Lipid production in nannochloropsis gaditana is doubled by decreasing expression of a single transcriptional regulator

ABSTRACT 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. Access through your

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CHARACTERIZATION AND RNA-SEQ TRANSCRIPTOMIC ANALYSIS OF A _SCENEDESMUS OBLIQNUS_ MUTANT WITH ENHANCED PHOTOSYNTHESIS EFFICIENCY AND LIPID PRODUCTIVITY Article Open access 03 June 2021 IRON

RESCUES GLUCOSE-MEDIATED PHOTOSYNTHESIS REPRESSION DURING LIPID ACCUMULATION IN THE GREEN ALGA _CHROMOCHLORIS ZOFINGIENSIS_ Article Open access 18 July 2024 A GUANIDINE-DEGRADING ENZYME

CONTROLS GENOMIC STABILITY OF ETHYLENE-PRODUCING CYANOBACTERIA Article Open access 26 August 2021 ACCESSION CODES PRIMARY ACCESSIONS SEQUENCE READ ARCHIVE * SRP096211 REFERENCES * Goncalves,

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Littler, D.S.) 349–375 (Cambridge University Press, 1985). Download references ACKNOWLEDGEMENTS 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. AUTHOR INFORMATION Author notes * Tom J Carlson: Deceased. AUTHORS

AND AFFILIATIONS * Synthetic Genomics Inc., La Jolla, California, USA Imad Ajjawi, John Verruto, Moena Aqui, Leah B Soriaga, Jennifer Coppersmith, Kathleen Kwok, Luke Peach, Elizabeth

Orchard, Ryan Kalb, Weidong Xu, Tom J Carlson, Kristie Francis, Katie Konigsfeld, Judit Bartalis, Andrew Schultz, William Lambert, Ariel S Schwartz, Robert Brown & Eric R Moellering

Authors * Imad Ajjawi View author publications You can also search for this author inPubMed Google Scholar * John Verruto View author publications You can also search for this author

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Francis View author publications You can also search for this author inPubMed Google Scholar * Katie Konigsfeld View author publications You can also search for this author inPubMed Google

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inPubMed Google Scholar * William Lambert View author publications You can also search for this author inPubMed Google Scholar * Ariel S Schwartz View author publications You can also search

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can also search for this author inPubMed Google Scholar CONTRIBUTIONS I.A. and E.R.M. conceived the study and designed experiments. R.B. provided technical advice. E.O. and R.K. designed the

productivity assays. L.B.S. and A.S.S. performed computational and bioinformatics analyses. M.A., J.V., J.C., L.P., J.B., A.S., W.X., T.J.C., K.F., W.L., K. Kwok and K. Konigsfeld performed

the experiments. I.A. and E.R.M. wrote the manuscript with support from all authors. CORRESPONDING AUTHORS Correspondence to Imad Ajjawi or Eric R Moellering. ETHICS DECLARATIONS COMPETING

INTERESTS 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 SUPPLEMENTARY FIGURE 1 TAG INDUCTION OF _ZNCYS_-KO FOR CULTURES GROWN IN

BATCH MODE ON SM-NO3−. 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.

SUPPLEMENTARY FIGURE 6 DOSE-DEPENDENT EFFECT OF CYCLOHEXIMIDE TREATMENT ON FAME/TOC. 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. SUPPLEMENTARY FIGURE 7 DIEL LIGHT PROFILES USED IN THIS STUDY. Incident irradiance profiles for batch growth assessment (A), and the

Semi-Continuous Productivity Assay (B). SUPPLEMENTARY FIGURE 8 DIAGRAMS OF VECTOR CONSTRUCTS USED IN THIS STUDY. 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. SUPPLEMENTARY INFORMATION SUPPLEMENTARY TEXT AND FIGURES Supplementary Figures 1–9, Supplementary Tables 1–9 and Supplementary Notes 1–3

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is doubled by decreasing expression of a single transcriptional regulator. _Nat Biotechnol_ 35, 647–652 (2017). https://doi.org/10.1038/nbt.3865 Download citation * Received: 08 November

2016 * Accepted: 05 April 2017 * Published: 19 June 2017 * Issue Date: July 2017 * DOI: https://doi.org/10.1038/nbt.3865 SHARE THIS ARTICLE Anyone you share the following link with will be

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