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Objective To construct high-yield engineering strains of Halomonas campaniensis XH26 by introducing five recombinant plasmids (pHX01-pHX05), each carrying the P tac promoter and combinations of the genes asd, lysC, ectA, ectB, and ectC. This metabolic engineering strategy was coupled with the response surface methodology (RSM) for optimization of the culture conditions, thereby enhancing ectoine accumulation. Methods The recombinant plasmids were conjugally transferred from Escherichia coli S17-1(λ-pir) into H. campaniensis XH26, with positive transconjugants selected via gentamicin (50 μg/mL). Recombinant strains were induced with 0.2 mmol/L IPTG, and ectoine accumulation was quantified by HPLC. Critical nutritional variables—NaCl, peptone, l-glutamate, and glucose—were optimized through one-factor-at-a-time experiments, Plackett-Burman design, and Box-Behnken design. Results Five recombinant strains (XH26/pHX01-XH26/pHX05) were successfully constructed. Culture in the MG medium revealed that strain XH26/pHX04 (overexpressing asd-lysC-ectA-ectB) achieved the highest ectoine titer of (1.32±0.04) g/L. Strains XH26/pHX05 and XH26/pHX03 achieved the ectoine titer of (1.19±0.07) g/L and (1.07±0.08) g/L, respectively, while XH26/pHX02 yielded a lower titer of (1.02±0.14) g/L. The medium composition optimized by RSM was composed of 116.08 g/L NaCl, 16.30 g/L peptone, 169.57 g/L l-glutamate, and 15.53 g/L glucose. Under these optimized conditions, the titer of ectoine produced by XH26/pHX04 increased to (1.81±0.02) g/L, representing a significant increase of 301.56% compared with that of the wild-type strain XH26. Conclusion This study demonstrates that using H. campaniensis as a chassis and overexpressing a key gene combination (asd, lysC, ectA, ectB) under a strong promoter, synergized with culture medium optimization via RSM, can significantly boost the ectoine yield of recombinant strains. The findings provide a robust technical framework for the subsequent industrial production of ectoine.
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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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