Construction of A High-efficiency Homologous Recombination and Uridine/Uracil Auxotroph Strain<italic/> of <i>Aspergillus oryzae</i> RIB40

GAO Yuqing; ZHANG Haojie; ZHANG Danfeng; PAN Yutian; ZHANG Feng

doi:10.13386/j.issn1002-0306.2022040179

Volume 44 Issue 1

Dec. 2022

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Science and Technology of Food Industry > 2023 > 44(1): 200-207. > DOI: 10.13386/j.issn1002-0306.2022040179

GAO Yuqing, ZHANG Haojie, ZHANG Danfeng, et al. Construction of A High-efficiency Homologous Recombination and Uridine/Uracil Auxotroph Strain of Aspergillus oryzae RIB40[J]. Science and Technology of Food Industry, 2023, 44(1): 200−207. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2022040179.

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Construction of A High-efficiency Homologous Recombination and Uridine/Uracil Auxotroph Strain of Aspergillus oryzae RIB40

1.
The Engineering Technological Center of Fungus Active Substances of Fujian Province, Minnan Normal University, Zhangzhou 363000, China
2.
College of Biological Sciences and Technology, Minnan Normal University, Zhangzhou 363000, China

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Received Date: April 17, 2022
Available Online: October 30, 2022

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Abstract

Objective: An auxotrophic strain with high efficiency of homologous recombination was developed in Aspergillus oryzae RIB40. Methods: The AopyrG mutant of A. oryzae RIB40 was firstly obtained using a homologous recombination technique and the 5-fluoroorotic acid (5-FOA) selection for the uridine/uracil auxotroph. To disrupt the ∆Aoku70 gene, the Aoku70 gene of AopyrG mutant was replaced with A. fumigatus pyrG gene. Then, under the screening effect of 5-FOA, the AfpyrG gene of the ∆Aoku70 strain was deleted via genome self-cyclization. To test the utilization of the resulting strain, the mCherry gene, a red fluorescent protein gene, was integrated with the histone H2B gene in the ∆Aoku70∆AopyrG strain. The subcellular localization of red fluorescent protein was detected by laser confocal microscope. Results: An auxotrophic mutant ∆Aoku70∆AopyrG with high homologous recombination efficiency was established via disruption of the A. oryzae RIB40 genes Aoku70 and AopyrG. The red fluorescence was observed in the nucleus of the fungal cell, indicating the utility of the ∆Aoku70∆AopyrG strain. Conclusion: The ∆Aoku70∆AopyrG strain can be used as a recipient strain with high-efficiency homologous recombination and uridine/uracil auxotroph for future gene modification in A. oryzae RIB40.
- Aspergillus oryzae RIB40,
- uridine/uracil auxotroph,
- pyrG,
- ku70,
- fluorescence localization

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[1]	刘丽萍, 刘丽华. 米曲霉研究进展与应用[J]. 中国调味品,2008(4):28−32. [LIU L P, LIU L H. Study and application of Aspergillus oryzae indifferent domains[J]. China Condiment,2008(4):28−32. doi: 10.3969/j.issn.1000-9973.2008.04.003
[2]	马萍苹, 鄢莉, 张佳兰. 米曲霉液态发酵香菇残次品产蛋白酶条件优化[J]. 中国酿造,2019,40(9):211−215. [MA P P, YAN L, ZHANG J L. Optimization of liquid state fermentation conditions for production protease with Lentinus edodes residues by Aspergillus oryzae[J]. China Brewing,2019,40(9):211−215.
[3]	罗雯, 郭建, 樊君, 等. 酱油酿造中复合米曲霉发酵制曲研究[J]. 中国调味品,2022,47(4):164−166. [LUO W, GUO J, FAN J, et al. Study on koji making by fermentation of compound Aspergillus oryzae in brewing of soy sauce[J]. China Condiment,2022,47(4):164−166. doi: 10.3969/j.issn.1000-9973.2022.04.031
[4]	阮露晨. 无痕敲除构建米曲霉尿嘧啶营养缺陷型菌株[D]. 天津: 天津科技大学, 2020 RUAN L C. Construction of Aspergillus oryzae uracil auxotrophy strain by seamless knockout[D]. Tianjin: Tianjin University of Science and Technology, 2020.
[5]	MACHIDA M, ASAI K, SANO M, et al. Genome sequencing and analysis of Aspergillus oryzae[J]. Nature,2005,438(7071):1157−1161. doi: 10.1038/nature04300
[6]	ZHAO G Z, YAO Y P, QI W, et al. Draft genome sequence of Aspergillus oryzae strain 3.042[J]. Eukaryotic Cell,2012,11(9):1178. doi: 10.1128/EC.00160-12
[7]	ZHAO G Z, YAO Y P, WANG C L, et al. Comparative genomic analysis of Aspergillus oryzae strains 3.042 and RIB40 for soy sauce fermentation[J]. International Journal of Food Microbiology,2013,164(2-3):148−154. doi: 10.1016/j.ijfoodmicro.2013.03.027
[8]	ZHAO G Z, YAO Y P, CHEN W, et al. Comparison and analysis of the genomes of two Aspergillus oryzae strains[J]. Journal of Agricultural and Food Chemistry,2013,61(32):7805−7809. doi: 10.1021/jf400080g
[9]	侯丽华, 卢嵩, 王檬, 等. 低盐固态工艺条件下米曲霉3.042和米曲霉RIB40酿造酱油发酵性能的比较[J]. 中国调味品,2014,39(8):1−8. [HOU L H, LU S, WANG M, et al. Comparison of fermentation performance between A. oryzae 3.042 and A. oryzae RIB40 in low-salt solid-state fermentation of soy sauce[J]. China Condiment,2014,39(8):1−8. doi: 10.3969/j.issn.1000-9973.2014.08.001
[10]	王莹. 米曲霉RIB40和3.042在酱油酿造中发酵性能的对比[D]. 天津: 天津科技大学, 2014 WANG Y. Fermentation performance comparison of Aspergillus oryzae RIB40 and 3.042 in soy sauce[D]. Tianjin: Tianjin University of Science and Technology, 2014.
[11]	SUN Y, NIU Y, HE B, et al. A dual selection marker transformation system using Agrobacterium tumefaciens for the industrial Aspergillus oryzae 3.042[J]. Workld Journal of Microbiolgy and Biotechnology,2019,29(2):230−234. doi: 10.4014/jmb.1811.11027
[12]	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. doi: 10.12161/j.issn.1005-6521.2021.03.031
[13]	刘雪. 米曲霉原生质体的制备及pyrG缺失株的构建研究[D]. 南昌: 南昌大学, 2012 LIU X. Study on preparation of protoplast and construction of pyrG disruption strains in Aspergillus oryzae[D]. Nanchang: Nanchang University, 2012.
[14]	WELD R J, PLUMMER K M, CARPENTER M A, et al. Approaches to functional genomics in filamentous fungi[J]. Cell Research,2006,16(1):31−44. doi: 10.1038/sj.cr.7310006
[15]	ZHANG F, XU G, GENG L, et al. The stress response regulator AflSkn7 influences morphological development, stress response, and pathogenicity in the fungus Aspergillus flavus[J]. Toxins (Basel),2016,8(7):202. doi: 10.3390/toxins8070202
[16]	EDYTA S, TANIA N, ELIZABETH O C, et al. Fusion PCR and gene targeting in Aspergillus nidulans[J]. Nature Protocols,2006,1(6):3111−3120. doi: 10.1038/nprot.2006.405
[17]	于潇淳. 表达蛋白酶的米曲霉工程菌构建及发酵特性研究[D]. 沈阳: 沈阳农业大学, 2018 YU X C. Study on the construction of engineered Aspergillus oryzae for expressing proteases and their effect on the fermentation[D]. Shenyang: Shenyang Agricultural University, 2018.
[18]	CHEEVADHANARAK S, SAUNDERS G, RENNO D, et al. Transformation of Aspergillus oryzae with a dominant selectable marker[J]. Journal of Biotechnology,1991,19(1):117−122. doi: 10.1016/0168-1656(91)90079-B
[19]	KUBODERA T, YAMASHITA N, NISHIMURA A. Pyrithiamine resistance gene (ptrA) of Aspergillus oryzae: Cloning, characterization and application as a dominant selectable marker for transformation[J]. Bioscience, Biotechnology, and Biochemistry,2000,64(7):1416−1421. doi: 10.1271/bbb.64.1416
[20]	SHIMA Y, ITO Y, HATABAYASHI H, et al. Five carboxin-resistant mutants exhibited various responses to carboxin and related fungicides[J]. Bioscience, Biotechnology, and Biochemistry,2011,75(1):181−184. doi: 10.1271/bbb.100687
[21]	卢园萍, 肖婷婷, 尚俊军, 等. 双孢蘑菇萎锈灵抗性基因定点突变的载体构建与遗传转化[J]. 菌物学报,2021,40(12):3256−3264. [LU Y P, XIAO T T, SHANG J J, et al. Construction of a vector containing a point-mutated carboxin-resistance gene and genetic transformation of Agaricus bisporus[J]. Mycosystema,2021,40(12):3256−3264. doi: 10.13346/j.mycosystema.210347
[22]	ORTEGA-ESCALANTE J A, KWOK O, MILLER S M. New selectable markers for Volvox carteri transformation[J]. Protist,2019,170(1):52−63. doi: 10.1016/j.protis.2018.11.002
[23]	PUNT P J, OLIVER R P, DINGEMANSE M A, et al. Transformation of Aspergillus based on the hygromycin B resistance marker from Escherichia coli[J]. Gene,1987,56(1):117−124. doi: 10.1016/0378-1119(87)90164-8
[24]	吴琴琴, 孙敏, 陈雨, 等. 米曲霉功能基因组研究策略和进展[J]. 生物技术通报,2019,35(8):186−192. [WU Q Q, SUN M, CHEN Y, et al. Strategies and advances in functional genomics of Aspergillus oryzae[J]. Biotechnology Bulletin,2019,35(8):186−192. doi: 10.13560/j.cnki.biotech.bull.1985.2019-0226
[25]	BINH C T, THAI H D, HA B T V, et al. Establishment of a new and efficient Agrobacterium-mediated transformation system in the nematicidal fungus Purpureocillium lilacinum[J]. Microbiol Res,2021,249:126773. doi: 10.1016/j.micres.2021.126773
[26]	李娟, 邱睿, 张盈盈, 等. 根癌农杆菌介导的尖孢镰刀菌遗传转化体系构建[J]. 中国烟草学报,2022:1−10. [LI J, QIU R, ZHANG Y Y, et al. Construction of agrobacterium tumefaciens-mediated genetic transformation system of tobacco Fusarium oxysporum[J]. Acta Tabacaria Sinica,2022:1−10.
[27]	曹旸. 谢瓦氏曲霉间型变种 ∆ku70 菌株的构建及功能分析[D]. 武汉: 华中农业大学, 2013 CAO Y. Construction and study of ∆ku70 strain in Aspergillus chevalieri var. Intermedius[D]. Wuhan: Huazhong Agricultural University, 2013.
[28]	李达, 沈雪莲, 李少华, 等. Ku70基因稳定敲除HeLa株的建立及其生物学功能研究[J]. 生物技术通讯,2018,29(2):155−161. [LI D, SHEN X L, LI S H, et al. Establishment and biological function of Ku70 gene stably knockout HeLa cell lines[J]. Letters in Biotechnology,2018,29(2):155−161. doi: 10.3969/j.issn.1009-0002.2018.02.001
[29]	NINOMIYA Y, SUZUKI K, ISHII C, et al. Highly efficient gene replacements in Neurospora strains deficient for nonhomologous end-joining[J]. Proceedings of the National Academy of Sciences,2004,101(33):12248−12253. doi: 10.1073/pnas.0402780101
[30]	JIN H, LEE B, LUO Y, et al. FOXL2 directs DNA double-strand break repair pathways by differentially interacting with Ku[J]. Nat Commun,2020,11(1):2010. doi: 10.1038/s41467-020-15748-1
[31]	KOH C M J, LIU Y, MOEHNINSI, et al. Molecular characterization of KU70 and KU80 homologues and exploitation of a KU70-deficient mutant for improving gene deletion frequency in Rhodosporidium toruloides[J]. BMC Microbiology,2014,14(1):50. doi: 10.1186/1471-2180-14-50
[32]	TU J L, BAI X Y, XU Y L, et al. Targeted gene insertion and replacement in the basidiomycete Ganoderma lucidum by inactivation of non-homologous end joining using CRISPR/Cas9[J]. Applied and Environmental Microbiology,2021,87(23):1510−1521.

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Science and Technology of Food Industry

Construction of A High-efficiency Homologous Recombination and Uridine/Uracil Auxotroph Strain of Aspergillus oryzae RIB40

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