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Genetic engineering of group 2 sigma factor SigE widely activates expressions of sugar catabolic genes in Synechocystis species PCC 6803.

ABSTRACT: Metabolic engineering of photosynthetic organisms is required for utilization of light energy and for reducing carbon emissions.Control of transcriptional regulators is a powerful approach for changing cellular dynamics, because a set of genes is concomitantly regulated. Here, we show that overexpression of a group 2 ? factor, SigE, enhances the expressions of sugar catabolic genes in the unicellular cyanobacterium, Synechocystis sp. PCC 6803. Transcriptome analysis revealed that genes for the oxidative pentose phosphate pathway and glycogen catabolism are induced by overproduction of SigE. Immunoblotting showed that protein levels of sugar catabolic enzymes, such as glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glycogen phosphorylase, and isoamylase, are increased. Glycogen levels are reduced in the SigE-overexpressing strain grown under light. Metabolome analysis revealed that metabolite levels of the TCA cycle and acetyl-CoA are significantly altered by SigE overexpression. The SigE-overexpressing strain also exhibited defective growth under mixotrophic or dark conditions. Thus, SigE overexpression changes sugar catabolism at the transcript to phenotype levels, suggesting a ? factor-based engineering method for modifying carbon metabolism in photosynthetic bacteria.

SUBMITTER: Osanai T

PROVIDER: S-EPMC3162455 | biostudies-other | 2011 Sep

REPOSITORIES: biostudies-other

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Genetic engineering of group 2 sigma factor SigE widely activates expressions of sugar catabolic genes in Synechocystis species PCC 6803.

Osanai Takashi T Oikawa Akira A Azuma Miyuki M Tanaka Kan K Saito Kazuki K Hirai Masami Yokota MY Ikeuchi Masahiko M

The Journal of biological chemistry 20110711 35

Metabolic engineering of photosynthetic organisms is required for utilization of light energy and for reducing carbon emissions.Control of transcriptional regulators is a powerful approach for changing cellular dynamics, because a set of genes is concomitantly regulated. Here, we show that overexpression of a group 2 σ factor, SigE, enhances the expressions of sugar catabolic genes in the unicellular cyanobacterium, Synechocystis sp. PCC 6803. Transcriptome analysis revealed that genes for the o ...[more]

PMID: 21757761

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Project description:In recent years, there has been an increased interest in the research and development of sustainable alternatives to fossil fuels. Using photosynthetic microorganisms to produce such alternatives is advantageous, since they can achieve direct conversion of carbon dioxide from the atmosphere into the desired product, using sunlight as the energy source. Squalene is a naturally occurring 30-carbon isoprenoid, which has commercial use in cosmetics and in vaccines. If it could be produced sustainably on a large scale, it could also be used instead of petroleum as a raw material for fuels and as feedstock for the chemical industry. The unicellular cyanobacterium Synechocystis PCC 6803 possesses a gene, slr2089, predicted to encode squalene hopene cyclase (Shc), an enzyme converting squalene into hopene, the substrate for forming hopanoids. Through inactivation of slr2089 (shc), we explored the possibility to produce squalene using cyanobacteria. The inactivation led to accumulation of squalene, to a level over 70 times higher than in wild type cells, reaching 0.67 mg OD750(-1) L(-1). We did not observe any significant growth deficiency in the Δshc strain compared to the wild type Synechocystis, even at high light conditions, suggesting that the observed squalene accumulation was not detrimental to growth, and that formation of hopene by Shc is not crucial for growth under normal conditions, nor for high-light stress tolerance. Effects of different light intensities and growth stages on squalene accumulation in the Δshc strain were investigated. We also identified a gene, sll0513, as a putative squalene synthase in Synechocystis, and verified its function by inactivation. In this work, we show that it is possible to use the cyanobacterium Synechocystis to generate squalene, a hydrocarbon of commercial interest and a potential biofuel. We also report the first identification of a squalene hopene cyclase, and the second identification of squalene synthase, in cyanobacteria.

Project description:BackgroundMetabolic engineering and synthetic biology of cyanobacteria offer a promising sustainable alternative approach for fossil-based ethylene production, by using sunlight via oxygenic photosynthesis, to convert carbon dioxide directly into ethylene. Towards this, both well-studied cyanobacteria, i.e., Synechocystis sp PCC 6803 and Synechococcus elongatus PCC 7942, have been engineered to produce ethylene by introducing the ethylene-forming enzyme (Efe) from Pseudomonas syringae pv. phaseolicola PK2 (the Kudzu strain), which catalyzes the conversion of the ubiquitous tricarboxylic acid cycle intermediate 2-oxoglutarate into ethylene.ResultsThis study focuses on Synechocystis sp PCC 6803 and shows stable ethylene production through the integration of a codon-optimized version of the efe gene under control of the Ptrc promoter and the core Shine-Dalgarno sequence (5'-AGGAGG-3') as the ribosome-binding site (RBS), at the slr0168 neutral site. We have increased ethylene production twofold by RBS screening and further investigated improving ethylene production from a single gene copy of efe, using multiple tandem promoters and by putting our best construct on an RSF1010-based broad-host-self-replicating plasmid, which has a higher copy number than the genome. Moreover, to raise the intracellular amounts of the key Efe substrate, 2-oxoglutarate, from which ethylene is formed, we constructed a glycogen-synthesis knockout mutant (ΔglgC) and introduced the ethylene biosynthetic pathway in it. Under nitrogen limiting conditions, the glycogen knockout strain has increased intracellular 2-oxoglutarate levels; however, surprisingly, ethylene production was lower in this strain than in the wild-type background.ConclusionMaking use of different RBS sequences, production of ethylene ranging over a 20-fold difference has been achieved. However, a further increase of production through multiple tandem promoters and a broad-host plasmid was not achieved speculating that the transcription strength and the gene copy number are not the limiting factors in our system.

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Genetic engineering of group 2 sigma factor SigE widely activates expressions of sugar catabolic genes in Synechocystis species PCC 6803.

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Genetic engineering of group 2 sigma factor SigE widely activates expressions of sugar catabolic genes in Synechocystis species PCC 6803.

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