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Transcription analysis of central metabolism genes in Escherichia coli. Possible roles of sigma38 in their expression, as a response to carbon limitation.

ABSTRACT: The phosphoenolpyruvate: carbohydrate transferase system (PTS) transports glucose in Escherichia coli. Previous work demonstrated that strains lacking PTS, such as PB11, grow slow on glucose. PB11 has a reduced expression of glycolytic, and upregulates poxB and acs genes as compared to the parental strain JM101, when growing on glucose. The products of the latter genes are involved in the production of AcetylCoA. Inactivation of rpoS that codes for the RNA polymerase sigma(38) subunit, reduces further (50%) growth of PB11, indicating that sigma(38) plays a central role in the expression of central metabolism genes in slowly growing cells. In fact, transcription levels of glycolytic genes is reduced in strain PB11rpoS(-) as compared to PB11. In this report we studied the role of sigma(70) and sigma(38) in the expression of the complete glycolytic pathway and poxB and acs genes in certain PTS(-) strains and their rpoS(-) derivatives. We determined the transcription start sites (TSSs) and the corresponding promoters, in strains JM101, PB11, its derivative PB12 that recovered its growth capacity, and in their rpoS(-) derivatives, by 5'RACE and pyrosequencing. In all these genes the presence of sequences resembling sigma(38) recognition sites allowed the proposition that they could be transcribed by both sigma factors, from overlapping putative promoters that initiate transcription at the same site. Fourteen new TSSs were identified in seventeen genes. Besides, more than 30 putative promoters were proposed and we confirmed ten previously reported. In vitro transcription experiments support the functionality of putative dual promoters. Alternatives that could also explain lower transcription levels of the rpoS(-) derivatives are discussed. We propose that the presence if real, of both sigma(70) and sigma(38) dependent promoters in all glycolytic genes and operons could allow a differential transcription of these central metabolism genes by both sigma subunits as an adaptation response to carbon limitation.

SUBMITTER: Olvera L

PROVIDER: S-EPMC2759082 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Transcription analysis of central metabolism genes in Escherichia coli. Possible roles of sigma38 in their expression, as a response to carbon limitation.

Olvera Leticia L Mendoza-Vargas Alfredo A Flores Noemí N Olvera Maricela M Sigala Juan Carlos JC Gosset Guillermo G Morett Enrique E Bolívar Francisco F

PloS one 20091019 10

The phosphoenolpyruvate: carbohydrate transferase system (PTS) transports glucose in Escherichia coli. Previous work demonstrated that strains lacking PTS, such as PB11, grow slow on glucose. PB11 has a reduced expression of glycolytic, and upregulates poxB and acs genes as compared to the parental strain JM101, when growing on glucose. The products of the latter genes are involved in the production of AcetylCoA. Inactivation of rpoS that codes for the RNA polymerase sigma(38) subunit, reduces f ...[more]

PMID: 19838295

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Project description:Beta (?)-carotene (C40H56; a provitamin) is a particularly important carotenoid for human health. Many studies have focused on engineering Escherichia coli as an efficient heterologous producer of ?-carotene. Moreover, several strains with potential for use in the industrial production of this provitamin have already been constructed via different metabolic engineering strategies. In this study, we aimed to improve the ?-carotene-producing capacity of our previously engineered E. coli strain ZF43?gdhA through further gene deletion and metabolic pathway manipulations. Deletion of the zwf gene increased the resultant strain's ?-carotene production and content by 5.1 and 32.5%, respectively, relative to the values of strain ZF43?gdhA, but decreased the biomass by 26.2%. Deletion of the ptsHIcrr operon further increased the ?-carotene production titer from 122.0 to 197.4 mg/L, but the provitamin content was decreased. Subsequently, comparative transcriptomic analysis was used to explore the dynamic transcriptional responses of the strains to the blockade of the pentose phosphate pathway and inactivation of the phosphotransferase system. Lastly, based on the analyses of comparative transcriptome and reduction cofactor, several strategies to increase the NADPH supply were evaluated for enhancement of the ?-carotene content. The combination of yjgB gene deletion and nadK overexpression led to increased ?-carotene production and content. The best strain, ECW4/p5C-nadK, produced 266.4 mg/L of ?-carotene in flask culture and 2,579.1 mg/L in a 5-L bioreactor. The latter value is the highest reported from production via the methylerythritol phosphate pathway in E. coli. Although the strategies applied is routine in this study, the combinations reported were first implemented, are simple but efficient and will be helpful for the production of many other natural products, especially isoprenoids. Importantly, we demonstrated that the use of the methylerythritol phosphate pathway alone for efficient ?-carotene biosynthesis could be achieved via appropriate modifications of the cell metabolic functions.

Dataset Information

Transcription analysis of central metabolism genes in Escherichia coli. Possible roles of sigma38 in their expression, as a response to carbon limitation.

Publications

Transcription analysis of central metabolism genes in Escherichia coli. Possible roles of sigma38 in their expression, as a response to carbon limitation.

Similar Datasets

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