Method to assemble and integrate biochemical pathways into the chloroplast genome of Chlamydomonas reinhardtii.
Biotechnol Bioeng
; 109(11): 2896-903, 2012 Nov.
Article
em En
| MEDLINE
| ID: mdl-22674415
ABSTRACT
Recombinant protein expression in the chloroplasts of green algae has recently become more routine; however, the heterologous expression of multiple proteins or complete biosynthetic pathways remains a significant challenge. Here, we show that a modified DNA Assembler approach can be used to rapidly assemble multiple-gene biosynthetic pathways in yeast and then integrate these assembled pathways at a site-specific location in the chloroplast genome of the microalgal species Chlamydomonas reinhardtii. As a proof of concept, this method was used to successfully integrate and functionally express up to three reporter proteins (AphA6, AadA, and GFP) in the chloroplast of C. reinhardtii. An analysis of the relative gene expression of the engineered strains showed significant differences in the mRNA expression levels of the reporter genes and thus highlights the importance of proper promoter/untranslated region selection when constructing a target pathway. This new method represents a useful genetic tool in the construction and integration of complex biochemical pathways into the chloroplast genome of microalgae and should aid current efforts to engineer algae for biofuels production and other desirable natural products.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Chlamydomonas reinhardtii
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Redes e Vias Metabólicas
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Genoma de Cloroplastos
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Engenharia Metabólica
Idioma:
En
Ano de publicação:
2012
Tipo de documento:
Article