The phosphite oxidoreductase gene, ptxD as a bio-contained chloroplast marker and crop-protection tool for algal biotechnology using Chlamydomonas.
Appl Microbiol Biotechnol
; 104(2): 675-686, 2020 Jan.
Article
em En
| MEDLINE
| ID: mdl-31788712
ABSTRACT
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 set-up 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 bio-available 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 TrpâOpal 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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01-internacional
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MEDLINE
Assunto principal:
Oxirredutases
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Fosfatos
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Biotecnologia
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Proteínas Recombinantes
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Cloroplastos
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Chlamydomonas reinhardtii
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Fosfitos
Idioma:
En
Ano de publicação:
2020
Tipo de documento:
Article