Abstract
BackgroundIn bioengineering, growth of microorganisms is limited because of environmental and industrial stresses during fermentation. This study aimed to construct a nisin-producing chassis Lactococcus lactis strain with genome-streamlined, low metabolic burden, and multi-stress tolerance characteristics.ResultsThe Cre-loxP recombination system was applied to reduce the genome and obtain the target chassis strain. A prophage-related fragment (PRF; 19,739 bp) in the L. lactis N8 genome was deleted, and the mutant strain L. lactis N8-1 was chosen for multi-stress tolerance studies. Nisin immunity of L. lactis N8-1 was increased to 6500 IU/mL, which was 44.44% higher than that of the wild-type L. lactis N8 (4500 IU/mL). The survival rates of L. lactis N8-1 treated with lysozyme for 2 h and lactic acid for 1 h were 1000- and 10,000-fold higher than that of the wild-type strain, respectively. At 39 ℃, the L. lactis N8-1 could still maintain its growth, whereas the growth of the wild-type strain dramatically dropped. Scanning electron microscopy showed that the cell wall integrity of L. lactis N8-1 was well maintained after lysozyme treatment. Tandem mass tags labeled quantitative proteomics revealed that 33 and 9 proteins were significantly upregulated and downregulated, respectively, in L. lactis N8-1. These differential proteins were involved in carbohydrate and energy transport/metabolism, biosynthesis of cell wall and cell surface proteins.ConclusionsPRF deletion was proven to be an efficient strategy to achieve multi-stress tolerance and nisin immunity in L. lactis, thereby providing a new perspective for industrially obtaining engineered strains with multi-stress tolerance and expanding the application of lactic acid bacteria in biotechnology and synthetic biology. Besides, the importance of PRF, which can confer vital phenotypes to bacteria, was established.
Highlights
In bioengineering, growth of microorganisms is limited because of environmental and industrial stresses during fermentation
The growth curves of the wild-type and mutant strains were obtained under different concentrations of nisin, and revealed that there were no significant differences in growth between the mutant and wild-type strains at 4000 IU/mL (Fig. 2d), 5000 IU/ mL (Fig. 2e), and 6000 IU/mL (Fig. 2f ) nisin concentrations. These results indicated that the wild-type and mutant strains could grow in GM17 medium with high concentration of nisin [6500 IU/mL (Fig. 2g), 7000 IU/ mL (Fig. 2h), and 7500 IU/mL (Fig. 2i)]; the wild-type strain started to grow was 15–20 h later than the mutant strain, suggesting that L. lactis N8-1 took less time to overcome the side effects of nisin and start to grow
After treatment for 120 min, the survival rate of L. lactis N8-1 was markedly higher (1000-fold) than that of the control strain (Fig. 3b). These results demonstrated that loss of prophage-related fragment (PRF) conferred L. lactis N8-1 lysozyme tolerance
Summary
Growth of microorganisms is limited because of environmental and industrial stresses during fermentation. Researchers have adapted strains with stress tolerance through different strategies, e.g., random mutagenesis, global transcription machinery engineering (gTME), global regulator overexpression and genome editing. Random mutagenesis has been extensively used to improve the acid tolerance of microbial cells. The survival rate of E. coli had been noted to increase by 10- to 100-fold at pH 2.5 by overexpressing the global regulator H-NS [7]. Li et al overexpressed sHSP20 in E. coli BL21 cells and increased its survival period at 50 °C by almost 2 h [8], while deletion of ADY2 improved the growth of S. cerevisiae under acetic acid, ethanol, and hydrogen peroxide stresses [9]
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