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ARTICLE
Cloning, Mutagenesis, and Characterization of the
Microalga Parietochloris incisa Acetohydroxyacid
Synthase, and its Possible Use as an Endogenous
Selection Marker
Omer Grundman,1 Inna Khozin-Goldberg,1 Dina Raveh,2 Zvi Cohen,1
Maria Vyazmensky,2 Sammy Boussiba,1 Michal Shapira2
1
Microalgal Biotechnology Laboratory, French Associates Institute of Agriculture and
Biotechnology of Drylands, The Jacob Blaustein Institutes for Desert Research,
Ben-Gurion University of the Negev, Sede-Boker Campus, Midreshet Ben-Gurion 84990,
Israel; telephone: 972-8-6563478; fax: 972-8-6596742; e-mail: [email protected]
2
Department of Life Sciences, Ben-Gurion University of the Negev, Israel
ABSTRACT: Parietochloris incisa is an oleaginous fresh water
green microalga that accumulates an unusually high content
of the valuable long-chain polyunsaturated fatty acid (LCPUFA) arachidonic acid within triacylglycerols in cytoplasmic lipid bodies. Here, we describe cloning and mutagenesis of the P. incisa acetohydroxyacid synthase (PiAHAS)
gene for use as an herbicide resistance selection marker for
transformation. Use of an endogenous gene circumvents the
risks and regulatory difficulties of cultivating antibioticresistant organisms. AHAS is present in plants and microorganisms where it catalyzes the first essential step in the
synthesis of branched-chain amino acids. It is the target
enzyme of the herbicide sulfometuron methyl (SMM),
which effectively inhibits growth of bacteria and plants.
Several point mutations of AHAS are known to confer
herbicide resistance. We cloned the cDNA that encodes
PiAHAS and introduced a W605S point mutation (PimAHAS). Catalytic activity and herbicide resistance of the
wild-type and mutant proteins were characterized in the
AHAS-deficient E. coli, BUM1 strain. Cloned PiAHAS wildtype and mutant genes complemented AHAS-deficient bacterial growth. Furthermore, bacteria expressing the mutant
PiAHAS exhibited high resistance to SMM. Purified PiAHAS
wild-type and mutant proteins were assayed for enzymatic
activity and herbicide resistance. The W605S mutation was
shown to cause a twofold decrease in enzymatic activity and
in affinity for the Pyruvate substrate. However, the mutant
exhibited 7 orders of magnitude higher resistance to the
SMM herbicide than that of the wild type.
Biotechnol. Bioeng. 2012;109: 2340–2348.
ß 2012 Wiley Periodicals, Inc.
Correspondence to: I. Khozin-Goldberg
Received: 27 November 2011; Revision received: 4 March 2012;
Accepted: 23 March 2012
Accepted manuscript online 4 April 2012;
Article first published online 17 April 2012 in Wiley Online Library
(http://onlinelibrary.wiley.com/doi/10.1002/bit.24515/abstract)
DOI 10.1002/bit.24515
2340
Biotechnology and Bioengineering, Vol. 109, No. 9, September, 2012
KEYWORDS: acetohydroxyacid synthase; SMM; Parietochloris incisa; site-directed mutagenesis
Introduction
Microalgae are one of the richest sources of long-chain
polyunsaturated fatty acids (LC-PUFAs). The green freshwater microalga Parietochloris incisa (Trebouxiophyceae) is
of special interest for microalgal biotechnology because of its
ability to accumulate extraordinary high amounts of LCPUFA arachidonic acid (ARA)-rich triacylglycerols (TAG)
(Bigogno et al., 2002a). When cultivated under nitrogen
starvation, the fatty acid (FA) content of the alga is over 35%
of dry weight; ARA constitutes about 60% of total FAs, and
over 90% of cell ARA is deposited in TAG (KhozinGoldberg et al., 2002), making it the richest plant source of
ARA.
The aim of the current study is to develop a platform for
genetic transformation of P. incisa and for the development
of an herbicide-insensitive alga. Use of antibiotic resistance
genes as selection markers in transformation presents
numerous environmental and health risks, as well as
regulatory difficulties that define the organism as genetically
modified (GM) (Bradford et al., 2005; Redenbaugh and
McHughen, 2004). We will therefore base our future
selection system on mutation of the endogenous P. incisa
acetohydroxyacid synthase (PiAHAS) gene to confer
herbicide resistance (Haughn and Somerville, 1988).
AHAS is present only in bacteria, fungi, and plants
where it catalyzes the first step in the biosynthesis of the
branched-chain amino acids (BCAAs), valine, leucine, and
isoleucine. Plant and green algal AHAS are localized in the
chloroplast (Jones et al., 1985) and fungal AHAS in the
mitochondria (Cassady et al., 1972; Ryan and Kohlhaw,
1974), although the genes may be present in the nuclear or
ß 2012 Wiley Periodicals, Inc.
organelle genome (Lapidot et al., 1999; Ohta et al., 1997;
Reith and Munholland, 1993). In cases of nuclear-encoded
genes, the enzyme is transported to the target subcellullar
compartment by an additional, poorly conserved, N-terminal
targeting peptide (Grula et al., 1995; Hattori et al., 1992;
Mazur et al., 1987). AHAS is the target enzyme of the
herbicide sulfometuron methyl (SMM) that effectively
inhibits growth of bacteria, yeast, plants, and algae.
Mutant forms of AHAS exhibit herbicide resistance and
serve as dominant selectable markers for nuclear transformation of yeast (Gysler et al., 1990), higher plants (Ott et al.,
1996), and green algae (Kovar et al., 2002). In red algae,
the chloroplast-encoded AHAS has been successfully used
as a selection marker for chloroplast transformation of
Porphyridium sp. (Lapidot et al., 2002). The molecular basis
for most of the characterized AHAS-herbicide-resistances is
due to a single or double amino acid change from the wildtype enzyme sequence. To date, at least 17 different amino
acid substitutions in AHAS are known to confer resistance to
growth inhibiting herbicides (Zhou et al., 2007). In tobacco,
a resistant mutant with a single amino acid change of
Tryptophan 557, within a conserved region of AHAS, was
found to be insensitive to inhibition by two sulfonylurea
herbicides, chlorsulfuron, and SMM (Chaleff and Mauvais,
1984). Corresponding Trp residue mutations were shown to
be important for AHAS SMM resistance of Escherichia coli
(Chipman et al., 1998), Mycobacterium tuberculosis (Choi et
al., 2010), Brassica napus (Hattori et al., 1995), and the red
microalga Porphyridium sp. (Lapidot et al., 1999). A similar
conserved Trp residue was shown in this work to be present
in the PiAHAS gene.
Use of AHAS as a selection marker has several advantages:
first, an herbicide-insensitive alga can be advantageous
for controlling microbial and foreign algal contaminations
in large-scale growth systems. Second, use of an endogenous
gene does not classify the organism as GM. Third
endogenous genes do not require codon optimization, thus
avoiding potential post-transcription and post-translation
difficulties. And finally, this alga does not seem capable of
developing spontaneous resistance to the SMM herbicide,
which would prevent selection of false transformation
events. Here, we report the cloning of the cDNA that
encodes PiAHAS, site-directed mutagenesis of the gene to
obtain herbicide resistance, in vivo and in vitro characterization of both wild-type and mutant recombinant enzymes
in the presence and the absence of herbicide.
Materials and Methods
Algal Growth Conditions
P. incisa was isolated and maintained in the Microalgal
Biotechnology Laboratory (Watanabe et al., 1996). Axenic
cultures of P. incisa were cultivated on BG-11 nutrient
medium (Stanier et al., 1971) in 250-mL Erlenmeyer glass
flasks in an incubator shaker at a speed of 170 rpm, under an
air/CO2 atmosphere (99:1, v/v), controlled temperature
(258C), and illumination (115 mmol quanta m2 s1)
(Bigogno et al., 2002b).
Construction of a P. incisa cDNA Library
One microgram of total RNA was reverse-transcribed into
cDNA using a VersoTM cDNA kit (ABgene, Surrey, UK),
according to the manufacturer’s instructions. Each 20 mL
reaction mix contained 1 mg of total RNA, 300 ng of random
hexamers and 125 ng of anchored oligo-dT, dNTP mix
(500 mM each), cDNA synthesis buffer, RT enhancer, and
Verso enzyme mix. Following cDNA synthesis at 428C for
1 h, reactions were stopped by heating at 958C for 2 min and
cDNA was diluted 10-fold with PCR grade water.
Cloning of the PiAHAS cDNA
Our strategy for cloning the PiAHAS gene was based on the
high degree of evolutionary conservation of the protein. In
order to identify partial sequence of the AHAS enzyme of P.
incisa, several known amino acid and nucleotide sequences
of related green algae and other higher plants were aligned,
using ClustalW (www.ebi.ac.uk/clustalw) to identify conserved motifs in the enzyme. The conserved ‘‘blocks’’ were
used to design two oligonucleotide primer sets, AHAS01
and AHAS02, for cloning partial PiAHAS sequences (Fig. 1).
All the primers used for PCR and sequencing are listed in
Table I. All the primers were designed by Primer3, version
0.4.0 (www.frodo.wi.mit.edu) software and checked with
NetPrimer (www.premierbiosoft.com/netprimer). PCR amplifications were carried out using the first strand cDNA as a
template, primers, and 2 PCR ReddyMixTM Master Mix
(ABgene). PCR amplification was as follows: denaturation
at 948C for 3 min, followed by 32 cycles of 948C for 30 s,
608C for 90 s, and 728C for 1 min, and a final extension cycle
of 728C for 10 min. All amplified products were cloned
into pGEM T-easy plasmid (Promega, Madison, WI) and
sequenced. The cloned fragments were analyzed by BLASTX.
The amplified fragments were used for the design of a
third primer set, AHAS03. PCR amplifications were carried
out using the first strand cDNA as a template, primers and
2 PCR ReddyMixTM Master Mix. Touch-Down PCR
(TD-PCR) amplification was as follows: denaturation at
A
C.
V.
C.
B.
P.
reinhardtii
carteri
variabilis
napus
patens
B
IGTDAFQETP
IGTDAFQETP
IGSDAFQETP
IGTDAFQETP
IGTDAFQETP
**:*******
211
210
158
192
123
MLGMHGTV
MLGMHGTV
MLGMHGTV
MLGMHGTV
MLGMHGTV
********
C
356
355
303
337
268
RAHTYLG
RAHTYLG
RAHTYLG
RAHTYLG
RAHTYLG
*******
D
595
593
542
571
502
VLPMIP
VLPMIP
VLPMIP
VLPMIP
VLPMIP
******
659
684
635
655
586
Figure 1. Multiple sequence alignment of five AHAS sequences from green
algae (C. reinhardtii, C. variabilis, and of V. carteri.), moss (P. patens subsp.) and higher
plant (B. napus), showing the conserved ‘‘blocks’’ (A–D), used for primer design.
Grundman et al.: P. incisa AHAS Site-Directed Mutagenesis
Biotechnology and Bioengineering
2341
Table I.
Oligonucleotides used in this study.
Oligo
AHAS01-F
AHAS01-R
AHAS02-F
AHAS02-R
AHAS03-F
AHAS03-R
3’AHAS GSP
5’AHAS GSP
PiAHAS-full F
PiAHAS-full R
AHAS-Mut-F
AHAS-Mut-R
pAH29-empty-F
pAH29-empty-R
PiAHAS-ORF
PiAHAS-trunc
PiAHAS-R
Sequence
Tm (8C)
AACCGGCGCACACGTACCTGG
CGGGGATCATGGGCAGCACG
GGCACCGAIGCITTICAIGAIAC
ACGGTGCCITGCATICCIAICAT
AGATCACCAAGCACAACTTCCT
ATGACAAAGTCG GGGAAGATGT
ACGCTGGACGAGAGCCACATCTTC
CCCTGTGATGGCAACAAGCGGAAC
ATAGTCGACAGCATGCAAGGCACTATG
AATGTCGACCTGCGCCTTAGTACTCG
GCATGGTGGTCCAGTcGGAGGACCGCTTCTACA
TGTAGAAGCGGTCCTCCgACTGGACCACCATGC
TATAGTCGACGCGCAAAAGGAATATAAAAA
TATAGGATCCCATAGTTAGTTCCCCGTCC
GGATCCATGCAAGGCACTATG
GGATCCAATGAGCTGGTGGC
GTCGACCTGCGCCTTAGTACTC
67
67
68
69
60
60
69
69
68
69
79
79
65
70
63
61
66
The restriction sites are italicized. Start and stop codons are underlined. Mutant nucleotides are in lower case and bolded.
948C for 2 min, followed by 30 cycles of 948C for 30 s, 558C
for 60 s, and 688C for 1.5 min, and a final extension cycle of
688C for 10 min. The complete internal fragment was used
for gene-specific primers (GSPs) design to clone the fulllength cDNA of the PiAHAS gene, employing the 30 and 50
rapid amplification of the cDNA ends (RACE) method,
using a BD smart RACE cDNA Amplification Kit (BD
Biosciences, Clontech, Palo Alto, CA) according to the
manufacturer’s manual. Two sets of RACE-cDNAs were
synthesized for 30 -end and 50 -end amplification. The
synthesized cDNA was used for PCR amplification of the
30 -cDNA end using the Universal Primer A Mix (UPM) and
the 30 -AHAS GSP. The PCR amplification was carried out
using the BD AdvantageTM 2 PCR Kit (BD Biosciences,
Clontech). PCR amplification was as follows: denaturation
at 948C for 2 min, followed by 30 cycles of 948C for 30 s,
638C for 30 s, and 728C for 2 min, and a final extension cycle
of 728C for 10 min; the reaction was terminated at 108C. For
the synthesis of the 50 -cDNA end of AHAS, 50 -AHAS GSP
was used as a reverse primer and UPM as a forward primer
and TD-PCR was employed: denaturation at 948C for 2 min,
followed by five cycles of 948C for 30 s, 708C for 40 s, and
728C for 2.5 min, followed by 5 cycles of 948C for 30 s, 688C
for 40 s, and 728C for 2.5 min, and finally 25 cycles of 948C
for 30 s, 648C for 40 s, and 728C for 2.5 min.
To clone the full length PiAHAS cDNA, an additional
set of primers, PiAHAS-full, was designed based on the
cDNA ends. The primers contained the start and stop
codons and had SalI restriction sites for future ligations.
PCR amplifications were carried out using the first strand
cDNA as a template, primers, and the PfuUltra DNA
polymerase (Stratagene, La Jolla, CA). PCR amplification
was as follows: denaturation at 948C for 3 min, followed by
30 cycles of 948C for 30 s, 658C for 60 s and 728C for 3 min,
and a final extension cycle of 728C for 10 min. The full gene
was ligated and cloned into pGEM T-easy plasmid.
2342
Biotechnology and Bioengineering, Vol. 109, No. 9, September, 2012
Site-Directed Mutagenesis of the PiAHAS Gene
A specific point mutation was designed for substitution of
the PiAHAS Trp605 with Serine. Mutagenesis of the PiAHAS
wild type cDNA was performed with the QuikChange sitedirected mutagenesis kit (Stratagene). A complementary
oligonucleotide primer set, AHAS-Mut, was designed with
the intended point mutation. PCR amplification was carried
out using the pGEM T-easy plasmid with the AHAS gene as
a template, primers and the proofreading PfuUltra DNA
polymerase (Stratagene). PCR amplification was as follows:
denaturation at 948C for 1 min, followed by 12 cycles of
948C for 30 s, 558C for 60 s, and 728C for 5 min, and a final
extension cycle of 728C for 10 min. After the PCR reaction,
the parental DNA template was digested with DpnI restriction
enzyme. The PCR amplified plasmid was separated on agarose
gel, extracted, and inserted into E. coli competent cells.
Ampicillin resistant colonies were selected from which the
plasmid was extracted. The extracted plasmid was sequenced
for confirmation of the desired point mutation.
PiAHAS Functional Expression in Bacteria
E. coli K12 strain BUM1 was kindly provided by Professor Z.
Barak, Ben-Gurion University, Israel. BUM1 is a recA
mutant of strain CU9090, which does not express any AHAS
enzymes and cannot grow on minimal medium lacking
isoleucine or valine (Ibdah et al., 1996). This strain also
requires proline and thiamin, regardless of AHAS expression. Transformation of BUM1 cells was achieved using a
standard heat shock protocol. BUM1 cells were grown in LB
or in M9 minimal medium (7 mg mL1 Na2HPO4;
3 mg mL1 KH2PO4; 0.5 mg mL1 NaCl; 1 mg mL1
NH4Cl; 0.12 mg mL1 MgSO4; 0.35 mg mL1 thiamin–
HCl; 2 mg mL1 glucose) supplemented with 200 mg mL1
proline. Where appropriate, the M9 medium was
supplemented with the BCAAs valine, leucine, and isoleucine (150 mg mL1 of each) or with 50 mM SMM. SMM was
a gift of Dr. J. V. Schloss, (then of E. I. DuPont and Co.,
Central R&D Department, Wilmington, DE). BUM1
transformants were propagated and kept on LB medium
supplemented with 100 mg mL1 Ampicillin. For functional
expression assays, the cells were washed and plated on M9
agar plates and incubated for 48 h at 378C.
The expression vector pAH29 (Lawther et al., 1981)
was also obtained from Professor Z. Barak. This plasmid
contains the ilvGM genes (i.e., the entire coding region
of E. coli AHASII large and small subunits) under the native
bacterial promoter of the ilvGMEDA operon, ilvEp (Lopes
and Lawther, 1986). The pAH29 plasmid was used as a
template for amplification of the plasmid backbone with
pAH29-empty primer set, without the ilvGM coding region.
To clone the PiAHAS cDNA, three primers were designed.
PiAHAS wild type and mutant genes were amplified with
the forward primers, PiAHAS-ORF and PiAHAS-trunc,
designed to include and exclude the estimated 90 amino-acids
chloroplast targeting peptide, respectively. The forward
primers were also designed to include BamHI restriction
site. The reverse primer PiAHAS-R included a SalI
restriction site and was designed based on the 30 -end of
the gene.
PiAHAS in Vitro Enzyme Assay
To overexpress the hexahistidine (6 His)-tagged PiAHAS
genes in E. coli strain JM109, we used the plasmids pQE-WT
and pQE-MUT. These plasmids were constructed by
inserting the BamHI-SalI fragment containing the cloned
PiAHAS wt and mutant genes into plasmid pQE30 (Qiagen,
Hilden, Germany). These plasmids express PiAHAS fused at
its N-terminus to the pQE30 6xHis leader. pQE-WT and
pQE-MUT were transformed into E. coli for expression. The
transformed cells were grown, IPTG induced, and harvested
as described previously (Hill et al., 1997). Bacterial cells were
disrupted by sonication in a binding buffer with 20 mM
imidazol, 0.5 M sodium chloride, 50 mM sodium dihydrogen phosphate (pH 8.0), 20 mM FAD, and 50 mL g1 cells of
protease inhibitor cocktail (Sigma–Aldrich, Rehovot,
Israel). After 30 min of centrifugation at 20,000g, the
supernatant was loaded on a 1.5 8-cm column of Ni2þnitrilotriacetatic acid-agarose (Qiagen) previously washed
with the binding buffer. The column was then washed with
80 mL of the binding buffer, and the His-AHAS protein
eluted with 0.4 M imidazol in the binding buffer. The
fractions were dialyzed against 50 mM potassium phosphate
buffer (pH 7.6), containing 20 mM FAD. The protein was
concentrated for storage at 208C by dialysis against same
buffer, containing 50% glycerol. The AHAS catalytic activity
was determined as previously described (Bar-Ilan et al.,
2001). The reactions were carried for 20 min at 378C in
0.1 M potassium phosphate buffer (pH 7.6), containing
10 mM magnesium chloride, 0.1 mM ThDP, 75 mM FAD,
5 mM EDTA, 1 mM DTT with 100 mM pyruvate as
substrate, except where otherwise indicated. The activity
is expressed in units (U) (1 U ¼ 1 mmol of acetolactate
formed min1). Km for pyruvate and Ki for SMM were
determined by varying the concentration of the factor in
question. Protein concentration was determined by the dyebinding method (Bradford, 1976), with bovine serum
albumin as standard. Km calculations were fit with the
program ‘‘Sigma-Plot’’ to Michaelis–Menten equation. For
Ki calculations, the lines were fit to equations:
V ¼ Vo Ki =ðKi þ ½SMMÞ, for wild-type;
V ¼ Vf þ ðVo Vf Þ Ki =ðKi þ ½SMMÞ, for mutant.
V is the rate of acetolactate formation by AHAS;
Vo and Vf are beginning and final rates of acetolactate
formation;
[SMM] is concentration of SMM;
Results
Cloning of the PiAHAS cDNA
Multiple sequence alignment, by ClustalW, of the AHAS
protein sequences of the green algae Chlorella variabilis
(EFN51096.1), Chlamydomonas reinhardtii (AAB88292.1),
and Volvox carteri (AAC04854.1), the moss Physcomitrella
patens subsp. (XP_001759950.1) and the higher plant
Brassica napus (AAA62705.1) showed four conserved
sequence blocks, designated A–D (Fig. 1). The first set of
primers, AHAS01, amplified a 250 bp region of the Cterminal part of the gene between Block C and Block D. The
second set of primers, AHAS02, was of a degenerate nature
in which inosine was introduced at positions of nonconserved nucleotides, amplified a 500 bp region of the Nterminal part of the gene between Block A and Block B. The
amplified products were found to contain partial AHAS
coding sequences. Based on these cloned sequences, we
designed a third primer pair, AHAS03, which was used to
amplify a single band of about 1,200 bp in size, extending
from Block A to Block D. This complete 1,200 bp fragment
was cloned and sequenced and found to encode the PiAHAS
gene. The 50 - and 30 -ends of the PiAHAS cDNA were
amplified from the RACE Ready cDNA. RACE-PCR enabled
amplification and cloning of the gene ends, including 800 bp
of the 30 -untranslated region (UTR) and 80 bp of the
50 -UTR. The clones were sequenced and PiAHAS-full
primer set was designed and used to amplify the complete
2,100 bp AHAS coding sequence (GeneBank accession
number JN817966).
Prediction of PiAHAS Chloroplast-Targeting Signal
The putative PiAHAS protein was found to be 70–75%
identical to the AHAS proteins of the green algae
C. variabilis, C. reinhardtii, and of V. carteri. When these
Grundman et al.: P. incisa AHAS Site-Directed Mutagenesis
Biotechnology and Bioengineering
2343
proteins were aligned by ClustalW (Fig. 2), we found low
conservation in the N-terminus between these different
green algae. The first 100 amino acids of the PiAHAS Nterminus showed poor similarity to the other proteins,
particularly by the presence of a unique polyQ repeat. We
analyzed the encoded protein for the presence of possible
chloroplast transit peptides using ChloroP and TargetP
prediction software (www.cbs.dtu.dk). Neither predicts
plastidial localization for PiAHAS.
PiAHAS Site-Directed Mutagenesis
pGEM-T Easy vector, harboring PiAHAS cDNA, was
used as a template for site-directed mutagenesis by PCR
C.
V.
C.
P.
reinhardtii
carteri
variabilis
incisa
-MKALRSGTAVARGQAGCVSP----APRPVPMSSQAMIPSTSSPAARAPARSGRRALAVS
--MALRFCPTAAP-PRGCGTP----IQHPVLLLPHKALLPYSTAASRQAARPARVCVTAY
-----------------------------------------------------------M
MQGTMRPTAGALQQTVGCWHVPAGIPHAQQALRGRILPEELKQRCSATKPRAARQSAVTA
C.
V.
C.
P.
reinhardtii
carteri
variabilis
incisa
AKLADG-SRRMQS------------------EEVRRAKEVAQAALAKDSPADWVDRYGSE 96
AKLADGSARRMQS------------------EEVRRAKEVAQAALAKESPADWVDRFGSE 95
AKDFSNKANKASK------------------AELEAARQAAQASLASEPPVEWVDRFNGQ 43
AKLAEGKAGTPSRSLRQQPAAPQQQQQQQDSNELVALREAAKASLSSPAPAEWVDRFGSE 120
** .. :
.
*:
::.*:*:*:. .*.:****:..:
C.
V.
C.
P.
reinhardtii
carteri
variabilis
incisa
PRKGADILVQALEREGVDSVFAYPGGASMEIHQALTRSDRITNVLCRHEQGEIFAAEGYA
PRKGADILIQCLEREGVDNVFAYPGGASMEIHQALTRSDRITNVLCRHEQGEIFSAEGYA
ARKGSDILVQALEREGVDTLFAYPGGASMEIHQALTRSDSIRNILCRHEQGEIFAAEGYA
PRKGADILVQCLEREGAFRVFAYPGGASMEIHQALTRSGIIRNILCRHEQGEIFAAEGYA
.***:***:*.*****. :******************. * *:**********:*****
156
155
103
180
C.
V.
C.
P.
reinhardtii
carteri
variabilis
incisa
KAAGRVGVCIATSGPGATNLVTGLADAMMDSIPLVAITGQVPRRMIGTDAFQETPIVEVT
KASGRVGVCIATSGPGATNLVTRLDDAMMDSITLIAITGQVPRRMIGTDAFQETPIVEVT
KVTGRVGVCIATSGPGATNLVTGLADALLDSVPLVAITGQVPRKLIGSDAFQETPIVEVT
KCTGDVGVCIATSGPGATNLVTGLADAMLDSVPLVAITGQVPRKMIGTDGFQETPIVEVT
* :* ***************** * **::**:.*:********::**:*.**********
216
215
163
240
C.
V.
C.
P.
reinhardtii
carteri
variabilis
incisa
RAITKHNYLVLDIKDLPRVIKEAFYLARTGRPGPVLVDVPKDIQQQLAVPDWEAPMSITG
RAITKHNYLVLDIKDLPRVIKEAFYLARTGRPGPVLVDVPKDIQQQLAVPDWDSPMSITG
RQITKHNFLVMDVKDIPRIIKEAFYLARTGRPGPVLVDVPKDVQQTLDVPDWDSPMTISA
RQITKHNFLVMDLDDLPRIMKEAFYLARTGRPGPVLVDVPKDIQQQLAVPDWDTPMAISG
* *****:**:*:.*:**::**********************:** * ****::**:*:.
276
275
223
300
C.
V.
P.
C.
reinhardtii
carteri
incisa
variabilis
YISRLPPPVEESQVLPVLRALQGAAKPVIYYGGGCLDAQAELREFAARTGIPLASTFMGL
YISRLPPPVEEYKMIPVLRAIQSATKPIIYYGGGCLDARNELREFAARTGIPLASKFMGL
YMSRLPAPPNPSQLAAVVRALKEAKRPTLYVGGGALDSSAELREFVRLTGIPVAQTLMGL
YMSRLPPPPQEAQLQQVLDAIRGSKRPALYVGGGCVDSAAEVIEFVQHTGIPVAQTLMAL
*:****.* : :: *: *:: : :* :* ***.:*: *: **. ****:*..:*.*
336
335
360
283
C.
V.
C.
P.
reinhardtii
carteri
variabilis
incisa
GVVPSTDPNHLQMLGMHGTVFANYAVDQADLLVALGVRFDDRVTGKLDAFAARARIVHID
GVVPAEDPNHLQMLGMHGTVAANYAVDQADLLVALGVRFDDRVTGRLDAFASRARIVHVD
GSFPEQDPLALQMLGMHGTVAANFAVNEADLLLAFGARFDDRVTGKLEAFAANARIVHID
GTFPEEDPLALQMLGMHGTVYANYAVNDSDLLLAFGVRFDDRVTGKLEAFASRACIVHID
* .* ** ********** **:**:::***:*:*.********:*:***:.* ***:*
396
395
343
420
C.
V.
C.
P.
reinhardtii
carteri
variabilis
incisa
IDAAEISKNKTAHVPVCGDVKQALSHLNRLLAAEPLPADKWAGWRAELAAKRAEFPMRYP
IDAAEISKNKTAHVPVCGDVKQALRHLNRMLEAEPL-SDRFVAWRAELAAKRAEFPLRYP
IDPAEIHKNKDAHIPVCADIKPALQILNRLLSQTPMDRSGYADWVAEVMAMKEENPLAYP
IDPAEICKNKEAHIPICADLRASLIALNELLRRDPLPEGAFADWRAAIEAKKQEFPMTFP
**.*** *** **:*:*.*:: :* **.:*
*: . :. * * : * : * *: :*
456
454
403
480
C.
V.
C.
P.
reinhardtii
carteri
variabilis
incisa
QRDDAIVPQHAIQVLGEETQGEAIITTGVGQHQMWAAQWYPYKETRRWISSGGLGSMGFG
QRDDAIVPQYAIQVLGEETKGEVIITTGVGQHQMWAAQWYPYKEPRRWISSGGLGSMGFG
QHDDVIMPQWAIEVLYEESKGDAIITTGVGQHQMWAAQYYKFREPRRWATSGGLGSMGFG
ERDDVIIPQRAIQMLYEETNGEAIISTGVGQHQMWAAQWYQYNEPRRWVTSGGLGSMGFG
::**.*:** **::* **::*:.**:************:* :.*.*** :**********
516
514
463
540
C.
V.
C.
P.
reinhardtii
carteri
variabilis
incisa
LPAALG-AAVAFDGKNGRPKKTVVDIDGDGSFLMNVQELATIFIEKLDVKVMLLNNQHLG
LPAALG-AAVAFDGKQGREKRIVVDIDGDGSFLMNVQELATVFIEKLDVKVMILNNQHLG
LPSALG-AAAAFDGRDGRPSKLVVDIDGDGSFIMNCQELATASVEQLGTKVFILNNQYLG
LPSALGAAAVAYDGTDGRPKKVVVDIDGDGSFLMNCQELATAAVEGLETKIMILNNQHLG
**:*** **.*:** :** .: **********:** ***** :* * .*:::****:**
575
573
522
600
C.
V.
C.
P.
reinhardtii
carteri
variabilis
incisa
MVVQWEDRFYKANRAHTYLGKRESEWHATQDEEDIYPNFVNMAQAFGVPSRRVIVKEQLR
MVVQWEDRFYKANRAHTYLGKREAEWHATGDEEDIYPNFVGMARSFGVPSMRVIRKEDLR
MVMQWEDRFYKANRAHTYLGRREGEYQVTGNVQDIFPDFVKMADAFKVPAKRVTHPSELR
MVVQWEDRFYKANRAHTYLGHRANEYHTTLDESHIFPDFVMMAKSCGVPGRRVIKPEELR
**:*****************:* *::.* : ..*:*:** ** : **. **
.:**
635
633
582
660
C.
V.
C.
P.
reinhardtii
carteri
variabilis
incisa
GAIRTMLDTPGPYLLEVMVPHIEHVLPMIPGGASFKDIITEGDGTVKY-GANRTMLDTPGPYLLEVMVPHIEHVLPMIPGGATFKDIITEGDGSVKY-AAIREMLDTPGPYLLDVMVPHIQHVLPMIPGGGSFKDIITKGDGTDVYFV
GAIREMLDTPGPFLLDVMVPHVEHVLPMIPGGGSFKDIITKGDGRDEY-.* * *******:**:*****::*********.:******:***
*
Figure 2.
55
53
1
60
683
681
632
708
Multiple sequence alignment of PiAHAS with selected green algae AHAS proteins (C. reinhardtii, C. variabilis, and of V. carteri). The poorly conserved N-terminus is
estimated to be involved in protein plastidial trafficking (highlighted grey). The black triangle points to start of the ‘‘truncated’’ PiAHAS gene form. Mutated Trp residue, involved in
the herbicide binding and resistance, is shown as conserved (highlighted black).
2344
Biotechnology and Bioengineering, Vol. 109, No. 9, September, 2012
amplification with AHAS-Mut primers, carrying the desired
W605S mutation. The reaction resulted in pGEM-T Easy,
containing the mutagenized PiAHAS insert (PimAHAS).
The mutation was verified by DNA sequencing, and the
nucleotide sequence was deposited in the GeneBank by
accession number JN817967.
Growth Complementation of AHAS-Deficient E. coli by
PiAHAS and PimAHAS
The expression vector pEp-empty, an empty plasmid that
retains the native ilvEp promoter and ATG start codon, was
created by amplification of pAH29 plasmid with the pAH29empty primer set, and included BamHI and SalI restriction
sites for future fusion with the algal genes. Three forms of
PiAHAS genes were used for this experiment: PiAHAS full
open reading frame (ORF), truncated PiAHAS without the
estimated chloroplast targeting peptide (from Serine 91),
and PimAHAS containing the point mutation. The three
forms were amplified and digested with BamHI and
SalI. Each PiAHAS form was inserted into the pEp-empty,
under the native bacterial promoter to form three
constructs: pEp-ORF, pEp-truncated, and pEp-mut. A
vector containing only the Ep, pEp-empty, was used as a
negative control. The four constructs, together with pAH29
as a positive control, were transformed into E. coli BUM1
competent cells. The transformed cell lines were plated on
three types of agar plates: M9, M9 supplemented with
BCAAs, and M9 supplemented with 50 mM SMM. The
plates were incubated for 48 h (Fig. 3) and bacterial growth
was determined. All the cell lines were able to grow on the
BCAAs supplemented medium. The M9 plates, without
BCAAs, were used to select for bacteria with functional
AHAS activity. The host strain, BUM1, transformed with the
pEp-empty vector was unable to grow on these plates. In
contrast, bacteria transformed with the different PiAHAS
genes were able to grow on the selective medium, as were
cells transformed with pAH29. This result indicates that
the algal PiAHAS gene can functionally complement the
bacterial mutation, and that the first 90 amino acids are not
required for enzymatic activity. To test whether PimAHAS
conferred SMM resistance, we added 50 mM SMM to the M9
plates. Growth was totally inhibited in the bacteria
transformed with the wild type PiAHAS genes, whereas
those carrying the mutant gene were not affected by the
herbicide. Bacteria transformed with pAH29 were able to
grow slowly in the presence of 50 mM SMM, probably due to
higher level of SMM resistance of the bacterial AHASII
enzyme (Steinmetz et al., 2010).
Biochemical Assay of PiAHAS Activity in-vitro
P. incisa genes encoding the wild-type and mutant forms of
AHAS were cloned into the pQE30 expression plasmid and
expressed in E. coli JM109 cells as N-terminal hexahistidinetagged proteins. The proteins were expressed in soluble form
and were purified using Niþ-chelating column chromatography. The purified proteins were analyzed by 12% SDS–
PAGE (Fig. 4) and their weight was determined as about
75 kDa, the expected size of the putative PiAHAS. Enzymatic
parameters for interaction of the enzyme with its substrate
and with the herbicide inhibitor were determined (Table II).
Enzymatic activity of the wild type and mutant forms of
AHAS in the presence of different Pyruvate substrate
concentrations was assayed with 6.9 mg mL1 of PiAHAS
wild-type protein and 11.5 mg mL1 of mutant protein
(Fig. 5) and the specific activity and Km were calculated. The
specific activity of the purified PiAHAS was 3 U mg1,
whereas the tested mutant W605S showed a twofold
decrease. The inhibition of the P. incisa wild type and
mutant enzymes, by different SMM concentrations, was also
assayed with 8.3 mg mL1 of PiAHAS wild-type protein and
23 mg mL1 of mutant protein (Fig. 6). The wild type form
was strongly inhibited by very low concentration of SMM
and its Ki was determined to be 0.15 mM. W605S
substitution on the other hand, resulted in strong resistance
to this herbicide, even at high concentrations of 200 mM.
The Ki was determined to be >30 106, 7 orders of
magnitude higher than that of the wild type.
Figure 3. Functional complementation of BUM1 strain by cloned PiAHAS genes. A single colony from each transformed cell lines was streaked onto M9 supplemented with
valine, leucine, and isoleucine (A), M9 (B), and M9 supplemented with 50 mM SMM (C) agar plates and incubated at 378C for 48 h. The sections are: 1. pEp-empty, 2. pAH29 (ilvGM), 3.
pEp-ORF, 4. pEp-truncated and 5. pEp-mut.
Grundman et al.: P. incisa AHAS Site-Directed Mutagenesis
Biotechnology and Bioengineering
2345
Specific activity, U*mg-1
3.0
2.5
2.0
1.5
1.0
.5
0.0
0
50
100
150
200
250
300
Pyruvate, mM
Figure 5.
Pyruvate dependence for the purified PiAHAS wild-type and its
mutant. The reactions for AHAS wild-type (*) and its mutant (*) were carried
out at 378C for 20 min in pH 7.6.
SDS–PAGE analysis of purified wild-type and mutant PiAHAS. Purified
proteins were analyzed by 12% SDS–PAGE gel stained with Coomassie blue. Lanes:
Protein marker (M); wild-type PiAHAS; mutant PiAHAS. Over-expression of 75 kD
recombinant protein is visible.
Discussion
In this study, the gene encoding the PiAHAS was successfully
isolated and cloned. The gene was mutated at a Trp residue
located at the active site of AHAS and known to confer SMM
resistance in bacteria (Chipman et al., 1998; Choi et al.,
2010), plants (Hattori et al., 1995), and algae (Lapidot et al.,
1999). We have focused on this mutation in the current
work but other mutations of AHAS conferring resistance to
SMM and related sulfonylurea herbicides in microalgae
(Kovar et al., 2002) could be characterized in the future in
the same manner. The wild type and mutant genes were
functionally expressed in AHAS-deficient bacteria. The
genes were shown to complement AHAS activity in vivo and
the mutant form was shown to confer SMM resistance. In
green algae and higher plants, AHAS is encoded in the
nucleus and the protein is targeted to the chloroplast by
Table II.
120
100
80
Activity, %
Figure 4.
N-terminal chloroplast-targeting signal (Jones et al., 1985).
Commonly used for higher plants prediction software, such
as ChloroP and TargetP, did not identify the protein as
plastidial targeted, suggesting a unique, species-specific,
chloroplast targeting sequence in P. incisa. Here, we
demonstrated that a truncated protein, lacking the first
90 amino-acids of the N-terminus, retained its enzymatic
activity in vivo (Fig. 3). This suggests that this poorly
conserved peptide is indeed likely to be a targeting peptide.
The wild type and mutant proteins were cloned into pQE30
vector, expressed as his-tagged proteins, isolated and
characterized in-vitro. The molecular weight of the
PiAHAS protein was determined to be 75 kDa by SDS–
PAGE (Fig. 4), comparable to that of other green algae such
60
40
Kinetic parameters for wild-type and mutant AHASa.
20
Parameter
Wild-type
Mutant
Specific activity, U mg1
Km for pyruvate, mM
kcat/Km, M1 s1
Ki for SMM, mMb
3.00 0.05
49.4 2.1
83
0.15 0.01
1.46 0.03
71.7 2.9
25
>30 106
a
The kinetic parameters were determined as described under Materials
and Methods Section at 378C and pH 7.6.
b
The concentration of pyruvate was 100 mM.
2346
Biotechnology and Bioengineering, Vol. 109, No. 9, September, 2012
0
0
50
100
150
200
250
SMM, µM
Figure 6.
Inhibition by SMM of the purified PiAHAS wild-type (*) and its mutant
(*). The reactions were carried out at 378C and pH 7.6.
as C. variabilis (69 kDa) and C. reinhardtii (74 kDa). We
have shown that the gene encodes an active protein. The
W605S mutation caused a twofold decrease in enzymatic
activity and in the affinity to the Pyruvate substrate,
compared to that of the wild type. Previous reports of E. coli
and M. tuberculosis AHAS reported a similar observation of
this particular mutation in the active site that was attributed
to involvement of this Trp residue in substrate preference.
Tryptophan plays the important role in the preference of
second substrate by forming hydrophobic interactions with
the substrates methyl groups. Therefore the replacement
hydrophobic Trp for hydrophilic serine leads to significant
changes in all other catalytic properties (Choi et al., 2010;
Engel et al., 2004). On the other hand, as predicted, the
mutant showed 7 orders of magnitude higher resistance to
the SMM herbicide than the wild type. E. coli AHASII
appeared to be more resistant to inhibition by SMM than the
wild type PiAHAS. This observation is consistent with Ki
reports of AHASII for SMM that are an order of magnitude
higher than of PiAHAS (1.1 0.1 mM and 0.15 0.01 mM,
respectively) (Steinmetz et al., 2010).
Transformation reports of mutant forms of AHAS as a
selection marker commonly use cloned genes from naturally
occurring herbicide resistant mutants (Kovar et al., 2002;
Lapidot et al., 2002; Li et al., 1992; Ray et al., 2004). We were
not able to isolate spontaneous resistant mutations in
P. incisa and this work introduces a tool for simple
assessment of different, artificially induced, mutations for
enzymatic activity and resistance to different inhibitors.
Previous works have demonstrated that cloned animal or
plant genes, including AHAS, are able to complement
bacterial mutations (Davidson and Niswander, 1983;
Goddard et al., 1983; Smith et al., 1989). This work
demonstrates the ability of bacterial cells to also synthesize
functional algal proteins and suggests a simple heterologous
gene characterization system. In conclusion, this mutant
gene form can be used as an endogenous, non-antibiotic,
environmentally safe selection marker for future P. incisa
genetic transformation that will result in herbicide-insensitive algae.
We thank Prof. Z. Barak (Life Science Department, Ben-Gurion
University, Beer-Sheva, Israel) for the helpful discussions and for
providing us the materials to carry out the AHAS characterization.
BGU VP for Research Support Fund for providing financial support.
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