Comprehensive screening of industrially relevant components at genome scale using a high-quality gene overexpression collection of Corynebacterium glutamicum

• Published in: Trends in biotechnology (Regular ed.) Vol. 43; no. 1; pp. 220 - 247
• Main Authors: Liu, Jiao, Zhao, Xiaojia, Cheng, Haijiao, Guo, Yanmei, Ni, Xiaomeng, Wang, Lixian, Sun, Guannan, Wen, Xiao, Chen, Jiuzhou, Wang, Jin, An, Jingjing, Guo, Xuan, Shi, Zhenkun, Li, Haoran, Wang, Ruoyu, Zhao, Muqiang, Liao, Xiaoping, Wang, Yu, Zheng, Ping, Wang, Meng, Sun, Jibin
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.01.2025; Elsevier Limited
• Subjects: amino acid biomanufacturing; Amino acids; Bacteria; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biotechnology; Capillary electrophoresis; Carbohydrates; Cloning; Collection; Controllability; Corynebacterium glutamicum; Corynebacterium glutamicum - genetics; Corynebacterium glutamicum - metabolism; DNA methylation; DNA repair; E coli; Enzymes; Escherichia coli - genetics; Escherichia coli - metabolism; functional components; Gene amplification; Gene expression; Gene Expression Regulation, Bacterial - genetics; gene overexpression collection; Genetic engineering; Genome, Bacterial - genetics; Genomes; hyperosmotic tolerance; Industrial Microbiology - methods; Integrated circuits; Internal Medicine; Interrogation; Lysine; Metabolic Engineering - methods; Microorganisms; Mutation; Plasmids; Proteins; Screening; Transcription factors; amino acid biomanufacturing; functional components; Corynebacterium glutamicum; hyperosmotic tolerance; gene overexpression collection
• ISSN: 0167-7799; 1879-3096; 1879-3096
• DOI: 10.1016/j.tibtech.2024.09.020

A high-quality gene overexpression collection of Corynebacterium glutamicum was constructed with a highly automated biofoundry.Functional components for enhanced stress tolerance and amino acid transport were comprehensively screened at a genome scale.Transcriptional factors regulating cell division and energy metabolism and DNA repair proteins removing m6G methylations were found to be important for hyperosmotic tolerance.Application of the newly screened exporters facilitates the highest l-threonine production by C. glutamicum achieved thus far. Development of efficient microbial strains for biomanufacturing requires deep understanding of the biology and functional components responsible for the synthesis, transport, and tolerance of the target compounds. A high-quality controllable gene overexpression strain collection was constructed for the industrial workhorse Corynebacterium glutamicum covering 99.7% of its genes. The collection was then used for comprehensive screening of components relevant to biomanufacturing features. In total, 15 components endowing cells with improved hyperosmotic tolerance and l-lysine productivity were identified, including novel transcriptional factors and DNA repair proteins. Systematic interrogation of a subset of the collection revealed efficient and specific exporters functioning in both C. glutamicum and Escherichia coli. Application of the new exporters was showcased to construct a strain with the highest l-threonine production level reported for C. glutamicum (75.1 g/l and 1.5 g/l·h) thus far. The genome-scale gene overexpression collection will serve as a valuable resource for fundamental biological studies and for developing industrial microorganisms for producing amino acids and other biochemicals. [Display omitted] This study reports the automated construction of a high-quality arrayed gene overexpression strain collection and high-throughput screening of biological components that can be used for developing industrial strains for biomanufacturing. This strain collection and screening strategy can be readily used to discover new components with various functions. A Corynebacterium glutamicum strain with the highest l-threonine production level (75.1 g/l and 1.5 g/l·h) was also engineered using the newly identified biological components and tested in 5-l bioreactors. To meet the requirement of industrialization, further genetic modification and process engineering are necessary to improve the l-threonine production level in C. glutamicum. Therefore, the current Technology Readiness Level (TRL) of this production technology lies between 3 and 4. A high-quality gene overexpression collection of the industrial workhorse Corynebacterium glutamicum containing 3049 strains was produced. To demonstrate its application, a comprehensive screening of industrially relevant components at a genome scale was conducted. New components for tolerance and transport were identified and used for developing industrial amino acid-producing strains.