Transformation of the cyanobacterium Synechocystis sp. PCC 6803 as a tool for genetic mapping: optimization of efficiency

Kufryk, Galyna

doi:10.1016/s0378-1097(01)00540-7

Cited by 25 publications

(34 citation statements)

References 17 publications

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“…However, before a conclusion can be drawn about the generality of this observation, more of these mutants should be analyzed. The overall genetic stability of Synechocystis is limited primarily by the frequency of occurrence of point mutations (20). Whether or not the impairment of the expression of the functional transhydrogenase in SAA016 is also dependent on this mechanism or in contrast de- pends on other molecular mechanisms of spontaneous mutagenesis will require more extensive screening of revertants of the type identified in this study.…”

Section: Resultsmentioning

confidence: 89%

Engineering a Cyanobacterial Cell Factory for Production of Lactic Acid

Angermayr

Paszota

Hellingwerf

2012

Appl Environ Microbiol

165

144

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Metabolic engineering of microorganisms has become a versatile tool to facilitate production of bulk chemicals, fuels, etc. Accordingly, CO 2 has been exploited via cyanobacterial metabolism as a sustainable carbon source of biofuel and bioplastic precursors. Here we extended these observations by showing that integration of an ldh gene from Bacillus subtilis (encoding an L-lactate dehydrogenase) into the genome of Synechocystis sp. strain PCC6803 leads to L-lactic acid production, a phenotype which is shown to be stable for prolonged batch culturing. Coexpression of a heterologous soluble transhydrogenase leads to an even higher lactate production rate and yield (lactic acid accumulating up to a several-millimolar concentration in the extracellular medium) than those for the single ldh mutant. The expression of a transhydrogenase alone, however, appears to be harmful to the cells, and a mutant carrying such a gene is rapidly outcompeted by a revertant(s) with a wild-type growth phenotype. Furthermore, our results indicate that the introduction of a lactate dehydrogenase rescues this phenotype by preventing the reversion.C oncerns about shrinking supplies of fossil fuel and about climate change have been a strong incentive during the past decade for the development of sustainable alternatives to provide fuel and chemical feedstock. Because of these concerns, several different ways to produce solar biofuel have been proposed. Initially the proposed procedures were based on the conversion of agricultural crops into short-chain alcohols. This unfortunately results in a direct competition for resources between the energy sector and food supply. Therefore, second-generation types of processes were proposed, in which one aims at the conversion of the triglyceride fraction from, e.g., green algae into biodiesel, which relaxes the competition between energy and the food supply. Nevertheless, the conversion of CO 2 into biofuel, driven by solar energy, is still rather indirect even in this second-generation approach: CO 2 is first converted into the complex building blocks of biomass, from which subsequently the triglyceride fraction must be extracted and converted into biodiesel.For these reasons, a third-generation type of process has been proposed, in which CO 2 is converted into biofuel directly, accomplished by the introduction of genes encoding a metabolic-e.g., fermentative-pathway into a cyanobacterium, resulting in the emergence of a "photofermentative" chimera. For several biofuel and chemical feedstock products, including ethanol, ethylene, isoprene, D-lactic acid, glucose, sucrose, isobutyraldehyde, and 1-butanol, the proof of principle of this approach has been provided (see references 3,8,10,24,25,28, and 39 and references therein). Ideally, this process would be carried out in such a way that the majority of the CO 2 that is fixed in the cyanobacterial CalvinBenson cycle is converted into the selected product. This implies that the central metabolism in the selected chimera should be largely redirected tow...

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Section: Resultsmentioning

confidence: 89%

Engineering a Cyanobacterial Cell Factory for Production of Lactic Acid

Angermayr

Paszota

Hellingwerf

2012

Appl Environ Microbiol

165

144

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“…Transformation of Synechocystis sp. strain PCC 6803 cells with the vector pFM02 was performed as described previously (21). The selection of mutants was carried out in plates initially supplemented with 25 g/l kanamycin.…”

Section: Methodsmentioning

confidence: 99%

An AbrB-Like Protein Regulates the Expression of the Bidirectional Hydrogenase in Synechocystis sp. Strain PCC 6803

Oliveira

Lindblad

2008

J Bacteriol

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In the unicellular cyanobacterium Synechocystis sp. strain PCC 6803, the pentameric bidirectional Ni-Fe hydrogenase (HoxEFUYH) is the sole enzyme involved in hydrogen metabolism. Recent investigations implicated the transcription factor LexA in the regulation of the hox genes in this cyanobacterium, suggesting the factor to work as an activator. In this work, we show evidence that LexA cannot account exclusively for the regulation of the hox genes in this cyanobacterium. Therefore, we investigated which additional transcription factors interact in and may regulate the expression of the hox genes in Synechocystis sp. strain PCC 6803. By using DNA affinity assays, a transcription factor with similarity to the transition state regulator AbrB from Bacillus subtilis was isolated. Electrophoretic mobility shift assays showed that the AbrB-like protein specifically interacts with the promoter region of the hox genes as well as with its own promoter region. In addition, results obtained with two genetically modified strains of Synechocystis sp. strain PCC 6803, one with a not fully segregated inactivation mutation of the abrB-like gene and the other overexpressing the same abrB-like gene, suggest that this transcription factor functions as a regulator of hox gene expression.Two different Ni-Fe hydrogenases have been characterized physiologically, biochemically, and on the molecular level in cyanobacteria: one is named uptake hydrogenase and catalyzes the consumption of H 2 produced by the nitrogenase during N 2 fixation, and the other is termed bidirectional hydrogenase and its function is still under debate (32,37,38).Over the years, it has continuously been shown by reverse transcription-PCR and Northern blot analysis (2,3,4,15,23,27,33,38) that the transcription of cyanobacterial hydrogenases varies under different environmental conditions. Nevertheless, an understanding of which key regulators are involved is just emerging and the inherent signal transduction pathways certainly deserve better attention. Therefore, it is imperative to study the mechanisms controlling the expression of both cyanobacterial hydrogenases more closely in order to acquire an improved comprehension of their functions and also to develop novel tools for enhancing a sustainable and reliable production of H 2 from cyanobacteria (38).Recently, the first reports on the transcription factors involved in the regulation of cyanobacterial hydrogenases became available. NtcA, the nitrogen control regulator in cyanobacteria, has been suggested to mediate the transcription of the uptake hydrogenase in different cyanobacterial strains, Nostoc punctiforme PCC 73102 (24), Gloeothece sp. strain ATCC 27152 (27), and Lyngbya majuscula CCAP 1446/4 (23), as well as that of the hydrogenase maturation proteins in Lyngbya majuscula CCAP 1446/4 (15). A LexA-related protein, which has been proposed to not be involved in the classical regulation of DNA repair genes (11), was shown to interact in two different regions of the promoter of the hox genes in Synechocystis...

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“…Size fractionation and isolation of the restriction fragments were performed as previously described (20). The transformation procedure was optimized (19) to maximize the number of transformants tolerant of low light intensity. Transformant colonies appeared after 16 to 21 days upon growth at 2 mol photons m Ϫ2 s Ϫ1 and at a temperature of 30°C.…”

Section: Methodsmentioning

confidence: 99%

Sll1717 Affects the Redox State of the Plastoquinone Pool by Modulating Quinol Oxidase Activity in Thylakoids

Kufryk

Vermaas

2006

J Bacteriol

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A Synechocystis sp. strain PCC 6803 mutant lacking CtaI, a main subunit of cytochrome c oxidase, is not capable of growing at light intensities below 5 mol photons m ؊2 s ؊1 , presumably due to an overreduced plastoquinone pool in the thylakoid membrane. Upon selection for growth at light intensities below 5 mol photons m ؊2 s ؊1 , a secondary mutant was generated that retained the CtaI deletion and had fully assembled photosystem II complexes; in this secondary mutant (pseudorevertant), oxygen evolution and respiratory activities were similar to those in the wild type. Functional complementation of the original CtaI-less strain to low-light tolerance by transformation with restriction fragments of genomic DNA of the pseudorevertant and subsequent mapping of the pseudoreversion site showed that the point mutation led to a Ser186Cys substitution in Sll1717, a protein of as-yet-unknown function and with a predicted ATP/GTP-binding domain. This mutation caused a decrease in the plastoquinone pool reduction level of thylakoids compared to that observed for the wild type. Based on a variety of experimental evidence, the most plausible mechanism to cause this effect is an activation of plastoquinol oxidation in thylakoids by the quinol oxidase CydAB that occurs without upregulation of the corresponding gene and that may be caused by an increased CydAB activity in thylakoids, conceivably due to altered CydAB sorting between cytoplasmic and thylakoid membranes. Sll1717 appears to be unique to Synechocystis sp. strain PCC 6803 and has a close homologue encoded in the genome of this organism. The transcript level of sll1717 is low, which suggests that the corresponding protein is regulatory rather than structural.

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Transformation of the cyanobacterium Synechocystis sp. PCC 6803 as a tool for genetic mapping: optimization of efficiency

Cited by 25 publications

References 17 publications

Engineering a Cyanobacterial Cell Factory for Production of Lactic Acid

Engineering a Cyanobacterial Cell Factory for Production of Lactic Acid

An AbrB-Like Protein Regulates the Expression of the Bidirectional Hydrogenase in Synechocystis sp. Strain PCC 6803

Sll1717 Affects the Redox State of the Plastoquinone Pool by Modulating Quinol Oxidase Activity in Thylakoids

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