地衣芽胞杆菌(Bacillus licheniformis)DW2生长和杆菌肽合成代谢在能量及前体利用方面存在竞争关系,为了提高杆菌肽的合成效率,在20 L发酵罐水平研究了ccpN基因敲除对杆菌肽合成与细胞生长的调控效应。结果显示:菌株DW2ΔccpN杆菌肽峰值效价(1 337 U/mL)比菌株DW2峰值效价(1 024 U/mL)提高30.6%。但是,其峰值生物量(3.39×1010 CFU/mL)仅为菌株DW2峰值生物量(5.56×1010 CFU/mL)的61%。另外,DW2ΔccpN胞外氨基酸浓度在36 h时、溢流有机酸总量在22~27 h时分别比DW2提高43.8%和24.7%~37.6%。同时,细胞对数期能量代谢(citZ、icd、zwf、cydA)、氨基酸转运(relA、brnQ、vpr、yvbW)以及丙酮酸转化(pycA、pckA、pyk)和hprK等13个基因的转录组表达(TPM值)也上调2.0~18.4倍,说明菌株DW2ΔccpN的溢流代谢增强,为对数后期提供了更多的碳源和能源分子;细胞严谨响应增强使CodY的调控活性下降,降低了细胞的生长速率,提高了氨基酸转运能力,增加了ATP和NADPH的供应,最终提高了杆菌肽的合成效率,为杆菌肽发酵生产提供了重要参考。
The growth of Bacillus licheniformis DW2 and the bacitracin anabolism are competitive in energy and precursors utilization. In order to improve the efficiency of bacitracin synthesis by B. licheniformis DW2, the regulatory effect of ccpN knockout in two strains was studied in 20 L bioreactor. The results showed that the peak bacitracin production of DW2ΔccpN (1 337 U/mL) was 30.6% higher than that of DW2 (1 024 U/mL). However, the maximum biomass of DW2ΔccpN (3.39×1010 CFU/mL) was only 61% of DW2 (5.56×1010 CFU/mL). In addition, the extracellular amino acids content of DW2ΔccpN (36 h) and the total amount of overflow organic acids (22-27 h) were 43.8% and 24.7%-37.6% higher than that of DW2, respectively. Meanwhile, the transcriptome expression (TPM value) of exponential phase including energy metabolism (citZ, icd, zwf, cydA), amino acid transport (relA, brnQ, vpr, yvbW), pyruvate conversion (pycA, pckA, pyk) and hprK was also enhanced by 2.0-18.4 times than that of DW2, respectively. The results suggested that in the strain DW2ΔccpN ,the overflow metabolism was enhanced, providing more carbon sources and energy molecules in the logarithmic period; the stringent response was increased, reducing the regulatory activity of CodY, decreasing the cell growth rate and increasing the amino acid transport capacity and the supplies of ATP and NADPH were improved, which ultimately enhanced bacitracin synthesis efficiency, providing important references for bacitracin fermentation production.
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