Sustainable and high-level microbial production of plant hemoglobin in Corynebacterium glutamicum

Biotechnology for Biofuels and Bioproducts

Research

Yanbing Shen¹  Bo Xin¹  Yingyu Zhou²  Mengmeng Wang²  Xuan Guo³  Jiao Liu³  Xiaomeng Ni³  Jiuzhou Chen³  Wenjuan Zhou³  Zhong Shi⁴  Jibin Sun⁵  Yu Wang⁵  Ping Zheng⁵  Ning Gao⁶ 

[1] College of Biotechnology, Tianjin University of Science and Technology, 300222, Tianjin, China;College of Biotechnology, Tianjin University of Science and Technology, 300222, Tianjin, China;Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 300308, Tianjin, China;Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 300308, Tianjin, China;Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 300308, Tianjin, China;National Technology Innovation Center of Synthetic Biology, 300308, Tianjin, China;Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 300308, Tianjin, China;National Technology Innovation Center of Synthetic Biology, 300308, Tianjin, China;University of Chinese Academy of Sciences, 100049, Beijing, China;Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 300308, Tianjin, China;University of Chinese Academy of Sciences, 100049, Beijing, China;

关键词: Hemoglobin;    Heme;    Heterologous expression;    Corynebacterium glutamicum;    Meat analogs;   

DOI  :  10.1186/s13068-023-02337-9

 received in 2023-02-15, accepted in 2023-05-03,  发布年份 2023

来源: Springer

【 摘 要 】

BackgroundPlant hemoglobin shows great potential as a food additive to circumvent the controversy of using animal materials. Microbial fermentation with engineered microorganisms is considered as a promising strategy for sustainable production of hemoglobin. As an endotoxin-free and GRAS (generally regarded as safe) bacterium, Corynebacterium glutamicum is an attractive host for hemoglobin biosynthesis.ResultsHerein, C. glutamicum was engineered to efficiently produce plant hemoglobin. Hemoglobin genes from different sources including soybean and maize were selected and subjected to codon optimization. Interestingly, some candidates optimized for the codon usage bias of Escherichia coli outperformed those for C. glutamicum regarding the heterologous expression in C. glutamicum. Then, saturated synonymous mutation of the N-terminal coding sequences of hemoglobin genes and fluorescence-based high-throughput screening produced variants with 1.66- to 3.45-fold increase in hemoglobin expression level. To avoid the use of toxic inducers, such as isopropyl-β-d-thiogalactopyranoside, two native inducible expression systems based on food additives propionate and gluconate were developed. Promoter engineering improved the hemoglobin expression level by 2.2- to 12.2-fold. Combination of these strategies and plasmid copy number modification allowed intracellular production of hemoglobin up to approximately 20% of total protein. Transcriptome and proteome analyses of the hemoglobin-producing strain revealed the cellular response to excess hemoglobin accumulation. Several genes were identified as potential targets for further enhancing hemoglobin production.ConclusionsIn this study, production of plant hemoglobin in C. glutamicum was systematically engineered by combining codon optimization, promoter engineering, plasmid copy number modification, and multi-omics-guided novel target discovery. This study offers useful design principles to genetically engineer C. glutamicum for the production of hemoglobin and other recombinant proteins.

【 授权许可】

CC BY   
© The Author(s) 2023

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