| In this dissertation, Lactobacillus casei Zhang was used as an initial strain, and an acid-resistant evolved strain was obtained by adaptive evolution. The physiological responses and metabolic regulations of the parental strain and the evolved strain during acid stress were investigated by using microbial physiology and proteomic approaches, and based on this, two strategies including biochemical and metabolic engineering were proposed and the acid tolerance of L. casei was significantly improved. The main results were described as follows:1) Adaptive evolution improved the growth performances of L. casei under acid stress, and the evolved strains generated through evolution for 70 d, exhibited 60% and 10% increases in biomass and the average specific growth rate, respectively. Moreover, the acid tolerance of the evolved strains increased with the extension of evolution time. After evolution for 70 d, the tolerance to lactic acid and hydrochloric acid stress increased 638- and 3.5-fold, respectively. In addition, the tolerances to simulated gastrointestinal juice and bile salt were evaluated, and the results showed that the evolved strains displayed significantly higher tolerance to transit and bile salt tolerance.2) The stress responses between the parental strain and the acid-resistant mutant during acid stress were compared based on the micro-environment and cell membrane analysis. Acid stress resulted in the decrease of pHi, and the mutant exhibited higher pHi, H+-ATPase, and intracellular ATP pool. The mutant maintained pHi homeostasis by utilizing the H+-ATPase to expel H+ at the expense of ATP consumption. Moreover, higher amounts of intracellular aspartate, alanine, arginine and glutamate were detected in the mutant when shocked for 1 h during acid stress. Analysis of the cell membrane fluidity showed that acid stress led to the decrease of fluidity, and the mutant possessed higher fluidity than that of the parental strain. In response to acid stress, the cells increased the ratio of unsaturated fatty acids to saturated ones (U/S Ratio), carbon chain length and the cyclopropane fatty acid distribution. The mutant displayed 1.32- and 1.52-fold increases in U/S Ratio and cyclopropane fatty acid content, when compared with the parental strain. In addition, transmission electron microscopy analysis showed that the mutant maintained a more intact cell membrane after environmental acidification, which contributed to decrease the effuse of intracellular materials and influx of deleterious materials and alleviate the damage induced by acid stress.3) A comparative proteomic approach based on 2D-DIGE and i-TRAQ was conducted to investigate the expression difference of proteins between the parental strain and the mutant during acid stress. Acid stress invoked the changes in cellular metabolism and the mutant exhibited higher metabolic activity in glycolytic pathway, peptidoglycan synthesis and aspartate and arginine metabolism in order to protect cells against acid damage. In addition, proteins involved in DNA replication, RNA synthesis and translation were down-regulated, while DNA repair proteins (Muts, RecO) and some chaperones (GroL, DnaJ and DnaK) were significantly up-regulated during acid stress.4) The protective mechanisms of aspartate on the acid tolerance in L. casei were investigated. Exogenous aspartate improved the growth performance and acid tolerance of L. casei during acid stress. When cultured in the presence of aspartate at pH 4.3 for 64 h, the biomass of L. casei (Asp+) increased 1.7-fold, compared to L. casei (Asp-) (without aspartate addition). The cells were challenged at pH 3.3 for 3 h, and the survival rate increased 43-fold in the presence of aspartate. Meanwhile, compared with L. casei (Asp-), L. casei (Asp+) maintained higher pHi, intracellular ATP pool and NH4+ concentration based on physiological analysis. In addition, analysis of the metabolomics data showed that aspartate increased the metabolic flux of glycolytic metabolism, and L. casei (Asp+) maintained higher amounts of 6-P-glucose, 6-P-frucose, 3-P-glycerate and lactic acid. In addition, L. casei (Asp+) accumulated higher levels of aspartate, arginine, ornithine, glutamate, threonine, valine, isoleucine, leucine, oleic acid and cyclopropane fatty acid. Validation of the gene expression related to aspartate metabolism indicated that genes argG and argH involved in arginine metabolism were up-regulated, while genes asnH and asdA involved in asparagines and alanine metabolism were down-regulated during acid stress. Interestingly, the expressions of genes argG and argH in the presence of aspartate during acid stress were significantly higher than that of the control (without aspartate addition).5) A DNA repair protein (RecO) from L. casei Zhang was introduced into L. lactis NZ9000, and the effects of RecO production on the acid and other environmental tolerances were investigated. Expression of the RecO protein in NZ9000 resulted in a marked improvement in growth performance during acid (pH 5.0), salt (3% NaCl), oxygen (0.1 mM H2O2) stresses, and the biomass of NZ-RecO increased 22.03%, 37.04% and 19.37%, respectively, when cultured at 30oC for 28 h. In addition, the survival rates of NZ-RecO during acid (pH 4.0) and salt (15% NaCl) stress enhanced 2.90- and 3.28-fold, respectively, compared with the control stain (NZ-Vector). The yield of biomass and lactic acid against glucose increased 50% and 43.48%, respectively, and lactic acid productivity increased 41.67%, when cultured in the presence of 3% NaCl for 28 h.
|
PDF Full Text Request