Catálogo de publicaciones - libros

Transformation of microalgae is a first step in their use for biotechnological applications involving foreign protein production or molecular modifications of specific cell metabolic pathways. Since the first reliable achievements of nuclear transformation in , other eukaryotic microalgae have become transformed with molecular markers that allow a direct selection. Different methods—glass beads, electroporation, particle bombardment, or Agrobacterium—and constructions have been set up in several organisms and successfully used. However, some problems associated with efficiency, integration, or stability of the transgenes still persist and are analysed herein. Though the number of microalgae species successfully transformed is not very high, prospects for transformation of many more are good enough on the basis of what has been achieved so far.

Cyanobacteria are a diverse and successful group of bacteria defined by their ability to carry out oxygenic photosynthesis. They occupy diverse ecological niches and are important primary producers in the oceans. Cyanobacteria are amenable to genetic manipulation. Some strains are naturally transformable. Many others have been transformed in the lab by conjugation or electroporation. The ability to transform cyanobacteria has been determinant in the development of the molecular biology of these organisms and has been the basis of many of their biotechnological applications. Cyanobacteria are the source of natural products and toxins of potential use and can be engineered to synthesize substances of biotechnological interest. Their high protein and vitamin content makes them useful as a dietary supplement. Because of their ability to occupy diverse ecological niches, they can be used to deliver to the medium substances of interest or as biosensors.

Diatoms are unicellular photoautotrophic eukaryotes that play an important role in ecology by fixing large amounts of CO in the oceans. Because they evolved by secondary endocytobiosis—a process of uptake of a eukaryotic alga into another eukaryotic cell—they have a rather unusual cell biology and genetic constitution. Diatoms are also of biotechnological interest since they produce highly unsaturated fatty acids. In addition they are able to form delicately ornate cell walls made of amorphous silica. Understanding and modifying the processes of biomineralization in diatoms might result in new nanotechnological processes. Therefore recent advances in molecular genomics and the development of genetic tools for diatoms might pave the way for biotechnological modification and utilization of diatoms. In this chapter I will briefly characterize these extraordinary organisms, give some insights into the actual advances in molecular biology of diatoms and present some examples for the potential future use of diatoms in algal biotechnology.

The chloroplast organelle of plant and algal cells contains its own genetic system with a genome of a hundred or so genes. Stable transformation of the chloroplast was first achieved in 1988, using the newly developed biolistic method of DNA delivery to introduce cloned DNA into the genome of the green unicellular alga . Since that time there have been significant developments in chloroplast genetic engineering using this versatile organism, and it is probable that the next few years will see increasing interest in commercial applications whereby high-value therapeutic proteins and other recombinant products are synthesized in the chloroplast. In this chapter I review the basic methodology of chloroplast transformation, the current techniques and applications, and the future possibilities for using the chloroplast as a green organelle factory.

The expression of functional proteins in heterologous hosts is a core technique of modern biotechnology. The transfer to a suitable expression system is not always achieved easily because of several reasons: genes from different origins might contain codons that are rarely used in the desired host or even bear noncanonical codons, or the genes might hide expression-limiting regulatory elements within their coding sequence. These problems can also be observed when introducing foreign genes into genomes of microalgae as described in a growing number of detailed studies on transgene expression in these organisms. Particularly important for the use of algae as photosynthetic cell factories is a fundamental understanding of the influence of a foreign gene’s codon composition on its expression efficiency. Therefore, the effect of codon usage of a chimeric protein on expression frequency and product accumulation in the green alga was analyzed. This fusion protein combines a constant region encoding the zeocin binding protein Ble with two different gene variants for the green fluorescent protein (GFP). It is shown that codon bias significantly affects the expression, but barely influences the final protein accumulation in this case.

Algae are dominant primary producers on the Earth and have a major impact on global productivity and biogeochemical cycling. There are still few algal genomes that have been completely characterized, and resources directed toward algal genomic sequencing are limited. However, it is also becoming evident that algae and prokaryotic picoplankton have a critical role in the fixation and sequestration of carbon, and so the interest in algal genomics is expanding. There are some algae for which full or near-full genome sequences have been secured; these genomes include those of the red alga , the green algae or chlorophytes and , the marine picoeukaryote (two different strains of have been sequenced), the diatoms and , and the haptophyte . There is also a full sequence for the vestigal ‘red’ algal genome of the nucleomorph of the Cyptomonad . In addition, numerous genomes of photosynthetic microbes, including marine and species have been sequenced. There have also been projects developed to define algal transcriptomes as determined by cDNA analysis, full genome sequences of numerous plastids, and the genomes of a variety of viruses that infect marine and freshwater algae. The recent efforts focused on acquiring and analyzing algal genome sequences have generated an influx of exciting data to a field that is in its infancy. In this review I discuss potential criteria for determining which organisms should be targeted for genome projects, successful forays into algal genomic sequencing, and some of the inferences generated from the analysis of the sequence information.

The unicellular alga has emerged during the last decades as a model system to understand gene functions, many of them shared by bacteria, fungi, plants, animals and humans. A powerful resource for the research community is the availability of complete collections of stable mutants for studying whole genome function. In the meantime other strategies might be developed; insertional mutagenesis has become currently the best strategy to disrupt and tag nuclear genes in allowing forward and reverse genetic approaches. Here, we outline the mutagenesis technique stressing the idea of generating databases for ordered mutant libraries, and also of improving efficient methods for reverse genetics to identify mutants defective in a particular gene.

Through advances in molecular and genetic techniques, protein expression in the chloroplasts of green algae has been optimized for high-level expression. Recombinant proteins expressed in algae have the potential to provide novel and safe treatment of disease and infection where current, high-cost drugs are the only option, or worse, where therapeutic drugs are not available due to their prohibitively high-cost to manufacture. Optimization of recombinant protein expression in chloroplasts has been accomplished by employing chloroplast codon bias and combinatorial examination of promoter and UTR combinations. In addition, as displayed by the expression of an anti-herpes antibody, the chloroplast is capable of correctly folding and assembling complex mammalian proteins. These data establish algal chloroplasts as a system for the production of complex human therapeutic proteins in soluble and active form, and at significandy reduced time and cost compared to existing production systems. Production of recombinant proteins in algal chloroplasts may enable further development of safe, efficacious and cost-effective protein therapeutics.

Microalgae account for most of the biologically sequestered trace metals in aquatic environments. Their ability to adsorb and metabolize trace metals is associated with their large surface:volume ratios, the presence of high-affinity, metal-binding groups on their cell surfaces, and efficient metal uptake and storage systems. Microalgae may bind up to 10% of their biomass as metals. In addition to essential trace metals required for metabolism, microalgae can efficiently sequester toxic heavy metals. Toxic heavy metals often compete with essential trace metals for binding to and uptake into cells. Recently, transgenic approaches have been developed to further enhance the heavy metal specificity and binding capacity of microalgae with the objective of using these microalgae for the treatment of heavy metal contaminated wastewaters and sediments. These transgenic strategies have included the over expression of enzymes whose metabolic products ameliorate the effects of heavy metal-induced stress, and the expression of high-affinity, heavy metal binding proteins on the surface and in the cytoplasm of transgenic cells. The most effective strategies have substantially reduced the toxicity of heavy metals allowing transgenic cells to grow at wild-type rates in the presence of lethal concentrations of heavy metals. In addition, the metal binding capacity of transgenic algae has been increased five-fold relative to wild-type cells. Recently, fluorescent heavy metal biosensors have been developed for expression in transgenic Chlamydomonas. These fluorescent biosensor strains can be used for the detection and quantification of bioavailable heavy metals in aquatic environments. The use of transgenic microalgae to monitor and remediate heavy metals in aquatic environments is not without risk, however. Strategies to prevent the release of live microalgae having enhanced metal binding properties are described.

This chapter summarizes the state-of-art in the field of green algal H-production and examines physiological and genetic engineering approaches by which to improve the hydrogen metabolism characteristics of these microalgae. Included in this chapter are emerging topics pertaining to the application of sulfur-nutrient deprivation to attenuate O-evolution and to promote H-production, as well as the genetic engineering of sulfate uptake through manipulation of a newly reported sulfate permease in the chloroplast of the model green alga . Application of the green algal hydrogenase assembly genes is examined in efforts to confer H-production capacity to other commercially significant unicellular green algae. Engineering a solution to the O sensitivity of the green algal hydrogenase is discussed as an alternative approach to sulfur nutrient deprivation, along with starch accumulation in microalgae for enhanced H-production. Lastly, current efforts aiming to optimize light utilization in transgenic microalgae for enhanced H-production under mass culture conditions are presented. It is evident that application of genetic engineering technologies and the use of transgenic green algae will improve prospects for commercial exploitation of these photosynthetic micro-organisms in the generation of H, a clean and renewable fuel.