A Novel Biocontainment Strategy Makes Bacterial Growth and Survival Dependent on Phosphite

Hirota, Ryuichi; Abe, Kenji; Katsuura, Zen-ichiro; Noguchi, Reiji; Moribe, Shigeaki; Motomura, Kei; Ishida, Takenori; Alexandrov, Maxym; Funabashi, Hiroaki; Ikeda, Takeshi; Kuroda, Akio

doi:10.1038/srep44748

Cited by 42 publications

(65 citation statements)

References 62 publications

(83 reference statements)

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“…PtxD has been introduced to Arabidopsis , tobacco and rice, allowing the use of phosphite as a dual fertilisation and weed control system 28 . PtxD and phosphite are also used as selectable markers in bacteria 20 , 34 , yeast 24 , plants 25 and algae 26 ; to provide competitive advantage in industrial fermentation processes 27 ; and in NADH-dependent biocatalysis for in situ cofactor regeneration 29 . So far these applications have only exploited PtxD, relying on non-specific phosphite uptake that requires media phosphite concentrations of ≥0.1 mM.…”

Section: Discussionmentioning

confidence: 99%

“…Furthermore, some technologies require that phosphite uptake is highly specific. For example, a recent study by Hirota et al 34 described the use of phosphite-dependent growth as a biocontainment strategy, which requires a phosphite transporter that does not transport phosphate. The authors introduced PtxD and the Ralstonia sp.…”

Section: Discussionmentioning

confidence: 99%

“…While the genetic and biochemical basis of phosphite and hypophosphite utilisation and the PtxD and HtxA enzymes have been characterised previously 18 , 23 , 32 , 33 , the mechanism of high-affinity ligand binding by the cognate ABC transporters is not well studied. The use of non-physiologically high concentrations (up to 1 mM) of P ligands in previous in vivo studies has hindered clear interpretation of the physiologically relevant specificity of reduced P compound transporters 18 , 19 , 30 , 34 . Here we elucidate the molecular basis of high-affinity phosphite and hypophosphite acquisition by determining the ligand-binding affinity and structure of the PBPs of phosphite and hypophosphite ABC transporters from environmental microorganisms.…”

Section: Introductionmentioning

confidence: 99%

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The molecular basis of phosphite and hypophosphite recognition by ABC-transporters

et al. 2017

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Inorganic phosphate is the major bioavailable form of the essential nutrient phosphorus. However, the concentration of phosphate in most natural habitats is low enough to limit microbial growth. Under phosphate-depleted conditions some bacteria utilise phosphite and hypophosphite as alternative sources of phosphorus, but the molecular basis of reduced phosphorus acquisition from the environment is not fully understood. Here, we present crystal structures and ligand binding affinities of periplasmic binding proteins from bacterial phosphite and hypophosphite ATP-binding cassette transporters. We reveal that phosphite and hypophosphite specificity results from a combination of steric selection and the presence of a P-H…π interaction between the ligand and a conserved aromatic residue in the ligand-binding pocket. The characterisation of high affinity and specific transporters has implications for the marine phosphorus redox cycle, and might aid the use of phosphite as an alternative phosphorus source in biotechnological, industrial and agricultural applications.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The molecular basis of phosphite and hypophosphite recognition by ABC-transporters

et al. 2017

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“…Though there is still the risk of horizontal gene transfer of the introduced pathways into environmental microorganisms, these pathways would be very unlikely to become a risk to either the ecological balance of local microorganism populations or human health. However, to completely eliminate such a risk, a biocontainment strategy for both the melamine operon as well as the Phi strategy, similar to that described for Phi usage in E. coli (Hirota et al, 2017) and Synechococcus sp. PCC 7942 (Motomura et al, 2018) can also be implemented by deleting the cognate ammonium, nitrate and phosphate transporter genes.…”

Section: Strain Mel5-a0935ptxd Is Able To Resist Deliberate Contaminamentioning

confidence: 99%

Growth and selection of the cyanobacterium Synechococcus sp. PCC 7002 using alternative nitrogen and phosphorus sources

Selão¹,

Włodarczyk²,

Nixon

et al. 2019

Preprint

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Cyanobacteria, such as Synechococcus sp. PCC 7002 (Syn7002), are promising chassis strains for "green" biotechnological applications as they can be grown in seawater using oxygenic photosynthesis to fix carbon dioxide into biomass. Their other major nutritional requirements for efficient growth are sources of nitrogen (N) and phosphorus (P). As these organisms are more economically cultivated in outdoor open systems, there is a need to develop cost-effective approaches to prevent the growth of contaminating organisms, especially as the use of antibiotic selection markers is neither economically feasible nor ecologically desirable due to the risk of horizontal gene transfer. Here we have introduced a synthetic melamine degradation pathway into Syn7002 and evolved the resulting strain to efficiently use the nitrogen-rich xenobiotic compound melamine as the sole N source. We also show that expression of phosphite dehydrogenase in the absence of its cognate phosphite transporter permits growth of Syn7002 on phosphite and can be used as a selectable marker in Syn7002. We combined these two strategies to generate a strain that can grow on melamine and phosphite as sole N and P sources, respectively. This strain is able to resist deliberate contamination in large excess and should be a useful chassis for metabolic engineering and biotechnological applications using cyanobacteria.

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“…However, whilst the very low abundance of Phi in the natural environment means that HGT of the ptxD marker to other microbial species is unlikely to confer any selective advantage, this could still compromise its use as for crop protection. For example, fungal pathogens of plants or microbial competitors of yeast, bacterial or microalgal platforms could acquire ptxD and then thrive under Phi-cultivation conditions (Hirota et al 2017).…”

Section: Introductionmentioning

confidence: 99%

The phosphite oxidoreductase gene, ptxD as a bio-contained chloroplast marker and crop-protection tool for algal biotechnology using Chlamydomonas

Changko

Rajakumar

Young

et al. 2019

Appl Microbiol Biotechnol

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Edible microalgae have potential as low-cost cell factories for the production and oral delivery of recombinant proteins such as vaccines, anti-bacterials and gut-active enzymes that are beneficial to farmed animals including livestock, poultry and fish. However, a major economic and technical problem associated with large-scale cultivation of microalgae, even in closed photobioreactors, is invasion by contaminating microorganisms. Avoiding this requires costly media sterilisation, aseptic techniques during setup and implementation of 'crop-protection' strategies during cultivation. Here, we report a strain improvement approach in which the chloroplast of Chlamydomonas reinhardtii is engineered to allow oxidation of phosphite to its bioavailable form: phosphate. We have designed a synthetic version of the bacterial gene (ptxD)-encoding phosphite oxidoreductase such that it is highly expressed in the chloroplast but has a Trp→Opal codon reassignment for bio-containment of the transgene. Under mixotrophic conditions, the growth rate of the engineered alga is unaffected when phosphate is replaced with phosphite in the medium. Furthermore, under non-sterile conditions, growth of contaminating microorganisms is severely impeded in phosphite medium. This, therefore, offers the possibility of producing algal biomass under non-sterile conditions. The ptxD gene can also serve as a dominant marker for genetic engineering of any C. reinhardtii strain, thereby avoiding the use of antibiotic resistance genes as markers and allowing the 'retro-fitting' of existing engineered strains. As a proof of concept, we demonstrate the application of our ptxD technology to a strain expressing a subunit vaccine targeting a major viral pathogen of farmed fish.

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A Novel Biocontainment Strategy Makes Bacterial Growth and Survival Dependent on Phosphite

Cited by 42 publications

References 62 publications

The molecular basis of phosphite and hypophosphite recognition by ABC-transporters

The molecular basis of phosphite and hypophosphite recognition by ABC-transporters

Growth and selection of the cyanobacterium Synechococcus sp. PCC 7002 using alternative nitrogen and phosphorus sources

The phosphite oxidoreductase gene, ptxD as a bio-contained chloroplast marker and crop-protection tool for algal biotechnology using Chlamydomonas

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