Evaluation of novel starch-deficient mutants of Chlorella sorokiniana for hyper-accumulation of lipids

When green algae are exposed to physiological stresses such as nutrient deprivation, growth is arrested and the cells channel fixed carbon instead into storage compounds, accumulating first starch granules and then lipid bodies containing triacylglycerides. In recent years there has been significant interest in the commercial exploitation of algal lipids as a sustainable source of biodiesel. Since starch and lipid biosynthesis involves the same C3 precursor pool, it has been proposed that mutations blocking starch accumulation should result in increased lipid yields, and indeed several studies have supported this. The fast-growing, thermotolerant alga Chlorella sorokiniana represents an attractive strain for industrial cultivation. We have therefore generated and characterized starch-deficient mutants of C. sorokiniana and determined whether lipid levels are increased in these strains under stress conditions. One mutant (ST68) is shown to lack isoamylase, whilst two others (ST3 and ST12) are defective in starch phosphorylase. However, we find no significant change in the accumulation or profile of fatty acids in these mutants compared to the wild-type, suggesting that a failure to accumulate starch per se is not sufficient for the hyper-accumulation of lipid, and that more subtle regulatory steps underlie the partitioning of carbon to the two storage products

The chloroplast of chlamydomonas reinhardtii as a testbed for engineering nitrogen fixation into plants

Eukaryotic organisms such as plants are unable to utilise nitrogen gas (N2) directly as a source of this essential element and are dependent either on its biological conversion to ammonium by diazotrophic prokaryotes, or its supply as chemically synthesised nitrate fertiliser. The idea of genetically engineering crops with the capacity to fix N2 by introduction of the bacterial nitrogenase enzyme has long been discussed. However, the expression of an active nitrogenase must overcome several major challenges: the coordinated expression of multiple genes to assemble an enzyme complex containing several different metal cluster co-factors; the supply of sufficient ATP and reductant to the enzyme; the enzyme’s sensitivity to oxygen; and the intracellular accumulation of ammonium. The chloroplast of plant cells represents an attractive location for nitrogenase expression, but engineering the organelle’s genome is not yet feasible in most crop species. However, the unicellular green alga Chlamydomonas reinhardtii represents a simple model for photosynthetic eukaryotes with a genetically tractable chloroplast. In this review, we discuss the main advantages, and limitations, of this microalga as a testbed for producing such a complex multi-subunit enzyme. Furthermore, we suggest that a minimal set of six transgenes are necessary for chloroplast-localised synthesis of an ‘Fe-only’ nitrogenase, and from this set we demonstrate the stable expression and accumulation of the homocitrate synthase, NifV, under aerobic conditions. Arguably, further studies in C. reinhardtii aimed at testing expression and function of the full gene set would provide the groundwork for a concerted future effort to create nitrogen-fixing crops

Cyanobacterial metabolites as a source of sunscreens and moisturizers: a comparison with current synthetic compounds

The recognition that ultraviolet radiation has harmful effects on the skin has led to the commercial development of inorganic and synthetic organic UV filters that can reduce the negative effects of exposure to sunlight. In addition, moisturizing chemicals are extensively used in personal care products to improve the ability of skin to retain water. Whilst current UV filter and moisturizing chemicals have clear beneficial qualities, they may also have adverse effects such as contact sensitivity, oestrogenicity and even tumorigenic effects on human skin. Furthermore, the accumulation of these chemicals in the aquatic environment could be potentially harmful. Consequently, there is interest in exploiting safer alternatives derived from biological sources, especially from photosynthetic organisms such as cyanobacteria which have developed mechanisms for coping with high UV irradiation and desiccation. In order to overcome the detrimental effects of UV radiation, these microorganisms produce UV screening compounds such as mycosporine-like amino acids and scytonemin, which are good candidates as alternatives to current synthetic UV filters. In addition, extracellular substances produced by some extremophilic species living in hyper-arid habitats have a high water retention capacity and could be used in cosmetic products as moisturizers. In this review, we present an overview of the literature describing the potential of cyanobacterial metabolites as an alternative source for sunscreens and moisturizers

Applications of Microalgal Biotechnology for Disease Control in Aquaculture

Aquaculture industries, and in particular the farming of fish and crustaceans, are major contributors to the economy of many countries and an increasingly important component in global food supply. However, the severe impact of aquatic microbial diseases on production performance remains a challenge to these industries. This article considers the potential applications of microalgal technology in the control of such diseases. At the simplest level, microalgae offer health-promoting benefits as a nutritional supplement in feed meal because of their digestibility and high content of proteins, lipids and essential nutrients. Furthermore, some microalgal species possess natural anti-microbial compounds or contain biomolecules that can serve as immunostimulants. In addition, emerging genetic engineering technologies in microalgae offer the possibility of producing 'functional feed additives' in which novel and specific bioactives, such as fish growth hormones, anti-bacterials, subunit vaccines, and virus-targeted interfering RNAs, are components of the algal supplement. The evaluation of such technologies for farm applications is an important step in the future development of sustainable aquaculture

Improving recombinant protein production in the Chlamydomonas reinhardtii chloroplast using vivid Verde Fluorescent Protein as a reporter

Microalgae have potential as platforms for the synthesis of high-value recombinant proteins due to their many beneficial attributes including ease of cultivation, lack of pathogenic agents, and low-cost downstream processing. However, current recombinant protein levels are low compared to other microbial platforms and stable insertion of transgenes is available in only a few microalgal species. We have explored different strategies aimed at increasing growth rate and recombinant protein production in the Chlamydomonas reinhardtii chloroplast. A novel fluorescent protein (vivid Verde Fluorescent Protein, VFP) was expressed under the control of the native atpA promoter/5'UTR element. VFP levels were detected by western blotting, with increased protein levels observed when co-expressed with a gene encoding the Escherichia coli Spy chaperone. We used these transformant lines to study the effect of temperature, light and media on recombinant protein production and cell growth. VFP levels and fluorescence, assessed by flow cytometry, allowed a determination of improved cultivation conditions as 30°C under mixotrophic mode. These conditions were tested for the accumulation of an antimicrobial endolysin (Cpl-1) of potential commercial interest, observing that the outcome obtained for VFP could not be easily replicated for Cpl-1. This study suggests that recombinant protein expression is product-specific and needs to be optimized individually

A Simple Technology for Generating Marker-Free Chloroplast Transformants of the Green Alga Chlamydomonas reinhardtii.

The availability of routine methods for the genetic engineering of the chloroplast genome of Chlamydomonas reinhardtii is allowing researchers to explore the use of this microalga as a phototrophic cell platform for synthesis of high value recombinant proteins and metabolites. However, the established method for delivering transforming DNA into the algal chloroplast involves microparticle bombardment using an expensive "gene gun". Furthermore, selection of transformant lines most commonly involves the use of a bacterial antibiotic resistance gene. In this chapter, we describe a simple and cheap delivery method in which cell-DNA suspensions are agitated with glass beads: a method that is more commonly used for nuclear transformation of Chlamydomonas. We also describe the use of plasmid expression vectors that target transgenes to a neutral site within the chloroplast genome between psbH and trnE2, and employ psbH as the selectable marker-thereby avoiding issues of unwanted antibiotic resistance genes in the resulting transgenic lines. Finally, we highlight a feature in our latest vectors in which the presence of a novel tRNA gene on the plasmid results in recognition within the chloroplast of UGA stop codons in transgenes as tryptophan codons. This feature simplifies the cloning of transgenes that are normally toxic to E. coli, serves as a biocontainment strategy restricting the functional escape of transgenes from the algal chloroplast to environmental microorganisms, and offers a simple system of temperature-regulated translation of transgenes

Characterization of Chlorella sorokiniana, UTEX 1230

This paper characterizes the strain Chlorella sorokiniana UTEX 1230 within a laboratory setting using a 1 L bubble column. The findings show that productivity can be trebled under mixotrophic conditions (from 0.2 g·L−1·d−1 to 0.66 g·L−1·d−1) with the addition of sodium acetate. The results also indicate that both the growth rate and final yield increase with the cultivation temperature, with most parameters showing an optimum in the range of 30–35 °C. The maximum specific growth rate was found to be in the region of 0.12 h−1 at a surface irradiance between 100–500 µE·m−2·s−1. This high growth rate makes the strain particularly suited to the rapid production of biomass, suitable for either whole cell bioprocessing or bioremediation. However, the relatively low lipid productivity (9.2 mg·L−1·d−1) confirms previous findings which would indicate poor applicability for biodiesel production. The strain shows greater promise in wastewater treatment applications with removal rates of nitrogen and phosphorus in the region of 37 and 30 mg·L−1·d−1 respectively. Furthermore, the findings show that a fed-batch strategy to inorganic nutrient loading can increase the final yield by around 50% compared to a conventional batch run. This is particularly interesting as fed-batch production techniques are rarely used within microalgal cultivation, so provide an interesting avenue for further investigation. Overall, the findings show that C. sorokiniana UTEX 1230 is a robust and fast-growing microalgal strain suitable both for the laboratory and scale-up

Cytosine deaminase as a negative selectable marker for the microalgal chloroplast: a strategy for the isolation of nuclear mutations that affect chloroplast gene expression

Negative selectable markers are useful tools for forward-genetic screens aimed at identifying trans-acting factors that are required for expression of specific genes. Transgenic lines harbouring the marker fused to a gene element, such as a promoter, may be mutagenized to isolate loss-of-function mutants able to survive under selection. Such a strategy allows the molecular dissection of factors that are essential for expression of the gene. Expression of individual chloroplast genes in plants and algae typically requires one or more nuclear-encoded factors that act at the post-transcriptional level, often through interaction with the 5' UTR of the mRNA. To study such nuclear control further, we have developed the Escherichia coli cytosine deaminase gene codA as a conditional negative selectable marker for use in the model green alga Chlamydomonas reinhardtii. We show that a codon-optimized variant of codA with three amino acid substitutions confers sensitivity to 5-fluorocytosine (5-FC) when expressed in the chloroplast under the control of endogenous promoter/5' UTR elements from the photosynthetic genes psaA or petA. UV mutagenesis of the psaA transgenic line allowed recovery of 5-FC-resistant, photosynthetically deficient lines harbouring mutations in the nuclear gene for the factor TAA1 that is required for psaA translation. Similarly, the petA line was used to isolate mutants of the petA mRNA stability factor MCA1 and the translation factor TCA1. The codA marker may be used to identify critical residues in known nuclear factors and to aid the discovery of additional factors required for expression of chloroplast genes

The algal chloroplast as a synthetic biology platform for production of therapeutic proteins

The chloroplast of Chlamydomonas reinhardtii and other microalgae represents an attractive new platform for the synthesis of recombinant therapeutics using synthetic biology (synbio) approaches. Transgenes can be designed in silico, assembled from validated DNA parts and inserted at precise and predetermined locations within the chloroplast genome to give stable synthesis of a desired recombinant protein. Numerous recent examples of different therapeutic proteins produced successfully in the C. reinhardtii chloroplast highlight the potential of this green alga as a simple, low-cost and benign host. Furthermore, some of the features of the alga may offer additional advantages over more-established microbial, mammalian or plant-based systems. These include efficient folding and accumulation of the product in the chloroplast; a lack of contaminating toxins or infectious agents; reduced downstream processing requirements; the possibility to make complex therapeutics such as immunotoxins; and the opportunity to use the whole alga as a low-cost oral vaccine. In this paper we review the current status of algal chloroplast engineering with respect to therapeutic proteins. We also consider future advances in synbio tools, together with improvements to recipient strains, which will allow the design of bespoke strains with high levels of productivity