RNAi‐based targeted gene knockdown in the model oleaginous microalgae Nannochloropsis oceanica

Abstract

Microalgae are promising feedstock for renewable fuels such as biodiesel, yet development of industrial oleaginous strains has been hindered by the paucity and inefficiency of reverse genetics tools. Here we established an efficient RNAi-based targeted gene-knockdown method for Nannochloropsis spp., which are emerging model organisms for industrial microalgal oil production. The method achieved a 40-80% success rate in Nannochloropsis oceanica strain IMET1. When transcript level of one carbonic anhydrase (CA) was inhibited by 62-83% via RNAi, mutant cells exhibited photosynthetic oxygen evolution (POE) rates that were 68-100% higher than wild-type (WT) at pH 6.0, equivalent to WT at pH 8.2, yet 39-45% lower than WT at pH 9.0. Moreover, the mutant POE rates were negatively correlated with the increase of culture pH, an exact opposite of WT. Thus, a dynamic carbon concentration mechanism (CCM) that is highly sensitive to pH homeostasis was revealed, where the CA inhibition likely partially abrogated the mechanism that normally deactivates CCM under a high level of dissolved CO . Extension of the method to another sequenced N. oceanica strain of CCMP 1779 demonstrated comparable performance. Finally, McrBC-PCR followed by bisulfite sequencing revealed that the gene knockdown is mediated by the CG, CHG and CHH types of DNA methylation at the coding region of the targeted gene. The efficiency, robustness and general applicability of this reverse genetics approach suggested the possibility of large-scale RNAi-based gene function screening in industrial microalgae.

The carbonic anhydrase CAH1 is an essential component of the carbon-concentrating mechanism in Nannochloropsis oceanica

“…Building a working model for CCM function in this organism would be aided by a complete list of possible carbonic anhydrases, so we independently searched for orthologs belonging to each of the families using blastp and tblastn. We identified two possible γ-type enzymes (SI Appendix, Table S1), corroborating recent findings reported by Wei et al (33); however, when aligned to known γ-type carbonic anhydrases, not all of the conserved residues typically necessary for activity were present (SI Appendix, Fig. S2).…”

“…Knockdown lines of the β-type carbonic anhydrase (11263-mRNA in CCMP1779) showed no defect in growth at ambient CO 2 , whereas experiments with RNAi lines exhibiting reduced transcript levels of a γ-type carbonic anhydrase (g2209 in the N. oceanica strain IMET1) indicate a possible role in repressing the CCM under low pH (33). The strong phenotype of the cah1 mutant (Fig.…”

“…Along with CAH1 (α type), there are at least two γ-type and two β-type carbonic anhydrases in N. oceanica CCMP1779 (SI Appendix, Table S1) (33). Knockdown lines of the β-type carbonic anhydrase (11263-mRNA in CCMP1779) showed no defect in growth at ambient CO 2 , whereas experiments with RNAi lines exhibiting reduced transcript levels of a γ-type carbonic anhydrase (g2209 in the N. oceanica strain IMET1) indicate a possible role in repressing the CCM under low pH (33).…”

A type-I diacylglycerol acyltransferase modulates triacylglycerol biosynthesis and fatty acid composition in the oleaginous microalga, Nannochloropsis oceanica

“…The RNAi vector construction for NoDGAT1A knockdown followed the procedures described by Wei et al [32], and the resulting vector is depicted in Additional file 1: Figure S16b. For the overexpression vector, NoDGAT1A coding sequence was driven by the Nannochloropsis ubiquitin extension protein promoter (Additional file 1: Figure S16c).…”

“…Nannochloropsis oceanica has been recognized as an emerging model oleaginous alga in the study of TAG metabolism because of its fast growth, high TAG content, available genome sequence, and established genetic tools [20, 23, 29–32]. Thirteen putative DGAT-encoding genes were identified in the genomes of two N. oceanica strains: IMET1 [23] and CCMP1779 [20].…”