Nannochloropsis gaditana, Master thesis

Analysis of Peroxisomal Lipid Metabolism in the Oleaginous Microalga Nannochloropsis

and Development of Synthetic Biology Tools for Genetic Engineering

Master’s thesis

by

Amit Kumar Sharma

Supervisor: Prof. Sigrun Reumann

Faculty of Science and TechnologyDepartment of Mathematics and Natural

SciencesUniversity of Stavanger

20. August 2014

Contents

• Introduction• Thesis goal• Methods• Result• Summary• Future work

Introduction

Algae• Algae are simple plants that can range from the

microscopic (microalgae), to large seaweeds (macroalgae).

• Most algae contain chlorophyll• Microalgae- cyanobacteria and green, brown and

red algae• Algae –high levels of oils, carbohydrates, sugars

and proteins• Base of aquatic food chain

Introduction

Introduction

• Grow fast• High biofuel yields• Consume CO2

• Do not compete with agriculture• Microalgal biomass can be used for fuel, feed and food • Purify wastewater

Why algae

Crop Oil yield (gallon/acre)

Corn 18

Cotton 35

Soybean 48

Mustard seed 61

Sunflower 102

Rapeseed/Canola 127

Jatropha 202

Oil palm 635

Algae

50g/m²/day at 50% triglycerides 1,200

Comparison of potential oil yields of algae. (Source: Pienkos & Darzins, 2009)

Introduction

• Unicellular• Autotrophic• Most species live in salt

water• High growth rate• Sequenced genomes for 2

species available• High lipid concentration (EPA

+ TAG) under stress conditions (Hoffmann et al., 2010)

Nannochloropsis sp.

EPA: Eicosapentaenoic acid.TAG: Triacylglycerol

Why Nannochloropsis gaditana?

Introduction

Biomass composition of N. gaditana (Source: Radakovits et al., 2012)

Comparison of N. gaditana lipid production rates with other algae (Source: Radakovits et al., 2012)

Introduction

• Modern-day algal research and industry focus production of high value algal products

• Require huge investment, algae experts, modified oleaginous strains

• Top 5 leading companies: Algenol Biofuels, Solix Biofuels, Sapphir Biofuels, Energy Solazyme.

• Biofuel scientist are engineering different strains of algae at the molecular level.

Development of Algal Industry

Introduction

• According to Fachy et. al. , “Lipids are hydrophobic or amphipathic small molecules that may originate entirely or in a part by carbanion–based condensations of thioesters and/or by carbocation-based condensations of isoprene units”.

• Algal lipids can be divided into two main groups: the non-polar lipids and the polar lipids.

Algal lipids

Introduction

• Functions: – Fatty acid β-oxidation– ROS metabolism– Photorespiration

• Two major targeting signals:– PTS1: Peroxisome Targeting

Signal 1 (C-terminal tripeptide)

– PTS2: Peroxisome Targeting Signal 2 (N-terminal nonapeptide)

Peroxisomes

Anatomy of peroxisome.UNSW Cell Biology, 2008(http://cellbiology.med.unsw.edu.au/units/science/lecture0806.htm)

Small (0.5-1 µm), single membrane-bound organelles

Introduction

• Synthetic biology is,– the design and construction of new biological

parts and systems, and– the re-design of existing, natural biological

systems for useful purposes.• Algae synthetic biology are lagging behind

compared to other model organisms• Tools for synthetic biology: cloning promoters

and reporter genes, transformation, bioinformatics.

Synthetic Biology

Predicted PTS1 proteins

Int.

#

Annotation Predicted

PTS1

Acronym Length

P2 arogenate dehydrogenase PKL> Ng_AroDH 785 aa

P3 peroxisomal glycolate oxidase SKI> Ng_GOX 250 aa

(from C-terminal)

Full length = 390 aa

P8 embryogenesis-associated protein

emb8

SRL> Ng_EAP 486 aa

P9 acyl- oxidase ARL> Ng_AOX 751 aa

Introduction

Thesis goals

1. Cloning of Full-lengths CDS or C-terminal exons of predicted PTS1 Proteins in N. gaditana.

2. Subcellular localization of predicted Nannochloropsis gaditana PTS1 proteins in onion epidermal cells.

3. Reproduction of nuclear transformation of Nannochloropsis oceanica using the vector pSELECT100.

4. Subcloning of the LDSP promoters from N. oceanica into pSELECT100 to create a 2nd expression cassette in pSELECT100.

5. Cloning of the LDSP promoter from N. gaditana into the pJET vector.

Methods

• Isolation of Genomic DNA from Nannochloropsis gaditana

• Cloning of 4 PTS1 predicted genes from N. gaditana

• Subcellular localization of predicted PTS1 proteins by fluorescence microscopy

• Genomic transformation of Nannochloropsis sp.• Cloning of inducible Nannochloropsis promoters

Approaches used

Methods

• Cloning of CDS into pJET1.2 cloning vector– 3 Full-length CDS of single exon genes;– 1 C-terminal exon.

• Sequence analysis• Sub-cloning of insert from pJET1.2 into plant

expression vector pCAT_EYFP.

Cloning of N. gaditana genes predicted to have PTS1

Methods

1. Coating gold particles with DNA– 40 ng/µl plasmid DNA– 50 µl 2.5 M CaCl2– 20 µl 0.1 M spermidine

2. Transient expression in onion epidermal cells– Biolistic transformation using a gene gun– 12 h dark incubation

3. Microscopy– Inverted fluorescence microscope (Nikon TE 2000u)

Subcellular localization of predicted PTS1 Proteins

Methods

• By electroporation

• Using pSelect100 vector– Linearized by BamHI– 2 antibiotic resistance genes:

Hygromycin B and Ampicillin

Based on the method described in Vieler et. al. (2012)

Genomic transformation of Nannochloropsis

pSELECT100 (C. Benning lab)

Methods

• Modification of pSELECT100 vector• Making an expression vector for N. oceanica• Making a cloning vector for N. gaditana

Cloning of inducible Nannochloropsis promoters

Extended pSELECT vector with 2nd expression cassette(Reumann lab)

Pre-existing pSELECT vector(Benning lab)

Results

PCR amplification of Nannochloropsis gaditana genes

• Ng_AroDH: 2355bp• Ng_GOX: 750 bp• Ng_ EAP: 1458 bp • Ng_AOX: 2253 bp

Results

Cloning of 4 N. gaditana genes into the pJET vector

• Ng_AroDH: 2355bp• Ng_GOX: 750 bp• Ng_ EAP: 1500 bp • Ng_AOX: 2253 bp

Transformed in E. Coli JM 109

Results

PCR and restriction digestion analysis of Plasmid

Plasmid analysis of inserts in pJET vector

• Ng_AroDH: 2355bp• Ng_GOX: 750 bp• Ng_ EAP: 1458 bp • Ng_AOX: 2253 bp

• Positive plasmids were sent for sequencing.• Sequencing result analyzed by blast and multiple

alingments• Resulting seqeunce was checked for PTS1

tripeptides.• Sequencing result was also checked for gene

specific primer.

Results

Sequence analysis of the insert in pJet vector

Results

• Restriction digestion of insert and vector pCAT• Ligation of digested sticky fragments• Analysis of colonies by colonies PCR• Analysis of plasmids by PCR and digestion

Sub-cloning of CDS/C-terminal exons in the back of EYFP in EYFP/pCAT vector

Analytical digestion of the destination vector EYFP-DECR/ pCAT

pCAT_EYFP_DECR: 4200 bpDECR: 1000 bp

Results

E coli colonies transformed with EYFP-Ng_EAP/pCAT on LB ampicillin platesDigestion and PCR analysis of plasmids, EYFP-Ng_EAP/pCAT and EYFP-Ng_GOX/pCAT

E coli colonies transformed with EYFP-Ng_EAP/pCAT on LB ampicillin plates

• Ng_GOX: 750 bp• Ng_ EAP: 1458 bp

Subcellular localization studies by transient expression in onion cells

Experimental validation of predicted PTS1 by in vivo subcellular targeting analysis via transient expression in onion cells

Results

Int.

#

Annotation Predicte

d PTS1

Acronym Length In vivo

subcellular

targeting

P2 Arogenate

dehydrogenase

PKL> Ng_AroDH 785 aa Plasmid not obtained

P3 Peroxisomal glycolate

oxidase

SKI> Ng_GOX 390 aa Peroxisomes

P8 embryogenesis-

associated protein emb8

SRL> Ng_EAP 486 aa Peroxisomes

P9 acyl- oxidase ARL> Ng_AOX 751 aa Peroxisomes

Subcellular localization of selected Nannochloropsis gaditana proteins carrying predicted PTS1 tripeptides

Result

Genomic transformation of N. oceanica CCMP1779

27

½ salinity f/2 agar plates containing 50 µg/ml Hygromycin B

N. Oceanica Negative controlPositive control

Results

• The pSELECT plasmid obtained from Prof. Dr. C. Benning contains only a single expression cassette.

• 2nd expression cassette: For subcellular localization studies (EYFP fusions) and to overexpress genes of interest in N. oceanica.

Subcloning of inducible Nannochloropsis promoters

Results

• Amplification of lipid droplet surface proteins from pSELECT 100 and N. gaditana

• Cloning of PCR product into pJET Vector

• Sub-cloning of the LDSP promoter from pJET into pSELECT100

Subcloning of inducible Nannochloropsis promoters

Preparative PCR to amplify LDSP promoter from pSELECT100 and N. gaditana

PRO_Noce_LDSP: 750 bpPRO_Nagd_LDSP: 1000 bp

Results

E. coli transformed with PRO_Ngad_LDSP/pJET (B) and PRO_Noce_LDSP/pJET (A) colonies on LB ampicillin plates

Plasmids analysis of the insert in pJET vector

PRO_Noce_LDSP: 750 bpPRO_Nagd_LDSP: 1000 bp

Subcloning of inducible Nannochloropsis promoters

Plasmid analysis of PRO_Noce_LDSP/pSELECT10E. coli transformed with PRO_Noce_LDSP/pSELECT100 colonies on LB ampicillin plates

Subcloning of inducible Nannochloropsis promoters

Manual analysis searching was done for the KpnI site of the forward subcloning primer (GTAGGTACC GAGGTCC TGTTTGGATT TTTTGCT) and the ClaI site of the reverse primer (CAGAAACTCT ATCTCAAGAT AAG ATCGATGCA):

Result

Sequencing analysis of PRO_Noce_LDSP/pSELECT100

Electropherogram read using Finch TV program

Summary

• 3 predicted PTS1 proteins of interest from Nannochloropsis gaditana were sub-cloned into pJET1.2:Ng_GOX, Ng_EAP and Ng_AOX was successful while Ng_ADH was not successful.

• Cloned genes were sub-cloned into the plant expression vector pCAT.

• The 3 predicted PTS1 proteins were localized to peroxisomes in onion epidermal cells.

• Reproduction of nuclear transformation of Nannochloropsis was successfully performed

• The LDSP promoter from pSELECT was cloned into pJET vector and sub-cloned into MCS of pSELECT100

• LDSP from Nannochloropsis gaditana successfully cloned into pJET1.2

Future work

1. Sub clone N. gaditana LDSP Promoter from pJET1.2 into pSELECT

2. Reproduction of nuclear transformation of N. gaditana with plasmids from Posewitz (plasmids obtained from the Posewitz lab)

3. Further construction of 2nd expression cassette in pSELECT

Acknowledgements

• Prof. S. Reumann• Manish Budathoki• Dmitry Kechasov • Dr. G. Chowdhary• Eli Drange Vee• All lab members

• University of Stavanger