Genetic engineering tools for the filamentous microalga Tribonema minus

Tribonema minus, a eukaryotic filamentous yellow-green alga, is widely regarded as an optimal candidate for the production of biofuels and high-value chemicals such as palmitoleic acid and eicosapentaenoic acid. To achieve artificial regulation of the biosynthetic pathways for target compounds in T. minus, it is crucial to establish a stable multi-gene genetic expression system. Genetic engineering provides an effective approach for regulating the expression of multiple genes. A crucial breakthrough in this context involves expanding the effective genetic tools, which enables the integrated use of promoters, terminators, resistance markers and reporter genes to express the endogenous or heterologous gene-of-interest. To this end, we have devised tools suitable for multi-gene expression in T. minus. These tools include promoters (Ptubulin, Phsp70A and Phsp90A), F2A peptide, resistance marker genes (nptII, aadA and ble) and an enhanced green fluorescent protein reporter gene (eGFP). The genetic elements incorporated into the tools have demonstrated efficacy in T. minus. The experimental findings indicate that the endogenous promoters identified in T. minus exhibit transcriptional activity, and are capable of driving enhanced green fluorescent protein gene (eGFP) expression and could result in the production of corresponding proteins that can be easily detected through fluorescence microscopy. The F2A peptide functions properly in T. minus, with its activity remaining unaffected by the location when the gene sequences preceding and following the F2A peptide are relatively short (<1 kbp). The precise cleavage of enhanced green fluorescent protein (eGFP) has been confirmed through immunoblotting experiments, which can be readily detected using fluorescence microscopy. The utilization of the F2A peptide not only decreases the number of promoters required but also mitigates gene silencing.