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A green light for engineered algae: redirecting metabolism to fuel a biotechnology revolution
Julian N Rosenberg1, George A Oyler1, Loy Wilkinson2 and Michael J Betenbaugh1
Microalgae have the potential to revolutionize biotechnology in a number of areas including nutrition, aquaculture, pharmaceuticals, and biofuels. Although algae have been commercially cultivated for over 50 years, metabolic engineering now seems necessary in order to achieve their full processing capabilities. Recently, the development of a number of transgenic algal strains boasting recombinant protein expression, engineered photosynthesis, and enhanced metabolism encourage the prospects of designer microalgae. Given the vast contributions that these solar-powered, carbon dioxide-sequestering organisms can provide to current global markets and the environment, an intensied focus on microalgal biotechnology is warranted. Ongoing advances in cultivation techniques coupled with genetic manipulation of crucial metabolic networks will further promote microalgae as an attractive platform for the production of numerous highvalue compounds.
Addresses 1 Department of Chemical & Biomolecular Engineering, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA 2 Coastal BioMarine, PO Box 6, Bridgewater, CT 06752, USA Corresponding authors: Rosenberg, Julian N (jrosenberg@jhu.edu), Oyler, George A (goyler@jhu.edu), Wilkinson, Loy (loywilkinson@coastalbiomarine.com) and Betenbaugh, Michael J (beten@jhu.edu)

[3,4]. Moreover, microalgae are unique because they combine the renewable energy-capturing ability of photosynthesis with the high yields of controlled microbial cultivation, making them potentially valuable organisms for economical, industrial-scale production processes in the 21st century. The overarching goal of microalgal biotechnology is to improve the productivity of these organisms in order to meet the demands of a rapidly growing market [5]. Although much of the industrys previous work has focused on cultivation techniques and species selection, novel microalgal applications are currently being brought to fruition with the help of genetic engineering. The aim of this review is to describe the ability of microalgae to synthesize useful products, examine methods of increasing yield through altered metabolism, and provide an outlook for the future of microalgal metabolic engineering.

Algaculture
Algae are a diverse group of aquatic, photosynthetic organisms, generally categorized as either macroalgae (i.e. seaweed) or microalgae, which are typically unicellular. Among the eukaryotic, green microalgae of the class Chlorophyceae, those most widely utilized for current commercial applications belong to the genera Chlamydomonas, Chlorella, Haematococcus, and Dunaliella. The study of these freshwater and marine algae has generated a wealth of information concerning their physiology, biochemistry, and cultivation [68]. In regard to genetic engineering, these species are amenable to rstly, nuclear transformation, necessary for metabolic control, secondly, chloroplastic transformation, for high levels of protein expression, and thirdly, more straightforward approaches to genetic modication compared to higher plants [9]. Diatoms, a group of silicon-rich microalgae, and prokaryotic cyanobacteria also offer substantial opportunities for metabolic engineering and biotechnology, but will not be examined extensively in this review [10,11]. As aquatic relatives of plants, microalgae thrive in aerated, liquid cultures where the cells have sufcient access to light, carbon dioxide, and other nutrients [7]. Algae are primarily grown photoautotrophically; yet, some species are able to survive heterotrophically by degrading organic substances like sugars [12]. Unlike terrestrial plants, microalgae do not require fertile land or irrigation
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Current Opinion in Biotechnology 2008, 19:430436 This review comes from a themed issue on Tissue, Cell and Pathyway Engineering Edited by William Bentley and Michael Betenbaugh Available online 6th September 2008 0958-1669/$ see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2008.07.008

Introduction
In recent years, microalgae have garnered interest for producing valuable molecules ranging from therapeutic proteins to biofuels. Nutritional and medical applications are especially tting for these organisms because many biomolecules expressed by microalgae are generally regarded as safe (GRAS) for human consumption [1,2]. Although numerous species produce useful compounds naturally, these unicellular organisms are also well suited for genetic manipulation and high-throughput analysis
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A green light for engineered algae: redirecting metabolism to fuel a biotechnology revolution Rosenberg et al. 431

marine algae even provide an alternative to freshwater use. Additionally, because algae consume carbon dioxide, large-scale cultivation can be used to remediate the combustion exhaust of power plants [13]. The most cost-effective way to farm microalgae is in large, circulating ponds; however, a serious weakness in the use of outdoor ponds is the risk of contamination by other microbes or indigenous algal species [13]. Alternatively, closed photobioreactors provide sterility and allow for much greater control over culture parameters such as light intensity, carbon dioxide, nutrient levels, and temperature [14]. Under optimal conditions, microalgal populations are capable of doubling within hours and achieving high cell densities, corresponding to as much as 60 g of heterotrophic biomass per liter and 5 g of photoautotrophic biomass per liter [15]. Furthermore, mixotrophic systems combine photoautotrophic and hetTable 1 Products synthesized by microalgae Product b-Carotene Microalgae Dunaliella

erotrophic conditions to facilitate high-density algal growth. Recent developments in heterotrophic cultivation and low-cost photobioreactors have provided additional economic advantages for the controlled growth of microalgae [12,16].

Commercial applications of microalgae

Successful commercial utilization of microalgae has been established in low-volume, high-value derivatives such as nutritional supplements, antioxidants, cosmetics, natural dyes, and polyunsaturated fatty acids (PUFA) [5] (Table 1). The worldwide annual production of algal biomass is estimated to be 5 million kilograms per year with a market value of about 330 USD per kilogram [17]. Approximately one-fth of this biomass is used to nourish the sh and shellsh that are cultivated in aquaculture hatcheries [15]. In recent years, microalgae have been investigated as an expression system for recombinant

Price (USD) 3003000/kg

Producer AquaCarotene (Washington, USA) Cognis Nutrition & Health (Australia) Cyanotech (Hawaii, USA) Nikken Sohonsha Corporation (Japan) Tianjin Lantai Biotechnology (China) Parry Pharmaceuticals (India) AlgaTechnologies (Israel) Bioreal (Hawaii, USA) Cyanotech (Hawaii, USA) Mera Pharmaceuticals (Hawaii, USA) Parry Pharmaceuticals (India) BlueBiotech International GmbH (Germany) Cyanotech (Hawaii, USA) Earthrise Nutritionals (California, USA) Phycotransgenics (Ohio, USA) Aquatic Eco-Systems (Florida, USA) BlueBiotech International GmbH (Germany) Coastal BioMarine (Connecticut, USA) Reed Mariculture (California, USA) BlueBiotech International GmbH (Germany) Spectra Stable Isotopes (Maryland, USA) Martek Biosciences (Maryland, USA) Spectra Stable Isotopes (Maryland, USA) BlueBiotech International GmbH (Germany) Cyanotech (Hawaii, USA) PharmaMar (Spain) Rincon Pharmaceuticals (California, USA) Cellana (Hawaii, USA) GreenFuel Technologies (Massachusetts, USA) LiveFuels, Inc. (California, USA) PetroAlgae (Florida, USA) Sapphire Energy (California, USA) Solazyme, Inc. (California, USA) Solix Biofuels (Colorado, USA)

Astaxanthin

Haematococcus

10,000/kg

Whole-cell dietary supplements

Spirulina Chlorella Chlamydomonas Tetraselmis Nannochloropsis Isochrysis Nitzschia Crypthecodinium Schizochytrium N/A Red Algae Cyanobacteria N/A Chlamydomonas Botryococcus Chlamydomonas Chlorella Dunaliella Neochloris

50/kg

Whole-cell aquaculture feed

70/L

Polyunsaturated fatty acids

60/g

Heavy isotope labeled metabolites Phycoerythrin (uorescent label) Anticancer drugs Pharmaceutical proteins Biofuels

100020,000/g 15/mg N/A N/A N/A

Over the years, algal biotechnology companies have brought a number of products to market, ranging from aquaculture feed to specialty chemicals. Currently, the development of pharmaceutical compounds and biofuels is a priority of the industry.

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432 Tissue, Cell and Pathyway Engineering

protein therapeutics and have the potential to be used as edible vaccines for sh and humans [18,19,20]. With predictions that crude oil prices will reach recordbreaking highs of 150200 USD per barrel [21], algalbased biofuels are gaining widespread attention. Renewable, carbon-neutral fuel applications exploiting algal components include transesterication of lipids to biodiesel [22,23], saccharication of carbohydrates to ethanol [24], gasication of biomass to syngas [25], cracking of hydrocarbons and isoprenoids to gasoline [26,27], and the direct synthesis of hydrogen gas [28,29]. Current economic modeling of algal biodiesel places the price of production in the range of 6.508.00 USD per gallon (Dr. Robert Wallace, NREL, personal communication). Some of the factors necessary to lower algal biofuel production costs include maximizing the content of lipids and other biofuel precursors, increasing the rate of cell growth, identifying chemical inducers of these metabolic changes, and implementing multistage growth systems. The two major driving forces impelling future commercial development of microalgae are the vitality of biological research in this area and the availability of funding for alternative energy sources. The blockbuster application for commercial production of microalgae is in nextgeneration biofuels, but on the way to this goal, a wealth

of collateral biotechnology opportunities are being realized.

Species selection for biodiesel production

Perhaps the most comprehensive evaluation of algal species was orchestrated by the US Department of Energys Aquatic Species Program (ASP) to develop microalgae as a source of biodiesel. Beginning in 1978, the ASP isolated microalgae from ponds and oceans, amassing a library of over 3000 strains. The constituents of this collection were then characterized and screened based on robustness, oil content, growth rate, and metabolic efciency [30]. Throughout this project, the ASP recognized that the key to unlocking protable commercialization of microalgae lies not only in species selection and optimal cultivation, but also in genetic and metabolic engineering.

Manipulating microalgal metabolism

Manipulation of metabolic pathways can redirect cellular function toward the synthesis of preferred products and even expand the processing capabilities of microalgae (Figure 1). One method of coercing microalgae employs specic environmental factors, such as nutrient regimens, to induce desired uxes in metabolism. As an alternative to manipulating culture conditions, metabolic engineering allows direct control over the organisms cellular machinery through mutagenesis or the introduction of transgenes.

Figure 1

Commercially important metabolic pathways in microalgae. This schematic representation depicts simplified cellular pathways involved in the biosynthesis of various products derived from microalgae. Although the chloroplast can act as a factory for protein and hydrogen production (solid blue), the nucleus plays a fundamental role in metabolic control (dotted red). Both of these organelles contain individual genomes, which offer the possibility for independent transgene incorporation (dashed blue and red). Current Opinion in Biotechnology 2008, 19:430436 www.sciencedirect.com

A green light for engineered algae: redirecting metabolism to fuel a biotechnology revolution Rosenberg et al. 433

Environmental factors affect metabolism

Whether conditions are favorable or adverse, the intricate metabolic pathways of a cell are heavily inuenced by its environment. In the case of microalgae, specialized cultivation can stimulate changes in metabolism, thereby providing a simple method of enriching biomass with a target metabolite.
Oil from algae

esized that this might be an innate algal mechanism to suppress transposons and viral invasion [34]. Researchers have recently shed light on this obstacle with the discovery of microRNA gene regulatory systems in unicellular algae [35]. Clearly, molecular approaches toward evading transgene silencing will be an important step in the metabolic engineering of algae. There are several techniques currently available for genetic transformation of microalgae. The simplest method involves agitating algal cells in a mixture of glass beads and DNA molecules [36]. The velocities of the cells and beads are sufcient to perforate the cell membrane upon collision. Only algae that lack a cell wall, either inherently or as a result of enzymatic degradation, can be transformed with this technique; however, micronlong silicon carbide whiskers have been used in lieu of glass beads to pierce cell walls [37]. Additionally, electroporation relies on pulses of electric current to permeabilize the cell membrane and has shown great success with cell wall decient organisms [3840]. Effective transformation can also be achieved by propelling microparticles of DNA-coated gold or tungsten at algal cells [4143]. One drawback to microparticle bombardment is its forceful nature comparable to shooting a baseball through a basketball at over 1000 miles per hour. A less aggressive approach involves the microbe Agrobacterium tumefaciens, which causes tumors in plants [44]. Through genetic manipulation of the bacteriums TDNA (transfer DNA), tumor-inducing genes can be replaced with useful gene constructs, which can then be introduced into microalgal cells [45]. Although the eld of microalgal genetic engineering has advanced signicantly over the past decade [46], routine transformation has only been achieved in a few algal species. Nonetheless, because of the current demand for microalgal commercialization, the library of transformable species is constantly expanding [4749].
Accomplishments of genetic engineering

For microalgae that are able to survive heterotrophically, exogenous carbon sources offer prefabricated chemical energy, which the cells often store as lipid droplets [31]. Heterotrophically cultivated Chlorella protothecoides has been shown to accumulate as much as 55% of its dry weight as oil, compared to only 14% in cells grown photoautotrophically [22]. Another natural mechanism through which microalgae can alter lipid metabolism is the stress response owing to a lack of bioavailable nitrogen [32]. Although nitrogen deciency appears to inhibit the cell cycle and the production of almost all cellular components, the rate of lipid synthesis remains higher, which leads to the accumulation of oil in starved cells [30]. Interestingly, nitrogen deprivation also promotes the accumulation of the antioxidant pigment astaxanthin in the green alga Haematococcus pluvialis [33]. Both of these adaptive responses help to ensure the cells survival during times of stress while lipids serve as energy stores, astaxanthin seems to play a role in the protection against reactive oxygen species. From an industrial standpoint, these lipid triggers can be useful for the production of polyunsaturated fatty acids (e.g. omega-3) and biodiesel from algal triacylglycerides. Despite the fact that specic uxes in metabolic pathways caused by heterotrophic growth and nitrogen starvation have yet to be elucidated, these two conditions are important for understanding the mechanisms of regulating fatty acid biosynthesis in green algae.
Genetic transformation technologies

Genetic transformation requires temporary permeabilization of the cell membrane in order to allow exogenous DNA molecules to enter the cell. During a successful transformation event, a DNA fragment is incorporated into the cells nuclear or chloroplastic genome and the cell remains viable afterward; however, most cells die as a result of cell membrane rupture. Even if the organism survives the initial wound, the exogenous gene may be recognized as foreign and become degraded. Furthermore, the positioning of the DNA fragment within the genome is arbitrary, which accounts for varying degrees of expression [3,9]. Other than Chlamydamonas reinhardtii and Volvox carteri, few green algae have demonstrated stable, long-term expression of transgenic proteins, despite exhibiting integration of the transgenic DNA. Some have hypothwww.sciencedirect.com

Although the wave caused by the genomic revolution has swept much of the scientic community, ripples are just now being felt in the ponds of microalgal researchers. Within the past decade, there have been several pioneering achievements in the genetic transformation of microalgae, each with an opportunity for commercial development.
Augmented lipid biosynthesis

In the interest of engineering an algal strain with high lipid content for biodiesel production, the ASP focused on overexpressing acetyl-CoA carboxylase (ACCase), an enzyme that catalyzes an initial metabolic step in lipid biosynthesis. In 1994, researchers were able to isolate the gene coding for ACCase in the diatom Cyclotella cryptica
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434 Tissue, Cell and Pathyway Engineering

[50]. With this, they developed expression vectors and a transformation protocol that enabled the rst attempt at metabolic engineering of a microalga [51]. Even though the overexpression of ACCase did not have a signicant effect on lipid synthesis, the tools and methodologies established by this work have set the stage for future genetic engineering efforts in microalgae [30].
Trophic conversion

these reasons, the production of proteins for vaccines is likely to become a widely used application of microalgal biotechnology [19].

Conclusions
In pursuit of improved nutraceutical agents, inexpensive protein therapeutics, and advanced biofuels, the inherently useful capacities of microalgae will only be enhanced by genetic and metabolic engineering. Recognizing the importance of these molecular approaches, the eld of microalgal biotechnology merits a continued investment of resources both in the basic and applied sciences in this emerging area. The rapid sequencing of algal genomes will facilitate the cloning and manipulation of genes and allow the power of -omics technologies (e.g. genomics, proteomics, and metabolomics) to be applied. For example, transcriptome analysis can identify key regulators of metabolism and enable the eventual manipulation of cellular pathways. Advances in genetic transformation techniques will permit ever more sophisticated forms of metabolic network engineering to be accomplished. In conjunction with the development of transgenic algae, methods to protect against unintended environmental consequences will be required. These mechanisms may include containment considerations in photobioreactor design and incorporation of suicide genes in the engineered organism. Ultimately, microalgae offer the potential to have a profound impact on the future welfare of the planet by addressing the pressing issues of alternative energy resources, global warming, human health, and food security.

Certain microalgae have the natural ability to metabolize sugars, while others rely strictly on photosynthesis. As a result of genetic engineering, some obligate photoautotrophs, formerly unable to partake in a sweet diet, have been given a taste of heterotrophy through the introduction of hexose transporters. Volvox carteri was one of the rst green algae to be transformed with the hexose/H+ symporter gene Hup1 from Chlorella and heterotrophic growth was veried by the assimilation of 14C-labeled glucose and glucosamine [52]. Similar trophic conversions have also been carried out in Chlamydomonas reinhardtii [53] and the diatom Phaeodactylum tricornutum [54], each demonstrating a fundamental change in metabolism caused by a single gene.
Engineered light-harvesting antennae

Physiological and biochemical adaptations for conditions of low light, including tightly stacked thylakoids and large light-harvesting antenna complexes (LHCs), allow microalgae to effectively capture light energy. As a result, high light intensity can overwhelm their photosystems and lead to the formation of reactive oxygen species, a reduction in photosynthetic efciency, and deleterious cell stress [55]. In order to avoid these consequences of photoinhibition, researchers have used RNA interference technology to downregulate the expression of LHC proteins in Chlamydomonas reinhardtii [56]. The resulting LHC mutant was indeed decient in mRNA and proteins of the entire LHC gene family and contained less tightly stacked thylakoids. Consequently, the strain exhibited a higher resistance to photodamage and increased light penetration in liquid culture. With these improvements, this newly engineered strain of algae offers more efcient conversion of solar energy to biomass.
Expression of recombinant proteins

References and recommended reading

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The development of rapid and reproducible chloroplast transformation techniques in Chlamydomonas reinhardtii combined with high-level expression systems shows promise for the production of edible, therapeutic proteins devoid of prion contamination [20]. As eukaryotic organisms, microalgae offer superior protein-folding mechanisms and post-translational modication systems that are not present in bacteria [9]. Furthermore, the time from initial transformation to large-scale production is surprisingly short perhaps only four to six months and the cost can be less than other protein expression platforms (e.g. mammalian cells and bacteria) [18]. For
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