Advances in using adaptive laboratory evolution technology for engineering of photosynthetic cyanobacteria / 生物工程学报

Cyanobacteria are the only prokaryotes capable of oxygenic photosynthesis, which have potential to serve as "autotrophic cell factories". However, the synthesis of biofuels and chemicals using cyanobacteria as chassis are suffered from poor stress tolerance and low yield, resulting in low economic feasibility for industrial production. Thus, it's urgent to construct new cyanobacterial chassis by means of synthetic biology. In recent years, adaptive laboratory evolution (ALE) has made great achievements in chassis engineering, including optimizing growth rate, increasing tolerance, enhancing substrate utilization and increasing product yield. ALE has also made some progress in improving the tolerance of cyanobacteria to high light intensity, heavy metal ions, high concentrations of salt and organic solvents. However, the engineering efficiency of ALE strategy in cyanobacteria is generally low, and the molecular mechanisms underpinning the tolerance to various stresses have not been fully elucidated. To this end, this review summarizes the ALE-associated technical strategies and their applications in cyanobacteria chassis engineering, following by discussing how to construct larger ALE mutation library, increase mutation frequency of strains and shorten evolution time. Moreover, exploration of the construction principles and strategies for constructing multi-stress tolerant cyanobacteria, and efficient analysis the mutant libraries of evolved strains as well as construction of strains with high yield and strong robustness are discussed, with the aim to facilitate the engineering of cyanobacteria chassis and the application of engineered cyanobacteria in the future.

Production of lactate from carbon fixation by cyanobacteria: development and prospect / 生物工程学报

Lactate is an important industrial chemical and widely used in various industries. In recent years, with the increasing demand for polylactic acid (PLA), the demand for lactate raw materials is also increasing. The contradiction between the high cost and the market demand caused by the heterotrophic production of lactate attracts researchers to seek other favorable solutions. The production of lactate from photosynthetic carbon fixation by cyanobacteria is a potential new raw material supply strategy. Based on the photosynthetic autotrophic cell factory, it can directly produce high optical purity lactate from carbon dioxide on a single platform driven by solar energy. The raw materials are cheap and easy to obtain, the process is simple and controllable, the products are clear and easy to separate, and the double effects of energy saving and emission reduction and production of high value-added products are achieved at the same time, which has important research and application value. This paper reviews the development history of cyanobacteria carbon sequestration to produce lactate, summarizes its research progress and encounters technical difficulties from the aspects of metabolic basis, metabolic engineering strategy, metabolic kinetics analysis and technical application, and prospects the future of this technology.

Photosynthesis at the far-red region of the spectrum in Acaryochloris marina

Acaryochloris marina is an oxygenic cyanobacterium that utilizes far-red light for photosynthesis. It has an expanded genome, which helps in its adaptability to the environment, where it can survive on low energy photons. Its major light absorbing pigment is chlorophyll d and it has α-carotene as a major carotenoid. Light harvesting antenna includes the external phycobilin binding proteins, which are hexameric rods made of phycocyanin and allophycocyanins, while the small integral membrane bound chlorophyll binding proteins are also present. There is specific chlorophyll a molecule in both the reaction center of Photosystem I (PSI) and PSII, but majority of the reaction center consists of chlorophyll d. The composition of the PSII reaction center is debatable especially the role and position of chlorophyll a in it. Here we discuss the photosystems of this bacterium and its related biology.

Técnicas moleculares para la detección de cianobacterias en los embalses Riogrande II y La Fe, Colombia / Molecular techniques for cyanobacteria detection at Riogrande II and La Fe water reservoirs, Colombia

In lentic water bodies as reservoirs occur eutrophication processes, originated mainly from human activities (i.e. agriculture, animal exploitation). This influx of nutrients in aquatic ecosystems could promote blooms of potentially toxic cyanobacteria. The purpose of this work is to detect the presence of cyanobacteria strains in water samples, using molecular techniques to help in preventive management of reservoirs dedicated to water purification. We used two molecular techniques to detect genes implied with the synthesis of hepatotoxic microcystins from potentially toxic cyanobacteria strains, and to evaluate the molecular diversity of cyanobacteria in water samples from two high-mountain reservoirs used for purification of drinking water for the metropolitan area of Medellin, Colombia. Between 2010-2011 collections of 12 water samples were taken and DNA extraction together with PCR and DGGE analyses where carried out. We amplified 22 sequences between 250-300bp of the genes mcyA and mcyE, and these sequences were related with several strains and cyanobacteria genera accessions from NCBI-GenBank databases. Moreover, sequence amplifications of the 16S small ribosomal RNA subunit - 16S rRNA- between 400-800bp were also performed in order to use them for the DGGE technique. The amplification products of DGGE were set in polyacrilamide gel with posterior denaturing electrophoresis, and the scanned images of the gel bands were analysed with the software GelCompar II. For Riogrande II and La Fe reservoirs we found 35 and 30 different DGGE bands, respectively, as a measurement of molecular diversity in these artificial ecosystems. Here, we demonstrated the utility of two molecular techniques for the detection of genes associated with toxicity and molecular diversity of cyanobacteria in reservoirs destined for drinking water in urban centers. We recommend strongly following with periodically molecular biology studies in these ecosystems combined with limnological and ecological data, as new tools for management of plants of water purification and for the prevention of potentially public health concerns. Rev. Biol. Trop. 62 (1): 403-419. Epub 2014 March 01.

En embalses, la eutrofización es consecuencia de procesos naturales y de actividades humanas, lo cual puede facilitar la aparición de afloramientos de cianobacterias potencialmente tóxicas. En este trabajo, se utilizaron dos técnicas moleculares en la detección de genes presentes en cepas potencialmente tóxicas de cianobacterias y evaluaciones de diversidad molecular de cianobacterias en los embalses para potabilización de agua, Riogrande II y La Fe, Colombia. Entre 2010-2011, se tomaron 12 muestras de agua en ambos embalses y se realizaron extracciones de ADN para un análisis de marcadores moleculares mediante PCR y DGGE. Se amplificaron secuencias entre 250-300pb de los genes mcyA y mcyE, implicados en la toxicidad de cepas de cianobacterias. Asimismo, se amplificaron secuencias de la región 16S del ARN ribosomal (422pb), para la técnica DGGE. Se corrieron geles de poliacrilamida en gradientes de desnaturalización, se realizó agrupamiento genético (UPGMA), y se separaron por patrones de bandeo las muestras de cada embalse evaluado. Se demuestra la utilidad de las técnicas moleculares en estudios relacionados con la búsqueda de genes asociados con toxicidad y diversidad molecular de cianobacterias en muestras de agua provenientes de embalses de agua con fines de potabilización para centros urbanos.

Variación espacial y estacional de grupos funcionales de bacterias cultivables del suelo de un bosque tropical seco en México / Spatial and seasonal variation of soil culturable-bacterial functional groups in a Mexican tropical dry forest

Microbial biomass and activity in soils are frequently studied in tropical dry forests, but scarce information is available about the relationships between functional bacterial groups and soil fertility, where relief interacts with rainfall seasonality. The culturable-bacterial groups and nutrients were studied during two consecutive years in soils from two topographic areas of different relief (hilltop vs hillslope) in a tropical dry forest from Chamela Jalisco, Mexico. We expected that seasonal and spatial variation in soil resources availability affects the abundance of functional culturable-bacterial groups. To evaluate this, fifteen soil cores (1kg), 0-5cm depth, were taken in the dry, early rainy and rainy seasons, from each of the ten replicate plots in hilltop and hillslope areas located in three microbasins. We found that hilltop soils were more organic and had higher concentration of labile C and total nutrient forms than hillslope soils, for which these soils had higher counts of colony-forming units (CFU) of total heterotrophic and P solubilizing bacteria. In both hilltop and hillslope soils, C and nutrient concentrations, as well as the counts of CFU of heterotrophic and P solubilizing bacteria generally decreased from the dry to the rainy season during the two study years. In contrast, the counts of CFU nitrifying and cellulolytic bacteria were higher at the hillslope than at the hilltop soils. The seasonal pattern of both groups was opposite to that of heterotrophic bacteria, presumably associated with a decrease in soil labile C and organic matter quality. In conclusion, our study suggests that available C appears to be the main factor that controls the structure of soil bacterial groups and soil fertility, where relief, rainfall seasonality and intraand inter-annual variations are critical factors that interactively modify bacterial dynamics related to soil C availability in the tropical dry forest.

Los bosques tropicales secos tienen una amplia heterogeneidad ambiental y por lo tanto se podría esperar una variación temporal y espacial en la abundancia y tipo de grupos bacterianos activos. Se evaluó, durante dos años consecutivos, el efecto de la estacionalidad de la lluvia sobre algunos grupos bacterianos cultivables y nutrimentos del suelo en dos posiciones topográficas (crestas vs laderas) en el bosque tropical seco, Chamela Jalisco, México. La hipótesis fue que la variación temporal y espacial de la disponibilidad de recursos afectaría la abundancia de los grupos bacterianos funcionales (heterótrofas totales, celulolíticas, solubilizadores de fosfatos y nitrificantes) del suelo. En las crestas, el suelo fue más orgánico y con mayor concentración de nutrimentos totales y C orgánico lábil que en la ladera, registrando más bacterias heterótrofas y solubilizadoras de fosfato. En ambas posiciones topográficas la concentración de C y nutrimentos, así como el número de bacterias heterótrofas y solubilizadoras de P, decrecieron de la estación seca a la lluviosa en ambos años. En contraste, las bacterias nitrificantes fueron mayores en el suelo de la ladera, y al igual que las celulolíticas, tuvieron un patrón estacional opuesto a las bacterias heterótrofas, asociado a la disminución del C lábil y de la calidad de la materia orgánica. En conclusión, se sugiere que el relieve, la estacionalidad y la variación inter-anual de la lluvia regulan interactivamente la relación entre el C disponible y la estructura de las comunidades bacterianas del suelo en el bosque tropical seco de Chamela.

True branched nostocalean cyanobacteria from tropical aerophytic habitats and molecular assessment of two species from field samples

Aerophytic cyanobacteria are commonly found growing on rocks, tree trunks and soil, but the diversity of these organisms is still poorly known. This complex group is very problematic considering the taxonomic arrangement and species circumscription, especially when taking into account tropical populations. In this work, 20 samples of aerophytic cyanobacteria from 15 distinct sampling sites were collected along a tropical rainforest area at the São Paulo State (Brazil). Samples were dried at room temperature after the collection, and once in the laboratory, they were rehydrated and analyzed. The taxonomic study resulted in the record and description of nine species of true-branched cyanobacteria pertaining to the genera Spelaeopogon (one specie), Hapalosiphon (two species) and Stigonema (six species). The similarity of the flora found when compared to surveys conducted in other geographical regions was relatively low. These differences could be addressed to ecological conditions of the habitats, to the extension of the area surveyed or even to taxonomic misinterpretations. The molecular assessment of 16S rDNA on the basis of field material was successful for two morphospecies, Hapalosiphon sp. and Stigonema ocellatum; however, their relationships with other populations and species revealed to be uncertain. The results of the floristic survey and of the molecular approach evidenced the fragile delimitation of some genera and species in the true branched group of cyanobacteria.

Las bacterias aerofíticas crecen comúnmente en las rocas, troncos de árboles y el suelo, pero la diversidad de estos organismos todavía es poco conocida. Este complejo grupo es muy problématico considerando la disposición taxónomica y la circunscripción de las especies, especialmente cuando se toman en cuenta las poblaciones tropicales. En esta investigación, 20 muestras de cianobacterias aerofíticas de 15 distintos sitios de muestro fueron recolectadas a lo largo de un área de selva tropical en el estado de São Paulo (Brasil). Las muestras fueron secadas a temperatura ambiente después de su recolecta, y una vez en el laboratorio, se hidrataron y analizaron. El estudio taxónomico resultó en un registro y descripción de nueve especies de cianobacterias con ramificaciones verdaderas pertenecientes al género Spelaeopogon (una especie), Hapalosiphon (dos especies) y Stigonema (seis especies). La similutud de la flora encontrada en comparación con las encuestas realizadas en otras regiones geográficas fue relativamente baja. Estas diferencias podrían deberse a las condiciones ecológicas de los hábitats, a la extensión de la zona de estudio o incluso a malas interpretaciones taxonómicas. La evaluación molecular del 16S rDNA del material de campo fue un éxito para dos morfoespecies, Hapalosiphon sp. y Stigonema ocellatum, sin embargo, sus relaciones con otras poblaciones y especies revelan que son inciertas. Los resultados del estudio florístico y de la aproximación molecular evidencian la débil delimitación de algunos géneros y especies en el grupo de cianobacterias con ramificaciones verdaderas.

Cyanobacterial defense mechanisms against foreign DNA transfer and their impact on genetic engineering

Cyanobacteria display a large diversity of cellular forms ranging from unicellular to complex multicellular filaments or aggregates. Species in the group present a wide range of metabolic characteristics including the fixation of atmospheric nitrogen, resistance to extreme environments, production of hydrogen, secondary metabolites and exopolysaccharides. These characteristics led to the growing interest in cyanobacteria across the fields of ecology, evolution, cell biology and biotechnology. The number of available cyanobacterial genome sequences has increased considerably in recent years, with more than 140 fully sequenced genomes to date. Genetic engineering of cyanobacteria is widely applied to the model unicellular strains Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942. However the establishment of transformation protocols in many other cyanobacterial strains is challenging. One obstacle to the development of these novel model organisms is that many species have doubling times of 48 h or more, much longer than the bacterial models E. coli or B. subtilis. Furthermore, cyanobacterial defense mechanisms against foreign DNA pose a physical and biochemical barrier to DNA insertion in most strains. Here we review the various barriers to DNA uptake in the context of lateral gene transfer among microbes and the various mechanisms for DNA acquisition within the prokaryotic domain. Understanding the cyanobacterial defense mechanisms is expected to assist in the development and establishment of novel transformation protocols that are specifically suitable for this group.

Experimental and bioinformatic approaches for analyzing and visualizing cyanobacterial nitrogen and hydrogen metabolism

Many cyanobacteria are capable of utilizing light energy for nitrogen fixation. As a by-product of this nitrogenase mediated catalysis, hydrogen gas is produced. Several approaches to increase hydrogen production from cyanobacteria exist. Usually, these approaches are non-targeted. Here we exemplify how DNA-microarray based gene-expression analysis and bioinformatic visualization techniques can be used to analyze nitrogen and hydrogen metabolism from the filamentous, heterocyst forming cyanobacterium Nostoc PCC 7120. We analyzed the expression of 1249 genes from major metabolic categories under nitrogen fixing and non-nitrogen fixing growth. Of the selected genes, 494 show a more than 2-fold expression difference in the two conditions analyzed. Under nitrogen-fixing conditions 465 genes, mainly involved in energy metabolism, photosynthesis, respiration and nitrogen-fixation, were found to be stronger expressed, whereas only 29 genes showed a stronger expression under non-nitrogen fixing conditions. To help understanding probe hybridization, all expression data were correlated with potential target secondary structures and probe GC-content. For the first time the expression of high light-induced stress proteins (HLIP-family) is shown to be linked to the nitrogen availability.

The upstream sequence of the phycocyanin b subunit gene from Arthrospira platensis regulates expression of gfp gene in response to light intensity

In cyanobacteria, few details are known of the mechanisms through which the expression of the light-harvesting pigment c-phycocyanin is regulated. In the present study, a 419 bp upstream sequence of the phycocyanin b subunit (cpcB) gene from Arthrospira platensis FACHB341 was fused with green fluorescent protein (gfp) gene, and a heterologous reporting system was built up to investigate the influence of light intensity on the expression of gfp gene, and the regulation function of different region of the upstream sequence of cpcB gene. Results showed that the upstream sequence of cpcB gene could drive the expression of gfp gene in Synechococcus sp. strain PCC7942, and the expression was influenced by light intensity, the lower the light intensity, the higher the GFP level. Deletion analysis revealed that a light-responsive element was located in the region -276 to-218, a promoter sequence was in the region -85 to -1, and two positive cis elements were in the -419 to -276 and the -218 to -130 regions, respectively.

Non-Darwinian and Darwinian prokaryotic and eukaryotic evolution--an enigma in cell biology conservation

Our theory is embarrassingly simple. What made today's prokaryotes and modern cyanobacteria so robust is the fact that in their origin, back in the Archean (3 billion years ago), selection did not play a central role in evolution, it had only a transitory role. Asexual reproduction, mutation, drift and sampling variance in local demes were more important especially when they were accompanied by population catastrophes, where millions perished. Metazoans are generally macroscopic, sexually reproducing, ecologically specialized organisms whose history is full of extinctions and radiations leading to morphological change. On the other hand, prokaryotes, thanks to their origin, avoid extinction because as a group they have slowly evolved as generalists. Specialization appears to be less important than ecological versatility and metabolic unspecialization. Modern cyanobacteria keep on using that strategy

Characterization of a phototolerant mutant of Synechocystis sp. PCC 6803 created by random mutagenesis of psbAII gene.

Photosensitivity and photosynthetic characteristics have been analyzed in wild type (KC) and its psbAII mutant (I6) of Synechocystis having three point amino acid substitutions, i.e., N322I, I326F and F328S, which are localized in the C-terminal extension of D1 protein of the photosystem II reaction center. Wild type and mutant cells show almost an identical growth pattern under normal/low light (30 mumol m-2s-1, 30 degrees C) liquid culture (BG-11) condition. However, upon shifting the cultures to high light (500 mumol m-2s-1, 30 degrees C), these two types of cells exhibit entirely different growth characteristics, i.e., the mutant cells continue to grow normally whereas, the control cells fail to adapt the light stress and eventually resulting in complete loss of the photosynthetic pigments. On the other hand, a quick loss in the Fv/Fm value with half--decay time of about 30 min is observed in the mutant, in contrast to 120-130 min in case of control, upon shifting to high light conditions. In spite of this, mutant cells are able to adapt and grow well under prolonged high light exposure even after losing a major part of the variable yield of chlorophyll fluorescence (Fv/Fm). The high light treatment also induced decrease in the level of D1 protein in the mutant. However, half-decay time for D1 is much longer (approximately 10 hr) than that of variable fluorescence. Thus, the mutant cells have shown an unique way for cell growth and maintenance under high light even after losing Fv/Fm and photosynthetic oxygen evolving capacity as well as D1 content to a great extent. Therefore, these results could extend an interesting insight to understand the coordination of physiological, biochemical and molecular mechanisms regulating phototolerance of the photosynthetic organisms.