PUB - Publikationen an der Universität Bielefeld

A sigma factor is one of the components of RNA polymerase holoenzyme and responsible for promoter recognition and transcription initiation. Bacteria usually possess multiple genes encoding sigma factors and strictly control their activity. Activation of a specific sigma factor alters the promoter recognition of the RNA polymerase holoenzyme and enhances the transcription of a specific set of genes with similar promoter sequences. Therefore, sigma factors can be used for activating expression of a set of genes at the same time and could be useful for strain development. In this study, the potential of sigma factors for strain development was examined in Corynebacterium glutamicum. This bacterium was first identified as a natural glutamate producer and has been used in industry for production of amino acids such as L-glutamate and L-lysine. Because of its genetic amenability, various strains have been developed to produce industrial relevant compounds using metabolic engineering. C. glutamicum possesses seven sigma factor genes, sigA, sigB, sigC, sigD, sigE, sigH and sigM. In order to test the effect of sigma factors on the cell behavior, all sigma factor genes were overexpressed in an IPTG-inducible system with different IPTG concentrations. Overexpression of each sigma factor gene affected the growth rate differently, and the effect was dependent on the IPTG concentration, thus, on the strength of expression. The strongest effect was observed for overexpression of sigD and sigH. Overexpression of sigH led to the strong yellow supernatant. HPLC analysis identified that this color was caused by riboflavin. Transcriptome analysis revealed increased expression of genes of riboflavin biosynthesis, the pentose phosphate pathway and of enzymes requiring FMN (flavin mononucleotide), FAD (flavin adenine dinucleotide) or NADPH as cofactor. Since riboflavin is a precursor of FMN, sigH overexpression was applied for the production of FMN. Balanced expression o f sigH and the bifunctional riboflavin kinase/FMN adenyltransferase gene ribF improved accumulation of riboflavin (20 ± 1 μM) and allowed for its conversion to FMN (33 ± 2 μM). sigD overexpression in C. glutamicum WT reduced foaming possibly due to secretion of anti-foaming compounds. Transcriptome analysis revealed that expression of genes encoding for proteins which are involved in the cell wall integrity such as mycomembrane synthesis increased as a consequence of sigD overexpression. TLC analysis revealed that sigD overexpression increased the content of trehalose dicorynomycolate, which is one of the components of the mycomembrane. In order to explore the potential of sigma factor for strain development, overexpression of sigma factor genes was performed in the recombinant lycopene producing strain LYC5, aiming at an increase of lycopene production. Among seven sigma factor genes, sigA was selected as the most effective gene for this purpose. Overexpression of sigA in the WT strain also increased production of decaprenoxanthin, which is naturally produced by C. glutamicum. Accumulation of lycopene and decaprenoxanthin in the sigA overexpressing strain was observed especially in the stationary phase. Transcriptome analysis identified many genes which expression was upregulated under sigA overexpression. Based on this transcriptome information, the supplement of thiamine and protocatechuic acid was found to further increase decaprenoxanthin production under sigA overexpression. In addition, the effect of sigA overexpression was successfully transferred to other carotenoid producing strains. In this study, it was proven that overexpression of sigma factor genes can perturb the transcriptome profile of C. glutamicum by artificially activating the native metabolic pathway and change cell physiology under normal conditions. Artificial activation of sigma factors was helpful to understand regulatory networks in bacteria by connecting genes at different loci with similar promoter sequences. In addition, overexpression of sigma factor and the following screening of the potent sigma factor gene for a specific purpose was shown to be an effective approach for strain development. Therefore, overexpression of genes encoding sigma factors is not only relevant to study the gene regulatory networks, but also effective in strain development of C. glutamicum with respect to production of value-added chemicals.