Genetic Engineering of H.

pluvialis by Nuclear and

Chloroplast Genome

Transformation

Revital S.G., Edo M., Stefan L., Aliza Z. and Sammy B.

16/09/2013 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation

Potential

16/09/2013 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 2

Haematococcus pluvialis (H. pluvialis) accumulate up to 4%

astaxanthin on a dry weight basis (Boussiba, 2000).

Limitations:

Low growth rate

Environmental stresses

These limitations might be mitigated, by genetic engineering

Goals and Achievements

16/09/2013 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 3

Nuclear:

pBS-PDS S/L

pBS-PDS S/L-ble 5’

pBS-PDS S/L-ble 3’

PDS S/L

PDS S/L-ble 5’

Chloroplast:

pUC-atpX-aadA-16S-23S

We have developed nuclear and chloroplast transformation

systems for genetic engineering of H. pluvialis.

Circular

Linear

Nuclear vectors introduced

16/09/2013 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 4

Transformed colonies

16/09/2013 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 5

Control pBS-PDS S pPlat-PDS

Transformed cultures

16/09/2013 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 6

Optimal conditions

HL

-N

PDS gene is present in several pBS-PDS

/linear PDS- transformed colonies

16/09/2013 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 7

1-3- pBS-PDS-S 14-pBS-PDS S

4-6- pBS-PDS-L 15-pBS-PDS L

7-9- PDS-S linear 16,17 - pBS-PDS-S/L+13

10-12- PDS-L linear 18-no DNA

13- H. pluvialis gDNA 19-spontaneous resistant

colony

M– marker

colo

nie

s

Mutation site as sequenced from

transformed colonies

16/09/2013 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 8

Polymorphism in PDS introns indicate

successful transformation

16/09/2013 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 9

pBS-PDS as a versatile tool for nuclear

transformation

16/09/2013 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 10

PDS* cassette serves as an efficient selection marker for

nuclear genome transformation.

linear and circular DNA are inserted efficiently.

Short and long promoter versions are equally effective.

Insertion of additional gene

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(psaD-ble cassette was driven from the pGenD-ble of Fischer and Rochaix, 2001)

ble gene is present in transformed colonies

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1-5- pBS-PDS-L-ble 3’ 18, 19- pBS-PDS-S-ble 5’/3’, respectively

6-8- pBS-PDS-S-ble 5’ 20, 21- pBS-PDS-L- ble 5’/3’, respectively

9-11- pBS-PDS-S-ble 3’ 22-H. pluvialis gDNA

12, 13- PDS-S-ble 5’ 23-no DNA

14-17- PDS-L-ble 5 M– marker

co

lonie

s

Insertion of ble via pBS-PDS-ble

16/09/2013 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 13

linear PDS-ble is sufficient for transformation.

Ble cassette was incorporated efficiently both at the 5’

and 3’ end of the PDS cassette in pBS-PDS.

PDS-ble-transformed colonies grew slower than PDS

transformed colonies. This may be due to the

heterologous nature of ble or to an inhibitory effect of ble

itself.

pBS-PDS is suitable for the insertion of two or more

transgenes.

Vector for Chloroplast Transformation

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pUC-atpX-aadA was kindly provided by (Goldschmidt-Clermont et al., 1991) based on the method presented in

(Gutiérrez et al., 2012).

aadA gene is present in transformed colonies

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1-4- pUC-atpX-aadA-16S-23S-transformed colonies

5– H. pluvialis gDNA

6- pUC-atpX-aadA-16S-23S

7- H. pluvialis gDNA+pUC-atpX-aadA-16S-23S

8– no DNA

M– marker

pUC-atpX-aadA-16S-23S is suitable for

chloroplast transformation

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The aadA cassette serves as efficient selection marker for

chloroplast transformation in H. pluvialis.

Insertion into other locations of the chloroplast genome

should be tested.

Other marker genes for chloroplast transformation

should be tested.

Transformation results

16/09/2013 Genetic Engineering of H. pluvialis by Chloroplast and Nuclear Genome Transformation 17

Vector

introduced

Bombarded

cells (*106)

Resistant

colonies*

Frequency of

resistant cells (*10-6)

Average (per plate)

+Standard Error

No DNA 3 1 0.33 0.5±0.25

pBS-PDS S 6 94 15.7 23.5±3.9

pBS-PDS L 6 111 18.5 27.75±2.8

PDS S linear 6 61 10.2 15.25±2.9

PDS L linear 6 88 14.7 22±5.5

pBS-PDS S-ble 3’ 6 29 4.83 7.25±2.4

pBS-PDS L-ble 3’ 6 58 9.67 14.5±1.25

pBS-PDS S-ble 5’ 6 39 6.5 9.75±1.6

pBS-PDS L-ble 5’ 6 5 0.83 1.25±0.7

PDS S-ble 5’ 6 18 3 4.5±0.6

PDS L-ble 5’ 6 25 4.17 6.25±1.6

No DNA 3 1 0.33 0.5±0.25

pUC-atpX-aadA-

16S-23S 6 20 3.3 5±3

Conclusions

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All transformation experiments with the various vectors were successful, both for the nuclear– and for the chloroplast genome.

Transformation frequencies of about 2*10-5 were achieved, compared to 2*10-6 reported before.

Transformation frequency of pBS-PDS S/L and linear PDS are similar.

Transformation frequency of vector+ ble cassette was lower than vector alone.

Transformation frequency of pUC-atpX-aadA-16S-23S was lower than of pBS-PDS S/L or linear PDS.

Concluding Remarks

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Genetic transformation both to the nuclear- and chloroplast genome of H. pluvialis is now established and can be routinely applied.

Draft genome sequence allowed us to identify and sub-clone essential genes for basic understanding and to improve the industrial performance of the high-value commercial alga by genetic engineering.

MBL and GIAVAP have established H. pluvialis as a model organism for experimental biology and industrial application.

Additional marker genes need to be tested both for the nuclear genome and for the chloroplast.

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Thank you!!!!