基于重组毕赤酵母的fusaruside生物合成 <sup>*</sup>

doi:10.13523/j.cb.20190702

中国生物工程杂志

2019, Vol. 39

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

研究报告

基于重组毕赤酵母的fusaruside生物合成 ^*

田园(

),李艳玲

山东第一医科大学(山东省医学科学院)生命科学学院泰安 271016

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

全文: PDF(1034 KB) HTML

摘要：

目的构建产fusaruside的毕赤酵母菌株,解决天然小分子免疫抑制剂fusaruside的来源问题。方法从禾谷镰刀菌Fusarium graminearum PH-1中扩增获得合成fusaruside的相关基因-3位去饱和酶[Δ3(E)-SD]和10位去饱和酶[Δ10(E)-SD]基因;并通过2A肽策略构建两种基因的共表达载体,转化到毕赤酵母GS115中进行双酶的诱导表达;对诱导后的毕赤酵母菌体进行甲醇和二氯甲烷的处理后,经高效液相色谱质谱联用仪(HPLC-MS)检测其中产物变化。结果 3位去饱和酶和10位去饱和酶在毕赤酵母中成功共表达,SDS-PAGE显示3位去饱和酶分子量约为48kDa,10位去饱和酶分子量约为65kDa;HPLC-MS显示重组酵母可以产生fusaruside。结论与fusaruside原产菌株镰刀菌相比,该酵母菌的发酵时间更短、产量更高,为fusaruside的进一步开发与应用奠定基础。

关键词： 毕赤酵母; 去饱和酶; 2A肽; 共表达; Fusaruside

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

收稿日期: 2018-12-20 出版日期: 2019-08-05

ZTFLH:

Q819

基金资助: * 国家自然科学基金(21602152);山东省自然科学基金(ZR2016BB01);山东省农业微生物重点实验室开放课题基金资助项目(SDKL2017015)

通讯作者: 田园 E-mail: tianyuan2005hit@163.com

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引用本文:

田园,李艳玲. 基于重组毕赤酵母的fusaruside生物合成 ^*[J]. 中国生物工程杂志, 2019, 39(7): 8-14.

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

链接本文:

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

表1 所用引物及2A肽基因序列

图1 目的基因PCR扩增电泳图

图2 Δ3(E)-sd-2a-Δ10(E)-sd的组合基因片段PCR扩增电泳图

图3 共表达质粒示意图及酶切鉴定结果

图4 转化子的PCR鉴定结果

图5 重组酵母菌体蛋白的SDS-PAGE分析

图6 重组酵母细胞提取物的HPLC-MS分析

图7 Fusaruside氢谱(CDCl3,400MHz)

表2 不同fusaruside产生菌之间产量对比

图8 Fusaruside完整生物合成途径[12]

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