Abstract
Transgenic microalgae have the potential to impact many diverse biotechnological industries including energy, human and animal nutrition, pharmaceuticals, health and beauty, and specialty chemicals. However, major obstacles to sophisticated genetic and metabolic engineering in algae have been the lack of well-characterized transformation vectors to direct engineered gene products to specific subcellular locations, and the inability to robustly express multiple nuclear-encoded transgenes within a single cell. Here we validate a set of genetic tools that enable protein targeting to distinct subcellular locations, and present two complementary methods for multigene engineering in the eukaryotic green microalga Chlamydomonas reinhardtii. The tools described here will enable advanced metabolic and genetic engineering to promote microalgae biotechnology and product commercialization.
Highlights
This strategy allowed for the selection of transgenic lines that efficiently express the transgene-of-interest, and this robust expression remains for many generations
We describe vectors that enable protein targeting to four important organelles: the nucleus, mitochondria, endoplasmic reticulum (ER), and chloroplast (Table 1)
To generate a mitochondria-targeting vector, the Nterminal mitochondria transit sequence (MTS) from the nuclear gene encoding the alpha subunit of the mitochondrial ATP synthase located in the mitochondrial matrix, was fused between ble-2A and mCherry (Figure 1B)
Summary
Microalgae have recently attracted attention as potential lowcost platform for the production of a broad range of commercial products including biofuels, nutraceuticals, therapeutics, industrial chemicals and animal feeds [1,2,3,4,5,6,7,8,9,10,11]; and genome engineering will enable and enhance algae-produced bio-products [1,5,6,12,13,14,15,16,17,18,19]. It is believed that the FMDV 2A sequence ‘‘self-cleaves’’ through ribosome-skipping during translation rather than a proteolytic reaction, and has been termed CHYSEL (cis-acting hydrolase element) [25,26]. This strategy allowed for the selection of transgenic lines that efficiently express the transgene-of-interest, and this robust expression remains for many generations. We demonstrated the utility of our pBle-2A vector with the expression and secretion of the valuable industrial enzyme, xylanase [23] This expression strategy enabled, for the first time, the robust expression of six fluorescent proteins (FPs) in the cytosol of green microalgae [24]. FPs have become essential research tools that have revolutionized many fields of biology
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