Lactic acid bacteria (LAB) are a group of Gram-positive, low GC content bacteria with lactic acid as their main acidic fermentation product. Moreover, LAB are generally recognized as safe (GRAS) microorganisms due to its health-promoting and probiotic properties. In food industry, LAB are widely used for food and beverage fermentation, bacteriocins and exopolysaccharides. In addition, as cell factories, LAB are the most promising microorganisms to produce various chemicals, including lactic acid, vitamins and antimicrobial peptide (nisin).
However, LAB suffer from various stress conditions including acid, osmotic and oxygen during fermentation processing . Among these environmental stresses, acid stress is one of the important survival challenges. During fermentation, the continuous accumulation of the target products causes the decrease of environmental pH, and the activity of intracellular enzymes are inhibited . Moreover, this will affect the normal growth and metabolism of microbial cells.
In response to acid stress, microbial cells have evolved various anti-acid stress response mechanisms including the maintenance of intracellular pH homeostasis, cell
membrane functionality, non-coding sRNAs and stress response proteins . On these basis, different kinds of anti-acid components have been elucidated through the application of system biology technologies.
In previous study, genome mutagenesis combined with high-throughput technologies was performed to screen acid tolerance strain. The mutant strain L. lactis WH101 showed remarkably 16000-fold higher survival rate than that of the parent strain after 5 h of acid shock at pH 4.0 . Meanwhile, it maintained higher ATP, NH4+ and intracellular pH levels compared to parent strain during acid stress. Next, comparative transcriptomics analysis was performed on L. lactis WH101 and L. lactis NZ9000 to investigated the response mechanisms of microbial cells during acid stress. Several interesting acid tolerance mechanisms were proposed. Carbohydrate metabolism and sugar transporters were strengthened to provide more energy. Amino acids metabolism and transporters were regulated to maintain intracellular pHi homeostasis and generate ATP. Fatty acid metabolism was also regulated to enhance the acid tolerance of cells. Moreover, transcriptional regulators and stress response proteins were also found to be related to acid stress . Based on the proposed acid tolerance mechanisms, one anti-acid component was selected.
In this study, we constructed MsmK overexpression strains in L. lactis NZ3900 which was named L. lactis NZ3900 (MsmK). Next, the gene encoding the fluorescence protein was co-expressed with msmK in L. lactis NZ3900，resulting in L. lactis NZ3900 (MsmK/GFP). The fluorescence intensity was used to characterize the survival rates of the engineered strain in simulated human body environment (pH 4.0) .
1. Nezhad MH, Hussain MA, Britz ML. 2015. Stress responses in probiotic Lactobacillus casei. Crit Rev Food Sci Nutr 55:740-749.
2. Wu CD, Huang J, Zhou RQ. 2014. Progress in engineering acid stress resistance of lactic acid bacteria. Appl Microbiol Biotechnol 98:1055-1063.
3. Zhang J, Caiyin Q, Feng WJ, Zhao XL, Qiao B, Zhao GR, Qiao JJ. 2016. Enhance nisin yield via improving acid-tolerant capability of Lactococcus lactis F44. Sci Rep 6:12.
4. Martani F, Berterame NM, Branduardi P. 2017. Microbial stress: From molecules to systems (Sitges, November 2015). New Biotechnology 35:30-34.
5. Bosma EF, Forster J, Nielsen AT. 2017. Lactobacilli and pediococci as versatile cell factories - Evaluation of strain properties and genetic tools. Biotechnol Adv 35:419-442.
6. Teusink B, Bachmann H, Molenaar D. 2011. Systems biology of lactic acid bacteria: a critical review. Microbial Cell Factories 10:17.