Hao Panlong, Liang Dongmei, Cao Lijie, Qiao Bin, Wu Hao, Caiyin Qinggele, Zhu Hongji, Qiao Jianjun
Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, China.
Key Laboratory of Systems Bioengineering, Ministry of Education Tianjin, Tianjin, 300072, China.
Appl Microbiol Biotechnol. 2017 Aug;101(15):6137-6153. doi: 10.1007/s00253-017-8365-7. Epub 2017 Jun 22.
Nisin fermentation by Lactococcus lactis requires a low pH to maintain a relatively higher nisin activity. However, the acidic environment will result in cell arrest, and eventually decrease the relative nisin production. Hence, constructing an acid-resistant L. lactis is crucial for nisin harvest in acidic nisin fermentation. In this paper, the first discovery of the relationship between D-Asp amidation-associated gene (asnH) and acid resistance was reported. Overexpression of asnH in L. lactis F44 (F44A) resulted in a sevenfold increase in survival capacity during acid shift (pH 3) and enhanced nisin desorption capacity compared to F44 (wild type), which subsequently contributed to higher nisin production, reaching 5346 IU/mL, 57.0% more than that of F44 in the fed-batch fermentation. Furthermore, the engineered F44A showed a moderate increase in D-Asp amidation level (from 82 to 92%) compared to F44. The concomitant decrease of the negative charge inside the cell wall was detected by a newly developed method based on the nisin adsorption amount onto cell surface. Meanwhile, peptidoglycan cross-linkage increased from 36.8% (F44) to 41.9% (F44A), and intracellular pH can be better maintained by blocking extracellular H due to the maintenance of peptidoglycan integrity, which probably resulted from the action of inhibiting hydrolases activity. The inference was further supported by the acmC-overexpression strain F44C, which was characterized by uncontrolled peptidoglycan hydrolase activity. Our results provided a novel strategy for enhancing nisin yield through cell wall remodeling, which contributed to both continuous nisin synthesis and less nisin adsorption in acidic fermentation (dual enhancement).
乳酸乳球菌发酵产生乳酸链球菌素需要低pH值来维持相对较高的乳酸链球菌素活性。然而，酸性环境会导致细胞生长停滞，并最终降低乳酸链球菌素的相对产量。因此，构建耐酸的乳酸乳球菌对于在酸性条件下发酵生产乳酸链球菌素至关重要。本文首次报道了D-天冬酰胺酰胺化相关基因（asnH）与耐酸性之间的关系。与野生型F44相比，在乳酸乳球菌F44（F44A）中过表达asnH导致其在酸胁迫（pH 3）期间的存活能力提高了7倍，并增强了乳酸链球菌素的解吸能力，这随后有助于提高乳酸链球菌素产量，在分批补料发酵中达到5346 IU/mL，比F44高出57.0%。此外，与F44相比工程菌F44A的D-天冬酰胺酰胺化水平有适度提高（从82%提高到92%）。通过基于细胞表面乳酸链球菌素吸附量的新开发方法检测到细胞壁内负电荷的相应减少。同时，肽聚糖交联度从36.8%（F44）增加到41.9%（F44A），由于肽聚糖完整性的维持，通过阻断细胞外H⁺可以更好地维持细胞内pH值，这可能是由于抑制水解酶活性的作用。acmC过表达菌株F44C进一步支持了这一推断，该菌株的特征是肽聚糖水解酶活性不受控制。我们的结果提供了一种通过细胞壁重塑提高乳酸链球菌素产量的新策略，这有助于在酸性发酵中持续合成乳酸链球菌素并减少乳酸链球菌素吸附（双重增强）。
