Biosynthesis of Fusaruside Based on Recombinant <i>Pichia pastoris</i>

doi:10.13523/j.cb.20190702

China Biotechnology

2019, Vol. 39

Issue (7): 8-14 DOI: 10.13523/j.cb.20190702

Biosynthesis of Fusaruside Based on Recombinant Pichia pastoris

Yuan TIAN(

),Yan-ling LI

College of Life Science, Shandong First Medical University & Shandong Academy of Medical Sciences, Taian 271016, China

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Abstract

Objective: A strain of Pichia pastoris producing fusaruside was constructed to solve the source problem of fusaruside, a selective immunosuppressive molecule.Method: Two related biosynthetic genes coding delta 3(E)-sphingolipid desaturase [Δ3(E)-SD] and delta 10(E)-sphingolipid desaturase [Δ10(E)-SD] were amplified from fungus Fusarium graminearum PH-1. 2A peptide based strategy was used to construct the co-expression vector. Then the 2A polyprotein construct was transformed into Pichia pastoris GS115 for induction. Finally, P. pastoris cells were extracted with methanol and dichloromethane, and the extract was detected by high performance liquid chromatography mass spectrometer (HPLC-MS).Result: The two desaturases were successfully co-expressed in P. pastoris. SDS-PAGE showed that the molecular weight of Δ3(E)-SD and Δ10(E)-SD were about 48kDa and 65kDa, respectively. HPLC-MS indicated that fusaruside could be produced by the recombinant yeast.Conclusion: Compared with fusarium which producing fusaruside, the engineered yeast had shorter fermentation time and higher yield, laying a foundation for further development and application of fusaruside.

Key words： Pichia pastoris Desaturase 2A peptide Co-expression Fusaruside

Received: 20 December 2018 Published: 05 August 2019

ZTFLH:

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Corresponding Authors: Yuan TIAN E-mail: tianyuan2005hit@163.com

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Cite this article:

Yuan TIAN,Yan-ling LI. Biosynthesis of Fusaruside Based on Recombinant Pichia pastoris. China Biotechnology, 2019, 39(7): 8-14.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20190702 OR https://manu60.magtech.com.cn/biotech/Y2019/V39/I7/8

Table 1 Primers used in this study and gene sequence coding 2A peptide

Fig.1 Agarose gel electrophoresis of PCR amplified target genes M: DNA marker;Δ3(E)-sd: PCR product with Δ3(E)-sd; Δ10(E)-sd: PCR product with Δ10(E)-sd

Fig.2 Agarose gel electrophoresis of PCR amplified Δ3(E)-sd-2a-Δ10(E)-sd combined gene M: DNA marker; 1: PCR product with Δ3(E)-sd-2a-Δ10(E)-sd

Fig.3 Schematic of co-expression plasmid and identification by digestion M:DNA marker;1:Product of double enzyme digestion

Fig.4 Identification of transformants by PCR M:DNA maker;1-8:PCR products with primer D3-F/D10-R

Fig.5 SDS-PAGE analysis of proteins in the recombinant yeast cells M:Protein marker;1:0h;2:12h;3:24h;4:36h;5:48h;6:60h;7:72h;8:84h

Fig.6 HPLC-MS analysis of extracts from the recombinant yeast cells

Fig.7 ¹H NMR of fusaruside (CDCl₃,400MHz)

Table 2 The contrast of the yield of fusaruside from different producing strains

Fig.8 Complete biosynthetic pathway of fusaruside


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