Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering

Mougiakos, Ioannis; Orsi, Enrico; Ghiffary, Mohammad Rifqi; Post, Wilbert; Maria, Alberto De; Adiego-Pérez, Belén; Kengen, S.W.M.; Weusthuis, Ruud A.; Oost, John van der

doi:10.1186/s12934-019-1255-1

Cited by 24 publications

(42 citation statements)

References 48 publications

(38 reference statements)

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“…1). In a recent study, we demonstrated that deletion of the phaB gene prevents PHB biosynthesis [35]. Here, we confirmed that the deletion of either the phaB gene (Rs265_∆phaB strain) or the combined deletion of the phaC1 and phaC2 genes (Rs265_∆phaC1∆phaC2 strain) prevents PHB formation both under nitrogen excess and nitrogen-limited conditions ( Fig.…”

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

confidence: 86%

“…Rs265 with deletion of phaB for PHB biosynthesis [35] Rs265-MVA_∆dxr Rs265 with chromosomally integrated MVA pathway operon and deletion of the endogenous MEP pahtway (dxr) [16] Rs265-MVA_∆dxr∆phaB Rs265 with chromosomally integrated MVA pathway operon and deletion of the endogenous MEP pahtway (dxr) and PHB biosynthetic pathway (phaB)…”

Section: Rs265_∆phabmentioning

confidence: 99%

“…Source of reference pBBR-ads pBBR1MCS-2 + crtE promoter and ads (amorphadiene synthase) [29] pBBR-MVA-ads pBBR1MCS-2 + crtE promoter controlling MVA enzymes and ads [28] pBBR_ Cas9_ΔphaB_HR pBBR1MCS-2 + codon harmonized cas9 sequence, sgRNA targeting phaB and 1 kb homologousrecombination flanks for recombination with phaB [35] Figures legends Figure 1. Network investigated in this work.…”

Section: Plasmid Descriptionmentioning

confidence: 99%

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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 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: 86%

Section: Rs265_∆phabmentioning

confidence: 99%

Section: Plasmid Descriptionmentioning

confidence: 99%

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Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides

Orsi

Mougiakos

Post

et al. 2020

Preprint

Self Cite

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“…1). In a recent study, we demonstrated that deletion of the phaB gene prevents PHB biosynthesis (31). Here, we con rmed that the deletion of either the phaB gene (Rs265_∆phaB strain) or the combined deletion of the phaC1 and phaC2 genes (Rs265_∆phaC1∆phaC2 strain) prevents PHB formation both under nitrogen excess and nitrogen-limited conditions ( Fig.…”

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

confidence: 70%

Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides

Orsi

Mougiakos

Post

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

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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“…Compared to the traditional double crossover method, these novel base editors greatly shortened the process duration (from 2 weeks/ gene to 1 week/gene), elevated the positive screening rate (from variable 0-40% to stable ~ 100%), increased the throughput (from single gene/round to three genes/ round), and eliminated the potential polar effect (from antibiotic marker integration to no-scar left). During the submission of this manuscript, an efficient Cas9-sgRNA based genome editing toolbox was published [34], which enabled knock-out, knock-in, and single nucleotide substitutions in Rhodobacter sphaeroides 265-9c (derivative of ATCC 35053). However, the efficiency of this system was fluctuant and spacer-dependent, and it had limited throughout because the cloning of different homologous upstream and downstream arm was required for each target.…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-deaminase enables robust base editing in Rhodobacter sphaeroides 2.4.1

Luo

Ge²,

Wang

et al. 2020

Microb Cell Fact

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Background: CRISPR/Cas9 systems have been repurposed as canonical genome editing tools in a variety of species, but no application for the model strain Rhodobacter sphaeroides 2.4.1 was unveiled. Results: Here we showed two kinds of programmable base editing systems, cytosine base editors (CBEs) and adenine base editors (ABEs), generated by fusing endonuclease Cas9 variant to cytosine deaminase PmCDA1 or heterodimer adenine deaminase TadA-TadA*, respectively. Using CBEs, we were able to obtain C-toT mutation of single and double targets following the first induction step, with the efficiency of up to 97% and 43%; while the second induction step was needed in the case of triple target, with the screening rate of 47%. Using ABEs, we were only able to gain A-to-G mutation of single target after the second induction step, with the screening rate of 30%. Additionally, we performed a knockout analysis to identify the genes responsible for coenzyme Q10 biosynthesis and found that ubiF, ubiA, ubiG, and ubiX to be the most crucial ones. Conclusions: Together, CBEs and ABEs serve as alternative methods for genetic manipulation in Rhodobacter sphaeroides and will shed light on the fundamental research of other bacteria that are hard to be directly edited by Cas9-sgRNA.

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Efficient Cas9-based genome editing of Rhodobacter sphaeroides for metabolic engineering

Cited by 24 publications

References 48 publications

Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides

Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides

Growth-uncoupled isoprenoid synthesis in Rhodobacter sphaeroides

CRISPR/Cas9-deaminase enables robust base editing in Rhodobacter sphaeroides 2.4.1

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