RESUMO
Riboswitches are RNA regulatory elements that bind specific ligands to control gene expression. Because of their modular composition, where a ligand-sensing aptamer domain is combined with an expression platform, riboswitches offer unique tools for synthetic biology applications. Here we took a mutational approach to determine functionally important nucleotide residues in the thiamine pyrophosphate (TPP) riboswitch in the THI4 gene of the model alga Chlamydomonas reinhardtii, allowing us to carry out aptamer swap using THIC aptamers from Chlamydomonas and Arabidopsis thaliana. These chimeric riboswitches displayed a distinct specificity and dynamic range of responses to different ligands. Our studies demonstrate ease of assembly as 5'UTR DNA parts, predictability of output, and utility for controlled production of a high-value compound in Chlamydomonas. The simplicity of riboswitch incorporation in current design platforms will facilitate the generation of genetic circuits to advance synthetic biology and metabolic engineering of microalgae.
Assuntos
Chlamydomonas/metabolismo , Engenharia Metabólica/métodos , Riboswitch/genética , Regiões 5' não Traduzidas , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/metabolismo , Expressão Gênica , Mutagênese , Tiamina Pirofosfato/metabolismo , Raios UltravioletaRESUMO
Microalgae are regarded as promising organisms to develop innovative concepts based on their photosynthetic capacity that offers more sustainable production than heterotrophic hosts. However, to realize their potential as green cell factories, a major challenge is to make microalgae easier to engineer. A promising approach for rapid and predictable genetic manipulation is to use standardized synthetic biology tools and workflows. To this end we have developed a Modular Cloning toolkit for the green microalga Chlamydomonas reinhardtii. It is based on Golden Gate cloning with standard syntax, and comprises 119 openly distributed genetic parts, most of which have been functionally validated in several strains. It contains promoters, UTRs, terminators, tags, reporters, antibiotic resistance genes, and introns cloned in various positions to allow maximum modularity. The toolkit enables rapid building of engineered cells for both fundamental research and algal biotechnology. This work will make Chlamydomonas the next chassis for sustainable synthetic biology.
Assuntos
Chlamydomonas reinhardtii/metabolismo , Fotossíntese , Plasmídeos/metabolismo , Biologia Sintética/métodos , Biotecnologia , Chlamydomonas reinhardtii/genética , Expressão Gênica , Genes Reporter/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Plasmídeos/genética , Regiões Promotoras GenéticasRESUMO
Through metabolic engineering cyanobacteria can be employed in biotechnology. Combining the capacity for oxygenic photosynthesis and carbon fixation with an engineered metabolic pathway allows carbon-based product formation from CO(2), light, and water directly. Such cyanobacterial 'cell factories' are constructed to produce biofuels, bioplastics, and commodity chemicals. Efforts of metabolic engineers and synthetic biologists allow the modification of the intermediary metabolism at various branching points, expanding the product range. The new biosynthesis routes 'tap' the metabolism ever more efficiently, particularly through the engineering of driving forces and utilization of cofactors generated during the light reactions of photosynthesis, resulting in higher product titers. High rates of carbon rechanneling ultimately allow an almost-complete allocation of fixed carbon to product above biomass.
