Advanced Materials Research Vols 347-353 (2012) pp 2438-2442 Online: 2011-10-07 © (2012) Trans Tech Publications, Switzerland doi:10.4028/www.scientific.net/AMR.347-353.2438

Molecular Cloning and Characterization Analysis of pyruvate phosphate dikinase from Dunaliella parva

Changhua Shang 1,a , Shunni Zhu 1,b , Zhenhong Yuan 1,c and Zhongming Wang 1,d* 1 Key Laboratory of Renewable Energy and Natural Gas Hydrate, Chinese Academy of Sciences, Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou, 510640, China No.2, Nengyuan Rd, Wushan, Tianhe District, Guanzhou. aemail: [email protected], bemail: [email protected], cemail: [email protected], demail: [email protected] *Corresponding author: Zhongming Wang

Keywords: Dunaliella parva; pyruvate phosphate dikinase; ; sequence analysis

Abstract. Pyruvate phosphate dikinase (PPDK) catalyzes the reversible conversion of AMP, phosphoenolpyruvate (PEP) and pyrophosphate (PPi) to ATP, pyruvate and inorganic phosphate (Pi). It is a key in gluconeogensis and photosynthesis that is responsible for reversing the reaction performed by pyruvate in Embden-Meyerhof-Parnas glycolysis. A cDNA clone for the Dunaliella parva PPDK was isolated by . Then the 3'-RACE and 5'-cDNA amplification were conducted based on the obtained sequence. The molecular characterization of the PPDK gene was described.The Dunaliella parva PPDK gene cDNA sequence was 3249 bp, which contained 2595 bp coding region and 654 bp 3'-untranslated regions. The deduced sequence of Dunaliella parva PPDK showed significant homology to the known PPDK from Volvox carteri and Chlamydomonas reinhardtii . This study provided foundation for further research on the function analysis and overexpression of PPDK . To our knowledge this is the first reported.

Introduction Oil-rich microalgae have been proven to be a promising alternative source of lipids producing biodiesel [1-4]. Nevertheless, significant challenges remain in the cost of microalgal biodiesel production. One problem was that the lipid content of microalgae was unsatisfactory for the requirement of commercial production. One of the solution was increasing lipid content in cells. Lipid overproduction using microalgae by genetic engineering approaches was a burgeoning trend. Our research focused on the enhancement of lipid production in green alga using genetic engineering approaches. Because there was correlation between the photosynthesis and lipid accumulation, therefore we planed to enhance the lipid production of green alga through the enhancement of the photosynthesis. Pyruvate phosphate dikinase (PPDK ; EC 2.7.9.1 ) catalyzes the reversible conversion of AMP, phosphoenolpyruvate (PEP) and pyrophosphate (PPi) to ATP, pyruvate and inorganic phosphate (Pi) [5]. It is a key enzyme in gluconeogensis and photosynthesis [5]. This enzyme is part of the C 4 -dicarboxylic acid cycle, for the fixation of atmospheric CO 2 in mesophyll cells and subsequent transport (as malate) to bundle sheath cells for photosynthesis [6-8]. The result of Wang et al. indicated that the two known to limit C 4 photosynthesis, increase of PPDK, not Rubisco content, corresponds to the recovery and maintenance of photosynthetic capacity [9]. The studies of Hatch and Furbank et al. suggested that PPDK was a rate-limiting enzyme in the C 4 cycle, the PPDK activity is regulated in a light-dependent manner so the total pathway can function optimally in CO 2 assimilation [10-12].

All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of Trans Tech Publications, www.ttp.net. (ID: 152.14.136.77, NCSU North Carolina State University, Raleigh, USA-09/04/15,07:32:04)

Advanced Materials Research Vols. 347-353 2439

The PPDK gene have been cloned from various species, including Giardia lamblia , Entamoeba histolytica , Trypanosoma cruzi [13-15]. In view of a possible quantitative correlation between PPDK expression levels, photosynthesis, and lipid accumulation, it is of interest to study the PPDK-catalyzed committed step at the molecular level. As a first step to better understanding of the role of PPDK in photosynthesis and lipid accumulation in Dunaliella parva , we report for the first time the cloning and characterization of a new PPDK gene from this commercially important microalgae.

Materials and Methods Cell culture conditions. Dunaliella parva FACHB-815, was obtained from the Freshwater Algae Culture Collection of the Institute of Hydrobiology, Chinese Academy of Sciences, and maintained on Dunaliella medium at 25˚C under light illumination of 2000lux.

Total RNA extraction and 1st strand cDNA synthesis. Total RNA was isolated from Dunaliella parva , using a RNAiso Plus reagent (Takara Co. Ltd., China). RNA concentration was determined spectrophotometrically. were reversely transcribed with PrimeScript Reverse Transcriptase, and an 3′ RACE Adaptor primer was used according to protocol of 3′-Full RACE Core Set Ver.2.0 (Takara Co . Ltd., China). 1 µ g total RNA was used for the reverse reaction.

Cloning of a PPDK cDNA fragment from Dunaliella parva. A cDNA clone for the Dunaliella parva PPDK was isolated by sequencing. This clone showed extensive homology with a number of known PPDK genes , providing strong evidence that the obtained sequence encoded part of the Dunaliella parva PPDK cDNA.

Cloning of PPDK 3'-cDNA by rapid amplification of cDNA ends (RACE). Total RNA was used to synthesize the first-strand cDNA using a 3'-Full RACE Core Set Ver 2.0 Kit (Takara Co. Ltd., China ). 3'-RACE was conducted with PPDK (w) and PPDK (n) (Table 1) as gene specific outer and inner primers.

The first cloning of the 5'-cDNA sequence. Based on highly conserved amino acid regions of known PPDK , a degenerate primers, PPDK (s1) (Table 1) encoding MPGMMDT was synthesized. According to the obtained cDNA clone sequence, a specific primer PPDK(a1t) was synthesized. PPDK (s1) and PPDK(a1t) were used for amplification of partial 5'-cDNA sequence of PPDK .

The second cloning of the 5'-cDNA sequence. Based on the obtained partial 5'-cDNA sequence, a specific primer PPDK(a2t) was synthesized. Based on highly conserved amino acid regions of known PPDK, a degenerate primers, PPDK (s2) (Table 1) encoding LLGGKGA was synthesized. PPDK (s2) and PPDK(a2t) were used for amplification of partial 5'-cDNA sequence of PPDK.

Table 1 Primers used in this study Primers Primer sequences (5′→3′ ) PPDK(w) CACTCCCGCTTGGACTCACTG PPDK(n) ATCCCTCTGGTCGGCATTGTG PPDK(s1) ATGCCHGGCATGATGGACAC PPDK(a1t) CCATCCATCGCCCTGAAGAG PPDK(s2) CTSCTSGGCGGCAAGGGCGC PPDK(a2t) GTGCTGCCTTGACCCTCT

2440 Renewable and Sustainable Energy

Results and Discussion A cDNA clone for the Dunaliella parva PPDK was isolated by sequencing. A Blastx search revealed that the cloned sequence (613 bp) was homologous to a number of known PPDK genes, providing strong evidence that the obtained sequence encoded part of the Dunaliella parva PPDK cDNA. The 3' end cDNA of Dunaliella parva PPDK was amplified by 3'-RACE using PPDK(w) and PPDK(n) based on the 613 bp cDNA fragment. The 3' end cDNA of PPDK is 1173 bp, which contained a 519 bp coding region, 654 bp of 3'-untranslated regions, and a poly A tail 15 bp. The 5' end cDNA of PPDK was amplified by PPDK(s1) and PPDK(a1t) first, then amplified by PPDK(s2) and PPDK(a2t). The length of two amplification were 1544 bp and 433 bp respectively. The total cDNA sequence was assembled from the obtained sequence, which include 2595 bp coding region and 654 bp 3'- untranslated regions (Fig. 1). NCBI-CD search revealed that there are three mainly domains predicted in the Dunaliella parva PPDK (Fig. 2). PPDK_N superfamily was a PEP/pyruvate binding domain, PEP-utilizers domain was a "swivelling" beta/beta/alpha domain which is thought to be mobile in all known to contain it. Pyruvate_kinase superfamily was a domain that regulates glycolysis through binding of the substrate, phosphoenolpyruvate, and one or more allosteric effectors. Blastx search revealed that the Dunaliella parva PPDK was homologous to a number of known PPDK genes, The highest similarities were recorded with green algae Volvox carteri and Chlamydomonas reinhardtii . Oleaginous algae have been recognized as promising alternative lipid/oil feedstocks for biofuel production. However, algae-based biofuels are limited by feedstock supply. Lipids are more energy dense, which have frequently been applied as fuel feedstocks. The former studies indicated the feasibility of enhancing photosynthesis through overexpression of PPDK gene [5-12]. In this study, we cloned and characterized the gene encoding PPDK of Dunaliella parva . Multiple sequence alignments revealed that the deduced amino acid sequence of the Dunaliella parva PPDK gene shared high homology with that of many algal species and other organisms. This study provided foundation for further research on the function analysis and overexpression of PPDK genes. Our next work should conduct the correlation analysis among the overexpression of PPDK gene, the photosynthesis and lipid accumulation.

Conclusions In conclusion, our data show partial PPDK cDNA was cloned from Dunaliella parva , which includes 2595 bp coding region and 654 bp 3'-untranslated regions. The characterization of PPDK shows it should be functional and can be used to enhance photosynthesis of transgenic algae by it's overexpression in cells.

Acknowledgements This work was financially supported by the comprehensive strategic cooperation project between Guangdong Province and Chinese Academy of Sciences (No. 2010A090100010 ), National Key Technology R&D Program of the 12th Five-year Plan of China (No. 2011BAD14B03 ) and Foundation of Key Laboratory of Renewable Energy and Natural Gas Hydrate, Chinese Academy of Sciences (No. y107j6).

Advanced Materials Research Vols. 347-353 2441

References [1] Y. Chisti, Biotechnol. Adv. 25 (2007) 294 -306 [2] Y. L i, M. Horsman, N. Wu, C.Q. Lan, N. Dubois-Calero, Biotechnol. Prog. 24 (2008) 815-820 [3] B. Wang, Y. Li, N. Wu, C.Q. Lan, Appl. Microbiol. Biotechnol. 79 (2008) 707-718 [4] T.L. Walker, S. Purton, D.K. Becker, C. Collet, Plant Cell Rep. 24 (2005) 629-641 [5] D.J. Pocalyko, L.J. Carroll, B.M. Martin, P.C. Babbitt, D. Dunaway-Mariano, Biochemistry 29 (1990) 10757-10765 [6] F.D. MacDonald, B.B. Buchanan, Carbon Dioxide Assimilation, in: J. Amesz (Eds.), Photosynthesis (New Comprehensive Biochemistry), Elsevier Science Ltd, Amsterdam, 1987, pp. 175-197 [7] J. Sheen, Annu Rev Plant Physiol Plant Mol Biol 50 (1999) 187-217 [8] R.A. Maldonado, A.H. Fairlamb, Mol. Biochem. Parasitol. 112 (2001) 183-191 [9] D. Wang, A.R. Jr. Portis, S.P. Moose, S.P. Long, Plant Physiol. 148 (2008) 557-567 [10] M.D. Hatch, Biochim. Biophys. Acta 895 (1987) 81-106 [11] C.G. Hocking, J.W. Anderson, Phytochemistry 25 (1986) 1537-1543 [12] C.J. Chastain, J.P. Fries, J.A. Vogel, C.L. Randklev, A.P. Vossen, S.K. Dittmer, E.E. Watkins, L.J. Fiedler, S.A. Wacker, K.C. Meinhover, G. Sarath, R. Chollet, Plant Physiol. 128 (2002) 1368-1378 [13] X.M. Feng, L.J. Cao, R.D. Adam, X.C. Zhang and S.Q. Lu: Biochem. Biophys. Res. Commun. V ol. 367(2) (2008) , p. 394-398. [14] M.Varela-Gómez, R. Moreno-Sánchez, J.P. Pardo, R. Perez-Montfort, J. Biol. Chem. 279 (2004) 54124-54130. [15] H. Acosta, M. Dubourdieu, W. Quiñones, A. Cáceres, F. Bringaud, J.L. Concepción, Comp. Biochem. Physiol. B, Biochem. Mol. Biol. 138 (2004) 347-356

Figure 2 The deduced domain of the Dunaliella parva PPDK protein There are three mainly domains predicted in the Dunaliella parva PPDK protein, including a conserved PPDK_N superfamily at position 1~317 amino acid, a PEP-utilizers domain at 403~484 amino acid and a Pyruvate_kinase superfamily at 497~850 amino acid

2442 Renewable and Sustainable Energy

Figure 1 The partial cDNA sequence of PPDK gene from Dunaliella parva Renewable and Sustainable Energy 10.4028/www.scientific.net/AMR.347-353

Molecular Cloning and Characterization Analysis of Pyruvate Phosphate Dikinase Gene from Dunaliella parva 10.4028/www.scientific.net/AMR.347-353.2438

DOI References [1] Y. Chisti, Biotechnol. Adv. 25 (2007) 294-306. http://dx.doi.org/10.1016/j.biotechadv.2007.02.001 [3] B. Wang, Y. Li, N. Wu, C.Q. Lan, Appl. Microbiol. Biotechnol. 79 (2008) 707-718. http://dx.doi.org/10.1007/s00253-008-1518-y [4] T.L. Walker, S. Purton, D.K. Becker, C. Collet, Plant Cell Rep. 24 (2005) 629-641. http://dx.doi.org/10.1007/s00299-005-0004-6 [5] D.J. Pocalyko, L.J. Carroll, B.M. Martin, P.C. Babbitt, D. Dunaway-Mariano, Biochemistry 29 (1990) 10757-10765. http://dx.doi.org/10.1021/bi00500a006 [7] J. Sheen, Annu Rev Plant Physiol Plant Mol Biol 50 (1999) 187-217. http://dx.doi.org/10.1146/annurev.arplant.50.1.187 [8] R.A. Maldonado, A.H. Fairlamb, Mol. Biochem. Parasitol. 112 (2001) 183-191. http://dx.doi.org/10.1016/S0166-6851(00)00362-5 [9] D. Wang, A.R. Jr. Portis, S.P. Moose, S.P. Long, Plant Physiol. 148 (2008) 557-567. http://dx.doi.org/10.1104/pp.108.120709 [10] M.D. Hatch, Biochim. Biophys. Acta 895 (1987) 81-106. http://dx.doi.org/10.1016/S0304-4173(87)80009-5 [11] C.G. Hocking, J.W. Anderson, Phytochemistry 25 (1986) 1537-1543. http://dx.doi.org/10.1016/S0031-9422(00)81205-4 [12] C.J. Chastain, J.P. Fries, J.A. Vogel, C.L. Randklev, A.P. Vossen, S.K. Dittmer, E.E. Watkins, L.J. Fiedler, S.A. Wacker, K.C. Meinhover, G. Sarath, R. Chollet, Plant Physiol. 128 (2002) 1368-1378. http://dx.doi.org/10.1104/pp.010806 [14] M. Varela-Gómez, R. Moreno-Sánchez, J.P. Pardo, R. Perez-Montfort, J. Biol. Chem. 279 (2004) 54124- 54130. http://dx.doi.org/10.1074/jbc.M401697200