류코노스톡 메센테로이데스 균주의 향산된 젖산 내성 연구
Study on enhanced lactic acid tolerance of Leuconostoc mesenteroides subsp. mesenteroides strains
- 주제(키워드) lactic acid , acid tolerance , leuconostoc
- 발행기관 아주대학교
- 지도교수 이평천
- 발행년도 2016
- 학위수여년월 2016. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000021742
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
To improve acid tolerance in lactic acid bacteria, Leuconostoc mesenteroides subsp. mesenteroides KCTC 3718 producing only D-lactic acid was challenged with exogenously-supplied lactic acid. During the progress of the adaptive evolution to 100 g/L of lactic acid concentration, we had selected mutant strains exhibiting high half maximal inhibitory concentration (IC50) and lactic acid production in fermentation. Two mutant strains, LMS100A and LMS100B, isolated at 100 g/L lactic acid condition significantly increased by 2.1-fold the IC50 values compared to wild type. In fed-batch fermentation, LMS100A and LMS100B achieved 113.4 g/L and 99.6 g/L of the D-lactic acid productions, which is higher than 67.0 g/L lactic acid of wild type. In order to investigate mechanisms of the acid tolerance and changes between mutant strains, omics analysis were performed for wild type, LMS50, LMS60, LMS70A and LMS70B in which fitness probably exhibited a fast increase under acid stress. Analysis of the physiological data showed that the mutant cells retained higher intracellular ammonia concentrations and redox power, but not intracellular ATP levels. Moreover, the increased contents of F0F1 ATPase, phosphosglycerate mutase and several amino acids including alanine, glutamate, asparagine, valine and tyrosine may contribute to maintain intracellular pH homeostasis. In acid stress, results presented in the manuscript also suggested that F0F1 ATPase epsilon subunit deleted C-terminal structure may increase ATP hydrolysis activity to extrude proton from the cells and improve cell viability.
more목차
1. introduction 1
2. Materials and Methods 6
2.1 Bacterial strains 6
2.2 Strain mutagenesis and mutant screening 6
2.3 Measurement of lactic acid tolerance 7
2.4 Production and analysis of D-lactate 8
2.5 2D gel electrophoresis and analysis 9
2.6 Analysis of metabolites 10
2.7 Estimation of intracellular ATP, NADH and ammonia 11
2.8 Genome sequencing and comparison 13
2.9 Computational three-dimensional AtpC protein structure prediction 13
2.10 Heterologous expression and acid tolerance response in E. coli 14
2.11 Construction of optical D-lactic acid producing Lactobacillus plantarum 14
3. Results 18
3.1 Adaptive evolution of Leuconostoc mesenteroides KCTC 3718 18
3.2 Overproduction of D-lactic acid with mutant strains 21
3.3 Proteomic analysis to compare with wild type and mutant strains 26
3.4 Alteration of amino acids in mutant strains enhanced acid tolerance 29
3.5 Measurement of intracellular ATP, redox balance and ammonia 31
3.6 Effect of F0F1ATPase on acid tolerance 35
3.7 Construction of only D-lactic acid producing Lactobacillus plantarum to confirm validation of acid tolerance 39
4. Discussion 43
5. References 51
6. Abstract in Korean 57
7. Acknowledgement 59
