High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp

Abstract

Algae have reemerged as potential next-generation feedstocks for biofuels, but strain improvement and progress in algal biology research have been limited by the lack of advanced molecular tools for most eukaryotic microalgae. Here we describe the development of an efficient transformation method for Nannochloropsis sp., a fast-growing, unicellular alga capable of accumulating large amounts of oil. Moreover, we provide additional evidence that Nannochloropsis is haploid, and we demonstrate that insertion of transformation constructs into the nuclear genome can occur by high-efficiency homologous recombination. As examples, we generated knockouts of the genes encoding nitrate reductase and nitrite reductase, resulting in strains that were unable to grow on nitrate and nitrate/nitrite, respectively. The application of homologous recombination in this industrially relevant alga has the potential to rapidly advance algal functional genomics and biotechnology.

Fig. 1.

Composition of transformation constructs. The VCP1 gene from Nannochloropsis sp. consists of a promoter (Prom), the coding region, and a 3′ untranslated region. The VCP2 locus consists of two identical coding sequences (dubbed VCP2 and VCP2′) driven by a central, bidirectional promoter element (VCP2-Prom bidirectional) and each followed by a 3′ UTR. In the C2 transformation construct, the Sh ble gene conferring resistance to zeocin is fused to the bidirectional promoter from VCP2 and the 3′ UTR from VCP1. In the NT7 cassette, the bidirectional promoter is truncated.

Fig. 2.

KO of nitrate reductase and nitrite reductase genes by HR in Nannochloropsis sp. W2J3B. Structures of NR-KO (A) and NiR-KO (B) transformation constructs (TC), wild-type (Wt) genes, and HR products. Each KO construct consists of a left flank (red) and a right flank (orange) separated by the NT7 selection marker cassette (green). A target region (purple) of each gene is replaced by the NT7 cassette when HR occurs. Primer positions (arrows) for PCR analysis and expected PCR product sizes for Wt and KO mutants are indicated.

Fig. 3.

Analysis of transformants obtained with NR-KO and NiR-KO transformation constructs. (A, B, D, and E) Sixteen randomly selected zeocin-resistant transformants obtained with each TC were resuspended in medium lacking a nitrogen source and spotted on plates containing ammonium (A and D) or nitrate (B and E) as a sole nitrogen source. Putative NR-KO and NiR-KO mutants bleach, because they cannot use nitrate as a nitrogen source. (C and F) PCR analysis of the transformants shown in B and E. Genomic DNA was amplified by PCR with the primer pairs indicated in Fig. 2. Transformants shown by PCR analysis to have undergone HR at the NR or NiR locus are marked with an asterisk in B and E, respectively.

Fig. 4.

Growth of wild-type, NR-KO (NR1 and NR2), and NiR-KO (NiR1 and NiR2) strains with different nitrogen sources. Cells in mid-log phase were washed in nitrogen-free medium, resuspended in media with the indicated nitrogen sources, and allowed to grow to early stationary phase. Results are expressed relative to the wild type in 1 mM NH₄Cl, and SDs for three independent cultures of each strain are indicated.