Cyanobacteria: Omics and Manipulation
"a treasure trove of valuable information" (Biospektrum)
Caister Academic Press
Dmitry A. Los
Institute of Plant Physiology, Russian Academy of Sciences, Botanicheskaya Street 35, 127276 Moscow, Russia
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In this era of the 'Green Planet', cyanobacteria are ideally placed for exploitation as microbial cell factories, both for carbon capture and storage and for the sustainable production of secondary metabolites and biofuels. The application of omics technologies to cyanobacterial research has yielded a wealth of new information. However for today's busy researchers, trawling through the literature to stay abreast of current developments can be extremely time-consuming. By compiling and summarising the most important topics on cyanobacterial omics and manipulation, the authors of this book provide the reader with a timely overview of the field.
Topics covered: The cyanobacterial core-genome with a focus on secondary metabolites; cyanobacterial evolution; genomics of NRPS/PKS biosynthetic gene clusters; RNA-seq based transcriptomic analysis of single cyanobacterial cells; transcriptomics of the responses: genes, sensors, and molecular triggers; transcriptomic and proteomic analysis of diurnal cycles in nitrogen-fixing cyanobacteria; proteomic analysis of post translational modifications; metabolic engineering and systems biology for free fatty acid production; isoprene production; ethanol production: impact of omics of the model organism Synechocystis on yield enhancement; engineering of alkane production; photoautotrophic polyhydroxyalkanoate production.
This cutting-edge text will serve as a valuable resource for all those working in this field and is recommended for all microbiology libraries.
"some very well written and highly informative chapters. Thus ... the book is a treasure trove of valuable information " from Biospektrum (2017)
Table of contents
1. The Cyanobacterial Core-genome: Global and Specific Features with a Focus on Secondary Metabolites
Stefan Simm, Enrico Schleiff and Rafael Pernil
Cyanobacteria are a prolific source of natural products and produce a vast array of compounds, including many notorious toxins as well as natural products of huge interest to pharmaceutical and biotechnological industries. Genome mining has enabled the identification and characterization of natural product gene clusters, and mechanisms that are unique to cyanobacteria, or rarely seen in other organisms, have been discovered. Many cyanobacterial secondary metabolites are cyanotoxins, which show a broad range of chemical structures and biological activities, but in addition to toxin production, also several NRPS and PKS gene clusters are devoted to important cellular processes in cyanobacteria such as iron uptake and nitrogen fixation. Most of the biosynthetic clusters identified here have unknown end products, highlighting the power of genome mining for the discovery of new secondary metabolites. These studies show that cyanobacteria encode a huge variety of cryptic gene clusters involved in the production of natural products, and the known chemical diversity to date is likely to be only a fraction of the true biosynthetic capabilities of this fascinating and ancient group of organisms. Furthermore, mechanistic insights obtained from the biochemical studies of cyanobacterial pathways can inspire the development of concepts for the design of bioactive compounds by synthetic-biology approaches in the future. Here, we survey the biosynthetic pathways of the top five most researched cyanobacterial species with the most extensive literature, including Microcystis aeruginosa NIES-843, Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, Arthrospira platensis NIES-39 and Synechococcus elongatus PCC 7942. These analyses have enabled the identification of biosynthetic gene clusters for structurally diverse metabolites, including non-ribosomal peptides, polyketides, ribosomal peptides, terpenes and fatty acids. We highlight the unique enzyme mechanisms that were elucidated or can be anticipated for the individual products, but further include different classes of secondary metabolites from cyanobacteria other than NRPS and PKS.
2. Genome-wide Analysis of Cyanobacterial Evolution: The Example of Synechococcus
Cyanobacteria are an ancient group of prokaryotes with an immense impact on the evolution of other organisms and the global ecosystem. In this chapter, I will focus on a genetically diverse yet morphologically similar group of cyanobacteria merged under the name Synechococcus. Together with Prochlorococcus, Synechococcus may be the most abundant cyanobacterium and amongst the most ancient organisms on Earth. However, the evolutionary trajectories of Synechococcus are entangled, resulting in polyphyletic relationships based on whole-genome data in phylogenetic reconstruction. Moreover, there is a high variability of the genome composition, size, and complexity within Synechococcus, potentially accounting for and being an example of speciation patterns within cyanobacteria, which may be largely driven by genome alterations (e.g., Horizontal Gene Transfer). In conclusion, I will show that the cyanobacterial evolution is a dynamic process, characterized by drastic genome alterations and gene transfer (both homologous and non-homologous), often resulting in convergent evolutionary patterns as typified by Synechococcus.
3. Genomics of NRPS/PKS Biosynthetic Gene Clusters in Cyanobacteria
Claire Pancrace, Muriel Gugger and Alexandra Calteau
Cyanobacteria are well known producers of a broad range of natural products of diverse chemical structures. Many of these compounds are the products of non-ribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) pathways. In the last 15 years, genome mining of biosynthetic gene clusters has become a key methodology for studying NRPS/PKS biosynthetic gene clusters in cyanobacteria. This particularly rich phylum revealed an unexpected diversity for these clusters with only 20% of them that could be linked to a known natural product. The present chapter reviews recent literature on genomics of NRPS/PKS biosynthetic gene clusters in cyanobacteria from strategies of genome mining to comparative genomics. We then tackle the gap from prediction to characterization of natural products before discussing the benefits of combined genomic and metagenomic investigations. Finally we propose future trends for the study of these natural products in cyanobacteria to overcome the current gap between genome mining and natural products discovery.
4. RNA-seq Based Transcriptomic Analysis of Single Cyanobacterial Cells
Zixi Chen, Jiangxin Wang, Lei Chen and Weiwen Zhang
Gene-expression heterogeneity among individual cells will eventually determine the fate of a bacterial population. We describe the first bacterial single-cell RNA sequencing (RNA-seq), BaSiC RNA-seq, a method integrating RNA isolation, cDNA synthesis and amplification, and RNA-seq analysis of the whole transcriptome of single cells of the cyanobacterium Synechocystis sp. PCC 6803, which typically contain approximately 5-7 femtogram of total RNA per cell. We applied the method to 3 Synechocystis single cells at 24 h and 3 single cells at 72 h after a nitrogen-starvation stress treatment, as well as their bulk-cell controls for the same conditions, to determine the heterogeneity upon the environmental stress. With 82-98% and 31-48% of all putative Synechocystis genes identified in single cells of 24 and 72 h, respectively, the results demonstrated the method could achieve good identification of the transcripts in single bacterial cells. In addition, the preliminary results from nitrogen-starved cells also showed a possible increasing gene-expression heterogeneity from 24 h to 72 h after nitrogen starvation. Moreover, preliminary analysis of single-cell transcriptomic datasets revealed that genes from the 'Mobile elements' functional category have the most significant increase of gene-expression heterogeneity under stress, which was further confirmed by single-cell RT-qPCR analysis of gene expression in 24 randomly selected cells.
5. Transcriptomics of Cyanobacterial Stress Responses: Genes, Sensors, and Molecular Triggers
Maria A. Sinetova, Anna A. Zorina, Kirill S. Mironov and Dmitry A. Los
Systemic analysis of transcriptomes of the cyanobacterium Synechocystis sp. PCC 6803 revealed that all stress-induced genes can be separated into two groups: one is clustered around heat-shock- and another around cold-shock-inducible genes. At the initial stages of stress (in a range of 15-20 min) genes induced by heat and cold stress never overlap. Genes for so-called heat shock proteins (HSPs) are induced by various stressors, e.g. heat, salt, hyperosmotic environment, reactive oxygen species (ROS), changes in light intensity and quality, or in the redox potential of the photosynthetic electron transport chain components. The number of specifically heat-induced genes is rather limited and their functions are mostly unknown. Cold-stress-induced genes also overlap, but with different sets of genes induced by all above mentioned stressors with the exception of heat stress. The analysis suggests that ROS (in particular, H2O2) and redox changes of the components of the photosynthetic electron chain may function as universal triggers for stress responses in cyanobacteria.
6. Transcriptomic and Proteomic Analysis to Understand Systems-level Properties of Diurnal Cycles in Nitrogen-fixing Cyanobacteria
Uma K. Aryal and Louis A. Sherman
Photosynthetic cyanobacteria are important model organisms for many studies and, like plants, they have developed complex strategies for regulating metabolism during alternating light-dark cycles. Transcriptomics and proteomics technologies are now routinely applied for systems-level analysis of cyanobacteria. Although transcriptional regulation is a mature field, analysis of the cyanobacterial proteome is in its infancy. Thus, this review will concentrate on proteomics and focus on new technological improvements that have been applied to cyanobacteria. Proteins are the key functional units of the cell and their functions are controlled and driven by post-translational modifications (PTMs), and protein-protein interactions. We will emphasize research on the diazotrophic, unicellular cyanobacteria of the genus Cyanothece, since these strains perform two disparate types of metabolism: photosynthesis, which functions during the light and nitrogen fixation, which functions during the dark. Transcriptional features varied dramatically throughout the 24 hr diel cycle. However, proteome analysis revealed that proteins are much more stable throughout the light and dark and provides clues as to how cells conserve energy by keeping proteins within a modest range during the light-dark cycle. We emphasize that the development of new methods and mass spectrometers that enhances the performance of the proteomic workflow will be critical for future research, which should focus on PTMs, endogenous protein complexes and protein-protein interactions.
7. Proteomic Analysis of Post Translational Modifications in Cyanobacteria
Qian Xiong, Zhuo Chen and Feng Ge
Cyanobacteria are a diverse group of Gram-negative bacteria and the only prokaryotes capable of oxygenic photosynthesis. Recently, cyanobacteria have attracted great interest due to their crucial roles in global carbon and nitrogen cycles and their ability to produce clean and renewable biofuels. To survive at various environmental conditions, cyanobacteria have developed a complex signal transduction network to sense environmental signals and implement adaptive changes. The post-translational modifications (PTMs) systems play important regulatory roles in the signaling networks of cyanobacteria. Mass spectrometry (MS)-based proteomic technologies have been applied to identify PTMs in a systematic manner. Although the proteomic studies of PTMs carried out in cyanobacteria were limited, these data have provided clues to elucidate their sophisticated sensing mechanisms that contribute to their evolutionary and ecological success. This chapter aims to summarize the current status of PTM studies and recent publications regarding PTM proteomics in cyanobacteria, and discuss the novel developments and applications for the analysis of PTMs in cyanobacteria. Challenges, opportunities, and future perspectives in the proteomics studies of PTMs in cyanobacteria are also discussed.
8. Metabolic Engineering and Systems Biology for Free Fatty Acid Production in Cyanobacteria
Anne M. Ruffing
Free fatty acids (FFAs) are essential cellular components and also potential precursors for biofuel production. The modification of cyanobacteria for FFA production allows for the production of this high density energy molecule from CO2 and sunlight as the main carbon and energy sources. Efforts to engineer cyanobacteria for FFA production have provided a proof-of-concept demonstration for this approach, yet FFA yields are too low to support industrial-scale production for biofuel applications. This chapter highlights previous successes in engineering cyanobacterial FFA production, possible targets for future metabolic engineering efforts, and the many challenges that must be overcome. Due to the essential nature of fatty acid biosynthesis, modification of this major metabolic pathway includes interactions with many other pathways of carbon metabolism and complex regulatory mechanisms. In addition, cyanobacterial FFA production has been shown to result in cellular stress responses affecting growth, membrane integrity and composition, and photosynthesis. These complex interactions necessitate the use of systems biology approaches, such as omics and computational modeling, to understand and effectively manipulate cyanobacteria for enhanced FFA production. While there are few examples of the application of systems biology to FFA production in cyanobacteria, a review of technologies and tools developed for cyanobacteria is presented to guide future efforts in this area. The proposed integration of metabolic engineering and systems biology approaches may advance our understanding of cyanobacterial fatty acid biosynthesis and overcome current barriers in cyanobacterial FFA production.
9. Terpene Hydrocarbons Production in Cyanobacteria
A description is given on the development and state of the art of technologies for the renewable generation of terpene hydrocarbons to serve as renewable fuel for transportation, and chemicals for the synthetic chemistry industry, with cyanobacteria as the primary photocatalyst and isoprene (C5H8) and monoterpene (C10H16) hydrocarbons as the paradigm outputs. This approach integrates pivotal aspects of the Photosynthetic Biofuels concept: (a) Efficient sunlight utilization by cyanobacterial mass cultures; (b) Effective delivery and assimilation of quantities of CO2 to fast-growing cyanobacteria; (c) Paradigm of synthesis, spontaneous product separation from the biomass, and isoprene and β-phellandrene (monoterpene) sequestration and harvesting; (d) Application of novel low-cost photobioreactors for the successful and cost-effective implementation of (a) through (c); and (e) Evaluation of isoprene (C5H8) and monoterpene (C10H16) hydrocarbons as fuel molecules, compared to alkanes and alcohols. The effort advocates the development and application of designer cyanobacterial strains, which can grow in mass culture under bright sunlight conditions and demonstrate efficient sunlight conversion and utilization. Metabolic engineering approaches endow and enhance the yield of isoprene and monoterpene hydrocarbons generated in photosynthesis, relative to the biomass accumulated. Gaseous/aqueous two-phase photobioreactors are used for efficient conversion of sunlight and CO2 into isoprene or monoterpenes, molecules that are potential biofuels and highly valuable industrial chemicals. Following isoprene or monoterpene sequestration, the residual cyanobacterial biomass is used as feedstock of bacterial fermentations to generate biogas (a mixture of CH4 and CO2), with the residual biomass serving as bio-fertilizer. In terms of Energy Density, terpene hydrocarbons are favorably compared to alkanes, and are substantially better than similar size alcohols. The promise of a cyanobacterial 'Photosynthesis-to-Fuels' approach is the ability to transform the primary products of photosynthesis directly, in a single cyanobacterial cell, into commodity products for human industrial and domestic consumption.
10. Ethanol Production in Cyanobacteria: Impact of Omics of the Model Organism Synechocystis on Yield Enhancement
J. Tony Pembroke, Lorraine Quinn, Helen O'Riordan, Con Sheahan and Patricia Armshaw
Due to their phototrophic lifestyle, cyanobacteria have been investigated as a sustainable source of biofuel production. Model cyanobacteria, such as Synechocystis, have been metabolically engineered with heterologous genes encoding pyruvate decarboxylase and alcohol dehydrogenase to convert the metabolic intermediate pyruvate first to acetaldehyde and then to ethanol. The current state of the art suggests that there is some way to go before such a system can be exploited. Lessons from the genomics of the model organism and proteomic and transcriptomic analysis of the organism's response to ethanol production are being utilised to enhance productivity. These are informing a systems approach to rational modification for enhanced ethanol production, which will be a key to the biotechnological exploitation of such a system.
11. Engineering of Alkane Production in Cyanobacteria
Xuefeng Lu and Weihua Wang
Alkanes with defined carbon chain lengths possess higher energy density, low hygroscopicity and volatility, and compatibility with existing liquid fuel infrastructure, which are the predominant constituents of gasoline, diesel and jet fuels. Alkane biosynthesis is ubiquitous and biosynthetic pathways have been identified in cyanobacteria, photosynthetic microbes, which opens a door to engineer alkane production with high efficiency in cyanobacteria. Firstly, redirecting the carbon flux to fatty acids or acyl-acyl carrier protein (ACP) can provide larger precursor pool for further conversion to alkanes. In combination with the overexpression of alkane biosynthesis genes, alkane production can be significantly improved in engineered strains. Protein engineering for key enzymes in alkane biosynthesis pathways will further enhance the yield of alkanes. Secondly, system biology research on cyanobacteria will increase our knowledge about the metabolism in cyanobacteria and lead to significant improvements in strain modification for alkane production. Convenient and effective molecular tools for genetic engineering of cyanobacteria will expand the ability to engineer cyanobacteria for alkane production. It is significant and promising to directly utilize solar energy and convert carbon dioxide into alkanes, drop-in biofuels, in cyanobacteria.
12. Photoautotrophic Polyhydroxyalkanoate Production in Cyanobacteria
Ka-Kei Sam, Nyok-Sean Lau, Amirul Al-Ashraf Abdullah and Minami Matsui
Accumulation of recalcitrant plastics waste in the natural environment leading to environmental problems and still unresolved plastic litter issues are of significant environmental concern. Development and production of biobased and biodegradable plastics including polyhydroxyalkanoate (PHA) are rapidly expanding because these bioplastics can serve as alternatives for petrochemical based polymers. Industrial-scale PHA production currently depends on a fermentation process using heterotrophic bacteria. Economic competitiveness of this production process is limited by the cost of carbon feedstock. It is therefore desirable to develop production platforms based on carbon-neutral PHA producers with capability to fix atmospheric carbon dioxide by which carbon feedstock can be omitted, and at the same time reduce carbon dioxide emissions. Throughout this chapter, potentials of photosynthetic cyanobacteria as hosts for sustainable PHA production from renewable sunlight and atmospheric carbon dioxide are discussed. Some future trends for improving PHA productivity in photosynthetic cyanobacteria are also briefly described.
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(EAN: 9781910190555 9781910190562 Subjects: [bacteriology] [environmental microbiology] )