Taniguchi, Hironori: Exploring the potential of sigma factors for strain development in Corynebacterium glutamicum. 2016
Inhalt
- 1 Introduction
- 1.1 Strain development
- 1.2 Strain development using global regulators
- 1.3 Sigma factors
- 1.4 Transcription initiation mechanism by RNA polymerase holoenzyme
- 1.5 Sigma factor competition as a regulatory mechanism
- 1.6 Strain development using sigma factors
- 1.7 Corynebacterium glutamicum
- 1.8 Global regulators in C. glutamicum
- 1.9 Sigma factors of C. glutamicum
- 1.10 Objectives
- 2 Result
- 2.1 Exploring the role of sigma factor gene expression on production by Corynebacterium glutamicum: sigma factor H and FMN as example
- 2.1.1 Abstract
- 2.1.2 Introduction
- 2.1.3 Material and methods
- 2.1.3.1 Bacterial strains, plasmid and primer
- 2.1.3.2 Medium and growth condition
- 2.1.3.3 Riboflavin production experiments
- 2.1.3.4 Transcriptome analysis of sigH overexpressing strain using DNA microarray
- 2.1.3.5 Measurement of glucose-6-phosphate 1-dehydrogenase enzyme activities
- 2.1.3.6 FMN and FAD production experiments
- 2.1.4 Results
- 2.1.4.1 Effect of overexpressing sigma factor genes in C. glutamicum
- 2.1.4.2 Overexpression of sigH resulted in riboflavin secretion
- 2.1.4.3 Global gene expression changes due to sigH overexpression
- 2.1.4.4 FMN production by C. glutamicum established as proof-of-concept based on overexpression of endogenous genes sigH and ribF
- 2.1.5 Discussion
- 2.2 Characterization of the physiological role and the regulatory mechanism of sigma factor D in Corynebacterium glutamicum
- 2.2.1 Abstract
- 2.2.2 Introduction
- 2.2.3 Material and methods
- 2.2.3.1 Bacterial strains, plasmids and oligonucleotides
- 2.2.3.2 Medium and conditions for growth experiments
- 2.2.3.3 Photometric determination of supernatant turbidity
- 2.2.3.4 Confirmation of cell aggregation by microscopic and flow cytometric analysis
- 2.2.3.5 RNA extraction
- 2.2.3.6 RNA-seq analysis and real-time PCR analysis
- 2.2.3.7 Protein analysis in supernatant
- 2.2.3.8 Quantification of carbohydrate in acetone precipitates
- 2.2.3.9 Detection of TDCM by TLC
- 2.2.4 Results
- 2.2.4.1 Disruption and overexpression of sigD influenced the maximum growth rate
- 2.2.4.2 sigD overexpression influenced the characteristic of cell culture
- 2.2.4.3 sigD overexpression induced cell aggregation
- 2.2.4.4 SigD regulated transcription of several genes especially related to cell envelope integrity
- 2.2.4.5 sigD overexpression changed the pattern of secreted proteins
- 2.2.4.6 sigD overexpression altered the pattern of secreted metabolites
- 2.2.4.7 Overexpression of sigD increased the amounts of trehalose dicorynomycolate
- 2.2.4.8 cg0697 is a candidate for the anti-sigma factor of SigD
- 2.2.4.9 The cg0697 disrupted mutant showed a similar phenotype as the sigD overexpressing strain
- 2.2.4.10 Transcription of cg0697 is SigD-dependent and Cg0697 regulates SigD activity
- 2.2.4.11 Disruption of cg0697 led to similar transcriptome changes as sigD overexpression
- 2.2.4.12 Consensus promoter sequence for SigD
- 2.2.5 Discussion
- 2.3 Harnessing sigma factor gene expression for production in Corynebacterium glutamicum: sigma factor A and carotenoids as an example
- 2.3.1 Abstract
- 2.3.2 Introduction
- 2.3.3 Material and methods
- 2.3.3.1 Bacterial strains, plasmids and primers
- 2.3.3.2 Medium, growth conditions and growth rate comparison
- 2.3.3.3 Carotenoid extraction and quantification
- 2.3.3.4 Transcriptome analysis of the sigA overexpressing strain using DNA microarray
- 2.3.4 Results
- 2.3.4.1 The sigA overexpressing strain showed the best improvement of lycopene production
- 2.3.4.2 Overexpression of sigA improved lycopene accumulation especially in the stationary phase
- 2.3.4.3 Overexpression of sigA also influenced decaprenoxanthin accumulation of the wild type strain in the stationary phase
- 2.3.4.4 Global gene expression changed due to sigA overexpression
- 2.3.4.5 Addition of thiamine/PCA improved carotenoid production in the sigA overexpressing strain
- 2.3.4.6 The effect of sigA overexpression was transferable to the β-carotene producing strain
- 2.3.4.7 The effect of sigA overexpression is additive to the effect of the derepression of crt operon
- 2.3.5 Discussion
- 3 Discussion
- 3.1 sigH overexpression on production: riboflavin, FMN and further
- 3.2 Overexpression of a sigma factor gene sigD for biological understanding
- 3.3 Strain development using sigma factors: Carotenoids as targets
- 3.4 The effect of overexpression of different sigma factor genes
- 3.5 Strain development by overexpression of sigma factor genes
- 3.6 Potential use of sigma factors and more in the future
- 4 Supplemental Figures
- 5 References