Genetic improvement of the microalga Phaeodactylum tricornutum for boosting neutral lipid accumulation.

In order to realize the potential of microalgal biodiesel there is a need for substantial impetus involving interventions to radically improve lipid yields upstream. Nutrient stress and alteration to cultivation conditions are commonly used lipid enhancement strategies in microalgae. The main bottleneck of applying conventional strategies is their scalability as some of these strategies incur additional cost and energy. Novel lipid enhancement strategies have emerged to research forefront to overcome these challenges. In this review, the latest trends in microalgal lipid enhancement strategies, possible solutions and future directions are critically discussed. Advanced strategies such as combined nutrient and cultivation condition stress, microalgae–bacteria interactions, use of phytohormones EDTA and chemical additives, improving light conditions using LED, dyes and paints, and gene expression analysis are described. Molecular approaches such as metabolic and genetic engineering are emerging as the potential lipid enhancing strategies. Recent advancements in gene expression studies, genetic and metabolic engineering have shown promising results in enhancing lipid productivity in microalgae; however environmental risk and long term viability are still major challenges.

https://openscholar.dut.ac.za/bitstream/10321/2245/1/Singh_RSER_Vol55_Pg1-16_2016.pdf

Trends and novel strategies for enhancing lipid accumulation and quality in microalgae

Microalgae have been emerging as an important source for the production of bioactive compounds. Marine diatoms can store high amounts of lipid and grow quite quickly. However, the genetic and biochemical characteristics of fatty acid biosynthesis in diatoms remain unclear. Glycerophospholipids are integral as structural and functional components of cellular membranes, as well as precursors of various lipid mediators. In addition, diacylglycerol acyltransferase (DGAT) is a key enzyme that catalyzes the last step of triacylglyceride (TAG) biosynthesis. However, a comprehensive sequence-structure and functional analysis of DGAT in diatoms is lacking. In this study, an isoform of diacylglycerol acyltransferase type 2 of the marine diatom Phaeodactylum tricornutum was characterized. Surprisingly, DGAT2 overexpression in P. tricornutum stimulated more oil bodies, and the neutral lipid content increased by 35%. The fatty acid composition showed a significant increase in the proportion of polyunsaturated fatty acids; in particular, EPA was increased by 76.2%. Moreover, the growth rate of transgenic microalgae remained similar, thereby maintaining a high biomass. Our results suggest that increased DGAT2 expression could alter fatty acid profile in the diatom, and the results thus represent a valuable strategy for polyunsaturated fatty acid production by genetic manipulation.

https://www.mdpi.com/1660-3397/11/11/4558/pdf

Improvement of neutral lipid and polyunsaturated fatty acid biosynthesis by overexpressing a type 2 diacylglycerol acyltransferase in marine diatom Phaeodactylum tricornutum.

Microalgae comprise a biodiverse group of photosynthetic organisms that reside in water sources and sediments. The green microalgae Chlamydomonas reinhardtii was adopted as a useful model organism for studying various physiological systems. Its ability to grow under both photosynthetic and heterotrophic conditions allows efficient growth of non-photosynthetic mutants, making Chlamydomonas a useful genetic tool to study photosynthesis. In addition, this green alga can grow as haploid or diploid cells, similar to yeast, providing a powerful genetic system. As a result, easy and efficient transformation systems have been developed for Chlamydomonas, targeting both the chloroplast and nuclear genomes. Since microalgae comprise a rich repertoire of species that offer variable advantages for biotech and biomed industries, gene transfer technologies were further developed for many microalgae to allow for the expression of foreign proteins of interest. Expressing foreign genes in the chloroplast enables the targeting of foreign DNA to specific sites by homologous recombination. Chloroplast transformation also allows for the introduction of genes encoding several enzymes from a complex pathway, possibly as an operon. Expressing foreign proteins in the chloroplast can also be achieved by introducing the target gene into the nuclear genome, with the protein product bearing a targeting signal that directs import of the transgene-product into the chloroplast, like other endogenous chloroplast proteins. Integration of foreign genes into the nuclear genome is mostly random, resulting in large variability between different clones, such that extensive screening is required. The use of different selection modalities is also described, with special emphasis on the use of herbicides and metabolic markers which are considered to be friendly to the environment, as compared to drug-resistance genes that are commonly used. Finally, despite the development of a wide range of transformation tools and approaches, expression of foreign genes in microalgae suffers from low efficiency. Thus, novel tools have appeared in recent years to deal with this problem. Finally, while Chlamydomonas reinhardtii was traditionally used as a model organism for the development of transformation systems and their subsequent improvement, similar technologies can be adapted for other microalgae that may have higher biotechnological value.

/pdf/transgene-expression-in-microalgae-from-tools-to-1xyfvisy6k.pdf

Transgene Expression in Microalgae-From Tools to Applications.

In the wake of the uprising global energy crisis, microalgae have emerged as an alternate feedstock for biofuel production. In addition, microalgae bear immense potential as bio-cell factories in terms of producing key chemicals, recombinant proteins, enzymes, lipid, hydrogen and alcohol. Abstraction of such high-value products (algal biorefinery approach) facilitates to make microalgae-based renewable energy an economically viable option. Synthetic biology is an emerging field that harmoniously blends science and engineering to help design and construct novel biological systems, with an aim to achieve rationally formulated objectives. However, resources and tools used for such nuclear manipulation, construction of synthetic gene network and genome-scale reconstruction of microalgae are limited. Herein, we present recent developments in the upcoming field of microalgae employed as a model system for synthetic biology applications and highlight the importance of genome-scale reconstruction models and kinetic models, to maximize the metabolic output by understanding the intricacies of algal growth. This review also examines the role played by microalgae as biorefineries, microalgal culture conditions and various operating parameters that need to be optimized to yield biofuel that can be economically competitive with fossil fuels.

/pdf/recent-developments-in-synthetic-biology-and-metabolic-bypjlftmod.pdf

Recent developments in synthetic biology and metabolic engineering in microalgae towards biofuel production

Diatoms are important primary producers in the marine ecosystem. Currently it is difficult to genetically transform diatoms due to the technical limitations of existing methods. The promoter/terminator of the nitrate reductase gene of the model diatom Phaeodactylum tricornutum was cloned and used to drive chloramphenicol acetyltransferase (CAT) reporter gene expression. The construct was transferred by electroporation into P. tricornutum grown in medium lacking silicon. CAT expression was induced in transformed diatoms in the presence of nitrate, enabling growth in selective medium, and was repressed when ammonium was the only nitrogen source. Expression of CAT transcript and protein were demonstrated by RT-PCR and Western blot analysis, respectively. Our study is the first to report a successful genetic transformation of diatom by electroporation in an economical and efficient manner and provides a tightly regulated inducible gene expression system for diatom.

Transformation of diatom Phaeodactylum tricornutum by electroporation and establishment of inducible selection marker.

Plastids are ideal subcellular hosts for the expression of transgenes and have been successfully used for the production of different biopolymers, therapeutic proteins and industrial enzymes. Phaeodactylum tricornutum is a widely used aquatic feed species. In this study, we focused on developing a high-efficiency plastid expression system for P. tricornutum. In the plastid transformation vector, the site selected for integration was the transcriptionally active intergenic region present between the trnI and trnA genes, located in the IR (inverted repeat) regions of the plastid genome. Initially, a CAT reporter gene (encoding chloramphenicol acetyltransferase) was integrated at this site in the plastid genome. The expression of CAT in the transformed microalgae conferred resistance to the antibiotic chloramphenicol, which enabled growth in the selection media. Overall, the plastid transformation efficiency was found to be approximately one transplastomic colony per 1,000 microalgae cells. Subsequently, a heterologous gene expression cassette for high-level expression of the target gene was created and cloned between the homologous recombination elements. A TA cloning strategy based on the designed XcmI-XcmI sites could conveniently clone the heterologous gene. An eGFP (green fluorescent protein) reporter gene was used to test the expression level in the plastid system. The relatively high-level expression of eGFP without codon optimisation in stably transformed microalgae was determined to account for 0.12 % of the total soluble protein. Thus, this study presents the first and convenient plastid gene expression system for diatoms and represents an interesting tool to study diatom plastids.

https://link.springer.com/content/pdf/10.1007%2Fs10126-014-9570-3.pdf

Construction of Novel Chloroplast Expression Vector and Development of an Efficient Transformation System for the Diatom Phaeodactylum tricornutum

The invention discloses a marine microalgae chloroplast expression vector and application thereof. The marine microalgae chloroplast expression vector contains a homologous recombinant sequence I, a target gene expression case and a homologous recombinant sequence II, wherein the target gene expression case is positioned between the homologous recombinant sequence I and the homologous recombinant sequence II; 1st to 972nd bp of the homologous recombinant sequence I are 3' part of rns, and 973rd to 1,097th bp are 5' part of trnI; and 1st to 196th bp of the homologous recombinant sequence II are 3' part of trnA, and 197th to 1,483rd bp are 5' part of rnl. The homologous recombinant sequence ensures that the expression vector can be induced into the marine microalgae chloroplast genome by an electric shock method, which is advantageous to the way of inducing the vector into the chloroplast genome, greatly reduces conversion cost, and provides technical support for realizing that marine microalgae chloroplast is used as a bioreactor to produce high value-added products.

Cong-Cong Zhu

Papers

Transformation of diatom Phaeodactylum tricornutum by electroporation and establishment of inducible selection marker.

Construction of Novel Chloroplast Expression Vector and Development of an Efficient Transformation System for the Diatom Phaeodactylum tricornutum

Marine microalgae chloroplast expression vector and application thereof

Microalgae chloroplast carrier for efficient cloning and expression and application thereof