Lipid production in the industrial microalga Nannochloropsis gaditana exceeds that of model algal species and can be maximized by nutrient starvation in batch culture. However, starvation halts growth, thereby decreasing productivity. Efforts to engineer N. gaditana strains that can accumulate biomass and overproduce lipids have previously met with little success. We identified 20 transcription factors as putative negative regulators of lipid production by using RNA-seq analysis of N. gaditana during nitrogen deprivation. Application of a CRISPR–Cas9 reverse-genetics pipeline enabled insertional mutagenesis of 18 of these 20 transcription factors. Knocking out a homolog of fungal Zn(II)2Cys6-encoding genes improved partitioning of total carbon to lipids from 20% (wild type) to 40–55% (mutant) in nutrient-replete conditions. Knockout mutants grew poorly, but attenuation of Zn(II)2Cys6 expression yielded strains producing twice as much lipid (∼5.0 g m−2 d−1) as that in the wild type (∼2.5 g m−2 d−1) under semicontinuous growth conditions and had little effect on growth.
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This work was funded by ExxonMobil and Synthetic Genomics, Inc. We thank A. Withrow (Center for Advanced Microscopy at Michigan State University) for producing the TEM images and C. Packard, B. Scherer, E. Wang and the rest of the analytical team at SGI for processing FAME and TOC samples. This work is dedicated to our colleague Tom Carlson, who passed away during the preparation of this manuscript.
I.A., J.V., M.A., J.C., K. Kwok, L.P., E.O., R.K., K. Konigsfeld, A.S., W.L., R.B. and E.R.M. are employees of Synthetic Genomics, Inc. Synthetic genomics has filed patents related to this work, with I.A., J.V., M.A., L.B.S. and E.R.M. listed as inventors.
Integrated supplementary information
A) FAME/TOC comparison of knockout lines for 18 –N down-regulated transcription regulators (see Supplementary Table 1 for full gene annotation information). FAME/TOC values represent the average and standard deviation of 3 time-points from biological duplicates during batch growth. The knockout line in gene Naga_100104g1g (referred to as ZnCys-KO) selected for further study is shown in red. B) Confirmation of the increased FAME/TOC ratio in a second Cas9-mediated ZnCys-KO line 2; the ZnCys-KO line 1 was used in the remainder of the study and is referred to as ZnCys-KO. C) FAME (mg/L) and D) TOC values (mg/L) of ZnCys-KO and WT grown in batch mode on SM-NO3- (n=2) E) FAME profiles (as mol % of total FAME) showing most abundant fatty acid species for ZnCys-KO in N-replete conditions and WT in +N and -N. C# indicates the fatty acid carbon chain length and:# indicates the number of double bonds. F) TAG content normalized to TOC (g/g) as determined by LC-MS. See Materials and Methods for further details on growth conditions.
Supplementary Figure 2 Initial batch-mode assessment of ZnCys-attenuated lines (ZnCys-BASH-3, ZnCys-BASH-12 and ZnCys-RNAi-7) grown in nitrate-replete medium (SM-NO3−).
A) FAME (mg/L) and B) TOC (mg/L) measurements corresponding to days 3, 5 and 7 of the screen. TOC productivity values displayed in Fig 2C were derived from these values.
Supplementary Figure 3 Productivity assessment of ZnCys-KO and ZnCys-RNAi-7 grown in semicontinuous mode on nitrate-rich medium (SM-NO3−).
Daily (A) FAME (mg/L), (B) TOC (mg/L), and (C) C/N values derived from cellular N-content. (D) FAME and E) TOC productivities (g/m2/day) for WT and ZnCys-RNAi-7 calculated for the entire 13-day assay. ZnCys-KO failed to reach steady-state at a 30 % daily dilution scheme and essentially washed away as the run progressed, therefore lipid and biomass productivity values were not calculated for this line (N/A, not available). Due to the severe growth defect of ZnCys-KO on medium containing nitrate as the sole N source, this strain was scaled up in SM-NH4+/NO3- to obtain enough biomass for the assay, but grown on SM-NO3- medium for the duration of semi-continuous productivity assessment.
Supplementary Figure 4 Productivity assessment of ZnCys mutants grown in semicontinuous mode for 8 d on NO3−-rich medium (SM-NO3−).
A) Daily FAME and B) TOC (mg/L) measurements for ZnCys mutants (ZnCys-RNAi-7, ZnCys-BASH-12 and ZnCys-BASH-3) compared to their parental lines Ng-CAS9+ and WT. Productivity values displayed in Fig 3A were derived from these values. Error bars represent standard deviations for 3 biological replicates (n=3). See Materials and Methods section for a detailed description on the assay and productivity calculations. C) Cell counts for various strains. Shown is the average and standard deviation of biological triplicate cultures for three consecutive days under semi-continuous growth (N = 9; corresponding to days 6 through 8 in Figure S4A).
Supplementary Figure 5 Repression of the lipid-accumulation phenotype of ZnCys-KO by NH4+ supplementation.
Daily A) FAME (mg/L), B) TOC (mg/L) and C) FAME/TOC values of ZnCys-KO and WT grown in batch mode on medium supplemented with NH4+ (SM-NH4+/NO3-). Error bars are standard deviations of 2 biological replicates.
Cultures were grown as in Fig. 4, and treated with the indicated amount of cycloheximide at 0 h. FAME/TOC measurements were taken 48 h after treatment.
Incident irradiance profiles for batch growth assessment (A), and the Semi-Continuous Productivity Assay (B).
Vector used to generate the Nannochloropsis Cas9 expression strain Ng-Cas9+ (A), and the hygromycin resistance cassette used for generating Cas9-mediated insertional mutants in the Ng-Cas9+ background (B). The coding sequences (yellow) for BSD (blasticidin deaminase), Cas9, and GFP, are driven by the TCT_P, RPL24_P, and 4AIII_P endogenous promoters, and terminated by the EIF3_T, FRD_T, and GNPDA_T endogenous terminator sequences, respectively. See Supplementary Table 7 for a further description of gene sources for promoter and terminator elements.
Supplementary Figure 9 Selection of the Ng-CAS9+ editor line and validation of transgenic Cas9 protein expression.
(A) Flow cytometry histogram (using the Accuri c6 cytometer) showing fluorescence in the FL1-A channel to detect GFP fluorescence. Wild-type fluorescence is shown in the black trace, whereas Ng-CAS9+ is shown in red. Histograms are drawn in the Accuri c6 Sampler software using data collected from 50,000 events. (B) Western blot detection of transgenic Cas9 protein in the Ng-CAS9+ editor line.
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Ajjawi, I., Verruto, J., Aqui, M. et al. Lipid production in Nannochloropsis gaditana is doubled by decreasing expression of a single transcriptional regulator. Nat Biotechnol 35, 647–652 (2017). https://doi.org/10.1038/nbt.3865
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