Design of gene structure and expression of E. coli phytase and Aspergillus Niger phytase in Pichia pastoris yeast in order to increase plant phytate degradation.

Document Type : Research Paper

Authors

1 Ph.D. Student, Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

2 Professor, Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

3 Assistant Professor, School of Animal and Veterinary Science, The University of Adelaide, Roseworthy, South Australia, Australia.

4 Assistant Professor, Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran

Abstract

Phytases are classified as 3-phytases (E.C.3.1.3.8), 5-phytases (E.C. 3.1.3.72) and 6-phytases (E.C. 3.1.3.26) based on the position of the first phosphate residue removed from the myo-inositol ring of phytate. Yeasts are particularly suited to expression of foreign proteins for numerous features mean while some of them shows probiotic properties. Pichia pastoris, has been developed for the production of various recombinant proteins and growth into high cell densities in an inexpensive medium. Accordingly, Pichia pastoris is an appropriate secretory system for obtaining larger quantities of correct products, in compared to other host cells. The coexpression of digestive enzymes in a single recombinant cell system would thus be advantageous. The objective of this study is to determine whether combining fungal phytases (3-phytase) with bacterial phytase (6-phytase) was more effective than each phytase alone in degrading of plant phytate. we tested the new vector, aimed to clone and coexpress the phytase genes isolated from E. coli and aspergillus niger and transformed into Pichia pastoris. Evaluations were made for the biochemical properties of the active expressed phytase.
Methods:
The nucleotide sequence of phyA and appA2, 2a peptide and alpha factor were obtained from NCBI database. A coexpression system for the extracellular production of phytase of Aspergillus Niger (3-phytase) and phytase of E. coli (6-phytase) will be established in Pichia pastoris yeast. Plasmid that used in this study was pPIC9k. The genes for each enzyme are fused in-frame with the “a-factor” secretion signal and linked by the 2A-peptide-encoding sequence. After receiving the synthetic plasmid with fragments, plasmid that contained phyA and appA2 was cloned in DH5@ and after that digested by SacI and electrotransfered into Pichia pastoris. Positive cells are resuspended at BMMY containing methanol as an inducer. To follow the induction, methanol was injected into the culture every 24 hours in order to reach a definitive concentration of 0.5 percent. The recombinant protein was analyzed using sds-page.
Result:
The results showed that the recombinant phytase gene was transferred to Pichia pastoris and the results of PCR confirmed that. The molecular weight of the produced recombinant phytases was estimated to be around 45 and 80 kDa for appA and phyA, respectively. The recombinant protein has phytase activity equal to 160.97 U/ml
Conclusions;
The designed phytase genes containing phyA and appA were successfully cloned in DH5a and the recombinant plasmid was transferred to Pichia pastoris for high expression. Recombinant yeast will be used for further experiments.

Keywords

References

عرب پور رق آبادی زهرا، محمدآبادی محمدرضا، خضری امین (1400) الگوی بیانی ژن p32 در بافت‌های ران، دست، راسته و چربی پشت بره کرمانی. مجله بیوتکنولوژی کشاورزی 13(4)، 183-200.
محمدآبادی محمدرضا، اسدالله پور نعنایی حجت (1400) بیان ژن لپتین در بز کرکی راینی با استفاده از Real Time PCR. مجله بیوتکنولوژی کشاورزی 13(1)، 214-197.
محمدآبادی محمدرضا، حسن زاده داورانی فاطمه (1400) اهمیت تغذیه در  بیان ژن، همانندسازی، ترمیم و پیشگیری از آسیب  DNA. مجله بیوتکنولوژی کشاورزی 13(3)، 222-205.
محمدآبادی محمدرضا، خیرالدین حمید، لطیفی آیگین، بابنکو اولنا ایوانیونا (1401) پروفایل بیانی ژن  DNAH1در بافت بیضه بز کرکی راینی. مجله بیوتکنولوژی کشاورزی 14(3)، 243-256.
محمدآبادی محمدرضا، شبان جرجندی دیانا، عرب پور رق آبادی زهرا و همکاران (1401) نقش رازیانه بر بیان ژن DLK1 در بافت قلب گوسفند. مجله بیوتکنولوژی کشاورزی 14(2)، 155-133.
 
References
Arabpoor Raghabadi Z, Mohammadabadi MR, Khezri A (2021) The expression pattern of p32 gene in femur, humeral muscle, back muscle and back fat tissues of Kermani lambs. Agric Biotechnol J 13 (4), 183-200.
Cregg JM, Cereghino JL, Shi J, Higgins DR (2000) Recombinant protein expression in Pichia pastoris. J Mol Biotechnol 16, 23-52.                      
Cregg JM, Barringer KJ, Hessler AY, Madden KR (1985) Pichia pastoris as a host system for transformations. J Mol Cell Biol 5, 3376-3385.         
Ehrlich KC, Montalbano BG, Mullaney EJ et al. (1993). Identification and         cloning of a second phytase gene (phy B) from Aspergillus niger (ficuum). Biochem Biophysic Res Commun 195, 53–57.
Fidler AE, Lin JS, Lun S et al. (2003) Production of biologically active tethered ovine FSHβα by the methylotrophic yeast Pichia pastoris. J Mol Endocrinol 30, 213–25.
Greiner R, Konietzny U (2006) Phytase for Food Application. Food Technol and Biotechnol 4, 125–140
Heravi RM, Sankian M, Kermanshahi H et al. (2017) The Construction of a Probiotic Lactic Acid Bacterium Expressing Acid-Resistant Phytase Enzyme. International Journal of Biotechnology and Bioengineering, 9(8), 963-968.‏
Invitrogen (2005) Pichia Expression Kit, Protein Expression, A Manual of Methods for Expression of Recombinant Proteins in Pichia pastoris. Catalog no. K1710-01. http://www.invitrogen.com
Konietzny U, Greiner R (2004) Bacterial phytase: potential application, in vivo function and regulation of its synthesis. Braz. J. Microbiol 35, 12-18.
Kumar V, Sinha AK, Makkar HPS, Becker K (2010) Dietary roles of phytate and phytase in human nutrition: A review. Food Chem 120(4), 945–959.     
Lei GX, Porres JM, Mullaney EJ, Brinch-Pedersen H (2007) Phytase: Source, structure and application. Industerial enzymes. 505-529.         
Damasceno LM, Huang CJ, Batt CA (2012) Protein secretion in Pichia pastoris and advances in protein production. J Appl Microbiol Biotechnol 93, 31-39.
Li P, Anumanthan A, Gao XG et al. (2007) Expression of recombinant proteins in Pichia pastoris. J Appl Biochem Biotechnol 142, 105-124.
Liu JR, Yu B, Lin SH et al. (2005) Direct cloning of a xylanase gene from the mixed genomic DNA of rumen fungi and its expression in intestinal Lactobacillus reuteri. FEMS microbiol lett 251(2), 233-241.
Liu JR, Yu B, Zhao X, Cheng KJ (2007) Coexpression of rumen microbial β-glucanase and xylanase genes in Lactobacillus reuteri. Appl microbiol biotechnol 77, 117-124.
Liu R, Zhao B, Zhang Y et al. (2015) High-level expression, purification, and enzymatic characterization of truncated human plasminogen (Lys531-Asn791) in the methylotrophic yeast Pichia pastoris. BMC biotechnology 15(1), 1-8.
Mattanovich D, Branduardi P, Dato L et al. (2012) Recombinant protein production in yeasts. J Methods Mol Biol 824, 329-358.
Mohammadabadi MR, Kheyrodin H, Latifi A, Babenko OI (2022a) mRNA expression profile of DNAH1 gene in testis tissue of Raini Cashmere goat. Agric Biotechnol J 14 (3), 243-256.
Mohammadabadi MR, Asadollahpour Nanaei H (2021) Leptin gene expression in Raini Cashmere goat using Real Time PCR. Agric Biotechnol J 13 (1), 197-214.
Mohammadabadi MR, Shaban Jorjandy D, Arabpoor Raghabadi Z, et al. (2022b) The role of fennel on DLK1 gene expression in sheep heart tissue. Agric Biotechnol J 14 (2), 155-170.
Mohammadabadi MR, Hasanzadeh Davarani F (2021) The importance of nutrition in gene expression, replication, repair and prevention of DNA damage. Agric Biotechnol J 13 (3), 205-222.
Pakbaten B, Majidzadeh Heravi R, Kermanshahi et al. (2019) Production of phytase enzyme by a bioengineered probiotic for degrading of phytate phosphorus in the digestive tract of poultry. Probiotics Antimicrob Proteins 11, 580-587.‏
Prinz B, Schultchen J, Rydzewski R et al. (2004) Establishing a versatile fermentation and purification procedure for human proteins expressed in the yeasts Saccharomyces cerevisiae and Pichia pastoris for structural genomics. J Struct Funct Genomics 5, 29-44.
Roongsawang N, Promdonkoy P, Wongwanichpokhin M et al. (2010) Coexpression of fungal phytase and xylanase utilizing the cis-acting hydrolase element inPichia pastoris.  J FEMS Yeast Res 10 (2010), 909–916.
Rodriguez E, Han Y, Lei XG (1999) Cloning, Sequencing, and Expression of an Escherichia coli Acid Phosphatase/Phytase Gene (appA2) Isolated from Pig Colon. Biochem. Biophys Res Commun 257,117-123.
Selle PH, Ravindran V (2007) Microbial phytase in poultry nutrition. Anim Feed Sci and Technol 135(1), 1-41.
Tran TT, Hatti-Kaul R, Dalsgaard S, Yu S (2011) A simple and fast kinetic assay for phytases using phytic acid–protein complex as substrate. Anal biochem 410, 177-184.
Ullah AHJ, Gibson DM (1987) Extracellular phytase (E.C.3.1.3.8) from Aspergillus ficuum NRRL 3135: purification and characterization. Prep Biochem 17, 63–91.
Wang S, Yao Q, Tao J et al. (2007) Co-ordinate expression of glycine betaine synthesis genes linked by the FMDV 2A region in a single open reading frame in Pichia pastoris. Appl Microbiol Biot 77, 891–899.
Weaver JD, Ullah AH, Sethumadhavan K et al. (2009) Impact of assay conditions on activity estimate and kinetics comparison of Aspergillus niger PhyA and Escherichia coli AppA2 phytases. J Agric Food Chem 57(12), 5315-5320.‏
Yanke LJ, Bae HD, Selinger LB, Cheng KJ (1998). Phytase activity of anaerobic ruminal bacteria. Microbiol 144(6) 1565-1573.‏
Yokoyama S (2003) Protein expression systems for structural genomics and proteomics. J Currn Opin Chem Biol 7, 39-43.
Zhang AL, Luo JX, Zhang TY et al. (2009) Recent advances on the GAP promoter derived expression system of Pichia pastoris. J Mol Biol Rep 36, 1611-1619.
Zou LK, Wang HN, Pan X et al. (2008). Expression, purification and characterization of a phyA m-phyCs fusion phytase. J Zhejiang Univ Sci B 9, 536-545.‏
Agricultural Biotechnology Journal
Volume 16, Issue 1
February 2024
Pages 135-154

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