研究报告

基于代谢工程构建产β-胡萝卜素重组毕赤酵母

  • 刘洁 ,
  • 王宏涛 ,
  • 钱和 ,
  • 徐建中 ,
  • 张伟国
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  • 1(江南大学 生物工程学院,江苏 无锡,214122)
    2(工业生物技术教育部重点实验室(江南大学),江苏 无锡,214122)
    3(江南大学 食品学院,江苏 无锡,214122)
硕士研究生(张伟国教授和徐建中副教授为共同通讯作者,E-mail:zhangw9168@126.com;xujzl26@126.com)

收稿日期: 2020-01-02

  网络出版日期: 2020-06-24

基金资助

国家自然科学基金(31601459);国家轻工业技术与工程一流学科计划(LITE2018-07);江苏省高等学校学术计划项目;江苏省科技支撑计划(BE2018316)

Metabolically engineered Pichia pastoris for β-carotene production

  • LIU Jie ,
  • WANG Hongtao ,
  • QIAN He ,
  • XU Jianzhong ,
  • ZHANG Weiguo
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  • 1(School of Biological Engineering, Jiangnan University, Wuxi 214122, China)
    2(Key Laboratory of Industrial Biotechnology of Ministry of Education (Jiangnan University), Wuxi 214122, China)
    3(School of Food Science, Jiangnan University, Wuxi 214122, China)

Received date: 2020-01-02

  Online published: 2020-06-24

摘要

巴斯德毕赤酵母(Pichia pastoris)是工业上生产异源蛋白的良好平台,且具有合成β-胡萝卜素的潜力,为推进β-胡萝卜素工业化生产,克隆来自锁掷酵母(Sporidiobolus pararoseus)的类胡萝卜素相关基因,并整合到巴斯德毕赤酵母基因组中,产生类胡萝卜素生产菌株Pp-EYBI。通过优化β-胡萝卜素合成限速酶基因拷贝数并过表达MVA限速酶基因,增强前体物质的供应及流向β-胡萝卜素合成的通量,最终获得菌株Pp-EYBI+(YB)3H,其β-胡萝卜素产量为3.7 mg/gDCW。该研究首次克隆来自锁掷酵母的类胡萝卜素相关基因并实现其在毕赤酵母中的表达,为异源合成类胡萝卜素提供了新的思路,且有望通过巴斯德毕赤酵母实现工业生产β-胡萝卜素。

本文引用格式

刘洁 , 王宏涛 , 钱和 , 徐建中 , 张伟国 . 基于代谢工程构建产β-胡萝卜素重组毕赤酵母[J]. 食品与发酵工业, 2020 , 46(11) : 32 -37 . DOI: 10.13995/j.cnki.11-1802/ts.023242

Abstract

In order to promote the industrial production of β-carotene, carotenoid-related genes were cloned from Sporidiobolus pararoseus and integrated into the genome of Pichia pastoris and a carotenoid-producing strain Pp-EYBI was constructed. Meanwhile, the gene copy number of the rate-limiting enzyme for β-carotene synthesis was optimized and the gene of MVA rate-limiting enzyme was over-expressed to enhance the supply of precursor materials and the flux to β-carotene synthesis. The strain Pp-EYBI+(YB)3H was finally obtained, which yielded as high as 3.7 mg/g (dry cell weight) β-carotene. This lays a foundation for the industrial production of β-carotene by P. pastoris.

参考文献

[1] 郝俊光,李芳杰,陈静, 等.香蕉皮中酚类和类胡萝卜素的国外研究进展[J].食品与发酵工业,2019,45(12):265-271.
[2] BOHN T, DESMARCHELIER C, EL S N, et al. Beta-Carotene in the human body metabolic bioactivation pathways from digestion to tissue distribution and excretion[J].Proceedings of the Nutrition Society,2019,78(1):68-87.
[3] FRASER P D, BRAMLEY P. The biosynthesis and nutritional uses of carotenoids[J]. Progress in Lipid Research,2004,43(3):228-265.
[4] NZAMWITA M, DUODU K G, MINNAAR A. Stability of beta-carotene during baking of orange-fleshed sweet potato-wheat composite bread and estimated contribution to vitamin A requirements[J].Food Chemistry,2017,228:85-90.
[5] QIAN C, DECKER E A, XIAO H, et al. Physical and chemical stability of β-carotene-enriched nanoemulsions: influence of pH, ionic strength, temperature, and emulsifier type[J].Food Chemistry,2012,132(3):1 221-1 229.
[6] 王岩岩,邢建民,陈红歌. β-胡萝卜素合成的代谢工程研究进展[J].生物工程学报,2017,33(4):578-590.
[7] 张宁,张帝. 微生物源类胡萝卜素及基因工程产类胡萝卜素研究进展[J].浙江万里学院学报,2016,29(6):82-87.
[8] SANDMANN G. Combinatorial biosynthesis of carotenoids in a heterologous host: a powerful approach for the biosynthesis of novel structures[J].ChemBioChem,2002,3(7):629-635.
[9] MIJTS B N, SCHMIDT-DANNERT C. Engineering of secondary metabolite pathways[J].Current Opinion in Biotechnology,2003,14(6):597-602.
[10] MACAULEY-PATRICK S, FAZENDA M L, MCNEIL B, et al. Heterologous protein production using the Pichia pastoris expression system[J].Yeast,2005,22(4):249-70.
[11] LEE P C, YOON Y G, SCHMIDT-DANNERT C. Investigation of cellular targeting of carotenoid pathway enzymes in Pichia pastoris[J].Journal of Biotechnology,2009,140(3-4):227-233.
[12] ARAYA-GARAY J M, FEIJOO-SIOTA L, ROSA-DOS-SANTOS F, et al. Construction of new Pichia pastoris X-33 strains for production of lycopene and β-carotene[J].Appl Microbiology & Biotechnology,2012,93(6):2 483-2 492.
[13] KIM J K, KIM J I, LEE N K, et al. Extraction of β-carotene produced from yeast Rhodosporidium sp. and its heat stability[J]. Food Science and Biotechnology,2010,19(1):263-266.
[14] ZHAO X, SHI F, ZHAN W. Overexpression of ZWF1 and POS5 improves carotenoid biosynthesis in recombinant Saccharomyces cerevisiae[J].Letters in Applied Microbiology,2015,61(4):354-360.
[15] LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method [J]. Methods,2001,25(4):402-408.
[16] 王蕊,王林风,闫德冉,等. 毕赤酵母表达β-葡萄糖苷酶中试条件优化[J].食品科技,2015,40(12):15-19.
[17] 张晓龙,肖静,王瑞明. 毕赤酵母高密度发酵产β-甘露聚糖酶的工艺优化[J].湖北农业科学,2015,54(23):5 978-5 983.
[18] 陈龙军,陈济琛,林新坚,等. 环糊精酶基因在毕赤酵母中的组成型表达[J].福建农业学报,2017,32(1):82-86.
[19] 杨然,范光森,郦金龙,等. 重组毕赤酵母高产木聚糖酶菌株筛选及发酵条件优化[J].中国食品学报,2017,17(12):95-104.
[20] LI C J, LI B X, ZHANG N, et al. Comparative transcriptome analysis revealed the improved β-carotene production in Sporidiobolus pararoseus yellow mutant MuY9[J]. Journal of General and Applied Microbiology,2018,65(3):121-128.
[21] 范超,洪皓,李妍, 等.三孢布拉霉发酵生产类胡萝卜素的产业化关键点探讨[J].食品与发酵工业,2018,44(5):284-290.
[22] 孙明雪,刘继栋,堵国成, 等.调控酿酒酵母类异戊二烯合成途径强化芳樟醇合成[J].生物工程学报, 2013, 29(6):751-759.
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