Abstract
BackgroundNutrient limitation, such as nitrogen depletion, is the most widely used method for improving microalgae fatty acid production; however, these harsh conditions also inhibit algal growth significantly and even kill cells at all. To avoid these problems, we used artificial microRNA (amiRNA) technology as a useful tool to manipulate metabolic pathways to increase fatty acid contents effectively in the green microalga Chlamydomonas reinhardtii. We down-regulated the expression of phosphoenolpyruvate carboxylase (PEPC), which catalyzes the formation of oxaloacetate from phosphoenolpyruvate and regulates carbon flux.ResultsamiRNAs against two CrPEPC genes were designed and transformed into Chlamydomonas cells and amiRNAs were induced by heat shock treatment. The transcription levels of amiRNAs increased 16–28 times, resulting in the remarkable decreases of the expression of CrPEPCs. In the end, inhibiting the expression of the CrPEPC genes dramatically increased the total fatty acid content in the transgenic algae by 28.7–48.6%, which mostly increased the content of C16–C22 fatty acids. Furthermore, the highest content was that of C18:3n3 with an average increase of 35.75%, while C20–C22 fatty acid content significantly increased by 85–160%.ConclusionsOverall our results suggest that heat shock treatment induced the expression of amiRNAs, which can effectively down-regulate the expression of CrPEPCs in C. reinhardtii, resulting in an increase of fatty acid synthesis with the most significant increase occurring for C16 to C22 fatty acids.
Highlights
Nutrient limitation, such as nitrogen depletion, is the most widely used method for improving microalgae fatty acid production; these harsh conditions inhibit algal growth significantly and even kill cells at all
Mutant strains with the names such as aRP1.1 and aRP1.2 were derived from individual amicroRNA-PEPC1 transformants, and aRP2.1, aRP2.2, and aRP2.3 were from amicroRNA-PEPC2 transformants
The low contents of long-chain fatty acids such as C20:3, C22:2, and C22:6 were observed, they significantly increased from 85 to 160% according to GC– MS after 3 × heat shock (HS) (Table 3). These results suggest that HS treatment to induce the expression of artificial microRNA (amiRNA) can effectively down-regulate CrPEPC genes’ expression in C. reinhardtii, resulting in an increase of fatty acid synthesis with the most significant increase occurring for C16 to C22 fatty acids
Summary
Nutrient limitation, such as nitrogen depletion, is the most widely used method for improving microalgae fatty acid production; these harsh conditions inhibit algal growth significantly and even kill cells at all. To avoid these problems, we used artificial microRNA (amiRNA) technology as a useful tool to manipulate metabolic pathways to increase fatty acid contents effectively in the green microalga Chlamydomonas reinhardtii. The expression of acetyl coenzyme A carboxylase (ACCase) and fatty acid synthase (FAS) from higher plants in microalgae did not achieve the aim of dramatically increasing the oil content [9, 10]. Previous studies showed that there was a negative correlation between the activities of phosphoenolpyruvate carboxylase (PEPC), a key enzyme of the amino acid metabolic pathway, and lipid accumulation because of sharing its ACCase with a common substrate, pyruvate [11, 12].
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