Food and Fermentation Industries >

2022 , Vol. 48 >Issue 2: 15 - 19

DOI: https://doi.org/10.13995/j.cnki.11-1802/ts.027893

Construction and application of Aspergillus niger pyrG genetic transformation system

  • HU Jiangfeng ,
  • LIU Shuwen ,
  • YANG Yan ,
  • LI Linlin ,
  • LI Yingkai ,
  • ZHANG Jian
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  • 1(Key Laboratory of Industrial Fermentation Microbiology Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, China)
    2(Tianjin Economics and Trade School, Tianjin 300381, China)

Received date: 2021-04-28

  Revised date: 2021-05-15

  Online published: 2022-02-28

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Abstract

Few selective marker genes are used in the genetic operation of Aspergillus niger. However, genetic modification often involves the knockout or expression of many genes, and the limited selective marker genes are difficult to meet the needs. In this study, genetic transformation system was constructed using pyrG as selective marker in A. niger and the red fluorescent protein rfp in A. niger was expressed using this system. First, the pyrG gene was knocked out by targeted gene replacement using Agrobacterium tumefaciens mediated transformation. The stable genetic uridine/uracil auxotrophic strain A. niger ng-1 was obtained on medium containing 5-fluoroorotic acid and uridine/uracil. Then we constructed the rfp gene-expression plasmid which contained the rfp gene driven by the glyceraldehyde triphosphate dehydrogenase promoter gpdA and the selective marker pyrG. The plasmid was transferred to A. tumefaciens AGL1 and infected the A. niger ng-1, so as to obtain rfp gene-expression A. niger strains. This work showed that the genetic transformation system using the pyrG as a selective marker is effective and feasible in A. niger.

Key words： Aspergillus niger; pyrG; Agrobacterium tumefaciens-mediated transformation; selective marker; red fluorescent protein

Cite this article

HU Jiangfeng , LIU Shuwen , YANG Yan , LI Linlin , LI Yingkai , ZHANG Jian . Construction and application of Aspergillus niger pyrG genetic transformation system[J]. Food and Fermentation Industries, 2022 , 48(2) : 15 -19 . DOI: 10.13995/j.cnki.11-1802/ts.027893

References

[1] LI C, ZHOU J W, DU G C, et al.Developing Aspergillus niger as a cell factory for food enzyme production[J].Biotechnology Advances, 2020, 44:107630.
[2] 谢承佳, 庄琪.酱油制曲研究进展[J].中国调味品, 2017, 42(6):77-80.
XIE C J, ZHUANG Q.Research progress of koji making for fermented soy sauce[J].China Condiment, 2017, 42(6):77-80.
[3] 李莉. 用黑曲霉、绿色木霉制备酱油种曲的工艺参数确定[J].中国调味品, 2008, 33(9):42-44;52.
LI L.Ascertainment of optimum technology parameters for producting seed koji using Aspergillus niger and Trichoderma viride[J].China Condiment, 2008, 33(9):42-44;52.
[4] 孙士健, 王丽娟, 秦郦, 等.复合诱变筛选高产柠檬酸黑曲霉及其发酵研究[J].食品研究与开发, 2019, 49(17):194-200.
SUN S J, WANG L J, QIN L, et al.Compound mutation and screening Aspergillus niger with high yield of citric acid and its fermentation research[J].Food Research and Development, 2019, 49(17):194-200.
[5] 孙菁, 任文琪, 许勤虎, 等.以amdS为筛选标记的黑曲霉遗传转化体系的建立[J].中国农业科技导报, 2020, 22(9):179-187.
SUN J, REN W Q, XU Q H, et al.Transformation system of Aspergillus niger using amdS as a dominant selective marker[J].Journal of Agricultural Science and Technology, 2020, 22(9):179-187.
[6] ORTEGA-ESCALANTE J A, KWOK O, MILLER S M.New selectable markers for Volvox carteri transformation[J].Protist, 2019, 170(1):52-63.
[7] LIU J J, XIE Z P, SHIN H D, et al.Rewiring the reductive tricarboxylic acid pathway and L-malate transport pathway of Aspergillus oryzae for overproduction of L-malate[J].Journal of Biotechnology, 2017, 253:1-9.
[8] SUZUKI S, TADA S, FUKUOKA M, et al.A novel transformation system using a bleomycin resistance marker with chemosensitizers for Aspergillus oryzae[J].Biochemical and Biophysical Research Communications, 2009, 383(1):42-47.
[9] 王伯华. 橙色红曲菌中基于pyrG标记的同源转化系统的建立[D].南昌:南昌大学, 2010.
WANG B H.Development of a homologous transformation system for Monascus aurantiacus based on the pyrG gene[D].Nanchang:Nanchang University, 2010.
[10] HARTINGSVELDT W, MATTERN I E, ZEIJL C M J, et al.Development of a homologous transformation system for Aspergillus niger based on the pyrG gene[J].Molecular and General Genetics, 1987, 206(1):71-75.
[11] NGUYEN K T, HO Q N, PHAM T H, et al.The construction and use of versatile binary vectors carrying pyrG auxotrophic marker and fluorescent reporter genes for Agrobacterium-mediated transformation of Aspergillus oryzae[J].World Journal of Microbiology and Biotechnology, 2016, 32(12):1-9.
[12] UNKLES S E, CAMPBELL E I, DE RUITER-JACOBS Y M J T, et al.The development of a homologous transformation system for Aspergillus oryzae based on the nitrate assimilation pathway:A convenient and general selection system for filamentous fungal transformation[J].Molecular and General Genetics, 1989, 218(1):99-104.
[13] 李方方, 潘力, 曾沛斌.酱油工业生产菌沪酿3042基因工程转化体系的构建[J].食品工业科技, 2009,13(6):94-95;99.
LI F F, PAN L, ZENG P B.Construction of genetic engineering transforming system of Aspergillus oryzae HuNiang 3042 used in soy sauce industry[J].Science and Technology of Food Industry, 2009,13(6):94-95;99.
[14] THAI H D, NGUYEN B P T, NGUYEN V M, et al.Development of a new Agrobacterium-mediated transformation system based on a dual auxotrophic approach in the filamentous fungus Aspergillus oryzae[J].World Journal of Microbiology and Biotechnology, 2021, 37(6):92.
[15] BOEKE J D, TRUEHEART J, NATSOULIS G, et al.5-Fluoroorotic acid as a selective agent in yeast molecular genetics[J].Methods in Enzymology, 1987, 154:164-175.
[16] 陈英, 王培娟, 张文君, 等.mCherry红色荧光标记乳酸菌的融合表达系统构建及应用[J].生物工程学报, 2019, 35(3):492-504.
CHEN Y, WANG P J, ZHANG W J, et al.Construction and application of mCherry red fluorescent protein fusion expression system in lactic acid bacteria[J].Chinese Journal of Biotechnology, 2019, 35(3):492-504.
[17] ZHAO P, YE M H, WANG R Q, et al.Systematic identification and functional analysis of potato (Solanum tuberosum L.) bZIP transcription factors and overexpression of potato bZIP transcription factor StbZIP-65 enhances salt tolerance[J].International Journal of Biological Macromolecules, 2020, 161:155-167.
[18] 陈阳霞, 顾桉菁, 梁宇恒, 等.电穿孔介导马尔尼菲青霉转化体系的建立和优化[J].中国真菌学杂志, 2014, 9(1):12-15.
CHEN Y X, GU A J, LIANG Y H, et al.Electroporation-mediate transformation of Penicillium marneffei and optimization of the transforming conditions[J].Chinese Journal of Mycology, 2014, 9(1):12-15.
[19] 李载平. 分子克隆实验指南(第3版)[J].科学通报, 2002, 47(24):1 888.
LI Z P.Molecular cloning experiment guide(the 3rd edition)[J].Chinese Science Bulletin, 2002, 47(24):1 888.
[20] 黎明, 刘萌, 黄云雁, 等.根癌农杆菌介导的黑曲霉遗传转化体系的建立及优化[J].中国生物工程杂志, 2012, 32(1):56-63.
LI M, LIU M, HUANG Y Y, et al.Establishment and optimization of Agrobacterium tumefaciens mediated transformation system of Aspergillus niger[J].China Biotechnology, 2012, 32(1):56-63.
[21] 毕付提, 史亚楠, 张久祎, 等.米曲霉3.042尿苷/尿嘧啶营养缺陷型遗传转化体系的构建[J].食品研究与开发, 2021, 42(3):189-195.
BI F T, SHI Y N, ZHANG J Y, et al.Construction of Aspergillus oryzae 3.042 uridine/uracil auxotrophy genetic transformation system[J].Food Research and Development, 2021, 42(3):189-195.
[22] 裴静娴, 王月刚, 张艺军, 等.p53RFP基因启动子荧光素酶报告基因载体的构建及鉴定[J].广东医学, 2017, 38(2):170-172.
PEI J X, WANG Y G, ZHANG Y J, et al.Construction and identification of luciferase reporter plasmid containing p53RFP gene promoter[J].Guangdong Medical Journal, 2017, 38(2):170-172.
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