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[Objective] To address the scarcity of neutral sites in Corynebacterium glutamicum ATCC 13032 caused by high G+C content and short intergenic sequences, we established a computer-aided screening platform to identify and characterize efficient neutral sites suitable for heterologous gene integration, aiming to provide key component support for constructing high-performance cell factories. [Methods] A computational screening platform CgNSFinder was constructed to screen candidate neutral sites by integrating heuristic rules such as genomic annotation, neighborhood characteristics, length, and G+C content. The candidate sites were systematically characterized in terms of integration efficiency, cell adaptability, expression intensity, and stability through reporter gene mKate knock-in experiments. We integrated the amylase gene, lycopene synthesis gene cluster, and key L-lysine synthesis genes into the screened sites to verify their heterologous expression ability and application efficiency. [Results] A total of 96 candidate neutral sites were screened out, from which 24 efficient sites were confirmed by experimental characterization. Among them, 19 sites had an integration efficiency greater than 50%, and 22 sites had a fluorescence expression intensity within the range of 2 400-3 500 a.u. The NS3 site showed the best performance, and the lycopene titer reached 48.4 mg/L after multi-copy integration. The L-lysine-producing strain D301 constructed based on the screened sites achieved a titer of 49.51 g/L, a yield of 14.25 g/g, and a productivity of 1.53 g/(L·h) in a 5-L fermenter. [Conclusion] The established neutral site screening method is efficient and feasible. The screened neutral sites are characterized by high integration efficiency, low host interference, and stable expression. This study provides an important tool for metabolic engineering of C. glutamicum.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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