Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
Key Laboratory of Systems Bioengineering, Ministry of Education Tianjin, 300072, China.
Sci Rep. 2016 Jun 16;6:27973. doi: 10.1038/srep27973.
Traditionally, nisin was produced industrially by using Lactococcus lactis in the neutral fermentation process. However, nisin showed higher activity in the acidic environment. How to balance the pH value for bacterial normal growth and nisin activity might be the key problem. In this study, 17 acid-tolerant genes and 6 lactic acid synthetic genes were introduced in L. lactis F44, respectively. Comparing to the 2810 IU/mL nisin yield of the original strain F44, the nisin titer of the engineered strains over-expressing hdeAB, ldh and murG, increased to 3850, 3979 and 4377 IU/mL, respectively. These engineered strains showed more stable intracellular pH value during the fermentation process. Improvement of lactate production could partly provide the extra energy for the expression of acid tolerance genes during growth. Co-overexpression of hdeAB, murG, and ldh(Z) in strain F44 resulted in the nisin titer of 4913 IU/mL. The engineered strain (ABGL) could grow on plates with pH 4.2, comparing to the surviving pH 4.6 of strain F44. The fed-batch fermentation showed nisin titer of the co-expression L. lactis strain could reach 5563 IU/mL with lower pH condition and longer cultivation time. This work provides a novel strategy of constructing robust strains for use in industry process.
传统上，乳链菌肽是通过中性发酵过程中使用乳球菌 lactis 来工业化生产的。然而，乳链菌肽在酸性环境中表现出更高的活性。如何平衡 pH 值以促进细菌正常生长和乳链菌肽活性可能是关键问题。在本研究中，分别在乳球菌 F44 中引入了 17 个耐酸基因和 6 个乳酸合成基因。与原始菌株 F44 的 2810 IU/mL 乳链菌肽产量相比，过表达 hdeAB、ldh 和 murG 的工程菌株的乳链菌肽产量分别增加到 3850、3979 和 4377 IU/mL。这些工程菌株在发酵过程中表现出更稳定的细胞内 pH 值。乳酸产量的提高可以部分为生长过程中耐酸基因的表达提供额外的能量。在 F44 菌株中共同过表达 hdeAB、murG 和 ldh(Z)，乳链菌肽产量达到 4913 IU/mL。与 F44 菌株的存活 pH4.6 相比，工程菌株（ABGL）能够在 pH4.2 的平板上生长。补料分批发酵显示，共同表达的乳球菌菌株的乳链菌肽产量在较低 pH 值和较长培养时间下可达到 5563 IU/mL。这项工作为构建用于工业生产的稳健菌株提供了一种新策略。
