بهینه‌سازی بیان مسیر بیوسنتز فلاونوئید نارینجنین با استفاده از کشت سلولی یارُویا لیپولیتیکا

نویسندگان

1 دانشکده علوم کشاورزی، دانشگاه گیلان، رشت، ایران

2 دانشکده کشاورزی، دانشگاه ایلام، ایلام، ایران

3 دانشکده بیوتکنولوژی، دانشگاه جیانگ نان، جیانگ سو، چین

4 دانشکده مهندسی شیمی، بیوشیمی و محیط زیست، دانشگاه مریلند، بالتیمور، آمریکا

چکیده
امروزه استفاده از مخمر یارُویا لیپولیتیکا به دلیل برخورداری از ظرفیت تولید بالا، گلیکوزیله شدن در مقادیر کم، برخورداری از نشانگرهای مولکولی و ابزارهای ژنتیکی انحصاری به طور گسترده و برای سرعت بخشیدن به روند تولید ترکیبات گیاهی-دارویی و به عنوان یک میزبان سلولی بسیار مورد توجه قرار گرفته است. نارینجنین نه‌تنها به‌عنوان هسته مرکزی تولید فلاونوئیدهای مختلف از اهمیت زیادی برخوردار است، بلکه در گیاهان و نیز درمان بیماری ­های مختلف انسان نقش ویژه ­ای برعهده دارد. به‌همین منظور و برای تولید بهینه این ترکیب فلاونوئیدی ارزشمند، ژن­ های مهم و اصلی مسیر بیوسنتز نارینجنین از منابع مختلف گیاهی شناسایی شدند و پس از مقایسه الگوی بیان و به منظور تولید نارینجنین در میزبان موردنظر معرفی شدند. نتایج این تحقیق نشان داد که chs ژن کلیدی و مهم در تولید نارنجنین به‌شمار می­ رود و به‌همین منظور افزایش تعداد نسخه­ های این ژن در هر سازه ژنی مورد بررسی قرار گرفت. نتایج حاصل از تجزیه داده ­های HPLC نشان داد که بهینه‌سازی شرایط رشد و رفع موانع محدودکننده و نیز افزایش تعداد 5 نسخه ژن chs در هر سازه می­ تواند 14/7 برابر میزان نارینجنین را افزایش دهد و تولید آن را به 16/90 میلی­ گرم در لیتر برساند. این بررسی نشان داد که دانش کافی از ژن­های درگیر در مسیر بیوسنتزی فراورده موردنظر، طراحی مصنوعی این مسیر و نیز بهره ­گیری از مخمر Y. lipolytica به عنوان یک میزبان کارآمد و ارزان می ­تواند در تولید انبوه ترکیبات گیاهی-دارویی نقش به سزایی داشته باشد.

کلیدواژه‌ها


عنوان مقاله English

The optimization of Naringenin biosynthesis pathway using Yarrowia lipolitica cell culture

نویسندگان English

Monireh Marsafari 1
Habibollah Samizadeh Lahiji 1
Babak Rabiei 1
Ali Ashraf Mehrabi 2
Yongkun Lv 3
Peng Xu 4
1 Faculty of Agricultural Science, University of Guilan, Rasht, Iran
2 Faculty of Agriculture, University of Ilam, Ilam, Iran
3 Faculty of Biotechnology, Jiangnan University, Jiangsu, China
4 Department of Chemical, Biochemical and Environmental Engineering, University of Maryland Baltimore County, Baltimore, USA
چکیده English

Yarrowia lipolytica, as a good cell factory to speed up the production of plant pharmaceutical components, has been considered to be one of the most important and attractive micro-organisms in recent years, due to its high secretion capacity, limited glycosylation, large range of genetic markers and molecular tools. Naringenin, as a central core of flavonoids production, plays important roles both in plants and in the treatment of different types of human diseases. For this purpose, specific naringenin biosynthesis genes from different origins were selected and introduced after comparative expression profiling in Y. lipolytica. This research indicated that chs plays the main role in the production of naringenin, so the increase copy number of this gene in each construct was investigated. The HPLC results confirmed that the construct with 5 copy numbers of chs resulted in 7.14 fold increase of naringenin extracellular titer to 90.16 mg/L in shake flask cultures. The results reported in this study demonstrated that sufficient knowledge of genes involved in the specific biosynthesis pathway, synthetic gene pathway and using Y. lipolytica as a capable and cheap host could help bioengineers to produce significant amounts of pharmaceutical components.


کلیدواژه‌ها English

gene copy number
HPLC
metabolic engineering
metabolic flux
yeast host
Alberstein, M., Eisenstein, M. & Abeliovich, H. 2012. Removing allosteric feedback inhibition of tomato 4-coumarate:CoA ligase by directed evolution. Plant J. 69: 57-69.
Anson, J.G., Gilbert, H.J., Oram, J.D. & Minton, N.P. 1987. Complete nucleotide sequence of the Rhodosporidium toruloides gene coding for phenylalanine ammonia-lyase. Gene 58: 189-199.
Atanasov, A.G., Waltenberger, B., Pferschy-Wenzig, E.M., Linder, T., Wawrosch, C., Uhrin, P., Temml, V., Wang, L., Schwaiger, S., Heiss, E.H., Rollinger, J.M., Schuster, D., Breuss, J.M., Bochkov, V., Mihovilovic, M.D., Kopp, B., Bauer, R., Dirsch, V.M. & Stuppner, H. 2015. Discovery and resupply of pharmacologically active plant-derived natural products: A review. Biotechnol. Adv. 33: 1582-1614.
Chemler, J.A. & Koffas, M.A. 2008. Metabolic engineering for plant natural product biosynthesis in microbes. Curr. Opin. Biotechnol. 19: 597-605.
Christensen, A.B., Gregersen, P.L., Schroder, J. & Collinge, D.B. 1998. A chalcone synthase with an unusual substrate preference is expressed in barley leaves in response to UV light and pathogen attack. Plant Mol. Biol. 37: 849-857.
Falcone Ferreyra, M.L., Rius, S.P. & Casati, P. 2012. Flavonoids: biosynthesis, biological functions, and biotechnological applications. Front. Plant Sci. 3: 1-15.
Fowler, Z.L., Gikandi, W.W. & Koffas, M.A. 2009. Increased malonyl coenzyme A biosynthesis by tuning the Escherichia coli metabolic network and its application to flavanone production. Appl. Environ. Microbiol. 75: 5831-5839.
Fowler, Z.L. & Koffas, M.A. 2009. Biosynthesis and biotechnological production of flavanones: current state and perspectives. Appl. Microbiol. Biotechnol. 83: 799-808.
Ganesan, V., Li, Z., Wang, X. & Zhang, H. 2017. Heterologous biosynthesis of natural product naringenin by co-culture engineering. Synth. Syst. Biotechnol. 2: 236-242.
Gietz, R.D. & Schiestl, R.H. 2007. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat. Protoc. 2: 31-34.
Hermenean, A., Ardelean, A., Stan, M., Herman, H., Mihali, C.V., Costache, M. & Dinischiotu, A. 2013. Protective effects of naringenin on carbon tetrachloride-induced acute nephrotoxicity in mouse kidney. Chem. Biol. Interact. 205: 138-147.
Jiang, H., Wood, K.V. & Morgan, J.A. 2005. Metabolic engineering of the phenylpropanoid pathway in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 71: 2962-2969.
Jones, J.A., Toparlak, Ö.D. & Koffas, M.A.G. 2015. Metabolic pathway balancing and its role in the production of biofuels and chemicals. Curr. Opin. Biotechnol. 33: 52-59.
Koopman, F., Beekwilder, J., Crimi, B., van Houwelingen, A., Hall, R.D., Bosch, D., van Maris, A.J., Pronk, J.T. & Daran, J.M. 2012. De novo production of the flavonoid naringenin in engineered Saccharomyces cerevisiae. Microb. Cell Fact. 11: 155-170.
Leonard, E., Ajikumar, P., Thayer, K., Xiao, W., Mo, J., Tidor, B., Stephanopoulos, G. & Prather, K. 2010. Combining metabolic and protein engineering of a terpenoid biosynthetic pathway for overproduction and selectivity control. Proc. Natl. Acad. Sci. USA 107: 13654-13659.
Lian, J., Mishra, S. & Zhao, H. 2018. Recent advances in metabolic engineering of Saccharomyces cerevisiae: New tools and their applications. Metab. Eng. 50: 85-108.
Lv, Y., Edwards, H., Zhou, J. & Xu, P. 2019a. Combining 26s rDNA and the Cre-loxP system for iterative gene integration and efficient marker curation in Yarrowia lipolytica. ACS Synth. Biol. 8: 568-576.
Lv, Y., Marsafari, M., Koffas, M., Zhou, J. and Xu, P. 2019b. Optimizing oleaginous yeast cell factories for flavonoids and hydroxylated flavonoids biosynthesis. ACS Synth. Biol. 8: 2514-2523.
Mahomoodally, M.F., Gurib-Fakim, A. & Subratty, A.H. 2005. Antimicrobial activities and phytochemical profiles of endemic medicinal plants of mauritius. Pharm. Biol. 43: 237-242.
Panche, A.N., Diwan, A.D. & Chandra, S.R. 2016. Flavonoids: an overview. J. Nutr. Sci. 5: 1-15.
Pandy, A.K. 2007. Anti-staphylococcal activity of a pan-tropical aggressive and obnoxious weed Parthenium histerophorus: An in vitro study. Natl Acad Sci Lett. 30: 383-386.
Ribeiro, I.A., Rocha, J., Sepodes, B., Mota-Filipe, H. & Ribeiro, M.H. 2008. Effect of naringin enzymatic hydrolysis towards naringenin on the anti-inflammatory activity of both compounds. J. Mol. Catal. B-Enzym. 52-53: 13-18.
Santos, C.N.S., Koffas, M. & Stephanopoulos, G. 2011. Optimization of a heterologous pathway for the production of flavonoids from glucose. Metab. Eng. 13: 392-400.
Stafford, H.A., Runeckles, V.C. & Conn, E.E. 1974. Possible Multienzyme Complexes Regulating the Formation of C6-C3 Phenolic Compounds and Lignins in Higher Plants. Recent Adv. Phytochem. 8: 53-79.
Trantas, E.A., Koffas, M.A., Xu, P. & Ververidis, F. 2015. When plants produce not enough or at all: metabolic engineering of flavonoids in microbial hosts. Front. Plant Sci. 6: 7-23.
van Tunen, A.J., Koes, R.E., Spelt, C.E., van der Krol, A.R., Stuitje, A.R. & Mol, J.N. 1988. Cloning of the two chalcone flavanone isomerase genes from Petunia hybrida: coordinate, light-regulated and differential expression of flavonoid genes. Embo. J. 7: 1257-1263.
Wang, Y., Chen, S. & Yu, O. 2011. Metabolic engineering of flavonoids in plants and microorganisms. Appl. Microbiol. Biotechnol. 91: 949-956.
Winkel, B.S. 2004. Metabolic channeling in plants. Annu Rev Plant Biol. 55: 85-107.
Winkel-Shirley, B. 2001. Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell biology, and biotechnology. Plant Physiol. 126: 485-493.
Winkel-Shirley, B. 2002. Biosynthesis of flavonoids and effects of stress. Curr. Opin. Plant Biol. 5: 218-223.
Wong, L., Engel, J., Jin, E., Holdridge, B. & Xu, P. 2017. YaliBricks, a versatile genetic toolkit for streamlined and rapid pathway engineering in Yarrowia lipolytica. Metab. Eng. Commun. 5: 68-77.
Wu, J., Du, G., Zhou, J. & Chen, J. 2013. Metabolic engineering of Escherichia coli for (2S)-pinocembrin production from glucose by a modular metabolic strategy. Metab. Eng. 16: 48-55.
Wu, J., Zhou, T., Du, G., Zhou, J. & Chen, J. 2014. Modular optimization of heterologous pathways for de novo synthesis of (2S)-naringenin in Escherichia coli. PLOS ONE 9: e101492.
Xu, C., Chen, J., Zhang, J., Hu, X., Zhou, X., Lu, Z. & Jiang, H. 2013a. Naringenin inhibits angiotensin II-induced vascular smooth muscle cells proliferation and migration and decreases neointimal hyperplasia in balloon injured rat carotid arteries through suppressing oxidative stress. Biol. Pharm. Bull. 36: 1549-1555.
Xu, P., Bhan, N. & Koffas, M.A.G. 2013b. Engineering plant metabolism into microbes: from systems biology to synthetic biology. Curr. Opin. Biotechnol. 24: 291-299.
Xu, P., Qiao, K.J., Ahn, W.S. & Stephanopoulos, G. 2016. Engineering Yarrowia lipolytica as a platform for synthesis of drop-in transportation fuels and oleochemicals. Proc. Natl. Acad. Sci. USA 113: 10848-10853.
Zelcbuch, L., Antonovsky, N., Bar-Even, A., Levin-Karp, A., Barenholz, U., Dayagi, M., Liebermeister, W., Flamholz, A., Noor, E., Amram, S., Brandis, A., Bareia, T., Yofe, I., Jubran, H. & Milo, R. 2013. Spanning high-dimensional expression space using ribosome-binding site combinatorics. Nucleic Acids Res. 41: e98-e106.
Zha, W., Rubin-Pitel, S.B., Shao, Z. & Zhao, H. 2009. Improving cellular malonyl-CoA level in Escherichia coli via metabolic engineering. Metab Eng. 11: 192-198.
Zhang, H. and Stephanopoulos, G. 2013. Engineering E. coli for caffeic acid biosynthesis from renewable sugars. Appl. Microbiol. Biotechnol. 97: 3333-3341.
Zhang, W., Liu, H., Li, X., Liu, D., Dong, X.-T., Li, F.-F., Wang, E.-X., Li, B.-Z. & Yuan, Y.-J. 2017. Production of naringenin from D-xylose with co-culture of E. coli and S. cerevisiae. Eng. Life. Sci. 17: 1021-1029.
Zhu, Q. & Jackson, E.N. 2015. Metabolic engineering of Yarrowia lipolytica for industrial applications. Curr. Opin. Biotechnol. 36: 65-72.
Zhu, S., Wu, J., Du, G., Zhou, J. & Chen, J. 2014. Efficient synthesis of eriodictyol from L-tyrosine in Escherichia coli. Appl. Environ. Microbiol. 80: 3072-3080.
دوره 7، شماره 2 - شماره پیاپی 24
تابستان 1399
صفحه 133-144

  • تاریخ دریافت 18 خرداد 1405
  • تاریخ انتشار 18 خرداد 1405