代谢工程耦合响应面法优化盐单胞菌合成四氢嘧啶的关键技术

Metabolic engineering coupled with response surface methodology for optimization of ectoine synthesis in Halomonas

摘要:

目的 通过构建5种含P_tac启动子的重组质粒pHX01-pHX05 (组合基因asd、lysC、ectA、ectB、ectC)，导入坎帕尼亚盐单胞菌(Halomonas campaniensis) XH26，构建高产工程菌株，并结合响应面法(response surface methodology, RSM)优化培养条件提高四氢嘧啶(ectoine)的积累量。方法 重组质粒经大肠埃希氏菌(Escherichia coli) S17-1(λ-pir)接合转移至菌株XH26，利用庆大霉素(50 μg/mL)筛选阳性克隆子；以0.2 mmol/L IPTG诱导表达重组菌株，用HPLC检测四氢嘧啶的积累量；采用单因素试验、Plackett-Burman与Box-Behnken设计优化关键变量因素(NaCl、蛋白胨、l-谷氨酸钠、葡萄糖)。结果 构建了5株重组菌株(XH26/pHX01-pHX05)。采用MG培养基培养重组菌株，发现菌株XH26/pHX04 (asd-lysC-ectA-ectB)的四氢嘧啶积累量最高，达(1.32±0.04) g/L；菌株XH26/pHX05和菌株XH26/pHX03次之，四氢嘧啶积累量分别为(1.19±0.07) g/L和(1.07±0.08) g/L；而XH26/pHX02积累量较低，为(1.02±0.14) g/L。利用响应面法优化培养基的关键成分，确定最优条件为NaCl 116.08 g/L、蛋白胨16.30 g/L、l-谷氨酸钠169.57 g/L、葡萄糖15.53 g/L。在此条件下，重组菌株XH26/pHX04的四氢嘧啶积累量提高至(1.81±0.02) g/L，较野生型菌株XH26显著提高了301.56%。结论 以坎帕尼亚盐单胞菌为“底盘细胞”，利用强启动子组合过表达基因asd、lysC、ectA/B，辅以响应面法优化重组菌株的培养基条件，可显著提高重组菌株的四氢嘧啶积累量，为后续工业化生产提供了一定的技术参考依据。

Abstract:

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.