporting product formation could be unknown. Therefore, implementation of DBTL cycles in non-traditional microorganisms is an intriguing challenge for biotechnology. Succeeding in such a task would allow to investigate and assess non-traditional microorganisms as potential cell factories. Isoprenoid compounds: a successful example for the bioeconomy obtained via DBTL cycles The case of semi-synthetic artemisinin synthesis in yeast Probably the most scientifically relevant achievement obtained via DBTL cycles is the microbial synthesis of the antimalarial drug artemisinin (40). Such a compound is natively produced by the plant Artemisia annua, but dependence on yearly harvesting makes its production challenging (41). Therefore, alternatives like microbial synthesis were explored for feasible and costcompetitive artemisinin production (40, 41). This endeavour started with the implementation of a heterologous production pathway in E. coli (42), and was concluded a decade later by producing up to 25 g/L of artemisinic acid in Fld/Fd red Fld/Fd ox

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Functional replacement of isoprenoid pathways in Rhodobacter sphaeroides

Cited by 17 publications

References 38 publications

Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides

Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides

Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides

Racing-red chassis : Advancing Rhodobacter sphaeroides as platform for isoprenoid production

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