Boosting lipid productivity of the oleaginous microalga N. gaditana via genome engineering

Transcription

1 Boosting lipid productivity of the oleaginous microalga N. gaditana via genome engineering Imad Ajjawi, John Verruto, Moena Aqui, Eric Moellering and Robert Brown October 25th 216

2 Outline n Brief intro to the algal biofuel R&D program at Synthetic Genomics Inc. (SGI) n Identification of a novel transcriptional regulator of lipid accumulation via a transcriptomics approach n Establishment of a high-efficiency knockout pipeline in Nannochloropsis n Fine-tuning expression of the transcription factor to maximize lipid productivity n Transcriptomics and further characterization of the mutants to unveil a mechanistic understanding of the lipid phenotype 1

3 SGI s Algal Biofuel R&D Program R&D Program Developing Algae-based Biofuels R&D collaboration with ExxonMobil Competitive Advantages of Algae n 4-1x higher oil yields per acre than traditional crops n Can be grown in non-arable land on brackish/saline water n Short generation time contributes to high productivity which minimizes land utilization From: Algal Bio-oil n R&D program (~ 25 people) developing an algal biomass refinery feedstock n Lower greenhouse gas emissions as compared to petroleum diesel n Lower fresh water consumption than other biofuel options 2

4 Research on Algae has Focused on Model Organisms Our focus is to use model organisms to improve productivity n Increases in lipid productivity have been reported for C. reinhardtii, T. pseudonona and P. tricornutum however, these are model organisms with inherently low lipid productivities Favorable N. gaditana Attributes: n High lipid productivity n Easily transformed by electroporation n Small, compact genome (haploid, 3 Mb) n Attractive host for forward & reverse genetics Radakovits et al

5 Lipid accumulation at the expense of growth N-deprived Nannochloropsis gaditana Total organic Carbon TOC = Biomass proxy Fatty acid methyl esters FAME = Lipid proxy TOC (µg/ml) N -N Slow growth 5 1 Time (d) FAME (µg/ml) N High lipid +N 5 1 Time (d) n Engineering a strain that is capable of accumulating high lipid levels without compromising growth is very desirable a strain with high lipid productivity (vs accumulation) 4

6 Transcriptional Responses of N-deprived Microalgae The Lipid Trigger Hypothesis Transcription Factor n Under N-deprivation a massive cellular transcriptional response occurs suggesting that a master transcriptional regulator controls lipid accumulation aka the lipid trigger 5

7 Comparative Transcriptomics to Identify The Lipid Trigger Early time-point in the N response A FAME (µg/ml) C N RNA-seq +N 3 1 Time (h) B E FDR (-log1) N/+N Fold change (log 2 ) Transcriptomics Stats: n 363 up-regulated genes n 71 down-regulated genes n transcription factors up-regulated n 2 transcription factors down - regulated n With the assumption that transcriptional changes should precede metabolic changes, we selected the 3-hr time-point for mrna sequencing 6

8 Development of High-Throughput Knockout Pipeline All 2 down-regulated putative regulators were targeted for knockout n With the hypothesis that a negative regulator of lipid accumulation would be down-regulated, we targeted all 2 TFs for insertional knockout Ng-Cas9 + grna C HygR ZnCys () Prom HygR 2.5 Kb.5 Kb T Cas9 + D ZnCys-KO 3 Kb * 3 Kb Prom HygR Hyg resistant lines T Positive selection cassette F.5 Kb 1. Construction of a Cas9 mother Ng-Cas Transformation of 2 different transient in vitro synthesized grnas targeting the putative TFs. A hygromycin cassette was used as positive selection 3. PCR screen transformants for insertions at the targeted locus 7

9 A High-Efficiency K.O. Pipeline Was Developed Ng-Cas9 + is a highly efficient editor line n 18 of the 2 regulators targeted were successfully knocked out as confirmed by PCR, and screened for lipid productivity in a batch screen 8

10 Screening for High Lipid Accumulators Lipid and biomass batch screen A.9 BA CB.8 ZnCys-KO FAME/TOC ZnCys-KO ZnCys-KO Time (d) 8 Time (d) FAME (mg/l) D C TAG/TOC (g/g).6 TOC (mg/l) D E F ZnCys-KO Time (d) 4 (NO3) (-N).5 35 ZnCys-KO (NO3) 3 ZnCys-KO.4 n An outlier was clearly identified from the batch screen N n Several lines of evidence confirmed it was a high lipid 15.2 accumulator 1.1 Ch 5 M n However, this strain was very slow-growing 1 µm LD Ch C14: C16: C16:1 C18:1 C18:2 C2:4 C2:5 ZnCys-KO FAME (mol %) TOC (mg/l) 1 µm LD 9

11 Mutation in a locus encoding for a putative Zn 2 Cys 6 transcription factor Creation of weaker alleles by targeted UTR bashing and RNAi silencing A B Relative Normalized Expression BASH-3 65bp NLS Zn 2 Cys 6 RNAi RNAi n Generation of weaker alleles was confirmed by qrt-pcr ZnCys-KO C C FAME/TOC FAME/TOC qrt-pcr BASH-12 3bp FAME/TOC TOC productivity TOC (mg/l/day) n We hypothesized that generation of weaker alleles would result in a more moderate phenotype n A batch assay indicated that the attenuated lines had intermediate FAME/TOC levels when compared to and ZnCys-KO. n Additionally, the attenuated lines grew only slightly slower than.. 1

12 Productivity Assessment on a Semi-Continuous System Attenuated lines exhibited the highest lipid productivity values A 14 A 12 TOC Productivity FAME Productivity 6 FAME (mg/l) ZnCys-BASH-12 ZnCys-BASH-3 ZnCys-RNAi-7 Ng-Cas TOC productivity (g/m 2 /day) FAME productivity (g/m 2 /day) Time (d) C n All 3 attenuated lines showed higher lipid productivities than for over a week, with ZnCys-RNAi-7 being twice as productive as (1% lipid productivity over ) n Equally as important, biomass productivity of ZnCys-attenuated lines was only marginally lower than 11

13 Ammonium Supplementation Complements The Mutant Phenotype Growth on NH 4 an unexpected result A B C FAME (mg/l) ZnCys-KO TOC (mg/l) 8 ZnCys-KO FAME/TOC ZnCys-KO n Supplementation with NH 4 + abolished the mutant phenotype Time (d) Time (d) D TN (mg/l) N-levels on NO (NO3) RNAi-7 (NO3) ZnCys-KO ZnCys-RNAi ZnCys-KO Time (d) D Plastid NO - 3 NRT NO - 3 NR NO - 2 NAR NO - 2 NiR GS NH + 4 NH + 4 AMT NH + 4 GOGAT Gln α-kg 2Glu Glu α-kg C-metabolism GDH Time (d) 12

14 Further Investigating N-Assimilation Key genes in primary N-assimilation appear to be down-regulated C D D NRT2 NiR GOGAT1 Amt2 NR-KO (NO 3 ) ZnCys -KO RNAi-7 (NH 4 ) (NH 4 ) (NH 4 ) NR-KO (NH 4 ) ZnCys -KO (NO 3 ) RNAi-7 (NO 3 ) (NO 3 ) NR NO - NO - 3 NO - 3 NRT 2 NAR NO - 2 NH 4 + AMT NH 4 + Plastid NiR NH 4 + GS Gln α-kg Glu GOGAT α-kg 2Glu GDH Amt1 GS1 UreT NAR2 GS2 GDH GOGAT2 NAR1 NR C-metabolism n The NR-KO has a distinct response that differentiates it from all other samples n However, down-regulation of NRT2 and NiR could explain ZnCys-KO s growing pains on NO 3 Log 2 (fold change) 13

15 Transcriptomics of ZnCys Knockdown Strain Confirmed down-regulation of primary N-assimilation genes Transcriptomics Stats: n 1118 differentially expressed genes (2-fold, FDR <.5) n 79 up-regulated genes n 328 down-regulated n Protein synthesis n Lipid re-modeling Desaturases, elongases n N-assimilation n Photosynthesis n Light Harvesting Ø No changes in FAS or enzymatic steps leading to TAG biosynthesis suggesting that upregulation of these components is not required for the lipid phenotype 14

16 Transcriptomics of ZnCys Knockdown Strain Confirmed down-regulation of primary N-assimilation genes n Under semi-continuous assay conditions, most genes in primary N-assimilation appear to be down-regulated in ZnCys-RNAi-7 15

17 Transcriptomics of ZnCys Knockdown Strain No changes in FAS or enzymatic steps leading to TAG biosynthesis KAR1 KAS1 KAS3 HAD ENR ACCase CBB n Up-regulation of LDSP is consistent with its proposed role in TAG lipid droplet biogenesis n Up-regulation of desaturases and elongases may be a response to a sensed shortage in PUFAs (e.g. low C2:5 in ZnCys-RNAi-7) n Western blotting for FAS components indicated that upregulation of FAS is not required for the lipid phenotype 16

18 Transcriptomics of ZnCys Knockdown Strain Gene Ontology Analysis Over-represented Up-regulated GO categories B 1% % C in Other Biomass % C in FAMEs % C in Carbohydrate % C in Protein 75% 5% ~1% increase (lipids) 25% % ZnCys-BASH-12 ZnCys-RNAi-7 ~45% decrease (protein) n The up-regulated gene set revealed significant enrichment in components of protein synthesis, suggesting a compensatory response to the ~45 % decrease in C partitioning to protein observed in ZnCys-RNAi-7 17

19 Summary of Results Current Mechanistic Understanding Wild type ZnCys-KO ZnCys-attenuated lines ZnCys ZnCys ZnCys N-assimilation NH 4 + C-metabolism N-assimilation NH 4 + C-metabolism N-assimilation NH 4 + C-metabolism Protein ~4% Carbohydrate Lipid ~2% Protein ~5% Carbohydrate Lipid ~55% Protein ~2% Carbohydrate Lipid ~4% n We hypothesize that ZnCys positively controls N-assimilation, thus causing severe growth and protein accumulation restrictions in the knockout n Expression of ZnCys can be fine-tuned to increase C-allocation to lipid at the expense of protein 18

20 Acknowledgements 19