Genetic control of hydrogen metabolism in cyanobacteria

The development of methods for the use of phototrophic cyanobacteria as producers of molecular hydrogen via bioconversion of solar energy is a promising filed of hydrogen energetics. Optimization of hydrogen formation and release is based on studying the genetic control of hydrogen metabolism and the use of genetic approaches for obtaining efficient producer strains. Data on genes coding for the hydrogenases that are responsible for hydrogen uptake and production in cyanobacteria are summarized. Bioinformatic methods have been used to construct the scheme of the hydrogen metabolism gene network of nitrogen-fixing heterocystous cyanobacteria. The possible approaches to constructing the cyanobacterium strains producing molecular hydrogen that would be promising for photobiotechnology by mutagenesis and genetic engineering methods are discussed in terms of this model and analysis of the data on hydrogen-producing mutants.     

Progress and perspective on cyanobacterial glycogen metabolism engineering

As important oxygenic photoautotrophs, cyanobacteria are also generally considered as one of the most promising microbial chassis for photosynthetic biomanufacturing. Diverse synthetic biology and metabolic engineering approaches have been developed to enable the efficient harnessing of carbon and energy flow toward the synthesis of desired metabolites in cyanobacterial cell factories. Glycogen metabolism works as the most important natural carbon sink mechanism and reserve carbon source, storing a large portion of carbon and energy from the Calvin-Benson-Bassham (CBB) cycle, and thus is traditionally recognized as a promising engineering target to optimize the efficacy of cyanobacterial cell factories. Multiple strategies and approaches have been designed and adopted to engineer glycogen metabolism in cyanobacteria, leading to the successful regulation of glycogen synthesis and storage contents in cyanobacteria cells. However, disturbed glycogen metabolism results in weakened cellular physiological functionalities, thereby diminishing the robustness of metabolism. In addition, the effects of glycogen removal as a metabolic engineering strategy to enhance photosynthetic biosynthesis are still controversial. This review focuses on the efforts and effects of glycogen metabolism engineering on the physiology and metabolism of cyanobacterial chassis strains and cell factories. The perspectives and prospects provided herein are expected to inspire novel strategies and tools to achieve ideal control over carbon and energy flow for biomanufacturing.     

The molecular evolution and DNA profiling of toxic cyanobacteria

Rapid and sensitive methods for the detection and genetic characterization of cyanobacteria have been developed based on DNA amplification techniques. This article describes the molecular methods that have been used to characterize cyanobacteria and their use as tools to identify toxin-producing strains. Different species and strains were compared using restriction fragment length polymorphism (RFLP) of amplified fragments of the phycocyanin gene and the 16S-23S rRNA internal transcribed spacer.     

蓝细菌制氢研究进展

蓝细菌具有很低的营养需求,能够利用太阳能直接光解水产生氢能,利用蓝细菌产氢是理想的生物制氢方式之一。目前,蓝细菌氢的产率尚未达到实际应用的要求。蓝细菌产氢依赖于菌株的遗传背景和产氢的环境条件。对蓝细菌产氢生理、产氢速率、产氢的环境条件、菌株筛选和突变株构建以及在光生物反应器中产氢的特征作了综述,以期有利于蓝细菌产氢水平的提高。     

Application of molecular genetic and microbiological techniques in ecology and biotechnology of cyanobacteria

The review discusses the advances and problems in biotechnology and ecology of cyanobacteria and considers the possibilities of molecular genetic and microbiological techniques in this field. Due to the ease of cultivation, high growth rate, availability of synchronous cultures, and existence of numerous molecular genetic and microbiological techniques for various cyanobacterial strains, cyanobacteria—prokaryotic organisms that are ancient relatives of the chloroplasts—are model organisms in the studies of photosynthesis, dinitrogen fixation, cell division, hydrogen production, and in a number of other areas of basic and applied science. These techniques make possible deeper understanding of the role of cyanobacteria in various ecosystems and utilization of their potential in numerous applied projects, including production of molecular hydrogen, phycobiliproteins, and cyanophycin; formation of nanoparticles; removal of heavy metals from the environment; substrate biodegradation; manufacture of products for medicine and food industry; and solution of the problem of cyanobacterial toxins in freshwater and marine environments.     

Towards optimization of cyanobacteria as biotechnologically relevant producers of molecular hydrogen, a clean and renewable energy source

In discussions about alternatives to our current fossil energy sources, basic and applied research leading to biological production of molecular hydrogen utilizing cyanobacteria deserves serious attention. In these oxygenic phototrophic bacteria, hydrogen can be produced by the activity of either nitrogenases or reversible/bidirectional hydrogenases. Knowledge of the physiological and molecular basis of some of the processes involved in hydrogen metabolism in these peculiar microorganisms has increased during the last decade. However, further efforts are required in basic as well as applied research in order to obtain a clear impression of these processes and their regulation. This information might then constitute the basis for optimizing the efficiency of hydrogen evolution by cyanobacteria. Progress might be achieved by screening more cyanobacterial strains for their ability to produce and evolve hydrogen, by genetically manipulating specific strains as well as by improving the conditions for cultivation in bioreactors. Received: 17 February 1998 / Received revision: 24 April 1998 / Accepted: 27 April 1998     

基于糖原代谢调控的蓝细菌光合细胞工厂优化研究进展

蓝细菌是重要的光合自养微生物,也是最具潜力的光合微生物底盘之一,被广泛应用于光驱固碳细胞工厂的开发。糖原是蓝细菌最重要的天然碳汇物质,糖原代谢对蓝细菌光合碳流的分配和调控具有重要意义。为了优化蓝细菌光合细胞工厂的合成效能,驱动更多的光合碳流重定向至目标代谢产物的合成,已经有多种策略和方法被成功开发用于调控蓝细菌的糖原代谢和糖原含量。然而,作为具有全局效应的重要碳汇机制,针对糖原代谢的调控往往对蓝细菌底盘藻株的光合生理和代谢网络造成复杂的影响,在不同光合细胞工厂合成效能优化上取得的效果也不尽相同。文中梳理了蓝细菌糖原代谢工程的最新进展,对糖原代谢调控造成的生理、代谢影响进行了介绍和分析,进而对通过糖原代谢调控来优化光合细胞工厂效能的研究前景进行了展望。     

Promoting R &; D in Photobiological Hydrogen Production Utilizing Mariculture-Raised Cyanobacteria

This review article explores the potential of using mariculture-raised cyanobacteria as solar energy converters of hydrogen (H₂). The exploitation of the sea surface for large-scale renewable energy production and the reasons for selecting the economical, nitrogenase-based systems of cyanobacteria for H₂ production, are described in terms of societal benefits. Reports of cyanobacterial photobiological H₂ production are summarized with respect to specific activity, efficiency of solar energy conversion, and maximum H₂ concentration attainable. The need for further improvements in biological parameters such as low-light saturation properties, sustainability of H₂ production, and so forth, and the means to overcome these difficulties through the identification of promising wild-type strains followed by optimization of the selected strains using genetic engineering are also discussed. Finally, a possible mechanism for the development of economical large-scale mariculture operations in conjunction with international cooperation and social acceptance is outlined.     

Genetic techniques appropriate for the biotechnological exploitation of cyanobacteria

A large collection of genetic tools are available for those who wish to manipulate laboratory strains of cyanobacteria. These tools have not yet been applied to strains that might be used for more practical ends. Applications in which a natural ability of a cyanobacterial strain is exploited would require different genetic tools than those in which a cyanobacterial strain is called upon to produce a foreign product. DNA can be transferred into cyanobacteria by three methods: transformation, conjugation, and electroporation. With each, efficiency is greatly affected by restriction enzymes within the recipient cyanobacterium. Transferred DNA may be propagated in the recipient strain if carried on a replicating plasmid or if integrated into the host chromosome. Different means of integration offer different advantages. Methods are available to increase and control the expression of genes of interest. These genetic tools are discussed with reference to specific problems that might be faced in the exploitation of cyanobacteria.     

The impact of biotechnology on the dairy industry

The review focusses on the use of genetic techniques to manipulate bacteria that are important to the dairy industry. Both classical and molecular approaches have been used to improve strains involved in yoghurt and cheese production. Examples are provided of methods for; increasing efficiency of substrate conversion, regulating the production of flavour enhancing metabolites, and developing starter cultures resistant to bacteriophage and bacteriocin attack. The possible applications of these systems are discussed     

Molecular genetic analysis of new Anabaena strains isolated from a plant-cyanobacterial community

Ecosystems of rice paddies are good sources of new strains of heterocyst-forming cyanobacteria that can be used in biotechnological systems for production of photohydrogen. The morphological and physiological properties of two novel epiphytic strains of cyanobacteria, Anabaena sp. 182 and Anabaena sp. 281, were studied. DNA typing of these strains based on PCR amplification of hydrogenase-encoding genes and DNA analysis using RAPD and Rep primers was carried out. The properties of the genome of strain Anabaena sp. 281 differed considerably from those of two reference strains (Anabaena variabilis ATCC 29413 and Nostoc sp. PCC 7120) with sequenced genomes, whereas strain Anabaena sp. 182 was found to be a close relative of A. variabilis ATCC 29413. Due to a number of physiological and biochemical advantages, Anabaena sp. 182 may be considered a new promising model for molecular and genetic engineering studies aimed at the development of H₂ producers.     

Admixture as the basis for genetic mapping

Genetic mapping in natural populations is increasing rapidly in feasibility and accessibility. As with many areas in genetics, advances in molecular techniques and statistics are drastically altering how we can investigate inheritance in wild organisms. For ecology and evolution, this is particularly significant and promising, because many of the organisms of interest are not amenable to conventional genetic approaches. Admixture mapping falls within a family of statistical approaches that use natural recombination and linkage disequilibrium between genetic markers and phenotypes as the basis for mapping. Our aim in this review is to provide a snapshot of previous and ongoing research, existing methods and challenges, the nature of questions that can be investigated and prospects for the future of admixture mapping.     

Hydrogenases and hydrogen metabolism of cyanobacteria.

Cyanobacteria may possess several enzymes that are directly involved in dihydrogen metabolism: nitrogenase(s) catalyzing the production of hydrogen concomitantly with the reduction of dinitrogen to ammonia, an uptake hydrogenase (encoded by hupSL) catalyzing the consumption of hydrogen produced by the nitrogenase, and a bidirectional hydrogenase (encoded by hoxFUYH) which has the capacity to both take up and produce hydrogen. This review summarizes our knowledge about cyanobacterial hydrogenases, focusing on recent progress since the first molecular information was published in 1995. It presents the molecular knowledge about cyanobacterial hupSL and hoxFUYH, their corresponding gene products, and their accessory genes before finishing with an applied aspect--the use of cyanobacteria in a biological, renewable production of the future energy carrier molecular hydrogen. In addition to scientific publications, information from three cyanobacterial genomes, the unicellular Synechocystis strain PCC 6803 and the filamentous heterocystous Anabaena strain PCC 7120 and Nostoc punctiforme (PCC 73102/ATCC 29133) is included.     

微生物生态学中分子生物学方法及T-RFLP技术研究

根据微生物基因 (DNA)多态性来研究微生物的多样性 ,是建立在多聚酶链式反应 (PCR)基础之上分子生物学的新方法 ,克服了传统微生物培养方法的限制。从理论、实验及应用角度出发 ,介绍了几种在微生物生态学中应用较为广泛的分子生物学技术 ;详细阐述了微生物生态学中分子生物学的一种新研究方法---末端限制性片段长度多态性 (T -RFLP)技术 ,该技术作为一种研究微生物群落特征的理想方法已经越来越受到人们的重视。     

Comparative evaluation of different preservation methods for cyanobacterial strains

Maintaining pure cultures using preservation methods is of high importance for biotechnological purposes. However, preservation does not necessarily guarantee the genetic stability of these cultures. Therefore, preservation methods are currently needed to assure viability as well as genetic, physiological, and morphological integrity across storage periods. In this study, preservation of five isolates from the microalgae and cyanobacteria collection of the Plant Biology Department, Federal University of Viçosa, Minas Gerais, Brazil was investigated via monthly analyses of cell viability, biomass recovery, and contaminant concentrations over a period of 120 days. Lyophilization was adequate for both heterocystous cyanobacteria and other strains that were able to differentiate hormogones or to synthesize thick layers of exopolysaccharides. Lyophilization was also able to maintain cultures with low levels of contaminants. Dimethyl sulfoxide was relatively efficient, though some of the strains were susceptible to its cytotoxic effects. Our results demonstrated that cryopreservation with glycerol was the most efficient method. The ability to routinely preserve cyanobacterial strains reduces costs associated with maintaining large culture collections and reduces the risks of losing particular strains or species through contamination and genetic drift. The results obtained in this study are therefore discussed in the context of the efficiency of the methods and the current need to develop suitable methods for maintenance of cyanobacterial collections.     

Hydrogenases and Hydrogen Metabolism of Cyanobacteria

Cyanobacteria may possess several enzymes that are directly involved in dihydrogen metabolism: nitrogenase(s) catalyzing the production of hydrogen concomitantly with the reduction of dinitrogen to ammonia, an uptake hydrogenase (encoded by hupSL) catalyzing the consumption of hydrogen produced by the nitrogenase, and a bidirectional hydrogenase (encoded by hoxFUYH) which has the capacity to both take up and produce hydrogen. This review summarizes our knowledge about cyanobacterial hydrogenases, focusing on recent progress since the first molecular information was published in 1995. It presents the molecular knowledge about cyanobacterial hupSL and hoxFUYH, their corresponding gene products, and their accessory genes before finishing with an applied aspect—the use of cyanobacteria in a biological, renewable production of the future energy carrier molecular hydrogen. In addition to scientific publications, information from three cyanobacterial genomes, the unicellular Synechocystis strain PCC 6803 and the filamentous heterocystous Anabaena strain PCC 7120 and Nostoc punctiforme (PCC 73102/ATCC 29133) is included.     

Cyanobacterial H<Subscript>2</Subscript> production — a comparative analysis

Several unicellular and filamentous, nitrogen-fixing and non-nitrogen-fixing cyanobacterial strains have been investigated on the molecular and the physiological level in order to find the most efficient organisms for photobiological hydrogen production. These strains were screened for the presence or absence of hup and hox genes, and it was shown that they have different sets of genes involved in H₂ evolution. The uptake hydrogenase was identified in all N₂-fixing cyanobacteria, and some of these strains also contained the bidirectional hydrogenase, whereas the non-nitrogen fixing strains only possessed the bidirectional enzyme. In N₂-fixing strains, hydrogen was mainly produced by the nitrogenase as a by-product during the reduction of atmospheric nitrogen to ammonia. Therefore, hydrogen production was investigated both under non-nitrogen-fixing conditions and under nitrogen limitation. It was shown that the hydrogen uptake activity is linked to the nitrogenase activity, whereas the hydrogen evolution activity of the bidirectional hydrogenase is not dependent or even related to diazotrophic growth conditions. With regard to large-scale hydrogen evolution by N₂-fixing cyanobacteria, hydrogen uptake-deficient mutants have to be used because of their inability to re-oxidize the hydrogen produced by the nitrogenase. On the other hand, fermentative H₂ production by the bidirectional hydrogenase should also be taken into account in further investigations of biological hydrogen production.Abbreviations Chl chlorophyll - MV methyl viologen     

Characterization by genotypic methods of symbiotic Nostoc strains isolated from five species of Gunnera

The genetic diversity of ten symbiotic Nostoc strains isolated from different Gunnera species was investigated. The strains were analyzed using molecular methods with different taxonomic resolutions, including restriction fragment length polymorphisms (RFLP) of the PCR-amplified 16S ribosomal gene and the 16S-23S internal transcribed spacer (ITS) region combined with computer-assisted analyses. The functional gene hetR, assigned to heterocyst differentiation, was used for denaturing gradient gel electrophoresis. A high genetic diversity was observed among the isolates even in the conserved gene coding for the small ribosomal unit. No correlation was observed between clustering of cyanobacteria and the host species of Gunnera.