Introduction

Microalgae are a heterogenic group of eukaryotic and prokaryotic, unicellular, and colonial photosynthetic organisms that are highly important from an ecological standpoint. Microalgae primarily inhabit aquatic environments and can be found in waters extending from the tropics to the Polar Regions. Additionally, these organisms exhibit a wide tolerance range to different salinities, temperatures, pH levels, and nutrient availabilities (Borowitzka 1999; Cadoret et al. 2012; Singh et al. 2005; Spolaore et al. 2006).

Microalgae play a critical role in ecological terms, constituting the first link in the food chain. In particular, microalgae are the primary food source for zooplankton, which in turn, are a significant source of nutrition for higher aquatic organisms. Concomitant with this, close to half of worldwide photosynthesis and oxygen production is through microalgae. In fact, these organisms are strict photoautotrophs and necessarily require light energy for biological processes. Microalgae are extremely diverse, not only in terms of shape, size, and phylogeny, but also with regard to a wide range of highly valuable compounds. Currently, between 40,000 and 70,000 microalgae species have been identified from nine different phyla (Cadoret et al. 2012; Chisti 2007; Doan et al. 2011; Gimpel et al. 2015; Ghosh et al. 2016; Rindi et al. 2009). However, although many microalgae species have been identified, only a few have been successfully exploited for biotechnological ends (Olaizola 2003). The main cultured species are Chlorella and Spirulina, for human and animal consumption, and Dunaliella salina and Haematococcus pluvialis, for the extraction of β-carotene and astaxanthin, respectively. It is estimated that over 10,000 microalgae species remain undescribed and that these likely belong to biological groups completely different than those already defined (Sastre and Posten 2010).

The exploitation of microalgae for biotechnological purposes has increased in recent years, leading to diverse commercial applications for microalgae, including in pigments, nutritional supplements, antioxidants, fatty acids, cosmetics, and biofuels. These uses have resulted in the development of important pharmaceutical and nutraceutical products (Milledge 2011; Leu and Boussiba 2014; Pulz and Gross 2004; Qin et al. 2012). For example, microalgae are currently the primary sustainable source of carotenoids and omega-3 fatty acids (Borowitzka 2013; Leu and Boussiba 2014; Vílchez et al. 2011). Nutritional and biomedical applications are particularly adequate for microalgae as many of the biomolecules expressed by these organisms are generally recognized as safe for human consumption (Fletcher et al. 2007; Franklin et al. 2005; Walker et al. 2005).

The goals of this mini-review are to highlight the potential of microalgae as biotechnological “factories” for the production of commercially relevant metabolites and to describe alternative solutions for natural, sustainable, and environmentally friendly microalgae production. For this, two cutting-edge concepts in the field of genetically transforming microalgae will be discussed as new alternatives to transgenesis—cisgenesis and intragenesis (Abiri et al. 2015; Camacho et al. 2014; Espinoza et al. 2013; Holme et al. 2012; Singh et al. 2015).

Genetic engineering in microalgae

Microalgae have recently come to light as novel and attractive alternatives for producing recombinant proteins, including from therapeutic proteins to biofuels. Microalgae also have the capacity to over-express endogenous enzymes from particular metabolic pathways, thereby facilitating the accumulation of metabolites of interest, such as astaxanthin and polyunsaturated fatty acids (Adarme-Vega et al. 2012; Cadoret et al. 2012; Hamilton et al. 2014; Leu and Boussiba 2014; Steinbrenner and Sandmann 2006).

The high demand for, but productivity low, algae-based biotechnologies has promoted advanced research into the development of genetically engineered microalgae strains (Almaraz-Delgado et al. 2014). Transformation protocols exist for genetic manipulations of the nucleus (Kindle 1990), mitochondria (Remacle et al. 2006), and chloroplast (Boynton et al. 1988). Of these, chloroplasts have presented the best success in accumulating recombinant proteins for therapeutic uses, including oral vaccines, immunotoxins, and monoclonal antibodies, among others (Cadoret et al. 2008; Demurtas et al. 2013; Gimpel and Hyun 2015; Gregory et al. 2013; Jones et al. 2013; Jones and Mayfield 2013; Mussgnug 2015; Purton et al. 2013; Rasala and Mayfield 2015; Soria-Guerra et al. 2014; Specht and Mayfield 2014; Tran et al. 2013a, b).

To date, more than 30 different microalgae species have been successfully transformed (Radakovits et al. 2010), an encouraging figure for ongoing efforts to design algae that present selected characteristics. As compared with current systems used for producing recombinant proteins (e.g., terrestrial plants, mammalian cells, yeasts, and bacteria), microalgae present several comparative advantages. These advantages include low costs of production, ease of manipulation, and the fact that certain species of microalgae have been considered “generally regarded as safe” (GRAS) by the US Food and Drug Administration (Cadoret et al. 2008; Gutiérrez et al. 2012; Rasala and Mayfield 2015).

There is a growing interest in the use of microalgae as low-cost hosts for the synthesis of bioactive metabolites due to genomic, proteomic, and metabolomic advances in different species. Indeed, these advances have driven the development of novel technologies for microalgae genetic engineering (Doron et al. 2016; Hlavova et al. 2015). For instance, the manipulation of metabolic pathways can redirect cellular functions to the synthesis of determined products, giving rise to the direct control of a cellular organism through the introduction of extra copies of a particular enzyme (Gimpel et al. 2015; Henríquez et al. 2016; León et al. 2007). Innovation is further fueled by widespread interest in the commercial demand for the high-value compounds produced by microalgae, including for lutein from Chlorella protothecoides, β-carotene from D. salina, astaxanthin from H. pluvialis (Campenni et al. 2013; Kathiresan et al. 2015; Shi et al. 1997; Steinbrenner and Sandmann 2006; Sun et al. 2014; Ye and Jiang 2010), and biofuels (Burkart and Mayfield 2013; Ghosh et al. 2016; Gimpel et al. 2013; Glass 2015; Gouveia 2011; Grima et al. 2013; Hannon et al. 2010; Scranton et al. 2015).

Cisgenic and intragenic technologies

Genetic transformation is a technology used to produce recombinant proteins and accumulate commercially valuable metabolites. This technology is primarily applied in terrestrial plants (Abiri et al. 2015; Gupta et al. 2013; Halford 2012; Kempinski et al. 2015; Molesini et al. 2012; Napier et al. 2015; Ortiz Rios 2015; Ulukan 2011) and, to a lesser extent, in microalgae (Cui et al. 2014; Gregory et al. 2013; Liu et al. 2013, 2014; Rasala et al. 2010; Sharon-Gojman et al. 2014; Zheng et al. 2014; Zhong et al. 2011). The optimization of desired characteristics has led to the generation of genetically modified crops that do not compromise human or animal health and that are environmentally friendly. Likewise, significant efforts have been made to apply genetic transformation in higher plants through cisgenic and intragenic technologies to attain products similar to those naturally produced (Cardi 2016; Cotter et al. 2015; Espinoza et al. 2013; Haverkort 2015; Jacobsen and Schouten 2007; Mlalazi et al. 2012; Napier et al. 2015; Schaart et al. 2015; Vanblaere et al. 2011). These technologies are analogous to self-cloning and natural occurrence processes in which fragments of non-recombinant and recombinant DNA, respectively, from a sexually compatible organism are introduced to a host (Kasai et al. 2015).

Genetic transformation through transgenesis, cisgenesis, and intragenesis has been widely reviewed by a number of authors (Holme et al. 2012, 2013a, b; León et al. 2004; Schouten et al. 2006, 2008; Singh et al. 2015). Transgenesis consists in the transference of foreign genes or transgenes to a particular organism, leading to the expression of new characteristics. Furthermore, transgenesis involves the recombination of genetic material that cannot naturally hybridize (Gupta et al. 2013; Molesini et al. 2012; Ortiz Rios 2015). In turn, the terms cisgenesis and intragenesis are used to describe species genetically modified using genes and genetic elements exclusively originating from a sexually compatible donor, usually from the same or a phylogenetically related species (Holme et al. 2012; Mlalazi et al. 2012; Molesini et al. 2012). These concepts were introduced over a decade ago, and to date, certain crops have been modified by these techniques and have been widely accepted by society (Holme et al. 2013a, b).

Cisgenesis only involves the use of genes and genetic elements from the same species or from a cross-breedable species (Camacho et al. 2014). Full coding sequences, including gene introns with the corresponding promoter and terminator regions, should be used in the transformation, thereby preventing recombination with genetic material from foreign species (Holme et al. 2013a; Schouten et al. 2006). In turn, the concept of intragenesis implies the use of genes and regulatory elements from phylogenetically related and/or cross-breedable species (Camacho et al. 2014). Thus, the genes are manipulated and/or reordered in vitro, including intron removal and sense or antisense orientation. These manipulations are driven by promoter and terminator regions of other endogenous genes that do not necessarily correspond to the same cisgene (Holme et al. 2012; Molesini et al. 2012; Rommens et al. 2007). These types of technologies accelerate gene transference among sexually compatible species, circumventing the linkage drag associated with conventional interbreeding while favoring the exchange of genetic material. Moreover, intragenesis favors expression levels by using more efficient promotors, thus decreasing the instance of gene silencing. Figure 1 shows the three types of gene modification: transgenesis, cisgenesis, and intragenesis.

Fig. 1
figure 1

General diagram for genetic transformation: transgenesis, cisgenesis, and intragenesis in microalgae

Full size image

These new technologies are highly promising tools for algae-based biotechnologies since the resulting products can be considered natural and conserved from one generation to the next one. These features are particularly relevant to consider for the accumulation of omega-3 polyunsaturated fatty acids and carotenoids with nutraceutical applications (Shew et al. 2015; Sticklen 2015). Furthermore, diverse studies in microalgae demonstrate the underlying potential of metabolic engineering using endogenous genes for the accumulation of these bioactive compounds. Nevertheless, metabolic engineering is classified more as a partial intragenic technology as it contains selection markers and vector backbones.

Genes coding for key enzymes involved in the biosynthesis of fatty acids have been identified in a number of species (Adarme-Vega et al. 2012; Schuhmann et al. 2012), including Ostreococcus tauri (Wagner et al. 2010), Ostreococcus RCC809 (Vaezi et al. 2013), Ostreococcus lucimarinus (Petrie et al. 2010), Thalassiosira pseudonana (Tonon et al. 2005; Xu et al. 2013), Phaeodactylum tricornutum (Domergue et al. 2003), and in the model organism Chlamydomonas reinhardtii (Chi et al. 2008).

The overexpression of DGAT2 in P. tricornutum showed a significant increase in the proportion of polyunsaturated fatty acids, with EPA in particular increasing by 76.2 % (Niu et al. 2013). Likewise, the overexpression of delta-5-desaturase in the same microalgae resulted in a higher accumulation of EPA, specifically a 58 % increase (Peng et al. 2014). The coding sequences of delta-6-desaturase OtD6 and delta-5-desaturase OtElo5 cloned from O. tauri in P. tricornutum resulted in an increased accumulation of DHA (Hamilton et al. 2014). These three studies used the specific pPha-T1 nuclear vector of the P. tricornutum diatom (Zaslavskaia et al. 2000). Similarly, Nannochloropsis oceanica cells engineered with DGAT2 show significantly increased (53.1 %) saturated fatty acid content as compared with wild-type cells. On the other hand, monounsaturated fatty acids decreased by 52.9 %. Similarly, polyunsaturated fatty acids showed an apparent decrease of 74.6 %, including for arachidonic acid (C20:4) and EPA (C20:5) (Li et al. 2016).

The microalgae Chlorella zofingiensis and H. pluvialis are promising candidates for the genetic engineering of carotenoids, particularly considering the commercial relevance of these pigments (Henríquez et al. 2016). Various enzymes have been identified from the metabolic pathways of carotenoids, including phytoene synthase (PSY), phytoene desaturase (PDS), plastid terminal oxidase, lycopene cyclase (LCY-b), and β-carotene ketolase (CRTR-B) in H. pluvialis (Henríquez et al. 2016; Huang and Chen 2006a; Kajiwara et al. 1995; Lotan and Hirschberg 1995; Tan et al. 2007; Steinbrenner and Linden 2001, 2003). The mutated pds gene has been used in H. pluvialis as a dominant selection marker and reporter gene for transformation (Steinbrenner and Sandmann 2006).

Similarly, the genes involved in the biosynthesis of astaxanthin in C. zofingiensis include PSY (Cordero et al. 2011), PDS (Huang et al. 2008), LCY-b (Cordero et al. 2010), LCY-e (Cordero et al. 2012), BKT (CrtO) (Huang et al. 2006b), and CrtR-b (Chy-b) (Li et al. 2008). The endogenous pds gene of C. zofingiensis was mutated and transformed, conferring resistance to norflurazon, and transformants accumulated 54.1 % more astaxanthin (Liu et al. 2014).

Cisgenesis and intragenesis have promising applications in microalgae. In particular, these technologies represent possible alternatives for increasing the accumulation of commercially relevant metabolites through the overexpression of native enzymes, in accordance with the needs of consumers and in line with environmental conservation (Adarme-Vega et al. 2012).

Generation of cisgenic and intragenic microalgae

The generation of new products through biotechnological means should not present human health risks; therefore, the presence of a selectable marker gene is inconvenient (Espinoza et al. 2013; Henley et al. 2013; Tuteja et al. 2012; Wannathong et al. 2016). The development of novel tools has facilitated an evident change in obtaining genetically modified organisms (GMOs) free of foreign DNA. These tools include (i) site-specific mutations, (ii) the excision of the selectable marker, (iii) zinc-finger nuclease and transcription activator-like effectors (TALEs), and (iv) Clustered Regularly Interspersed Short Palindromic Repeats System (CRISPR/Cas9) systems.

Site-specific mutations

Considering photoautotrophic growth, chloroplast transformations have used non-photosynthetic receptor strains with the mutated endogenous genes atpB, psbA, and tscA as reporters for recovering photosynthetic activities (Doron et al. 2016). Based on herbicide resistance, the psbA gene from C. reinhardtii encoding the D1 protein for the photosystem I has been mutated to modify the binding site for various herbicides (Johanningmeier et al. 1987). Indeed, resistance to the herbicide metribuzin was a key trait for the direct selection of C. reinhardtii transformants (Przibilla et al. 1991). Similarly, and as previously mentioned, Steinbrenner and Sandmann (2006) mutated the pds gene by changing the leucine codon in position 504 for an arginine codon, which resulted in transformants exhibiting high resistance to the herbicide norflurazon. Furthermore, acquired resistance to the herbicide sulfometuron methyl has been demonstrated by mutating the AHAS gene, which codes for the enzyme acetohydroxyacid synthase (Lapidot 2002). On the other hand, and considering metabolic enzymes, reversing mutations of the arg7 and arg9 genes, which are both involved in arginine synthesis and respectively code for argininosuccinate lyase (Debuchy et al. 1989) and the N-acetyl ornithine aminotransferase protein (Remade et al. 2009), result in a restored ability to synthesize arginine. Likewise, other studies propose designing plasmids and using the endogenous pds gene as a dominant selectable marker for efficient nuclear transformations in H. pluvialis and C. zofingiensis (Sharon-Gojman et al. 2014). Genetic modifications using endogenous genes as selectable markers are an advancement towards successfully generating environmentally friendly cisgenic and transgenic microalgae.

Excision of selectable markers using direct repeats

Methods for the removal of selectable markers have already been described (Fischer et al. 1996). Selectable markers are inevitable in the selection of modified microalgae, resulting in the need to isolate stable transformants when cloning genes within the chloroplast. However, selectable markers can be eliminated when flanked by direct-repeat sequences that split the DNA sequence interspersed together with direct repeat copies. Excision of the selectable marker and restoration of the microalgal chloroplast genome is mediated by the native machinery of homologous recombination (Akbari et al. 2014; Day and Goldschmidt-Clermont 2011). Excision is a spontaneous process, where the frequency depends on the length of the direct repeat sequences, which should be between 400 and 650 base pairs (Fischer et al. 1996). Finally, a uniform population of chloroplast genomes free of the selectable marker will be produced.

According to Day and Goldschmidt-Clermont (2011), the split DNA is unstable and will be naturally lost. Also, the removed selection markers are not reintegrated nor does homologous recombination occur.

Roles of zinc-finger nucleases and transcription activator-like effector in editing the microalgal genome

Genes from and the genome of C. reinhardtii have been edited in situ by engineering nucleases via TALEs (Gao et al. 2014) and zinc-finger nucleases (Sizova et al. 2013). The principal difference between zinc-finger and TALE domains is the mechanism employed for recognizing DNA sequences. Zinc-finger modules always distinguish three base pairs, limiting the number of potential target sequences. In contrast, TALEs are composed of a variable number of four different DNA recognition domains, each of which specifically recognizes one of the four nucleotides. Therefore, 12–20 of these domains can fuse to create a TALE that binds to any desired target DNA sequence (Jinkerson and Jonikas 2015).

TALEs were originally obtained from Gram-negative, pathogenic plant bacteria of the Xanthomonas genus. These bacterial TALEs inject proteins into plant cells through a type III secretion system mechanism (SST3). The injected proteins are translocated to the nucleus and specifically bind with the promoter regions of certain genes in the host DNA. Consequently, the genes are activated, and the transcriptome is manipulated, which contributes to bacterial survival and colonization for promoting further infection (Boch and Bonas 2010; Bogdanove et al. 2010; Moscou and Bogdanove 2009; Romer et al. 2009). TALEs have become the effectors of choice for increasing transcription, surpassing other options, such as zinc-fingers and meganucleases, due to the relatively easy and low-cost of laboratory synthesis, high specificities for the selected sequences, and low cellular toxicity, among other noted advantages (Bogdanove and Voytas 2011; Gaj et al. 2013; de Lange et al. 2014; Moscou and Bogdanove 2009).

Artificially designed TALEs have been used for gene-specific activation in Chlamydomonas. Specifically, Gao et al. (2014) selected two endogenous C. reinhardtii genes, ARS1 and ARS2, as activation targets for TALE domains. Through the TALE domains, both target genes exhibited noticeably increased transcript and protein expressions, which were confirmed via protein activity by ARS colorimetric assays. This genome-editing technique has also been used in the model diatom P. tricornutum to interrupt the urease enzyme encoding gene as a method for clarifying the role of urease in the urea cycle and improving the molecular toolkit for diatoms (Weyman et al. 2016).

Zinc-finger nucleases involve a fusion of three to four zinc-finger DNA-binding modules to a FokI DNA cleavage domain, forming an artificial restriction enzyme (Jinkerson and Jonikas 2015). A pair of these molecular scissors can be tailored to target specific DNA sequences, where dimerization of the FokI domain activates the nuclease, provoking site-specific, double-stranded DNA breaks that enable genome editing (Townsend et al. 2009). In C. reinhardtii, this technology has been used to interrupt genes of interest (Sizova et al. 2013). Furthermore, a C. reinhardtii genome database exists that contains over 330,000 identified target sites, accounting for 93 % of all model genes (Reyon et al. 2012). This technology could be useful for redirecting particular metabolic pathways.

Role of CRISPR/Cas in editing the microalgal genome

Currently, the traditional methods for genome editing in higher plants are being replaced by CRISPR technology (Ahuja 2015; Cong et al. 2013; Horvath and Barrangou 2010; Jiang et al. 2014; Jinek et al. 2012; Marraffini and Sontheimer 2010; Puchta and Fauser 2014). The development of this cutting-edge technology in activating and/or inactivating native genes is ongoing and seeks to circumvent the use of exogenous genes, thereby creating crops that are more socially acceptable (Araki et al. 2014; Araki and Ishii 2015; Hartung and Schiemann 2014; Kanchiswamy et al. 2015; Voytas and Gao 2014). CAS9 is an RNA-guided DNA nuclease that has been successfully used for targeted mutations in eukaryotic genomes (Belhaj et al. 2013; Cong et al. 2013; Dominguez et al. 2016; Gaj et al. 2013; Jinkerson and Jonikas 2015; Mali et al. 2013; Mussgnug 2015). Similarly, a CRISPR/Cas system was recently identified and characterized in the genome of the freshwater cyanobacterium Microcystis aeruginosa (Yang et al. 2015). In microalgae, preliminary studies in C. reinhardtii indicate that CRISPR/Cas technology can be feasibly used for editing the nuclear genome (Greiner 2014; Jiang et al. 2014). However, stable transformants of C. reinhardtii expressing the CAS9 protein were unobtainable (Guihéneuf et al. 2016; Mussgnung 2015). Recently, this technology has been employed to efficiently generate stable targeted gene mutations in the marine diatom P. tricornutum (Nymark et al. 2016). Genetic engineering advancements in higher plants using endogenous genes and applying CRISPR/Cas technology ensure the generation of non-GMOs (Araki and Ishii 2015; Cardi 2016). These studies could serve as strategies for standard genome editing to create cisgenic and intragenic microalgae.

Regulatory considerations for the development of cisgenic and intragenic microalgae

In general, the regulatory frameworks established by international entities for the testing and development of genetically modified plants have been rather drastic (Camacho et al. 2014; Devos et al. 2014; Sundaramurthy 2010; Wijffels 2015; Willems et al. 2016). Worries regarding the dissemination of GMOs are linked to ethical considerations and apprehensions concerning health risks and the propagation of new gene combinations in the environment (Shew et al. 2015; Sticklen 2015). However, most nationally established regulations have been fundamentally based only on transgenic organisms, without giving consideration to the concepts of transgenesis, cisgenesis, or intragenesis (Molesini et al. 2012; Schouten et al. 2006). In particular, evaluations of GMOs are primarily based on regulations given in the still standing 2000 Cartagena Protocol on Biosafety (Hartung and Schiemann 2014). Worth considering is that in Japan, the self-cloning microalga Pseudochoricytis ellipsoidea is considered natural, not GMO, and corresponding experiments are not governed by GMO limitations outlined in the Cartagena Protocol on Biosafety (Lusser et al. 2012; USDA Japan Report 2014).

Today, more than ever, researchers are making significant efforts to generate GMOs, particularly for plants, which are environmentally friendly and safe for human and animal health. Cis- and intragenesis involve the exchange of DNA between interbreeding groups, due to which, imposed regulations should be similar to those applied to conventional breeding. Regulations on cisgenic and intragenic organisms are currently under debate in various countries, including in the European Union and USA, where regulations are close to being defined (Kasai et al. 2015). While cisgenic and intragenic crops have already been developed and field-tested, only one intragenic crop has been approved for commercial use, the Innate™ potato developed by Simplot Plant Sciences. Likewise, the Wageningen University and Research Centre has applied cisgenesis to develop a potato resistant to the late blight fungus Phytophthora infestans. To date, regulations are pending for considering these organisms separate from transgenic GMOs.

In the near future, the innate properties of microalgae will become important resources for biotechnological products. Therefore, governmental authorities should take note of ongoing advancements and duly consider microalgae produced through cisgenic and/or intragenic technologies. Furthermore, self-cloning technology is important for outdoor open-pond algae breeding, which is currently considered the most viable option for the large-scale cultivation of microalgae (Brennan and Owende 2010).

Conclusions

The application of cisgenesis and intragenesis in microalgae represents promising biotechnological tools for rapidly and securely advancing the commercial exploitation of attractive and highly valued compounds, particularly of polyunsaturated fatty acids and carotenoids. Furthermore, these technologies could become socially accepted and can be considered “consumer friendly.” Cis- and intragenesis have already been applied in crops, but microalgae remain to be exploited, despite the existence of the required genetic tools. Both technologies require that microalgae be transformed only with genomic sequences derived from the same or sexually compatible species, and it is worth noting that foreign sequences, such as selectable markers and vector backbones, would be absent from cis- and intragenic microalgae.

Cisgenesis and intragenesis have provided new opportunities for discussions between scientists, producers, and consumers regarding the safety of genetically modified crops. These discussions should serve as a basis for more flexible governmental regulations, which would likely lead to new crops generated through modern technologies. Finally, less restrictive regulations should reasonably exist for cisgenic and intragenic organisms considering that the gene pools used in these technologies are identical to conventional interbreeding. Indeed, these technologies could facilitate the scaling of environmentally safe open-culture systems.