Genetic Engineering Of Microalgae Research Articles

Advantages of Heterotrophic Microalgae as a Host for Phytochemicals Production.

Currently, most commercial recombinant technologies rely on host systems. However, each host has their own benefits and drawbacks, depending on the target products. Prokaryote host is lack of post-transcriptional and post-translational mechanisms, making them unsuitable for eukaryotic productions like phytochemicals. Even there are other eukaryote hosts (e.g., transgenic animals, mammalian cell, and transgenic plants), but those hosts have some limitations, such as low yield, high cost, time consuming, virus contamination, and so on. Thus, flexible platforms and efficient methods that can produced phytochemicals are required. The use of heterotrophic microalgae as a host system is interesting because it possibly overcome those obstacles. This paper presents a comprehensive review of heterotrophic microalgal expression host including advantages of heterotrophic microalgae as a host, genetic engineering of microalgae, genetic transformation of microalgae, microalgal engineering for phytochemicals production, challenges of microalgal hosts, key market trends, and future view. Finally, this review might be a directions of the alternative microalgae host for high-value phytochemicals production in the next few years.

Advances and challenges in genetic engineering of microalgae

AbstractKnowledge at the molecular level of microalgae has obtained great scientific interest in recent years, because of their potential for biofuel production, as well as other highly valued molecules. Recent advances in the area of the genetic manipulation of microalgae open an entirely new field of possible applications for these organisms as biorefineries. This review represents a compendium that gathers the main milestones that have marked the area of genetic engineering in microalgae during the last decade, from classical techniques for the transformation and expression of transgenes, to the most avant‐garde techniques currently used for gene silencing and DNA edition, as well as a discussion about the future perspectives in this field.

Agrobacterium-mediated genetic transformation of Dictyosphaerium pulchellum for the expression of erythropoietin

Recombinant proteins are extensively used for a growing number of fields in biology. However, microalgal species have not been widely adopted as cell factories for recombinant protein production. Unique metabolic properties, ease of cultivation, fast growth rates, and continuous progress in genetic engineering of microalgae have raised interest in the use of microalgae species for recombinant protein production. Here, we report an Agrobacterium-mediated genetic transformation system for the heterologous expression of a therapeutic protein, “erythropoietin,” in a nonmodel green microalga, Dictyosphaerium pulchellum. Hygromycin resistance gene (Hyg) was used as a selectable marker. The genetic transformation of D. pulchellum was performed in modified AF6 medium supplemented with 150 μM acetosyringone, co-cultivated for 48 h at 25 ± 2 °C and a light intensity of 18 ± 2 μmol photons m−2 s−1. Co-cultivation of D. pulchellum with Agrobacterium tumefaciens harboring the binary expression vector pCAMBIA1301-Hyg-EPO-Histag yielded hygromycin-resistant colonies on selective medium after 2–3 weeks. Gene integration into the D. pulchellum nuclear genome was confirmed by PCR amplification of T-DNA from the genomic DNA of hygromycin-resistant and wild-type strains. Interestingly, SDS-PAGE and subsequent Western blotting with His-tag monoclonal and anti-erythropoietin monoclonal antibodies revealed an EPO-specific signal slightly below 34 kDa. Furthermore, EPO gene expression and transgene copy numbers were estimated by quantitative real-time PCR. Approximately, 500 μg L−1 of extracellular recombinant erythropoietin protein was purified by His-tag affinity chromatography. The developed genetic transformation system would allow the metabolic engineering and a better alternative to produce recombinant therapeutic proteins from nonmodel freshwater microalgae species.

Advanced multigene expression system for Nannochloropsis salina using 2A self-cleaving peptides

Even though there has been much interest in genetic engineering of microalgae, its progress has been slow due to the difficulty and limitation of available techniques. Currently, genetic modification in most microalgal strains is confined to single gene transformation. Here, a multigene expression system for the oleaginous model strain Nannochloropsis salina was developed with glycine-serine-glycine spacer linked 2A self-cleaving peptides (2A) for the first time. An efficiency test of the four most widely used 2As revealed that two different types of 2As T2A and E2A have the best performance in N. salina with a maximum cleavage rate of nearly 45%. The system was able to express the linked sequence of the selection marker shble and the fluorescence protein sfCherry with intact functions. Because 2A enabled multigene expression in the single cassette form, the use of 2A also reduced the vector size, which along with the stronger promoter resulted in a 9-fold increase in the transformation efficiency. Furthermore, confirmative screening accuracy of more than 90% was observed. Hence, the 2A applied vector system is expected to be beneficial in microalgal research field because it enables multigene expression as well as offering improved transformation and screening efficiency.

Development of an alcohol-inducible gene expression system for recombinant protein expression in Chlamydomonas reinhardtii

Microalgae have been widely considered for the production of valuable products, such as lipid-based biofuel, value-added pigments, and anti-photo aging reagents. More recently, microalgae have been considered an alternative host for recombinant protein production because of their economic benefits and ecofriendly characteristics. Additionally, many microalgal strains identified to date are generally recognized as safe (GRAS); therefore, the use of microalgae-based technology is promising. However, basic studies on the genetic engineering of microalgae are rare, despite their importance. Particularly, inducible promoter systems that can be applied for strain engineering or recombinant protein production are rarely studied; hence, a number of challenging issues remain unsolved. Therefore, in this study, we focused on the development of a convenient and compact-inducible promoter system that can be used in microalgae. Based on previous success with plant systems, we employed the alcohol-inducible AlcR-PalcA system, which originates from the filamentous fungus, Aspergillus nidulans. This system comprises only two components, a regulatory protein, AlcR, and an inducible promoter, PalcA. Therefore, construction and transformation of the gene cassettes can be easily performed. Ethanol-dependent gene expression was observed in the transformants with no significant growth retardation or inducer consumption observed in the cells cultivated under optimized conditions.

Enhancement of biomass and lipid productivity by overexpression of a bZIP transcription factor in Nannochloropsis salina

Microalgae are considered as excellent platforms for biomaterial production that can replace conventional fossil fuel-based fuels and chemicals. Genetic engineering of microalgae is prerequisite to maximize production of materials and to reduce costs for the production. Transcription factors (TFs) are emerging as key regulators of metabolic pathways to enhance production of molecules for biofuels and other materials. TFs with the basic leucine zipper (bZIP) domain have been known as stress regulators and are associated with lipid metabolism in plants. We overexpressed a bZIP TF, NsbZIP1, in Nannochloropsis salina, and found that transformants showed enhanced growth with concomitant increase in lipid contents. The improved phenotypes were also notable under stress conditions including N limitation and high salt. To understand the mechanism underlying improved phenotypes, we analyzed expression patterns of predicted target genes involved in lipid metabolism via quantitative RT-PCR, confirming increases transcript levels. NsbZIP1 appeared to be one of type C bZIPs in plants that has been known to regulate lipid metabolism under stress. Taken together, we demonstrated that NsbZIP1 could improve both growth and lipid production, and TF engineering can serve as an excellent genetic engineering tool for production of biofuels and biomaterials in microalgae.

Microalgae engineering toolbox: Selectable and screenable markers

Engineering microalgae has opened a new era for plant biologists and biotechnologists. Microalgae had been proved as a promising candidate for the production of biopharmaceuticals, nutraceuticals, antioxidants, antimicrobial and antiviral compounds, in dyeing and food industry as well for biofuel production. Genetic transformation of some important microalgae has been successful, but several other potential microalgae species still need scientific attention. The success of the genetic transformation depends mainly on the utilization of the selectable and screenable markers. Like for other higher crop plants, several useful markers have been reported for microalgae transformation. In this follow-up, we compared different marker genes for genetic engineering of approximately all the industrially important microalgae. We have discussed the expression host, the targeted genome, appropriate selection agent, as well as the transformation method. Genetic transformation is an expensive and labor intensive process and this review will aid to shorten the time span by providing a database of appropriate markers for microalgae research which could serve as a guide for those involved in the genetic engineering of microalgae.

Molecular structure of photosynthetic microbial biofuels for improved engine combustion and emissions characteristics.

The metabolic engineering of photosynthetic microbes for production of novel hydrocarbons presents an opportunity for development of advanced designer biofuels. These can be significantly more sustainable, throughout the production-to-consumption lifecycle, than the fossil fuels and crop-based biofuels they might replace. Current biofuels, such as bioethanol and fatty acid methyl esters, have been developed primarily as drop-in replacements for existing fossil fuels, based on their physical properties and autoignition characteristics under specific combustion regimes. However, advances in the genetic engineering of microalgae and cyanobacteria, and the application of synthetic biology approaches offer the potential of designer strains capable of producing hydrocarbons and oxygenates with specific molecular structures. Furthermore, these fuel molecules can be designed for higher efficiency of energy release and lower exhaust emissions during combustion. This paper presents a review of potential fuel molecules from photosynthetic microbes and the performance of these possible fuels in modern internal combustion engines, highlighting which modifications to the molecular structure of such fuels may enhance their suitability for specific combustion regimes.

Gateway-Assisted Vector Construction to Facilitate Expression of Foreign Proteins in the Chloroplast of Single Celled Algae

With a rising world population, demand will increase for food, energy and high value products. Renewable production systems, including photosynthetic microalgal biotechnologies, can produce biomass for foods, fuels and chemical feedstocks and in parallel allow the production of high value protein products, including recombinant proteins. Such high value recombinant proteins offer important economic benefits during startup of industrial scale algal biomass and biofuel production systems, but the limited markets for individual recombinant proteins will require a high throughput pipeline for cloning and expression in microalgae, which is currently lacking, since genetic engineering of microalgae is currently complex and laborious. We have introduced the recombination based Gateway® system into the construction process of chloroplast transformation vectors for microalgae. This simplifies the vector construction and allows easy, fast and flexible vector design for the high efficiency protein production in microalgae, a key step in developing such expression pipelines.