Advanced methods for genetic engineering

The phytoene desaturase pds gene mu-tated in the codon for the amino acid at position 504 from leucine to arginine, yields an enzyme resistant to the herbicide norflurazon and is used as

Advanced methods for genetic engineering of Haematococcus pluvialis (Chlorophyceae,

Volvocales)

Article in Algal Research · April 2015

DOI: 10.1016/j.algal.2015.03.022

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Advanced methods for genetic engineering of Haematococcus pluvialis

(Chlorophyceae, Volvocales)

Microalgal Biotechnology Laboratory, French Associates, The Albert Katz Department of Dryland Biotechnologies, The Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Sede Boker Campus, 84990 Israel

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 23 September 2014

Received in revised form 8 March 2015

Accepted 28 March 2015

Available online xxxx

Keywords:

Genetic engineering

Transformation

Haematococcus pluvialis

Phytoene desaturase

An advanced shuttle-vector for efficient nuclear transformation and genetic engineering of Haematococcus pluvialis has been developed and tested by inserting linked trans-genes The phytoene desaturase (pds) gene mu-tated in the codon for the amino acid at position 504 (from leucine to arginine), yields an enzyme resistant to the herbicide norflurazon and is used as an endogenous selection marker for transformation of H pluvialis A novel cloning vector allowing insertion of additional genes, both at the 5′ and the 3′ end of the mutated pds, has been designed by inserting the selection marker into the cloning vector pBluescript II SK(−) to give pBS–pds fea-turing the genomic mutated pds including 2000 bp of its promoter In a second version pBS–pds short was

creat-ed, by shortening the promoter sequence to 1000 bp Unique restriction sites 5′ and 3′ of the selection marker have been reserved for insertion and simultaneous transformation with two transgenes Transformation

efficien-cy was significantly better than previously reported, achieving transformation frequencies of 2 × 10−5both with long and short promoters, as well as with linear constructs An expression cassette for the ble derived from vector pGenD-ble was inserted into pBS-pds either 5′ or 3′ of the pds, and successfully transformed into H pluvialis, resulting in engineered strains weakly expressing the ble mRNA driven by the Chlamydomonas reinhardtii PsaD promoter Both circular plasmids, as well as linear DNA fragments only consisting of the ble cassette fused to the pds selection marker were used successfully to engineer H pluvialis The plasmid constructs presented here, as well as the use of an endogenous dominant selection marker, represent a blueprint for the future success-ful production of safe, genetically modified microalgae, for the possible production of high value products or biofuels

© 2015 Elsevier B.V All rights reserved

1 Introduction

Microalgae have high areal biomass productivity and can

accumu-late significant concentrations of high value products, or over 50% of

dry weight as oil in the form of triacylglycerol (TAG) under adequate

conditions[30,31,33,41] The suitability of microalgae as a potential

source of biofuel has attracted increased interest in microalgal

biotech-nology[5,7,11,17,19,26,35,36,41] Microalgae produce a wide range of

high-value products including carotenoids and polyunsaturated fatty

acids (PUFAs), both constitutively in the frame of primary metabolism

or inductively as a secondary metabolism response to various stresses

[1,3,9,17] The production of such pharmaceutically and nutraceutically

important chemicals is one of the most promising approaches for the

commercial exploitation of microalgae However, algal strains and

pro-duction processes require significant improvements at various levels, to

compete successfully with products used in food, cosmetics,

aquacul-ture and agriculaquacul-ture, that are prepared synthetically or extracted from

other natural feedstock In general, estimated microalgal product prices

(fuels, carotenoids, PUFA, protein etc.) are about 3–5 times above the market prices for competing products from other sources[22] Thus,

sig-nificant advances in biomass and product accumulation rates, cheaper cultivation technologies and improved harvesting and product extrac-tion procedures are required for advancing the competitiveness of microalgal products[11,41] One of the main approaches for improving productivities of microalgae is successful genetic engineering using safe technologies facilitating outdoor cultivation and product marketing Most technologies of microalgae transformation have relied on the in-sertion of bacterial antibiotics markers driven by viral or algal promoter elements[2,14,21,29,39]

Successful nuclear transformation and metabolic engineering so far have been reported only in Chlamydomonas reinhardtii, Phaeodactylum tricornutum and Thalassiosira pseudonana[16,27,38]

The unicellular green alga Haematococcus pluvialis (H pluvialis) is regarded as the best natural source for the high-value red pigment astaxanthin, a carotenoid that is accumulated in cytoplasmic oil globules under various stress conditions[3] Accumulation of astaxanthin in

H pluvialis is positively correlated with lipid accumulation[34,42,43] Under nitrate deprivation, astaxanthin and fatty acid contents can reach

up to 4% and 40% of cell dry mass, respectively[42] In H pluvialis

⁎ Corresponding author.

E-mail address: sammy@bgu.ac.il (S Boussiba).

http://dx.doi.org/10.1016/j.algal.2015.03.022

Contents lists available atScienceDirect

Algal Research

j o u r n a l h o m e p a g e :w w w e l s e v i e r c o m / l o c a t e / a l g a l

astaxanthin serves as protective pigment against exposure to excessive

light[28,40] Natural astaxanthin from H pluvialis is a popular high

price nutraceutical product successfully produced in Israel at Kibbutz

Qetura using Ben-Gurion University's (BGU) proprietary technology[8]

The major commercial use of much cheaper synthetically produced

astaxanthin is as a colorant for aquaculture, primarily for salmonids[24]

Astaxanthin productivity of H pluvialis is limited by intrinsically

slow proliferation, sensitivity to environmental stresses and

contami-nants and slow induction of pigment accumulation[4,22], so that the

production price for natural astaxanthin is significantly higher than

the price of the synthetic compound Therefore, genetic engineering of

this alga for increased growth, resilience and carotenoid productivity

is a major goal in algal biotechnology, for more competitive pigment

production, in the face of growing competition and significantly larger

market opportunities in the aquaculture sector H pluvialis was

success-fully transformed by microparticle bombardment with the pPlat-pds

vector developed by Steinbrenner and Sandmann[37], which carries a

mutated copy of the pds conferring resistance to the herbicide

norflurazon Recently, chloroplast transformation in H pluvialis using

the C reinhardtii aadA expression cassette[12]was also reported[15]

However, robust nuclear transformation and successful genetic

engi-neering have not been demonstrated in this valuable algae species,

due to lack of suitable shuttle vectors and adequate transformation

fre-quencies We present here a novel transformation and expression

vec-tor for high efficiency transformation and simultaneous expression of

two additional transgenes in H pluvialis Based on an endogenous

dom-inant marker, the vector also permits the production of safe transgenic

algae strains that do not contain foreign DNA sequences

2 Materials and methods

2.1 H pluvialis strain

H pluvialis Flotow 1844 em Wille 1903, SCCAP K-0084

(Chlorophyceae, order Volvocales) was obtained from the Scandinavia

Culture Center for Algae and Protozoa (SCCAP) at the University of

Co-penhagen, Denmark

2.2 Growth conditions for algal cultures

H pluvialis algal cultures were grown from a cell concentration of

2 × 105cell × mL−1(determined with a hemocytometer) in 250 mL

flasks, each containing 100 mL of modified BG-11 medium[6]; theflasks

were held in a shaker (150 RPM) enriched with CO2(300 mL × min−1)

at 25°C, 110–140 μmol photon × m−2× s−1 Under these conditions the

cultures reach a stationary stage within one week

2.3 DNA manipulation and cloning

Total H pluvialis DNA was purified as described[32]with slight

modifications, or by using the DNeasy plant mini kit (Qiagen) after

breaking the cells in Retsch MM400 Mixer Mill using 5 mm metal

beads for several rounds of 40 s, 25 Hz or grinding by mortar and pestle

in the presence of liquid nitrogen PCR reactions were performed using

the following DNA polymerases: Go-Taq (Promega), Ex-Taq (TAKARA)

and PrimeStar HS (TAKARA) using the primers described inTable 1

2.4 Primer design

Primers were designed using the VectorNti advance 11.0

(Invitrogen, Life Technologies) software Alignments and sequencing

results were viewed by BioEdit Sequence Alignment Editor 7.0.5.3

PCR primers used for cloning and sub-cloning of pBS–pds and pBS–

pds–ble are shown inTable 1

2.5 Gel purification of DNA fragments DNA fragments were purified from agarose gels using the AccuPrep Gel purification kit (Bioneer), and ligated into plasmid DNA digested with compatible restriction sites, or by using the In-Fusion HD Cloning Kit (Clontech) according to the manufacturer's recommendations The plasmid was transformed into competent DH5α Escherichia coli (E coli) (Invitrogen, Life Technologies) which were than cultivated on solid LB agar media containing 100μg × mL−1ampicillin Linear trans-formation cassettes were extracted from plasmids by digestion with re-striction enzymes cutting on both sides of the desired insert and purification from gel (Fig 1)

2.6 Construction of pBS–pds long and short The pds cassette from pPlat–pds Mod4.1[37]was amplified using PrimeStar HS DNA polymerase and primers: pdsSHindIIIF, pdsLHindIIIF, for pBS–pds short (S) and long (L), respectively and pdsEcoRIR for both (Table 1), restricted by HindIII and EcoRI and ligated into pBluescript II SK(−) Ligated plasmid was transformed into DH5α E coli cells and cul-tivated on solid LB agar media containing 200μg × mL−1ampicillin Transformed colonies were verified by PCR with the abovementioned primers and by DNA sequencing in the DNA sequencing center of Ben-Gurion University (BGU), Israel pBS–pds long contains bases Nr 186 till 5825 of plasmid pPlat–pds (accession number DQ404589, the pds cassette including the full length promoter) pBS–pds short contains bases Nr 1212 till 5825 of plasmid pPlat–pds cassette including short version of promoter

2.7 Construction of pBS–pds–S/L–ble KpnI–XhoI The PsaD–ble cassette from pGenD–ble[10]was amplified using Ex-Taq DNA polymerase (TAKARA) and primers blepGenDKpnIF and blepGenDXhoIR (Table 1) PCR product was cut and cleaned from gel using the Bioneer AccuPrep Gel purification kit followed by ligation into pGem-T-easy and transformation into JM109 E coli cells Cells were cultivated on 100μg × mL−1ampicillin-LB agar plates Trans-formed colonies were verified by PCR with the abovementioned primers The PsaD–ble cassette was extracted with KpnI and XhoI restric-tion enzymes from extracted plasmids using the Bioneer AccuPrep Plas-mid Mini extraction kit Restricted PsaD–ble was ligated into KpnI–XhoI restricted pBS–pds S or Land transformed into E coli DH5α Transformed colonies were verified by restriction assay using EcoRI and BglII and DNA sequencing

2.8 Construction of pBS–pds-S/L-ble BamHI–XbaI The PsaD–ble cassette from pGenD–ble[10]was amplified using PrimeStar HS DNA polymerase (TAKARA) and primers blepGenDBamHIF

Table 1 Primers used for cloning and construction of the various transformation plasmids Restric-tion sites are marked with bold italic.

Name of plasmid Name of primer F/R Primer sequence pBS–pds S pdsSHindIIIF F ATAGAAGCTTCTGTACGCCATTGT

ACGCCG pdsEcoRIR R GCGAATTCTGACCCCATATCCGTT

ACTGCC pBS–pds L pdsLHindIIIF F ATAGAAGCTTGCACAGTTAGAGGC

GTGGTTGC pdsEcoRIR R GCGAATTCTGACCCCATATCCGTT

ACTGCC pBS–pds S/L–ble KpnI,

XhoI

blepGenDKpnIF F AGTGGTACCCACACACCTGC blepGenDXhoIR R CGGTCTCGAGAAGCTTGATT pBS–pds S/L–ble

BamHI, XbaI

blepGenDBamHIF F AGTGGATCCCACACACCTGC blepGenDXbaIR R CGGTCTAGAGAAGCTTGATT

and blepGenDXbaIR PCR product was cut and cleaned from gel using the

Bioneer AccuPrep Gel purification kit followed by ligation into

pGem-T-easy and transformation into DH5α E coli cells Cells were cultivated on

100μg × mL−1ampicillin-LB agar plates Transformed colonies were

ver-ified by PCR with the above mentioned primers Extracted plasmids using

the Bioneer AccuPrep Plasmid Mini extraction kit were used for

restric-tion of the PsaD–ble cassette with BamHI–XbaI restriction enzymes

Re-stricted PsaD–ble was ligated into BamHI–XbaI restricted pBS–pds S/L

and transformed into DH5α cells and cultivated on solid LB agar media

containing 100μg × mL−1ampicillin Transformed colonies were verified

by restriction assays with EcoRI, BglII and DNA sequencing

2.9 Transformation of H pluvialis

H pluvialis was transformed using the PDS-1000/He particle delivery

system (BioRad) according to the manufacturer's instructions 1μg

plas-mid or linear DNA was used to coat 0.6μm gold particles (BioRad)

Two-four days after diluting stationary H pluvialis culture (hereby, referred

as 2–4 days old), 2–10 million cells, were plated onto TAP medium

[13], 1.5% agarose and were bombarded from a distance of 6 cm using

1350 psi rupture disks Two separate bombardments were conducted

with each construct DNA was omitted from control and bombarded

once After 1 night of recovery, cells were removed from the bombarded

plates using TAP medium, and divided into two TAP plates

supplement-ed with either 2 or 5μM norflurazon Transformed colonies started to

appear after 8–28 days of incubation at optimal conditions of 20–

40μmol photon × m−2× s−1, 25°C

Transformed colonies were grown successfully for long time periods, both in the presence and absence of selection pressure in our lab 2.10 Isolation and quantification of total RNA

Isolation of total RNA was performed using the SV Total RNA isola-tion kit (Promega USA) Approximately 1 × 107cells were pelleted from 10 mL culture (1500 g, 10 min) Cells were then broken in a Retsch MM400 Mixer Mill using 5 mm metal beads for several rounds of 40 s,

25 Hz RNA samples were quantified using a Nano Drop (ND-1000, Thermo Scientific, USA) spectrophotometer and stored at −80°C 2.11 PCR and DNA sequencing

On-Colony PCR was applied on norflorazon resistant colonies as fol-lows: resistant colonies were picked and scraped from selection plates, mixed with ethanol and 5% Chelex®100 and then broken in Mini Beadbeater (Biospec products, OK, USA), using 2.5 mm glass beads for several rounds on ice Colonies were then lysed by incubation at 98°C for 10 min, centrifuged and used for PCR with primers corresponding

to 500 bp region including the mutation site of pds sequence: F-AGCT TGCTCTGCTGTGCCAG, R-GCTATTGCACCACTGGCTGC PCR product was extracted from gel as described previously and sent for sequencing The same procedure, excluding sequencing, was applied for trans-formed colonies bombarded with the PsaD–ble cassette containing con-structs with appropriate primers: F-CACACCTGCCCGTCTGCCTGA, R-TGCCGTCCGTCCACTGACC for full PsaD–ble cassette and F-CCCCACTG

Gene 1

A

B

pBS - pds long

8599 bp

pBS - pds short

7571 bp

pds short - Gene 1

pds short - Gene 2

pds short - Gene 1 +2

amp

amp

pds

pds

pds

pds

pds

Fig 1 A Maps of pBS–pds long (left) and pBS–pds short (right); the mutation L–R in the CDS of pds (green) conferring resistance to norflurazon is indicated in purple The map of pBS–pds short is presented with confirmed insertion options for transgenes 5′ and 3′ of the selection marker using the polylinker sequences bordering the pds cassette in pBS–pds pBS–pds long is of exactly the same structure with a 1000 bp promoter region longer B linear fragments of pds with insertion of Gene 1, Gene 2 or both (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

CTACTCACAACA, R-TTAGTCCTGCTCCTCGGCCA for full 408 bp ble coding

sequence (CDS) including the end of PsaD 5′ untranslated region (UTR)

2.12 cDNA preparation

cDNA was synthesized from total RNA by the Verso cDNA synthesis

kit (Thermo Scientific), according to the manufacturer's instructions

in-cluding treatment with DNase This cDNA was used as template for PCR

amplification with appropriate primers: F-TAGGACCCCACTGCTACTCA

CAA, R-TGCCGTCCGTCCACTGACC specific to the PsaD 5′ and 3′ UTR

cov-ering the full PsaD–ble cassette sequence

3 Results

3.1 Transformation with pPlat–pds

Nuclear transformation of H pluvialis, with pPlat–pds vector

harbor-ing the mutated pds conferrharbor-ing resistance to norflurazon, was done

ac-cording to the method described by Steinbrenner and Sandmann[37]

and yielded approximately 380 resistant colonies out of 18 × 107cells

No resistant colonies were obtained in control experiments, in which

the plasmid DNA was omitted Very high variability in number of

trans-formed colonies per plate was observed, indicating a central role of cells'

handling and recovery, such as: cultivation, plating, re-plating and

incu-bation conditions during the recovery period on the apparent

transfor-mation efficiency Conditions applied were as described inMaterials

and methods Testing bombardment with different rupture disc calibers

or bombardment distances yielded either equal or worse

transforma-tion frequencies The best relative transformatransforma-tion frequency was

achieved with two days old cells plating 3 × 106per plate Transformed

colonies were maintained through successive platings for more than

33 months and stable insertion of the mutated gene was verified by

ex-tended periods of cultivation both in the absence or presence of selective

pressure, and subsequent PCR amplification and sequencing of the pds

3.2 Construction and testing of pBS–pds long and short

An analysis of an approximately 1 kb fragment of the promoter

re-gion of pds revealed that it contains several repetitive elements In

order to examine whether these elements interfere with, or reduce

ex-pression of the gene, pBS–pds long and pBS–pds short (Fig 1) were

pro-duced containing either ~ 2000 or ~ 1000 bp of the promoter region

(GenBank accession numbers: KP869828 and KP869829), respectively

Transformation of H pluvialis was tested carefully using both constructs,

but no significant difference in transformation efficiency between the

short and the long promoter versions were detected, though signi

ficant-ly higher efficiencies than with pPlat–pds were achieved (Table 2) 3.3 Molecular architecture of pBS–pds

The pBS–pds vectors harboring mutated pds have been designed to contain several unique restriction sites for insertion of transgenes (Fig 1) Thus, a number of restriction sites, both at the 5′ and 3′ of the pds, allow insertion of additional transgenes for co-transformation using the mutated pds as selection marker (Fig 1A) The unique restric-tion sites provided at both sides of both inserts facilitate extracrestric-tion of the linear DNA cassette consisting of transgenes and the endogenous DNA marker (Fig 1B)

3.4 Transformation with pBS–pds and co-insertion of the PsaD–ble cassette The usefulness of this concept was tested by inserting the PsaD–ble cassette under the control of the C reinhardtii PsaD promoter from pGenD–ble[10] This cassette was excised and added upstream of the mutated pds using the KpnI and XhoI restriction sites, or downstream

of the selection marker using the BamHI and XbaI sites (Fig 2) Both ver-sions were produced using the pBS–pds short (S) or long (L) versions (Figs 1 and 2) Resulting plasmids were transformed successfully into

H pluvialis, yielding norflurazon- resistant colonies, though yielding lower transformation frequency (Table 2) Successful incorporation of the PsaD–ble cassette was verified by PCR amplification using primers specific to the ble inserted at the end of the PsaD 5′ and at the end of the ble CDS as drawn inFig 2 All colonies derived from transformation with pBS–pds S–ble BamHI, XbaI; pBS–pds L–ble BamHI, XbaI; pBS–pds

S–ble KpnI, XhoI; pBS–pds L–ble KpnI, XhoI; or with linear DNA fragments extracted from those plasmids, featured a PCR product corresponding to the size expected from the inserted ble with the possible exception of lane 7 (Fig 3), while no signal was obtained with control H pluvialis DNA The 100% yield of co-insertion of ble observed indicates that addi-tional genes linked to pds in pBS–pds can be transformed into H pluvialis with very high efficiency Also, insertion appears to occur very near to the end of the linear DNA fragments, since all resistant clones tested also featured the full size PsaD-ble cassette PCR product (conducted

on extracted gDNA, not shown) WT (wild type) DNA yielded no PCR product for this gene while a positive control PCR yielded the expected product (Fig 3) The ble gene was weakly transcribed into RNA tran-scripts, as confirmed by PCR amplification of cDNA template synthe-sized from extracted RNA of transformed colonies (Fig 4) The PCR product includes 986bp corresponding to a partial 5' PsaD UTR, the full ble CDS (intronless), and PsaD 3' UTR PCR transcript Moreover,

Table 2

Transformation frequencies achieved with various constructs Only one bombarded spontaneously resistant colony grew on 2μM norflurazon.

bombarded cells

No of resistant colonies a

Frequency of resistant colonies

Average no per plate + standard error

pBS–pds S pBS–pds with short (~1000 bp) promoter sequence 6 × 10 6

94 1.57 × 10−5 23.5 ± 3.9 pBS–pds L pBS–pds with full-length (~2000 bp) promoter sequence 6 × 10 6

111 1.85 × 10−5 27.75 ± 2.8 pds S linear pBS–pds S linearized with HindIII, EcoRI 6 × 10 6

61 1.02 × 10−5 15.25 ± 2.9 pds L linear pBS–pds L linearized with HindIII, EcoRI 6 × 10 6

88 1.47 × 10−5 22 ± 5.5 pBS–pds S–ble BamHI, XbaI pBS–pds S + PsaD–ble cassette at the 3′ end (BamHI,

XbaI) of pds

6 × 10 6

29 4.83 × 10−6 7.25 ± 2.4 pBS–pds L–ble BamHI, XbaI pBS–pds L + PsaD–ble cassette at the 3′ end (BamHI,

XbaI) of pds

6 × 10 6 58 9.67 × 10−6 14.5 ± 1.25 pBS–pds S–ble KpnI, XhoI pBS–pds S + PsaD–ble cassette at the 5′ end (KpnI, XhoI) of pds 6 × 10 6

39 6.5 × 10−6 9.75 ± 1.6 pBS–pds L–ble KpnI, XhoI c pBS–pds L + PsaD–ble cassette at the 5′ end (KpnI, XhoI) of pds 6 × 10 6

pds S–ble KpnI, XhoI pBS–pds S–ble linearized with KpnI, XbaI 6 × 10 6

18 3 × 10−6 4.5 ± 0.6 pds L–ble KpnI, XhoI pBS–pds L–ble linearized with KpnI, XbaI 6 × 10 6

25 4.17 × 10−6 6.25 ± 1.6 a

The number of resistant colonies is the total obtained by 2 shootings and per 4 selection plates totaling 6 · 10 6

cells.

b

No DNA — one shooting was applied on control in which bombarded cells were divided into two selection plates.

c pBS–pds L–ble KpnI, XhoI was excluded from analysis since one of the shooting yielded no resistant colonies in either selection plate, probably due to experimental error.

insertion of the PsaD–ble cassette did not result in noticeable resistance

to zeocin in most of the clones checked, but rather resulted in a lessfit,

slow growing phenotype of H pluvialis This indicates that ble is not

expressed sufficiently into a functional protein in those transformed

cells possibly due to unsuitable codon use This is not an unexpected

re-sult considering that resistance was not originally selected for, and that

zeocin resistance depends directly on the amount of protein expressed

3.5 Transformation of H pluvialis with linear constructs

The inserts of pBS–pds S or pBS–pds L with the ble added 5′ or 3′ to

the mutated pds selection marker gene, were extracted using restriction

endonucleases and these linear DNA fragments were used to

successful-ly transform H pluvialis at frequencies similar to circular plasmids

(Table 2) Norflurazon-resistant colonies were isolated and analyzed

The mutated DNA genotype was confirmed by PCR and DNA sequencing

(data not shown) The transgene inserted was confirmed by PCR of the

full-length CDS (Fig 3) and the full PsaD–ble cassette (data not shown)

No significant differences in transformation efficiencies between the

two fragments were observed, whether the PsaD–ble cassette was

inserted 5′ or 3′ of pds (Table 2) In all transformed colonies the PsaD–

ble cassette was inserted together with the mutated pds selection

mark-er as confirmed by PCR (Fig 3), and by confirming the presence of ble–

mRNA after PCR amplification of cDNA from transformed colonies

(Fig 4) This demonstrates that transgenes linked to the mutated pds

selection marker gene are efficiently incorporated into the genomes of transformed colonies

3.6 Confirmation of transgene incorporation Transformed colonies were maintained for more than 16 months with periodically successive transfers and stable insertion of the

mutat-ed pds was verified by extended periods of cultivation both in the ab-sence and preab-sence of norflurazon The pds and ble were amplified by PCR and partial region of pds including the mutation site and ble were subjected to DNA sequencing (seeFigs 3 and 5for representative re-sults) All the transformed norflurazon-resistant colonies revealed the DNA sequence of the donor plasmid derived from pPlat–pds The point mutation conferring norflurazon resistance was identified in all trans-formed colonies (GC instead of TG in position 5185–5186 in the

mutat-ed pds) (Fig 5A) Insertion of the foreign DNA was also confirmed by the presence of polymorphisms in the introns of the pds The sequence of pds in pPlat–pds was derived from H pluvialis strain NIES-144[37] While pds of transformed strains showed a sequence similar to that of the pPlat–pds, the WT strain (1844 em Wille K-0084 (SCCAP)) pds (GenBank accession number: KP826910) and a spontaneous norflurazon-resistant strain showed a different sequence (Fig 5B) Moreover, sequencing of partial PsaD 5' UTR, full ble CDS, and 3' UTR

of PsaD verified insertion of PsaD-ble cassette in all tested colonies (data not shown) In addition, Southern blot analysis using a ble specific

pBS - pds S- ble Kpn I

9393 bp

pBS - pds S- ble Bam H I

9397 bp

pds pds

ble

ble

Promoter short

Promoter short

psaD 5' UTR

( C reinhardtii )

psaD 5' UTR

( C reinhardtii )

Fig 2 Map of pBS–pds S–ble KpnI, XhoI at the 5′ end/BamHI, XbaI at the 3′ end The ble CDS of pGenD–ble is designated in yellow; the C reinhardtii PsaD promoter and 3′ UTR are designated

in pink Restriction sites are indicated Note that the ble cassette was inserted either at the 5′ of pds (KpnI, XhoI) or at the 3′end (BamHI, XbaI) PCR primers are indicated by F and R (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

500 bp

500 bp

Fig 3 On-colony PCR of ble in several pBS–pds–ble or linear pds–ble transformed colonies 407 bp PCR product corresponding to the full CDS of ble and the end of PsaD 5′ UTR was found in all transformed colonies tested 1–17 — various transformed colonies containing the following constructs: 1, 2, 3, 4, 5 — pBS–pds–L–ble BamHI, 6, 7, 8 — pBS–pds–S–ble KpnI, 9, 10, 11 — pBS–pds–S–ble BamHI, 12, 13 — pds–S–ble KpnI (linear), 14, 15, 16, 17 — pds–L–ble KpnI (linear),18, 19 — pBS–pds–S–ble KpnI/BamHI plasmids, respectively, 20, 21 — pBS–pds–L–ble KpnI/

— H pluvialis gDNA, 23 — no DNA, M — marker.

probe (corresponding to the full ble CDS and the 3′ UTR region of the PsaD–

ble cassette) was performed using isolated gDNA of transformed colonies

digested by BamHI and BglII At least one clear ble copy was detected in

the gDNA of six out of eight colonies tested (in three of them, additional

copy was detected); WT and empty vector negative control colonies did

not display a ble copy, as expected (data not shown) All together these

re-sults confirm the successful introduction of the pPlat–pds derived mutated

gene into the genome This confirms the usefulness of the pBS–pds

trans-formation vectors for stably inserting transgenes added both 3′ and 5′ to

the selection marker into the genome of H pluvialis

3.7 Transformation frequencies with various constructs

H pluvialis cells were bombarded with all DNA constructs described

inTable 2; resistant colonies appeared 8–28 days after selection on

norflurazon-TAP plates and were transferred onto fresh

norflurazon-TAP plates Transformation frequencies were determined based on the

number of colonies per plate recognized by eye

Between 18 and 111 resistant colonies were obtained per

bombard-ment of 6 × 106cells as described inMaterials and methods

Transfor-mation efficiencies of 3–18.5 × 10−6were achieved, depending on the

vector used (Table 2) This is more than an order of magnitude higher

than previously reported frequencies of transformation of H pluvialis

[37] Out of 3 × 106control cells plated onto selective medium, one

spontaneously resistant mutant colony was obtained on TAP agar plates

supplemented with norflurazon (Table 2) However, although this

colo-ny was spontaneously resistant to norflurazon, the partial pds sequence

tested was identical to the WT strain, not exhibiting the GC to TG

muta-tion (Fig 5A and B) Resistance is probably due to a different

mecha-nism Transformation frequencies of short versus long and linear

versus circular constructs were roughly the same However, addition

of ble transgene lowered transformation frequency Improvement of

transformation frequency was achieved by optimized cultivation and

recovery procedures and the use of gold particles for bombardment

in-stead of tungsten

4 Discussion

A large number of possible commercial applications for microalgae have been suggested and are being investigated However, algal strains and production processes for various pigments or PUFAs require signif-icant improvements to compete successfully with currently used prod-ucts Strain improvements by genetic engineering for higher product yields[11,22], and promoting the acceptability of transgenic algae in the market and with regulatory authorities[18]are key challenges in promoting the future success of microalgal biotechnology in the high value and bulk product markets In the specific case of H pluvialis, the specific challenge is reducing astaxanthin production costs by a factor

of 3–4, for accessing the far larger aquaculture feed market with natural pigment This could be achieved by increasing astaxanthin accumula-tion, enhancing biomass productivity by means of genetic engineering and simultaneously reducing cultivation costs by moving to cheaper open pond production[22] Thus, adequate methods for genetic engi-neering of this commercially important species are required

We have tested multiple approaches and DNA delivery techniques for establishing H pluvialis transformation Attempts using various vectors and selection marker genes using particle gun bombardment, electroporation or Agrobacterium tumefaciens-mediated genetic trans-formation (AtMGT) were all un-successful Neither transtrans-formation with pCambia 1301/2 (CAMV35S promoter and hyg selection marker), nor with pSP124S harboring the rbcS2 promoter and the Sh–ble selec-tion marker gene with an rbcS2 introns[25], nor with pGenD–ble, har-boring the PsaD promoter of C reinhardtii and the Sh–ble selection marker gene, was successful Attempts to reproduce the method

report-ed by Kathiresan et al.[20]using AtMGT were unsuccessful as well, pos-sibly due to the difference in the strain Only testing the method described by Steinbrenner and Sandmann[37]using the mutated pds gene, conferring resistance to norflurazon, proved effective at the end However, the pPlat–pds plasmid, kindly provided by G Sandmann, proved unpractical for any genetic engineering approach, and long UTR's appeared to interfere with standard molecular genetics

1 kb

1 kb

Fig 4 All transformed colonies except 14 exhibited the 986bp ble PCR transcript in moderate levels of expression cDNA served as template for PCR reaction 1–17 — various transformed colonies containing the following constructs: 17, 14–16 — pds–L–ble KpnI (linear),12, 13 — pds–S–ble KpnI (linear), 6–8 — pBS–pds–S–ble KpnI, 9–10 — pBS–pds–S–ble BamHI, 1–3 — pBS– pds–L–ble BamHI, 18 — H pluvialis gDNA, 19, 21 — pBS–pds–S–ble KpnI/BamHI plasmids, respectively, 20, 22 — pBS–pds–L–ble KpnI/BamHI plasmids, respectively, 23 — no DNA, M — marker.

technologies, possibly due to the presence of repetitive DNA elements.

We therefore designed an advanced shuttle vector in the form of pBS–

pds (Fig 1) featuring all requirements of an advanced shuttle vector

for co-transformation of at least two additional transgenes

Transforma-tion frequency using this vector was up to twenty-fold higher than

transformation with pPlat–pds (Table 2and[37]) In addition,

trans-formed strains displayed resistance due to introduction of the selection

marker for long time periods (up to 33 months), indicating high nuclear

stability of the developed vector

The new vector is designed such that linear DNA fragments

consisting of the endogenous selection marker and one or two linked

transgenes can easily be excised and used for transformation (Fig 1),

thus removing all plasmid backbone DNA (and possible antibiotic

markers) for safe transformation Such a vector structure can also

serve as a blue-print for successful manipulation of different high

value microalgae, as the pds and its mutation leading to norflurazon

re-sistance are essentially ubiquitous[23] One amino acid substitution

in pds makes Chlorella zofingiensis resistant to norflurazon and enhances

the biosynthesis of carotenoids including astaxanthin[23] As such, the

technologies presented here represent a game-changing progress in ad-dressing the challenges of advanced genetic engineering methods in microalgae, exemplified using the high value green alga H pluvialis In order to profit from possible benefits of transgenic microalgae, outdoor cultivation in open ponds must be applied Most recent advances in ge-netic engineering of microalgae are based on the use of bacterial antibi-otic markers that can represent significant regulatory obstacles for cultivation and marketing of the resulting algal biomass Microalgae are not included in most current regulatory EU documents concerning genetic engineering For most matters of biological safety microalgae can be considered microorganisms, such that transgenic microalgae would fall under regulations in the“Guidance Document of the EFSA ge-netically modified organism (GMO) Panel on the risk assessment of ge-netically modified microorganisms and their derived products intended for food and feed use” (http://www.efsa.europa.eu/en/efsajournal/pub/ 374.htm) According to those guidelines, nontoxic non-pathogenic GMO require cultivation in contained enclosures Outdoor cultivation without a lengthy licensing and testing period is not permitted Accord-ingly, so far no transgenic algae are cultivated outdoors anywhere

A

B

Fig 5 DNA sequence of 12 transformed colonies compared to the sequence of wild type and a spontaneous bombarded norflurazon resistant strain (bottom 4 rows) and pPlat–pds (first upper row) A The mutation site conferring norflurazon resistance is marked by a red box (WT: TG; mutant: GC) B Successful integration of mutated pds into the genome is indicated by polymorphism in pds intron Various polymorphisms between the BGU strain (samples 13 and 19) and the mutated pds gene used for transformation [37] are marked with black frames on the lower panel A spontaneously resistant colony (no 19) does not contain the mutation; resistance is probably due to a different mechanism (For interpretation of the references to color

in this figure legend, the reader is referred to the web version of this article.)

However, genetically modified microalgae may be used without

restric-tions if they are the product of mutagenesis, or self-cloning consisting in

the removal of nucleic acid sequences from a cell of an organism followed

by reinsertion of all or part of that nucleic acid sequence Therefore, the

use of endogenous dominant selection markers, such as: genes for

mutat-ed acetohydroxyacid synthase (ahas)[21]or mutated pds ([37], this paper)

for transformation of microalgae is an important prerequisite for the

cre-ation of safe and applicable transgenic microalgae We have

demonstrat-ed here that the cassette containing the selection marker and the

associated transgene can be easily excised from the plasmid and used

suc-cessfully for transformation and insertion of additional transgenes both 3′

and 5′ of the selection marker, to create self-cloned strains without

for-eign DNA inserted

Thus in conclusion, the transformation vector and technologies

pre-sented here allow not only more efficient transformation of microalgae,

but also the creation of self-cloned, safe transgenic algae by adding

en-dogenous genes intended for overexpression 5′ and 3′ to the

endoge-nous selection marker Such transformation technologies for metabolic

engineering of high value product accumulation, TAG or biomass

pro-ductivity are essential tools for enhancing the impact of algal

biotech-nology in the future

Acknowledgments

The research leading to these results has received funding from GIAV

AP Programme of the European Union's Seventh Framework

Pro-gramme (FP7) under REA grant agreement no 362044

This work was submitted for patenting, BGU-P-040-USP

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