Yihan Qin, Caixia Zhou, Weiqiong Jin, H. Yao, Hui Chen, Yujun Wan, Yirong Xiao, Zizhong Tang, Zhi Shan, Tongliang Bu, Hong Chen
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Meanwhile, a novel and efficient expression vector pBC-hygro.4 was constructed, which included the pyrG cassette gene, His-Tag, amyB promoter and terminator, and green fluorescent protein GFP marker. pBC-hygro.4 was successfully transferred to A. oryzae RIB40ΔpyrG via the PEG-CaCl2-mediated transformation method, and the stability of pBC-hygro.4 was evaluated by detecting the expression of the GFP reporter gene. Through phenotyping and sequencing verification, a uridine/uracil auxotrophic strains A. oryzae RIB40ΔpyrG were successfully generated. In addition, the developed vectors were fully functional for heterologous expression of the GFP fluorescent proteins in A. oryzae RIB40ΔpyrG and the recombinant A. oryzae RIB40ΔpyrG cultures indicated pronounced green fluorescence in the mycelia. Based on auxotrophic/nutritional markers, this study provides an effective method that can be applied to develop similar fungal transformation systems in other filamentous fungi, which will be beneficial for food-grade applications.","PeriodicalId":12347,"journal":{"name":"Food Biotechnology","volume":"35 1","pages":"310 - 326"},"PeriodicalIF":1.8000,"publicationDate":"2021-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Construction of Aspergillus Oryzae food-grade expression system based on auxotrophic markers\",\"authors\":\"Yihan Qin, Caixia Zhou, Weiqiong Jin, H. Yao, Hui Chen, Yujun Wan, Yirong Xiao, Zizhong Tang, Zhi Shan, Tongliang Bu, Hong Chen\",\"doi\":\"10.1080/08905436.2021.1979580\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"ABSTRACT Aspergillus oryzae is an ideal host for expressing heterologous and homologous genes. Due to the risk of antibiotics to food safety, it is banned for use in resistance screening with food-grade expression system. Therefore there merit and value in exploring the use of Aspergillus oryzae food-grade expression system based on auxotrophic markers. In this study uridine/uracil auxotrophic strains A. oryzae RIB40ΔpyrG were generated by ultraviolet mutagenesis of pyrG gene deletion which would then be used as a host for further transformation analysis and applications. Meanwhile, a novel and efficient expression vector pBC-hygro.4 was constructed, which included the pyrG cassette gene, His-Tag, amyB promoter and terminator, and green fluorescent protein GFP marker. pBC-hygro.4 was successfully transferred to A. oryzae RIB40ΔpyrG via the PEG-CaCl2-mediated transformation method, and the stability of pBC-hygro.4 was evaluated by detecting the expression of the GFP reporter gene. Through phenotyping and sequencing verification, a uridine/uracil auxotrophic strains A. oryzae RIB40ΔpyrG were successfully generated. In addition, the developed vectors were fully functional for heterologous expression of the GFP fluorescent proteins in A. oryzae RIB40ΔpyrG and the recombinant A. oryzae RIB40ΔpyrG cultures indicated pronounced green fluorescence in the mycelia. 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Construction of Aspergillus Oryzae food-grade expression system based on auxotrophic markers
ABSTRACT Aspergillus oryzae is an ideal host for expressing heterologous and homologous genes. Due to the risk of antibiotics to food safety, it is banned for use in resistance screening with food-grade expression system. Therefore there merit and value in exploring the use of Aspergillus oryzae food-grade expression system based on auxotrophic markers. In this study uridine/uracil auxotrophic strains A. oryzae RIB40ΔpyrG were generated by ultraviolet mutagenesis of pyrG gene deletion which would then be used as a host for further transformation analysis and applications. Meanwhile, a novel and efficient expression vector pBC-hygro.4 was constructed, which included the pyrG cassette gene, His-Tag, amyB promoter and terminator, and green fluorescent protein GFP marker. pBC-hygro.4 was successfully transferred to A. oryzae RIB40ΔpyrG via the PEG-CaCl2-mediated transformation method, and the stability of pBC-hygro.4 was evaluated by detecting the expression of the GFP reporter gene. Through phenotyping and sequencing verification, a uridine/uracil auxotrophic strains A. oryzae RIB40ΔpyrG were successfully generated. In addition, the developed vectors were fully functional for heterologous expression of the GFP fluorescent proteins in A. oryzae RIB40ΔpyrG and the recombinant A. oryzae RIB40ΔpyrG cultures indicated pronounced green fluorescence in the mycelia. Based on auxotrophic/nutritional markers, this study provides an effective method that can be applied to develop similar fungal transformation systems in other filamentous fungi, which will be beneficial for food-grade applications.
期刊介绍：
Food Biotechnology is an international, peer-reviewed journal that is focused on current and emerging developments and applications of modern genetics, enzymatic, metabolic and systems-based biochemical processes in food and food-related biological systems. The goal is to help produce and improve foods, food ingredients, and functional foods at the processing stage and beyond agricultural production.
Other areas of strong interest are microbial and fermentation-based metabolic processing to improve foods, food microbiomes for health, metabolic basis for food ingredients with health benefits, molecular and metabolic approaches to functional foods, and biochemical processes for food waste remediation. In addition, articles addressing the topics of modern molecular, metabolic and biochemical approaches to improving food safety and quality are also published.
Researchers in agriculture, food science and nutrition, including food and biotechnology consultants around the world will benefit from the research published in Food Biotechnology. The published research and reviews can be utilized to further educational and research programs and may also be applied to food quality and value added processing challenges, which are continuously evolving and expanding based upon the peer reviewed research conducted and published in the journal.
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