Takashi Osanai

Genetic Engineering of Group 2 σ Factor SigE Widely Activates Expressions of Sugar Catabolic Genes in Synechocystis Species PCC 6803

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 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 σ factor-based engineering method for modifying carbon metabolism in photosynthetic bacteria.

Increased Bioplastic Production with an RNA Polymerase Sigma Factor SigE during Nitrogen Starvation in Synechocystis sp. PCC 6803

Because cyanobacteria directly harvest CO2 and light energy, their carbon metabolism is important for both basic and applied sciences. Here, we show that overexpression of the sigma factor sigE in Synechocystis sp. PCC 6803 widely changes sugar catabolism and increases production of the biodegradable polyester polyhydroxybutyrate (PHB) during nitrogen starvation. sigE overexpression elevates the levels of proteins implicated in glycogen catabolism, the oxidative pentose phosphate pathway, and polyhydroxyalkanoate biosynthesis. PHB accumulation is enhanced by sigE overexpression under nitrogen-limited conditions, yet the molecular weights of PHBs synthesized by the parental glucose-tolerant and sigE overexpression strain are similar. Although gene expression induced by nitrogen starvation is changed and other metabolites (such as GDP-mannose and citrate) accumulate under sigE overexpression, genetic engineering of this sigma factor altered the metabolic pathway from glycogen to PHB during nitrogen starvation.

ChlH, the H subunit of the Mg-chelatase, is an anti-sigma factor for SigE in Synechocystis sp. PCC 6803

Although regulation of sigma factors has been intensively investigated, anti-sigma factors have not been identified in oxygenic photosynthetic organisms. A previous study suggested that the sigma factor, SigE, of the cyanobacterium Synechocystis sp. PCC 6803, a positive regulator of sugar catabolism, is posttranslationally activated by light-to-dark transition. In the present study, we found that the H subunit of Mg-chelatase ChlH interacts with sigma factor SigE by yeast two-hybrid screening, and immunoprecipitation analysis revealed that ChlH associates with SigE in a light-dependent manner in vivo. We also found that Mg2+ promotes the interaction of SigE and ChlH and determines their localization in vitro. In vitro transcription analysis demonstrated that ChlH inhibits the transcription activity of SigE. Based on these results, we propose a model in which ChlH functions as an anti-sigma factor, transducing light signals to SigE in a process mediated by Mg2+.

Capillary electrophoresis–mass spectrometry reveals the distribution of carbon metabolites during nitrogen starvation in Synechocystis sp. PCC 6803

Nitrogen availability is one of the most important factors for the survival of cyanobacteria. Previous studies on Synechocystis revealed a contradictory situation with regard to metabolism during nitrogen starvation; that is, glycogen accumulated even though the expressions of sugar catabolic genes were widely upregulated. Here, we conducted transcript and metabolomic analyses using capillary electrophoresis-mass spectrometry on Synechocystis sp. PCC 6803 under nitrogen starvation. The levels of some tricarboxylic acid cycle intermediates (succinate, malate and fumarate) were greatly increased by nitrogen deprivation. Purine and pyrimidine nucleotides were markedly downregulated under nitrogen depletion. The levels of 19 amino acids changed under nitrogen deprivation, especially those of amino acids synthesized from pyruvate and phosphoenolpyruvate, which showed marked increases. Liquid chromatography-mass spectrometry analysis demonstrated that the amount of NADPH and the NADPH/NADH ratio decreased under nitrogen depletion. These data demonstrate that there are increases in not only glycogen but also in metabolites downstream of sugar catabolism in Synechocystis sp. PCC 6803 under nitrogen starvation, resolving the contradiction between glycogen accumulation and induction of sugar catabolic gene expression in this unicellular cyanobacterium.

A response regulator Rre37 and an RNA polymerase sigma factor SigE represent two parallel pathways to activate sugar catabolism in a cyanobacterium Synechocystis sp. PCC 6803

Sugar catabolic genes are induced during nitrogen starvation in a cyanobacterium Synechocystis sp. PCC 6803, but the underlying regulatory mechanism still remains to be completely characterized. In this study, we showed by molecular genetics and transcriptome analyses that a response regulator Rre37 (encoded by sll1330), whose expression is enhanced by nitrogen depletion under the control of NtcA, activates transcript accumulation of sugar catabolic genes, such as gap1, pfkA (sll1196), glgP (slr1367) and glgX (slr1857), mainly during nitrogen starvation. Previously, we reported that a group-2 sigma factor SigE also positively regulates sugar catabolic genes in this strain. Phenotypic analyses using a single or double mutant lacking rre37 and/or sigE indicated that both SigE and Rre37 positively regulate sugar catabolic genes independently. These findings substantiated a regulatory network of sugar catabolic genes in this cyanobacterium.

SigC, the Group 2 Sigma Factor of RNA Polymerase, Contributes to the Late-stage Gene Expression and Nitrogen Promoter Recognition in the Cyanobacterium Synechocystis sp. Strain PCC 6803

We examined the role of SigC (Sll0184), a sigma factor of RNA polymerase (RNAP), in a unicellular cyanobacterium, Synechocystis sp. strain PCC 6803. On the inactivation of sigC, which is an Escherichia coli rpoD homolog, cells were viable but had a low survival rate in the stationary phase of growth under normal physiological conditions, indicating that SigC is a group 2 type sigma factor. In analyses of transcript and protein levels using the sigC knockout strain, it was found that expression of glnB, a nitrogen key regulatory gene, is controlled by SigC in the stationary phase. Primer extension revealed that the glnB nitrogen promoter (P2) was specifically recognized by SigC in the stationary phase under conditions of nitrogen starvation. In vitro studies with purified enzymes indicated effective transcription, on supercoiled DNA templates, from P2 by SigC-RNAP with NtcA which is an activator for nitrogen gene transcription. DNase I footprinting also indicated binding and related sites of NtcA and/or RNAP with SigC on the nitrogen promoter. The unique promoter architecture and the mechanism of transcription by RNAP with SigC are also discussed.

Group 2 sigma factors in cyanobacteria

Group 1 and group 2 sigma factors are sigma factors of bacterial RNA polymerase responsible for transcription from consensus-type promoters. Thus, these sigma factors form the framework for basic transcriptional regulation in bacteria. Cyanobacteria are known to have various group 2 sigma factors, typically more than 4, but only recently the particular function of each sigma factor is being elucidated. In response to environmental signals such as nutrients, light and temperature, cyanobacteria change their transcriptional profile first by activating specific transcription factors and subsequently by modifying the basic transcriptional machinery, which is often involved in the regulation of group 2 sigma factors. In this article, we give an overview of the composition and evolution of group 2 sigma factors in cyanobacteria and summarize what was presently revealed regarding their function.

Pathway-Level Acceleration of Glycogen Catabolism by a Response Regulator in the Cyanobacterium Synechocystis Species PCC 6803

Overexpressing a response regulator accelerates glycogen degradation and polyhydroxylbutyrate biosynthesis, revealing pathway-level control of primary metabolism in a unicellular cyanobacterium. Response regulators of two-component systems play pivotal roles in the transcriptional regulation of responses to environmental signals in bacteria. Rre37, an OmpR-type response regulator, is induced by nitrogen depletion in the unicellular cyanobacterium Synechocystis species PCC 6803. Microarray and quantitative real-time polymerase chain reaction analyses revealed that genes related to sugar catabolism and nitrogen metabolism were up-regulated by rre37 overexpression. Protein levels of GlgP(slr1367), one of the two glycogen phosphorylases, in the rre37-overexpressing strain were higher than those of the parental wild-type strain under both nitrogen-replete and nitrogen-depleted conditions. Glycogen amounts decreased to less than one-tenth by rre37 overexpression under nitrogen-replete conditions. Metabolome analysis revealed that metabolites of the sugar catabolic pathway and amino acids were altered in the rre37-overexpressing strain after nitrogen depletion. These results demonstrate that Rre37 is a pathway-level regulator that activates the metabolic flow from glycogen to polyhydroxybutyrate and the hybrid tricarboxylic acid and ornithine cycle, unraveling the mechanism of the transcriptional regulation of primary metabolism in this unicellular cyanobacterium.

Pleiotropic effect of sigE over‐expression on cell morphology, photosynthesis and hydrogen production in Synechocystis sp. PCC 6803

Over-expression of sigE, a gene encoding an RNA polymerase sigma factor in the unicellular cyanobacterium Synechocystis sp. PCC 6803, is known to activate sugar catabolism and bioplastic production. In this study, we investigated the effects of sigE over-expression on cell morphology, photosynthesis and hydrogen production in this cyanobacterium. Transmission electron and scanning probe microscopic analyses revealed that sigE over-expression increased the cell size, possibly as a result of aberrant cell division. Over-expression of sigE reduced respiration and photosynthesis activities via changes in gene expression and chlorophyll fluorescence. Hydrogen production under micro-oxic conditions is enhanced in sigE over-expressing cells. Despite these pleiotropic phenotypes, the sigE over-expressing strain showed normal cell viability under both nitrogen-replete and nitrogen-depleted conditions. These results provide insights into the inter-relationship among metabolism, cell morphology, photosynthesis and hydrogen production in this unicellular cyanobacterium.

Genetic manipulation of a metabolic enzyme and a transcriptional regulator increasing succinate excretion from unicellular cyanobacterium

Succinate is a building block compound that the U.S. Department of Energy (DOE) has declared as important in biorefineries, and it is widely used as a commodity chemical. Here, we identified the two genes increasing succinate production of the unicellular cyanobacterium Synechocystis sp. PCC 6803. Succinate was excreted under dark, anaerobic conditions, and its production level increased by knocking out ackA, which encodes an acetate kinase, and by overexpressing sigE, which encodes an RNA polymerase sigma factor. Glycogen catabolism and organic acid biosynthesis were enhanced in the mutant lacking ackA and overexpressing sigE, leading to an increase in succinate production reaching five times of the wild-type levels. Our genetic and metabolomic analyses thus demonstrated the effect of genetic manipulation of a metabolic enzyme and a transcriptional regulator on succinate excretion from this cyanobacterium with the data based on metabolomic technique.