Harnessing Sigma Factor Gene Overexpression for Production By Corynebacterium Glutamicum
Sigma factor is one of the component of RNA polymerase holoenzyme and responsible for promoter recognition and transcription initiation. Bacteria usually have multiple genes of sigma factor and strictly control their activities at the transcription, translation and post-translation level. Activation of a specific sigma factor alters the promoter recognition of RNA polymerase holoenzyme and enhances the transcription of a specific group of genes with the similar promoter sequence. Sigma factor can be a good candidate to activate a set of genes at the same time and may be helpful for engineering bacterium.
In this study, the potential of sigma factor gene expression for production 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 acid, especially glutamate and lysine. Because of its genetic amenability, various strains has been developed to produce industrial relevant compounds using metabolic engineering. C. glutamicum possesses 7 sigma factor genes, sigA, sigB, sigC, sigD, sigE, sigH and sigM.
Each of these sigma factors was overexpressed in two different background strains, the wild-type strain (WT) and the recombinant strain from lycopene production (Lyc5). Sigma factor genes were overexpressed in an IPTG-inducible system with different IPTG concentrations. Overexpression of each sigma factor gene influenced the growth rate differently and the effect was dependent on the IPTG concentration, thus, on expression strength. The strongest effect was observed for overexpression of sigD and sigH, respectively.
Notably, sigD overexpression in C. glutamicum WT also reduced foaming possibly due to secretion of anti-foaming compounds. Transcriptome analysis revealed that genes related to the cell wall integrity such as mycolate synthesis showed increased RNA levels as consequence of sigD overexpression.
Overexpression of sigH in C. glutamicum WT resulted in a phenotypic change, i.e. accumulation of a yellow compound in the supernatant, which was identified as riboflavin by HPLC analysis. Transcriptome analysis revealed increased expression of genes of riboflavin biosynthesis, the pentose phosphate pathway and of enzymes requiring FAD, FMN or NADPH as cofactor (Taniguchi and Wendisch, 2015). Since riboflavin is a precursor of FMN, sigH overexpression was harnessed as strategy for production of FMN. Balanced expression of 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).
Overexpression of sigA in the lycopene producing strain Lyc5 increased lycopene production. In addition, sigA overexpression in WT also increased biosynthesis and accumulation of the endogenous C50 carotenoid decaprenoxanthin. As consequence of sigA overexpression accumulation of lycopene and decaprenoxanthin continued into the stationary phase.
In this study, we showed that sigma factor gene overexpression is not only relevant to study the gene regulatory network, but pertains to C. glutamicum strain development with respect to production of value-added chemicals.
Taniguchi H & Wendisch VF (2015) Exploring the role of sigma factor gene expression on production by Corynebacterium glutamicum: sigma factor H and FMN as example. Front Microbiol 6: 740