Artificial miRNA inhibition of phosphoenolpyruvate carboxylase increases fatty acid production in a green microalga Chlamydomonas reinhardtii

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0779-z) contains supplementary material, which is available to authorized users.

Advances and challenges in genetic engineering of microalgae

“…Overall, the results suggest that heat shock treatment induced the expression of amiRNAs, which can effectively downregulate the expression of CrPEPCs in C. reinhardtii , resulting in an increase in fatty acid synthesis with the most significant increase occurring for C16 to C22 fatty acids (Wang et al . ).…”

“…At this point, it is very important to note the work conducted by Wang et al (2017). Nutrient 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 the cells.…”

Potential and Challenges of Improving Photosynthesis in Algae

“…The conversion of phosphoenolpyruvate (PEP) to oxaloacetate by PEPC (PEP carboxylase) preferentially diverts the carbon flow towards protein synthesis, and indeed PEPC down-regulation resulted in the over-expression of pyruvate kinase, which increased the levels of pyruvate and acetyl-CoA [142]. On the other hand, knock-down of a PEPC isoform by either artificial microRNA-mediated technology and CRISPRi in Chlamydomonas, and by RNAi in P. tricornutum, resulted in higher TAG accumulation [112,143,144].…”

Molecular Genetic Tools and Emerging Synthetic Biology Strategies to Increase Cellular Oil Content in Chlamydomonas reinhardtii

“…In Chlamydomonas, inhibiting PEPC1 activity by RNAi or CRISPRi-based techniques increased the oil content by 20%, probably by enhancing the flow of carbon to FA synthesis (Deng et al 2014, Kao andNg 2017). Down-regulating both PEPC isoforms (CrPEPC1 and CrPEPC2) by amiRNA resulted in a 29% to 48% increase in total FA levels, respectively, which was suggested to occur due to the flux of carbon away from the TCA cycle, and the subsequent increase in the availability of acetyl-CoA for FA biosynthesis (Wang et al 2017). Consistent with this hypothesis, the expression levels of TAG biosynthesis-related genes, such as DGAT2 and phosphatidate phosphatase (PAP2), were increased in CrPEPC1 RNAi strains (Deng et al 2014).…”