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High-Efficiency Homologous Recombination in the Oil High-efficiency homologous recombination in the SEE COMMENTARY oil-producing alga Nannochloropsis sp. Oliver Kiliana, Christina S. E. Benemanna, Krishna K. Niyogib,1, and Bertrand Vicka,1 aAurora Algae, Inc., Hayward, CA 94545; and bDepartment of Plant and Microbial Biology, University of California, Berkeley, CA 94720-3102 Edited by Elisabeth Gantt, University of Maryland, College Park, MD, and approved November 1, 2011 (received for review April 12, 2011) Algae have reemerged as potential next-generation feedstocks for netic manipulation and systematic functional analysis of its genome biofuels, but strain improvement and progress in algal biology (23) and has led to pioneering discoveries in eukaryotic biology. research have been limited by the lack of advanced molecular Although it resembles green algae of the genus Chlorella (24), tools for most eukaryotic microalgae. Here we describe the Nannochloropsis sp. is a eustigmatophyte that is related to dia- development of an efficient transformation method for Nanno- toms and brown algae (25). Like Chlorella but unlike Chlamy- chloropsis sp., a fast-growing, unicellular alga capable of accumu- domonas, Nannochloropsis is a robust industrial alga that has lating large amounts of oil. Moreover, we provide additional been extensively grown in outdoor ponds and photobioreactors evidence that Nannochloropsis is haploid, and we demonstrate for aquaculture (26, 27). Under nitrogen-starvation conditions, that insertion of transformation constructs into the nuclear ge- Nannochloropsis can accumulate oil exceeding 60% of its biomass nome can occur by high-efficiency homologous recombination. on an ash-free dry weight basis (27), making it an excellent can- As examples, we generated knockouts of the genes encoding ni- didate for biofuel production. In addition, Nannochloropsis is a trate reductase and nitrite reductase, resulting in strains that were rich source of high-quality protein (28) and eicosapentaenoic acid unable to grow on nitrate and nitrate/nitrite, respectively. The (29), an omega-3 fatty acid with numerous health benefits (30). application of homologous recombination in this industrially rele- Here we describe the development of a highly efficient trans- vant alga has the potential to rapidly advance algal functional formation system for Nannochloropsis. Moreover, we demon- genomics and biotechnology. strate that Nannochloropsis sp. exhibits very efficient HR, allowing us to reliably and rapidly perform targeted genetic manipulation esearch on eukaryotic algae has provided fundamental of this organism. These findings open the door for systematic Rinsights into many basic cellular processes, particularly pho- functional genomics and biotechnological improvement of this tosynthesis. Recently, algae have reemerged as potential next- industrial alga. generation feedstocks for advanced biofuels, such as biodiesel and other hydrocarbons (1–4), and have attracted considerable Results interest from both the private and public sectors. Many algae have Development of an Efficient Transformation System for Nannochloropsis. high photoautotrophic growth rates and can accumulate more Nannochloropsis sp. (strain W2J3B) grows rapidly on solid or than half their dry weight biomass as lipids (5), including tri- liquid media, with a doubling time of ∼14 h under laboratory acylglycerol and a number of high-value pharmaceutical and conditions (Materials and Methods). To establish a transfor- nutraceutical products (6). Compared with agricultural plants, mation system for Nannochloropsis sp., we used endogenous algae do not require arable land, and many species can be grown promoters and 3′ untranslated regions (3′ UTRs) from two un- using wastewater or salt water. However, because unimproved linked violaxanthin/chlorophyll a-binding protein (VCP) genes, algae are unlikely to possess all of the traits necessary for eco- VCP1 and VCP2 (Fig. 1). The protein encoded by the VCP1 gene nomic production of biofuels (1), robust molecular biology tools is nearly identical to a previously characterized light-harvesting are required for strain optimization (7). antenna protein from Nannochloropsis sp. (31). The VCP2 locus Eukaryotic algae comprise a diverse, polyphyletic group of or- consists of two identical copies of the VCP2 gene driven by a ganisms with members in four of the five eukaryotic supergroups bidirectional promoter (Fig. 1, SI Materials and Methods, and (8). One supergroup, the Plantae, includes rhodophytes (red al- Fig. S1). Using transformation constructs containing the Sh ble gae), glaucophytes, chlorophytes (green algae), and plants, which selectable marker gene, which confers resistance to zeocin (32), all contain plastids derived from primary endosymbiosis of a cya- we developed and optimized an efficient transformation method nobacterium (9). Photosynthetic plastids have been transferred to based on electroporation of intact Nannochloropsis sp. cells (SI other eukaryotic supergroups through secondary endosymbiosis Materials and Methods and Fig. S1). The highest transformation of green algae and red algae. For example, chromalveolate algae efficiency (∼2,500 transformants/μg of DNA) was obtained using such as diatoms, brown algae, eustigmatophytes, and most dino- flagellates contain a plastid derived from a red alga (10). The green alga Chlamydomonas reinhardtii is currently the Author contributions: O.K., K.K.N., and B.V. designed research; O.K. and C.S.E.B. per- eukaryotic model alga of choice, because of its genetic tractability formed research; O.K., K.K.N., and B.V. analyzed data; and O.K., K.K.N., and B.V. wrote (11), array of molecular tools (12, 13), and comprehensively an- the paper. notated nuclear genome sequence (14). Genome sequences of Conflict of interest statement: O.K., C.S.E.B., and B.V. are employees of Aurora Algae, Inc. K.K.N. is a member of the Scientific Advisory Board and has served as a consultant for several algae of ecological, economic, and/or phylogenetic sig- Aurora Algae, Inc. Patent applications have been filed for some of the technology dis- nificance have been determined recently (15). Transformation closed in this publication. methods have been developed for several algal species, most This article is a PNAS Direct Submission. notably the model diatom Phaeodactylum tricornutum (16, 17); Freely available online through the PNAS open access option. however, the molecular genetic tools available for these species Data deposition: The sequences reported in this paper have been deposited in the Gen- are generally quite limited in comparison with Chlamydomonas. Bank database (accession nos. JF957601, JF946490, JF946488, and JF946489). “ ” Although Chlamydomonas is often regarded as the green yeast See Commentary on page 20859. fi (18, 19), the lack of ef cient homologous recombination (HR) in 1To whom correspondence may be addressed. E-mail: [email protected] or bvick@ the nuclear genome of this alga (20–22) has been a major limita- aurorainc.com. tion for algal biology research. By contrast, the implementation of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. PLANT BIOLOGY HR in the yeast Saccharomyces cerevisiae has enabled precise ge- 1073/pnas.1105861108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1105861108 PNAS | December 27, 2011 | vol. 108 | no. 52 | 21265–21269 Downloaded by guest on September 30, 2021 Gene Knockouts by Homologous Recombination. To test for the occurrence and frequency of HR in Nannochloropsis sp.,we designed gene knockout (KO) constructs based on a modified zeocin-resistance cassette (NT7) with a truncated VCP2 pro- moter (Fig. 1). Nannochloropsis sp. is able to grow on medium containing nitrate, nitrite, or ammonium as the sole nitrogen source, and we chose the nitrate reductase (NR) and nitrite re- ductase (NiR) genes as targets for HR, because successful dis- ruption of these genes can easily be detected by growth on different nitrogen sources. Within the algal cell, nitrate is first reduced to nitrite by the action of NR and then further reduced Fig. 1. Composition of transformation constructs. The VCP1 gene from to ammonia by NiR. Thus, cells lacking NR can grow on nitrite Nannochloropsis sp. consists of a promoter (Prom), the coding region, and but not nitrate, whereas cells without NiR are not able to use a3′ untranslated region. The VCP2 locus consists of two identical coding nitrate or nitrite. Ammonia assimilation does not require NR or sequences (dubbed VCP2 and VCP2′) driven by a central, bidirectional pro- NiR, and thus cells with a defective NR or NiR gene can be moter element (VCP2-Prom bidirectional) and each followed by a 3′ UTR. In grown on ammonium as a nitrogen source. the C2 transformation construct, the Sh ble gene conferring resistance to We performed transformation with KO constructs containing ′ zeocin is fused to the bidirectional promoter from VCP2 and the 3 UTR from ∼1-kb flanking sequences that target the NR (Fig. 2A) and NiR VCP1. In the NT7 cassette, the bidirectional promoter is truncated. (Fig. 2B) genes. KO constructs were designed to replace 242 bp of the NR gene encoding a part of the molybdenum cofactor- a very high electric field strength within the electroporation cu- binding domain (cd02112) or 793 bp of the NiR gene encoding vette (∼11,000 V/cm field strength, 50 μF capacitance, and 500 the terminal nitrite/sulfite reductase ferredoxin-like half-domain Ohm shunt resistance; Materials and Methods, SI Materials and (pfam03460) with the NT7 selection marker
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