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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). 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