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Overexpression of the primary sigma factor gene sigA improved carotenoid production by Corynebacterium glutamicum: application to production of β-carotene and the non-native linear C50 carotenoid bisanhydrobacterioruberin

ABSTRACT: Corynebacterium glutamicum shows yellow pigmentation due to biosynthesis of the C50 carotenoid decaprenoxanthin and its glycosides. This bacterium has been engineered for production of various non-native cyclic C40 and C50 carotenoids such as β-carotene, astaxanthin or sarcinaxanthin. In this study, the effect of modulating gene expression more broadly by overexpression of sigma factor genes on carotenoid production by C. glutamicum was characterized. Overexpression of the primary sigma factor gene sigA improved lycopene production by recombinant C. glutamicum up to eight fold. In C. glutamicum wild type, overexpression of sigA led to two fold increased accumulation of the native carotenoid decaprenoxanthin in the stationary growth phase. Under these conditions, genes related to thiamine synthesis and aromatic compound degradation showed increased RNA levels and addition of thiamine and the aromatic iron chelator protocatechuic acid to the culture medium enhanced carotenoid production when sigA was overexpressed. Deletion of the gene for the alternative sigma factor SigB, which is expected to replace SigA in RNA polymerase holoenzymes during transition to the stationary growth phase, also increased carotenoid production. The strategy of sigA overexpression could be successfully transferred to production of the non-native carotenoids β-carotene and bisanhydrobacterioruberin. Production of the latter is the first demonstration that C. glutamicum may accumulate a non-native linear C50 carotenoid instead of the native cyclic C50 carotenoid decaprenoxanthin.

ORGANISM(S): Corynebacterium glutamicum Corynebacterium glutamicum ATCC 13032

PROVIDER: GSE86866 | GEO | 2016/09/14

SECONDARY ACCESSION(S): PRJNA342810

REPOSITORIES: GEO

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Project description:To obtain insight in the genome-wide response of heterologous carotenoid production in Saccharomyces cerevisiae, we have analyzed the transcriptome of S. cerevisiae strains overexpressing carotenogenic genes from the yeast Xanthophyllomyces dendrorhous. For this purpose, two strains producing different levels of carotenoids were grown in carbon-limited continuous cultures and genome-wide expression was analyzed. The strain producing low carotenoid levels did not exhibit a clear genome-wide transcriptional response, suggesting that low carotenoid levels do not result in cellular stress. Transcriptome analysis of a strain producing high carotenoid levels resulted in specific induction of genes involved in pleiotropic drug resistance (PDR). These genes encode ATP-binding cassette (ABC) type transporters and major facilitator transporters which are involved in secretion of toxic compounds out of cells. Our results suggest that production of high amounts of carotenoids in S. cerevisiae lead to toxicity and that these cells are prone to secrete carotenoids out of the cell. Indeed, secretion of ï¢-carotene into sunflower oil was observed upon addition of this hydrophobic solvent to the growth medium. Finally, it was observed that deletion of the ABC transporter pdr10, one of the induced PDR transporters, highly decreased the transformation efficiency of an episomal vector containing carotenogenic genes. The few colored transformants that were obtained had decreased growth rates and lower carotenoid production levels compared to control strains transformed with the same carotenogenic genes. These results indicate that Pdr10 might be specifically involved in carotenoid tolerance in S. cerevisiae strains. Experiment Overall Design: The genome wide transcriptional response of S. cerevisiae cells that heterologously produce carotenoids might provide information concerning the impact of carotenoid production on yeast physiology and might identify bottlenecks relevant for the production of these compounds. DNA microarray experiments have been proven to be a powerful tool to study the genome wide transcriptional response of S. cerevisiae to changes of the physiological state and the environment (for example 3,. Genomics approaches on cells producing heterologous metabolites to study their impact on yeast physiology have not been reported yet for S. cerevisiae. Additionally, most transcriptome studies with S. cerevisiae have been performed with cells grown in shake flasks cultures. The main drawback of shake flask cultivation is that the environment is continuously changing, which may be of high influence on carotenoid production, and interpretation of transcriptome data . Chemostat cultivation offers advantages for studies with DNA microarrays because it enables cultivation of microorganisms under tightly defined environmental conditions. An interlaboratory comparison of transcriptome data obtained in chemostat cultures has indeed demonstrated that the accuracy and reproducibility of this approach are superior to those obtained in previous studies with shake-flask cultures .

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Overexpression of the primary sigma factor gene sigA improved carotenoid production by Corynebacterium glutamicum: application to production of β-carotene and the non-native linear C50 carotenoid bisanhydrobacterioruberin

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