Sonication-based isolation and enrichment of Chlorella protothecoides chloroplasts for Illumina genome sequencing

With the increasing world demand for biofuel, a number of oleaginous algal species are being considered as renewable sources of oil. Chlorella protothecoides Krüger synthesizes triacylglycerols (TAGs) as storage compounds that can be converted into renewable fuel utilizing an anabolic pathway that is poorly understood. The paucity of algal chloroplast genome sequences has been an important constraint to chloroplast transformation and for studying gene expression in TAGs pathways. In this study, the intact chloroplasts were released from algal cells using sonication followed by sucrose gradient centrifugation, resulting in a 2.36-fold enrichment of chloroplasts from C. protothecoides, based on qPCR analysis. The C. protothecoides chloroplast genome (cpDNA) was determined using the Illumina HiSeq 2000 sequencing platform and found to be 84,576 Kb in size (8.57 Kb) in size, with a GC content of 30.8 %. This is the first report of an optimized protocol that uses a sonication step, followed by sucrose gradient centrifugation, to release and enrich intact chloroplasts from a microalga (C. prototheocoides) of sufficient quality to permit chloroplast genome sequencing with high coverage, while minimizing nuclear genome contamination. The approach is expected to guide chloroplast isolation from other oleaginous algal species for a variety of uses that benefit from enrichment of chloroplasts, ranging from biochemical analysis to genomics studies.     

Complete Chloroplast Genome Sequence of Omani Lime (Citrus aurantiifolia) and Comparative Analysis within the Rosids

The genus Citrus contains many economically important fruits that are grown worldwide for their high nutritional and medicinal value. Due to frequent hybridizations among species and cultivars, the exact number of natural species and the taxonomic relationships within this genus are unclear. To compare the differences between the Citrus chloroplast genomes and to develop useful genetic markers, we used a reference-assisted approach to assemble the complete chloroplast genome of Omani lime (C. aurantiifolia). The complete C. aurantiifolia chloroplast genome is 159,893 bp in length; the organization and gene content are similar to most of the rosids lineages characterized to date. Through comparison with the sweet orange (C. sinensis) chloroplast genome, we identified three intergenic regions and 94 simple sequence repeats (SSRs) that are potentially informative markers with resolution for interspecific relationships. These markers can be utilized to better understand the origin of cultivated Citrus. A comparison among 72 species belonging to 10 families of representative rosids lineages also provides new insights into their chloroplast genome evolution.     

高等植物叶绿体基因组转化的应用

叶绿体基因组转化技术由于其独特的优越性,现已成为植物基因工程的研究热点。本文简单介绍了叶绿体基因组转化技术的原理和方法;并重点综述了该技术在基础研究和实践中的应用。这些应用主要包括利用叶绿体基因组转化技术进行Rubisco的组装,叶绿体基因结构、转录、翻译和RNA编辑等研究;利用叶绿体作为生物反应器生产人生长激素、霍乱毒素抗体、聚羟基丁酸脂和生物弹性蛋白等;获得抗虫、抗病、抗除草剂和耐旱的转基因植物;以及降低转基因植物的外源基因扩散等。     

叶绿体基因组研究进展

作为植物细胞器的重要组成部分和光合作用的器官,叶绿体在生物进化的漫长历史中发挥了重要作用.伴随着生物技术的深入发展,人们发现叶绿体基因组结构和序列的信息在揭示物种起源、进化演变及其不同物种之间的亲缘关系等方面具有重要价值.与此同时,比核转化具有明显优势的叶绿体转化技术在遗传改良、生物制剂的生产等方面显示出巨大潜力,而叶绿体基因组结构和序列分析则是叶绿体转化的基石.基于叶绿体的这些重要作用,收集整理了有关的资料,从几个方面归纳了本领域最近的研究进展,希望能使读者对迅速发展的叶绿体基因组研究有更全面的了解,以及对叶绿体基因组在物种的进化、遗传、系统发育关系等方面的作用有更深刻的认识,同时也希望对叶绿体转化技术的研究和广泛应用产生积极作用.     

An exogenous chloroplast genome for complex sequence manipulation in algae

We demonstrate a system for cloning and modifying the chloroplast genome from the green alga, Chlamydomonas reinhardtii. Through extensive use of sequence stabilization strategies, the ex vivo genome is assembled in yeast from a collection of overlapping fragments. The assembled genome is then moved into bacteria for large-scale preparations and transformed into C. reinhardtii cells. This system also allows for the generation of simultaneous, systematic and complex genetic modifications at multiple loci in vivo. We use this system to substitute genes encoding core subunits of the photosynthetic apparatus with orthologs from a related alga, Scenedesmus obliquus. Once transformed into algae, the substituted genome recombines with the endogenous genome, resulting in a hybrid plastome comprising modifications in disparate loci. The in vivo function of the genomes described herein demonstrates that simultaneous engineering of multiple sites within the chloroplast genome is now possible. This work represents the first steps toward a novel approach for creating genetic diversity in any or all regions of a chloroplast genome.     

ALGAL TRANSGENICS IN THE GENOMIC ERA1

The last few years have witnessed significant advances in the field of algal genomics. Complete genome sequences from the red alga Cyanidioschyzon merolae and the diatom Thalassiosira pseudonana have been published, the genomes for two more algae (Chlamydomonas reinhardtii and Ostreococcus tauri) are nearing completion, and several others are in progress or at the planning stage. In addition, large‐scale cDNA sequencing projects are being carried out for numerous algal species. This wealth of genome data is serving as a powerful catalyst for the development and application of recombinant techniques for these species. The data provide a rich resource of DNA elements such as promoters that can be used for transgene expression as well as an inventory of genes that are possible targets for genetic engineering programs aimed at manipulating algal metabolism. It is not surprising therefore that significant progress in the genetic engineering of eukaryotic algae is being made. Nuclear transformation of various microalgal species is now routine, and progress is being made on the transformation of macroalgae. Chloroplast transformation has been achieved for green, red, and euglenoid algae, and further success in organelle transformation is likely as the number of sequenced plastid, mitochondrial, and nucleomorph genomes continues to grow. Importantly, the commercial application of algal transgenics is beginning to be realized, and algal biotechnology companies are being established. Recent work has shown that recombinant proteins of therapeutic value can be produced in microalgal species, and it is now realistic to envisage the genetic engineering of commercially important species to improve production of valuable algal products. In this article we review the recent progress in algal transgenics and consider possible future developments now that phycology has entered the genomic era.     

Do Red and Green Make Brown?: Perspectives on Plastid Acquisitions within Chromalveolates

The chromalveolate "supergroup" is of key interest in contemporary phycology, as it contains the overwhelming majority of extant algal species, including several phyla of key importance to oceanic net primary productivity such as diatoms, kelps, and dinoflagellates. There is also intense current interest in the exploitation of these algae for industrial purposes, such as biodiesel production. However, the evolution of the constituent species, and in particular the origin and radiation of the chloroplast genomes, remains poorly understood. In this review, we discuss current theories of the origins of the extant red alga-derived chloroplast lineages in the chromalveolates and the potential ramifications of the recent discovery of large numbers of green algal genes in chromalveolate genomes. We consider that the best explanation for this is that chromalveolates historically possessed a cryptic green algal endosymbiont that was subsequently replaced by a red algal chloroplast. We consider how changing selective pressures acting on ancient chromalveolate lineages may have selectively favored the serial endosymbioses of green and red algae and whether a complex endosymbiotic history facilitated the rise of chromalveolates to their current position of ecological prominence.     

小球藻的分子生物学研究进展

单细胞真核藻类—小球藻是一种重要的微藻资源。近年来,随着藻类生物技术的迅速发展,有大量关于小球藻的研究工作被报道。在分子生物学领域,研究则集中在小球藻的核基因组和叶绿体基因组、重要功能基因的克隆和分析以及以小球藻为载体的基因工程研究等方面,本文仅就此方面的进展作一综述。Abstract: Unicellular green alga Chlorella is a kind of important eukaryotic microalga. A lot of work about Chlorella was reported in recent years with the rapid development of algal biotechnique. In the area of molecular biology, studies of Chlorella focus on the nuclear and chloroplast genome, cloning and analysis of important genes and genetic engineering using Chlorella as vector. This review reports the research progress in these aspects.     

The chloroplast genome sequence of Syzygium cumini (L.) and its relationship with other angiosperms

Only a few studies to date have conducted comparative genomics in the Myrtaceae family. Here, we report the complete sequence and bioinformatics analysis of the chloroplast genome of Syzygium cumini (L.), one of the family members. The size of S. cumini cp genome was within the range of reported angiosperm chloroplast genomes. Comparison of S. cumini cpDNA sequence with previously reported partial sequences of S. cumini revealed several SNPs that resulted in non-synonymous mutations in maturase K and NADH-plastoquinone oxidoreductase subunit-5. These polymorphic characters might serve as intra-specific markers to address whether lineage sorting from polymorphic ancestry has occurred. Comparison of the S. cumini chloroplast genome with related dicots revealed an expansion in the intergenic spacer located between IRA/large single copy (LSC) border and the first gene of LSC region, driven by sequence of 54 bp. This type of variation in the intergenic regions can be utilized in the development of species-specific vectors for chloroplast genetic engineering. Several of the longer (30–40 bp) repeats were found to be conserved in other dicot species, suggesting that they might be widespread in angiosperm chloroplast genomes.     

叶绿体基因工程研究进展

叶绿体是植物细胞和真核藻类执行光合作用的重要细胞器,在叶绿体中表达外源基因比在细胞核中表达具有一些独特优势。叶绿体基因工程涉及叶绿体的基因组特征、转化系统的优点、转化过程及方法等方面,叶绿体基因工程在提高植物光合效率、改良植物特性、生产生物药物及改善植物代谢途径等方面已得到应用。尽管叶绿体基因工程还存在同质化难度高、标记基因转化效率较低、宿主种类偏少等问题,但作为外源基因在高等植物中表达的良好平台其仍然具有广阔的发展和应用前景。     

Antibiotic-free chloroplast genetic engineering - an environmentally friendly approach

Chloroplast genetic engineering offers several advantages over nuclear genetic engineering, including gene containment and hyperexpression. However, introducing thousands of copies of transgenes into the chloroplast genome amplifies the antibiotic resistance genes. Two recent articles report different and novel strategies to either remove antibiotic resistance genes or select chloroplast transformants without using these genes. This should eliminate their potential transfer to microorganisms or plants and ease public concerns about genetically modified crops.     

Method to assemble and integrate biochemical pathways into the chloroplast genome of Chlamydomonas reinhardtii

Recombinant protein expression in the chloroplasts of green algae has recently become more routine; however, the heterologous expression of multiple proteins or complete biosynthetic pathways remains a significant challenge. Here, we show that a modified DNA Assembler approach can be used to rapidly assemble multiple‐gene biosynthetic pathways in yeast and then integrate these assembled pathways at a site‐specific location in the chloroplast genome of the microalgal species Chlamydomonas reinhardtii. As a proof of concept, this method was used to successfully integrate and functionally express up to three reporter proteins (AphA6, AadA, and GFP) in the chloroplast of C. reinhardtii. An analysis of the relative gene expression of the engineered strains showed significant differences in the mRNA expression levels of the reporter genes and thus highlights the importance of proper promoter/untranslated region selection when constructing a target pathway. This new method represents a useful genetic tool in the construction and integration of complex biochemical pathways into the chloroplast genome of microalgae and should aid current efforts to engineer algae for biofuels production and other desirable natural products. Biotechnol. Bioeng. 2012; 109: 2896–2903. © 2012 Wiley Periodicals, Inc.     

Manipulating Fatty Acid Biosynthesis in Microalgae for Biofuel through Protein-Protein Interactions

Microalgae are a promising feedstock for renewable fuels, and algal metabolic engineering can lead to crop improvement, thus accelerating the development of commercially viable biodiesel production from algae biomass. We demonstrate that protein-protein interactions between the fatty acid acyl carrier protein (ACP) and thioesterase (TE) govern fatty acid hydrolysis within the algal chloroplast. Using green microalga Chlamydomonas reinhardtii (Cr) as a model, a structural simulation of docking CrACP to CrTE identifies a protein-protein recognition surface between the two domains. A virtual screen reveals plant TEs with similar in silico binding to CrACP. Employing an activity-based crosslinking probe designed to selectively trap transient protein-protein interactions between the TE and ACP, we demonstrate in vitro that CrTE must functionally interact with CrACP to release fatty acids, while TEs of vascular plants show no mechanistic crosslinking to CrACP. This is recapitulated in vivo, where overproduction of the endogenous CrTE increased levels of short-chain fatty acids and engineering plant TEs into the C. reinhardtii chloroplast did not alter the fatty acid profile. These findings highlight the critical role of protein-protein interactions in manipulating fatty acid biosynthesis for algae biofuel engineering as illuminated by activity-based probes.     

Fifty million years of herbivory on coral reefs: fossils,fish and functional innovations

The evolution of ecological processes on coral reefs was examined based on Eocene fossil fishes from Monte Bolca, Italy and extant species from the Great Barrier Reef, Australia. Using ecologically relevant morphological metrics, we investigated the evolution of herbivory in surgeonfishes (Acanthuridae) and rabbitfishes (Siganidae). Eocene and Recent surgeonfishes showed remarkable similarities, with grazers, browsers and even specialized, long-snouted forms having Eocene analogues. These long-snouted Eocene species were probably pair-forming, crevice-feeding forms like their Recent counterparts. Although Eocene surgeonfishes likely played a critical role as herbivores during the origins of modern coral reefs, they lacked the novel morphologies seen in modern Acanthurus and Siganus (including eyes positioned high above their low-set mouths). Today, these forms dominate coral reefs in both abundance and species richness and are associated with feeding on shallow, exposed algal turfs. The radiation of these new forms, and their expansion into new habitats in the Oligocene–Miocene, reflects the second phase in the development of fish herbivory on coral reefs that is closely associated with the exploitation of highly productive short algal turfs.     

Involvement of host regulatory pathways during geminivirus infection: a novel platform for generating durable resistance

Geminiviruses are widely distributed throughout the world and cause devastating yield losses in almost all the economically important crops. In this review, the newly identified roles of various novel plant factors and pathways participating in plant–virus interaction are summarized with a particular focus on the exploitation of various pathways involving ubiquitin/26S proteasome pathway, small RNA pathways, cell division cycle components, and the epigenetic mechanism as defense responses during plant–pathogen interactions. Capturing the information on these pathways for the development of strategies against geminivirus infection is argued to provide the basis for new genetic approaches to resistance.     

叶绿体转化及其用于疫苗表达研究的最新进展

随着植物转基因研究的不断深入,核基因组转化的转基因沉默现象严重影响了基因工程的应用效果。植物叶绿体遗传转化以叶绿体基因组为平台对植物进行遗传操作,外源基因定点整合及母性遗传特性能较好地解决"顺式失活"和"位置效应"等类的基因沉默问题和转基因逃逸等安全问题,成为植物基因工程发展的新方向,在工业、农业及医药生物领域发挥了重要作用,也为生产廉价、安全的植物疫苗提供了新思路。本文在简要介绍叶绿体转化的原理、转化方法与优势的基础上,重点综述了近年来通过该技术表达的一些重要的病毒抗原和细菌抗原。最后,对叶绿体转化技术在表达外源基因方面存在的问题进行分析。未来随着叶绿体基因表达、调控机制研究的逐渐深入及相关技术体系的日臻完善,叶绿体转化有望成为疫苗生产的生力军。     

Technical guide for genetic advancement of underdeveloped and intractable Clostridium

In recent years, the genus Clostridium has risen to the forefront of both medical biotechnology and industrial biotechnology owing to its potential in applications as diverse as anticancer therapy and production of commodity chemicals and biofuels. The prevalence of hyper-virulent strains of C. difficile within medical institutions has also led to a global epidemic that demands a more thorough understanding of clostridial genetics, physiology, and pathogenicity. Unfortunately, Clostridium suffers from a lack of sophisticated genetic tools and techniques which has hindered the biotechnological exploitation of this important bacterial genus. This review provides a comprehensive summary of biotechnological progress made in clostridial genetic tool development, while also aiming to serve as a technical guide for the advancement of underdeveloped clostridial strains, including recalcitrant species, novel environmental samples, and non-type strains. Relevant strain engineering techniques, from genome sequencing and establishment of a gene transfer methodology through to deployment of advanced genome editing procedures, are discussed in detail to provide a blueprint for future clostridial strain construction endeavors. It is expected that a more thorough and rounded-out genetic toolkit available for use in the clostridia will bring about the construction of superior bioprocessing strains and a more complete understanding of clostridial genetics, physiology, and pathogenicity.     

Genome complexity of harmful microalgae

During the past decade, next generation sequencing (NGS) technologies have provided new insights into the diversity, dynamics, and metabolic pathways of natural microbial communities. But, these new techniques face challenges related to the genome size and level of genome complexity of the species under investigation. Moreover, the coverage depth and the short-read length achieved by NGS based approaches also represent a major challenge for assembly. These factors could limit the use of these high-throughput sequencing methods for species lacking a reference genome and characterized by a high level of complexity. In the present work, the evolutionary history, mainly consisting of gene transfer events from bacteria and unicellular eukaryotes to microalgae, including harmful species, is discussed and reviewed as it relates to NGS application in microbial communities, with a particular focus on harmful algal bloom species and dinoflagellates. In the context of genetic population studies, genotyping-by-sequencing (GBS), an NGS based approach, could be used for the discovery and analysis of single nucleotide polymorphisms (SNPs). The NGS technologies are still relatively new and require further improvement. Specifically, there is a need to develop and standardize tools and approaches to handle large data sets, which have to be used for the majority of HAB species characterized by evolutionary highly dynamic genomes.