Microalgae constitute a diverse group of eukaryotic unicellular organisms that are of interest for pure and applied research. Owing to their natural synthesis of value-added natural products microalgae are emerging as a source of sustainable chemical compounds, proteins and metabolites, including but not limited to those that could replace compounds currently made from fossil fuels. For the model microalga, Chlamydomonas reinhardtii, this has prompted a period of rapid development so that this organism is poised for exploitation as an industrial biotechnology platform. The question now is how best to achieve this? Highly advanced industrial biotechnology systems using bacteria and yeasts were established in a classical metabolic engineering manner over several decades. However, the advent of advanced molecular tools and the rise of synthetic biology provide an opportunity to expedite the development of C. reinhardtii as an industrial biotechnology platform, avoiding the process of incremental improvement. In this review we describe the current status of genetic manipulation of C. reinhardtii for metabolic engineering. We then introduce several concepts that underpin synthetic biology, and show how generic parts are identified and used in a standard manner to achieve predictable outputs. Based on this we suggest that the development of C. reinhardtii as an industrial biotechnology platform can be achieved more efficiently through adoption of a synthetic biology approach.Significance StatementChlamydomonas reinhardtii offers potential as a host for the production of high value compounds for industrial biotechnology. Synthetic biology provides a mechanism to generate generic, well characterised tools for application in the rational genetic manipulation of organisms: if synthetic biology principles were adopted for manipulation of C. reinhardtii, development of this microalga as an industrial biotechnology platform would be expedited.

Thiamin (vitamin B 1 ) is an essential micronutrient needed as a cofactor for many central metabolic enzymes. Animals must have thiamin in their diet, whereas bacteria, fungi, and plants can biosynthesize it de novo from the condensation of a thiazole and a pyrimidine moiety. Although the routes to biosynthesize these two heterocycles are not conserved in different organisms, in all cases exogenous thiamin represses expression of one or more of the biosynthetic pathway genes. One important mechanism for this control is via thiamin-pyrophosphate (TPP) riboswitches, regions of the mRNA to which TPP can bind directly, thus facilitating fine-tuning to maintain homeostasis. However, there is little information on how modulation of riboswitches affects thiamin metabolism in vivo. Here we use the green alga, Chlamydomonas reinhardtii, which regulates both thiazole and pyrimidine biosynthesis with riboswitches in the THI4 (Thiamin 4) and THIC (Thiamin C) genes, respectively, to investigate this question. Our study reveals that regulation of thiamin metabolism is not the simple dogma of negative feedback control. Specifically, balancing the provision of both of the heterocycles of TPP appears to be an important requirement. Furthermore, we show that the Chlamydomonas THIC riboswitch is controlled by hydroxymethylpyrimidine pyrophosphate, as well as TPP, but with an identical alternative splicing mechanism. Similarly, the THI4 gene is responsive to thiazole. The study not only provides insight into the plasticity of the TPP riboswitches but also shows that their maintenance is likely to be a consequence of evolutionary need as a function of the organisms' environment and the particular pathway used.eukaryotic riboswitch | gene expression coordination | metabolic regulation | cross-talk

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.

Riboswitches are regulatory elements in messenger RNA to which specific ligands can bind directly in the absence of proteins. Ligand binding alters the mRNA secondary structure, thereby affecting expression of the encoded protein. Riboswitches are widespread in prokaryotes, with over 20 different effector ligands known, including amino acids, cofactors, and Mg(2+) ions, and gene expression is generally regulated by affecting translation or termination of transcription. In plants, fungi, and microalgae, riboswitches have been found, but only those that bind thiamine pyrophosphate. These eukaryotic riboswitches operate by causing alternative splicing of the transcript. Here, we review the current status of riboswitch research with specific emphasis on microalgae. We discuss new riboswitch discoveries and insights into the underlying mechanism of action, and how next generation sequencing technology provides the motivation and opportunity to improve our understanding of these rare but important regulatory elements. We also highlight the potential of microalgal riboswitches as a tool for synthetic biology and industrial biotechnology.