Abstract
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.
Highlights
Nisin, as an antimicrobial peptide with 34 residues, is known for its wide range of antimicrobial activity against gram-positive bacteria and shows antimicrobial activity against gram-negative bacteria especially when combined with a chelating agent, such as disodium EDTA1,2
The mechanisms of acid tolerance in bacteria can be classified into four categories: (I) Decrease of intracellular protons mainly involved in transmitting obstacle of extracellular H+, exportation of intracellular H+ and consumption of intracellular H+. (Ia) The solidification of cell wall, the change of cell membrane and the protection of trehalose can work by transmitting obstacles of extracellular H+ 15–20. (Ib) The mechanism of intracellular H+ consumption mainly includes decarboxylation, the generation of alkali and the protective effect of glutathione and histidine21–27. (Ic) Proton pumps plays a major role in exportation of intracellular H+
Studies shown that DNA synthesis and repair increased under acid stress31,32. (III) Regulatory factors include the global regulatory factors, two-component-regulating system and other regulatory factors. (IIIa) EvgS/EvgA is the main two-component signal transduction systems (TCS) in the E. coli, which can endow bacteria the sense ability of acid stress and is involved with the regulation of other genes33,34. (IIIb) The overexpression of some non-coding sRNAs can improve the ability against low pH environment significantly
Summary
As an antimicrobial peptide with 34 residues, is known for its wide range of antimicrobial activity against gram-positive bacteria and shows antimicrobial activity against gram-negative bacteria especially when combined with a chelating agent, such as disodium EDTA1,2. It is necessary to construct an acid tolerant strain which can maintain its growth activity in the relative acidic fermentation condition, and alleviate nisin degradation. Simultaneous overexpression of the non-coding sRNA, such as DsrA, RprA and ArcZ displayed an 8500-fold higher survival of E. coli to low pH, which provided a protective effect on carboxylic acid and oxygen stress. This study aims to construct an acid tolerant module in L. lactis, hoping to balance the optimal pH of cell growth and nisin activity, so as to obtain higher nisin yield. To reach this goal, some acid tolerant genes were transformed to L. lactis F44, and the nisin production was analyzed
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