Eukaryotic microalgae as hosts for light-driven heterologous isoprenoid production
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Eukaryotic microalgae hold incredible metabolic potential for the sustainable production of heterologous isoprenoid products. Recent advances in algal engineering have enabled the demonstration of prominent examples of heterologous isoprenoid production.
Isoprenoids, also known as terpenes or terpenoids, are the largest class of natural chemicals, with a vast diversity of structures and biological roles. Some have high-value in human-use applications, although may be found in their native contexts in low abundance or be difficult to extract and purify. Heterologous production of isoprenoid compounds in heterotrophic microbial hosts such as bacteria or yeasts has been an active area of research for some time and is now a mature technology. Eukaryotic microalgae represent sustainable alternatives to these hosts for biotechnological production processes as their cultivation can be driven by light and freely available CO2 as a carbon source. Their photosynthetic lifestyles require metabolic architectures structured towards the generation of associated isoprenoids (carotenoids, phytol) which participate in photon capture, energy dissipation, and electron transfer. Eukaryotic microalgae should, therefore, contain inherently high capacities for the generation of heterologous isoprenoid products. Although engineering strategies in eukaryotic microalgae have lagged behind the more genetically tractable bacteria and yeasts, recent advances in algal engineering concepts have demonstrated prominent examples of light-driven heterologous isoprenoid production from these photosynthetic hosts. This work seeks to provide practical insights into the choice of eukaryotic microalgae as biotechnological chassis. Recent reports of advances in algal engineering for heterologous isoprenoid production are highlighted as encouraging examples that promote their expanded use as sustainable green-cell factories. Current state of the art, limitations, and future challenges are also discussed.
KeywordsMicroalgae Chlamydomonas reinhardtii Phaeodactylum tricornutum Terpenoids Isoprenoids Cytochrome P450s
Geranyl pyrophosphate (synthase)
Farnesyl pyrophosphate (synthase)
Geranylgeranyl pyrophosphate (synthase)
Yellow fluorescent protein
Cyan fluorescent protein
Red fluorescent protein
Cytochrome P450 monooxygenase
Microalgae are fascinating organisms that aid in our understanding of fundamental aspects of the natural world, photosynthesis, the environment, and primary production. More recently, these organisms have been touted for their biotechnological potential as containable, unicellular green-cell factories for generating sustainable bio-production processes from photosynthesis (Wijffels et al. 2013). As carbon dioxide (CO2) is currently in abundance in the global environment, the capacity of microalgae, like higher plants, for photosynthetic growth using CO2 as a carbon source and (sun)light for energy is a powerful prospect for the development of sustainable biotechnological processes with these organisms (Melis 2012). In contained microbial-style cultivation concepts, microalgae are able to capture a freely available feedstock (CO2) and convert it into higher value bio-based products from light energy (Melis 2012; Larkum et al. 2012; Jakob et al. 2013; Fresewinkel et al. 2014). These products include the algal biomass itself or the array of natural products generated by different algal species. In addition to native products generated from microalgae, biotechnological processes are now aimed at increasing the product range produced by these organisms through genetic and metabolic engineering (Rosenberg et al. 2008). Expanding the native product portfolio of microalgal strains holds incredible potential for the development of customized, sustainable, light-driven bio-production concepts from these hosts (Specht et al. 2016).
Although promises of genetic engineering of microalgal strains have existed for three decades, mature engineering concepts have only recently been successfully demonstrated (Rasala et al. 2013; Karas et al. 2015; Lauersen et al. 2015b, 2016, 2018; Wichmann et al. 2018; D’Adamo et al. 2018). Practical examples of high-level algal genetic engineering have lagged behind other biotechnological hosts due to their seemingly recalcitrant genetic natures, which is especially true in members of the Chlorophyta like the model green microalga Chlamydomonas reinhardtii (Fuhrmann et al. 1999; Schroda et al. 2000; Shao and Bock 2008) and many Chlorella sp. (Yang et al. 2016). Increasing developments in genetic engineering tools and design concepts have led to maturing of algal engineering technologies (Scaife and Smith 2016). New trends are emerging in the genetic engineering of not only green microalgae (Crozet et al. 2018), but Bacillarophyceae (diatoms) as well to produce completely non-native chemical products (D’Adamo et al. 2018; Lauersen et al. 2018).
Current market prices of exemplary terpenoid products
Market price EUR/USD
Pogostemon cablin (patchouli)
5 g, $10b
Patchouli oil (up to 65% patchoulol)
5 g, $8b
Clearwood synthetic patchouli oil (engineered microbial production)
5 g, $6b
Potential biofuel replacement, chemical intermediate, food additive
Wide variety of plants, Abies grandis (grand fir tree)
Mixture of isomers (96%)
10 g, €24/$39c
Euphorbia peplus (milk weed)
1 mg, €136/$130a
Neutraceutical, weight loss
Coleus forskohlii (Plectranthus barbatus or Indian coleus)
25 mg, €503/$426a
500 mg, $21f
Cancer treatment (Paclitaxel)
Taxus brevifolia (Pacific yew)
50 mg, €265/$270d
Skincare products, chemical intermediate
Shark liver oil, an intermediate triterpenoid in all organisms
100 mL (98%), €56/$94a
Variety of plants including Mangifera (mango)
100 mg (94%), €501/$299a
Numerous medicinal applications
Isolated from the bark of birch trees,
25 mg (98%), €149/$146a
Antioxidant, aquaculture supplement
100 mg, €303/$331a
1.44 g, $87e
Antioxidant, dietary supplement, pigment
Common intermediate carotenoid from plants and algae, farmed from Dunaliela salina and carrots
5 g (97%), €67/$65a
3.75 g, $14f
Antioxidant, dietary supplement, pigment
Common intermediate carotenoid from plants and algae, farmed from marigold flowers
1 mg (97%), €306/$448a
1.20 g, $15f
The value of algal hosts for sustainable biotechnology and containable, light-driven bio-processes
Microalgal biomass is rich in many interesting products, for example, the capacity for some algae to accumulate a large percentage of their biomass as triacylglycerols (TAGs), similar to those found in plant oils, further spurred interest in their use for the generation of liquid biofuels (Hu et al. 2008). Algal biofuel technologies are still steadily developing; however, fuels represent bulk chemicals, which are sold at large volumes with low prices per unit. Algae are also natively able to produce products of mid- or high value and are already sources of natural isoprenoid pigments (Table 1). Economically relevant examples of this include beta-carotene produced from Dunaliela salina and astaxanthin produced from the microalga Haematococcus pluvialis (Lorenz and Cysewski 2000; Raja et al. 2007). The diatom Phaeodactylum tricornutum is rich in omega poly-unsaturated fatty acids (PUFAs) and the anti-oxidant carotenoid fucoxanthin, which are both valuable for human nutrition and aquaculture (Danielewicz et al. 2011; Eilers et al. 2016).
Isoprenoid metabolism and its structure in eukaryotic microalgae
The wide variety of chemical structures observed from isoprenoid secondary metabolites in nature are formed by the concerted efforts of terpene synthases (TPSs) which cleave the prenyl groups from isoprenoid carbon skeletons and participate in chemical reactions which guide these molecules to their functional forms (Fig. 4) (Buckingham et al. 1994; Kirby and Keasling 2009). Further functionalization of isoprenoid skeletons can be mediated by membrane bound cytochrome P450 enzymes (CYPs) which catalyze the addition of functional groups to isoprenoid backbones (Pateraki et al. 2015). Many medicines or fragrances of high value which come from plants are diterpenoids (C20) which have been extensively chemically decorated by concerted efforts of CYPs (Lin et al. 1996; Andersen-Ranberg et al. 2016; Pateraki et al. 2017).
General considerations for genetic engineering of eukaryotic microalgae
All plastids evolved from a single endosymbiotic event and the diversification of algal types observed today is the result of this primary, as well as secondary and tertiary endosymbiotic events following the uptake of plastids and consequent natural selection (McFadden 2001; McFadden and Van Dooren 2004; Keeling 2010; Archibald 2012). Eukaryotic microalgae are not a consistent phylogenetic group; rather this is an umbrella term for many different microorganisms that are the product of divergent evolutionary histories, endosymbiotic events and, with few exceptions, are capable of photosynthesis (McFadden 2001; Archibald 2012; Hallmann 2016). Therefore, to generalize engineering strategies across all microalgae is difficult as their genetic contexts are highly specific, variable, and often poorly understood. Chlorophyta, such as the model microalga C. reinhardtii, are highly divergent to the oleaginous model microalgae Nannochloropsis sp. (Eustigmatophyceae) or the model diatoms (Bacillarophyceae) Thalassiosira pseudonana, Fitsulifera solaris, or Phaeodactylum tricornutum. Eustigmatophyceae and Bacillarophyceae have evolved from secondary endosymbiosis; therefore, their nuclear context is completely different and unrelated to Chlorophyta (McFadden 2001). This has the practical consequence for the biotechnologist that every target algal strain requires customized genetic tools to enable reliable genetic engineering strategies.
Eukaryotic microalgae generally maintain nuclear, mitochondrial, and plastid genomes (Scala et al. 2002; Merchant et al. 2007; Radakovits et al. 2012). In C. reinhardtii, all three have been sequenced and demonstrated to be transformable (Kindle et al. 1989; Goldschmidt-Clermont 1991; Remacle et al. 2006; Merchant et al. 2007). Complete genomic resources are also available for the model diatom P. tricornutum (Scala et al. 2002) and several Nannochloropsis species (Radakovits et al. 2012; Vieler et al. 2012; Corteggiani Carpinelli et al. 2014). With the advent of low-cost sequencing technologies, more algal strains with fully sequenced genomes are regularly becoming available (Bogen et al. 2013; Jaeger et al. 2017). When combined with transcriptomic data, promoters from highly expressed genes can be combined with appropriate codon optimization to match the target genome (Jaeger et al. 2017). This information can be used to construct appropriate genetic tools for each respective host, which can now be readily generated through customized DNA synthesis services.
Challenges in genetic engineering of eukaryotic microalgae
Conducting reliable engineering in a microalgal host depends on several factors: are there promoters that can drive high rates of transcription for gene expression? What is known of the codon usage bias of the genome and is it flexible? How do DNA regulatory mechanisms like epigenetic silencing and intron densities hinder or support transgene expression? Finally, can the strain be reliably grown in the lab and selected for with antibiotic (or other) selection agents?
Although numerous reports exist of genetic transformation of microalgae (Yang et al. 2016; Hallmann 2016), the common adoption of eukaryotic microalgal engineering as a biotechnological platform and fundamental research topic has not met the focused energy and input at the scales seen for plant biotechnology. At the time of writing, a Google Scholar search of the keywords “plant transformation” gave 3,380,000 results while “algal transformation” gave only 378,000. There is also a very large difference in the levels of maturity of algal transformation in comparison to efforts in higher plants: although many reports exist describing transformation of an algal strain with a selectable marker, concentrated and reliable over-expression of target genes and pathways in mature engineering concepts are a rarity. In this work, only mature engineering strategies are discussed, as other reports summarize claimed transformation success in eukaryotic microalgal strains (Hallmann 2007, 2016).
Much work has already been done on recombinant protein over-expression in the chloroplast of C. reinhardtii (Specht et al. 2010; Purton et al. 2013; Carrera Pacheco et al. 2018; Dyo and Purton 2018), while algal metabolic engineering strategies involving multiple expression cassettes and novel product generation have focused on nuclear engineering (Lauersen et al. 2016, 2018; Wichmann et al. 2018; D’Adamo et al. 2018). In addition, mature chloroplast engineering of other model microalgae like the diatom P. tricornutum has not yet been reported; therefore, the discussion here will focus on nuclear engineering in C. reinhardtii and P. tricornutum.
Algal promoters and codon optimization
In higher plants and other eukaryotic systems, reliable viral promoters are well characterized and drive efficient transgene expression (Streatfield 2007; Black et al. 2017). Perhaps the only common feature across eukaryotic microalgae is that viral promoters have not been found to drive robust expression of transgenes [a comparative study of promoter expression rates was performed in C. reinhardtii by (Kumar et al. 2013)]. There is also a large practical difference for many gene targets between basal expression levels and true overexpression depending on their catalytic activity. Lack of strong heterologous promoters has led to the use of inherent promoters and corresponding 3′ untranslated regions (UTRs) from a target alga to mediate transgene expression (Mussgnug 2015). These elements are often identified via transcriptomic studies and used alone, or as synthetic fusions of their elements to drive reliable transgene expression. The tools available for C. reinhardtii nuclear engineering have been extensively reviewed previously and are now being developed into standardized parts (Mussgnug 2015; Scaife and Smith 2016; Crozet et al. 2018). Key examples of algal-originating promoters are the hybrid HSP70A-RBCS2-i1 (Lumbreras et al. 1998), native photosystem I reaction center subunit II [PsaD, (Neupert et al. 2009)], or newly developed synthetic promoters (Scranton et al. 2016) used for C. reinhardtii and the fucoxanthin chlorophyll a/c-binding protein B (FcpBp) promoter used for P. tricornutum (Siaut et al. 2007).
In addition to the need of specific algal promoter use, the nuclear genome of C. reinhardtii exhibits a relatively high native GC content (~ 64%, 68% in coding regions) and specific codon bias (Merchant et al. 2007). This has led to transgene design strategies where a coding sequence (CDS) must first be synthetically redesigned with optimal codon usage prior to transformation for expression from the nuclear genome of this host (Potvin and Zhang 2010). Codon optimization is an enabling technology which promoted the expression of antibiotic resistance cassettes and small fluorescent or bioluminescent reporters in C. reinhardtii (Fuhrmann et al. 1999; Shao and Bock 2008; Rasala et al. 2013; Lauersen et al. 2015b).
Foreign DNA integration and transgene expression limitations
Integration of foreign DNA in eukaryotic algal nuclear genomes largely occurs by non-homologous end joining (NHEJ) (Gumpel et al. 1994), with the exception of one report of homologous recombination (HR) in Nannochloropsis sp. (strain W2J3B) (Kilian et al. 2011). Although HR does occur in the nuclear genome of C. reinhardtii, its frequency is too low to be of practical use (Gumpel et al. 1994). NHEJ is a random DNA integration process of vector DNA into the algal genome which results in position effects on transgene expression. In addition, the action of inherent nucleases can cause digestion of vector DNA during transformation or physical forces can result in shearing. Consequently, colonies obtained through antibiotic selection screening can be false positives with no or low target gene of interest (GOI) expression (Lumbreras et al. 1998; Barahimipour et al. 2016; Weiner et al. 2018). In order to overcome screening limitations, GOI are often fused to a fluorescent or bioluminescent reporter, allowing detection of positive transformants at the agar plate level (Fuhrmann et al. 1999; Shao and Bock 2008; Rasala et al. 2013; Lauersen et al. 2013b, 2015b, 2016). Other strategies have used linkage of a GOI to a selection marker by a viral 2A peptide, which causes a skip in ribosome linkage of amino acids at a known position, resulting in two separate proteins being formed from a single mRNA (Rasala et al. 2012). Using this strategy, quantitative increases in selection pressure can correlate with increased selection of strains exhibiting higher GOI expression (Rasala et al. 2012, 2013). This mechanism is, however, not completely efficient, with a mixture of full-fusion and separate proteins detectible in Western blots (Rasala et al. 2013).
C. reinhardtii strains have also been generated which are less susceptible to repression of foreign transgene expression. The UVM4 and 11 strains (ultraviolet light mutagenized strains 4 and 11) generated by Juliane Neupert and Ralph Bock, have contributed greatly to the advancement of green-algal biotechnology over the past decade (Neupert et al. 2009). These strains are well-domesticated work-horses for C. reinhardtii nuclear engineering concepts which, through random mutagenesis, now exhibit more reliable transgene expression rates than other parental strains. This domestication is similar in concept to that conducted with Escherichia coli which has led to the use of only a few standardized strains for common laboratory practices. The mutagenesis steps which were used to generate UVM4/11 have been described as knocking out factors which repress nuclear transgene expression by an as of yet unknown mechanism. In our hands, UVM4 has been a reliable chassis for the investigation of gene expression and applied biotechnology from the nuclear genome of C. reinhardtii since 2011 (Lauersen et al. 2013a, b, 2015a, b, 2016, 2018; Wichmann et al. 2018; Baier et al. 2018).
Intron mediated enhancement is necessary to enable robust nuclear transgene expression in C. reinhardtii
The intron spreading transgene design strategy adds a further level of complexity to heterologous sequence design for expression from this algal host. However, it has been found to be necessary to facilitate robust and reliable expression of larger transgenes (greater than 1 kb) from its nuclear genome to levels detectible by Western blotting, if the protein is tolerated by the eukaryotic cell (Baier et al. 2018). Although currently only demonstrated in C. reinhardtii, this strategy will likely aid engineering efforts in other green microalgae as well, especially those which share evolutionary lineage with the Chlorophyta. Elucidating the regulatory mechanisms which introns contribute to controlling gene expression is a pressing topic for continued fundamental research initiatives with green microalgae and may be of interest to understanding the general mechanisms of gene expression in other eukaryotes as well (Gallegos and Rose 2015).
Other biotechnologically relevant model microalgae like the Bacillariophyceae (diatoms) or Eustigmatophyceae maintain lower genetic complexity than the green microalgae (Scala et al. 2002; Radakovits et al. 2012; Vieler et al. 2012). The model diatom P. tricornutum and members of the Nannochlorospis sp. have been found to have nuclear genomes with medium to low GC contents, 54% and ~ 33%, respectively, as well as average intron densities of 0.8 and 1.7 introns/gene, respectively (Scala et al. 2002; Corteggiani Carpinelli et al. 2014). Expression of transgenes in these hosts, therefore, is less likely to be dependent on complex codon and transgene optimization as has been demonstrated for C. reinhardtii (Siaut et al. 2007; Poliner et al. 2018a, b). Nevertheless, organism specific promoters are commonly used for engineering these hosts.
It is yet unclear how intron addition may affect transgene expression levels in these two organisms; however, this is certainly an interesting factor to be tested. An added benefit of intron addition into target transgenes may be to minimize risks of transgene escape from cultivation of engineered algal strains. Indeed, codon-optimised intron-containing transgenes, including those for antibiotic resistance, would not be expressed by prokaryotes in the environment if DNA transfer were to occur. Intron addition into synthetic transgenes can also be used to stabilize genetic constructs in bacteria during cloning when basal promoter activity cannot be avoided in the bacterial strain. Coding sequence disruption mediated by the presence of introns can allow the cloning of expression constructs for proteins toxic to bacteria, or those which would disrupt plasmid stability (Verruto et al. 2018).
Heterologous production of isoprenoids and algal cells as hosts
Many high-value isoprenoids come from plants and are found in their native context in small quantities or are difficult to separate and purify (Leavell et al. 2016). In order to overcome these limitations and generate isoprenoid products in a reliable manner, their production in heterologous microbial hosts has been developed into a mature technology (Kirby and Keasling 2009; Leavell et al. 2016). This is enabled by the modularity of isoprenoid metabolic pathways (Andersen-Ranberg et al. 2016). Once characterised, the specific TPSs and CYPs required to generate a target isoprenoid product from prenyl precursors can be expressed in biotechnologically relevant hosts and identical chemical products created from them (Chandran et al. 2011). This kind of heterologous isoprenoid bio-production has been extensively developed in heterotrophic bacteria and yeasts owing to their genetic tractability and capacities for growth in current fermentation infrastructures (Kirby and Keasling 2009). Significant advances have also been made in the genetically tractable photosynthetic cyanobacteria which have been recently reviewed elsewhere (Davies et al. 2015; Chaves and Melis 2018). In this report, focus is placed on eukaryotic algal engineering as relative newcomers in the field of green genetic engineering.
As heterologous host systems for plant TPS expression, eukaryotic green algae share distant evolutionary ancestry with land plants and may have favorable cellular environments for these enzymes compared to bacteria or yeasts. Microbial style cultivation of algal cells is conducted at temperatures similar to those used for plant growth, although usually with elevated CO2, which further encourages their use as hosts for expression of heterologous plant TPSs (Lohr et al. 2012). Their metabolism is structured towards production of precursors for photosynthetic associated isoprenoids and plastids contain ample reducing equivalents during light-driven growth (Melis 2012). Therefore, algal cells may be ideal hosts for the expression and production of heterologous plant metabolic pathways. Members of the Bacillarophyceae (diatoms) may also be ideal hosts for the production of isoprenoids derived from FPP and squalene. These organisms are spread throughout global oceans, maintain the cytosolic MVA pathway, and are rich in sterols which contribute to membrane fluidity in cold environments (Lohr et al. 2012). Sterols and triterpenes share the same C30 squalene precursor, making a strong case for the use of diatoms for triterpenoid production.
The focus of this report is on engineering non-native isoprenoid production from eukaryotic microalgal hosts. At the time of writing, four prominent examples of heterologous isoprenoid production from eukaryotic microalgal hosts exist in literature. One example of heterologous triterpenoid production has been shown in the model diatom P. tricornutum (D’Adamo et al. 2018), two examples of heterologous sesquiterpenoid production have been shown in the model green microalga C. reinhardtii (Lauersen et al. 2016; Wichmann et al. 2018), and a very recent report describes the use of this host also for the production of heterologous diterpenoids (Lauersen et al. 2018). Each one is discussed in detail in the following sections.
Heterologous triterpenoid production in the model diatom P. tricornutum
Lupeol is a base triterpenoid which can be further functionalized, first to the betulin intermediate, then to the pharmaceutical agent betulinic acid by concentrated activity of CYP enzymes (D’Adamo et al. 2018). CYPs require electron donors to provide the reducing power for their chemical reactions, a role which is naturally performed by cytochrome P450 oxidoreductases (CPRs) (Pateraki et al. 2015). In this work, the Medicago truncatula CYP716A12 and its corresponding reductase (MtCPR) were co-expressed in P. tricornutum in order to determine the capacity for functionalization of heterologous lupeol to the intermediate betulin. Two different approaches were investigated: (1) simultaneous co-transformation of the LjLUS with a zeocin resistance cassette, and the MtCYP716A12, as well as MtCPR coding sequences. (2) Serial transformation of a synthetic fusion of MtCY716A12 and MtCPR into an LjLUS expression strain and selection for both original LjLUS zeocin resistance and the CYP cassette nourseothricin (nat) resistance.
Both approaches were found to enable expression of the CYP and CPR enzymes as well as LjLUS, indicating the DNA uptake and construct expression in P. tricornutum is robust. Transforming multiple vectors in a single event saves time and logistical complexity of multiple rounds of selection, a feature which promotes the use of P. tricornutum for complex metabolic engineering strategies. Several strains could be isolated which converted small amounts of lupeol into the betulin intermediate, thus confirming activity of the CYP within the diatom. No aberrant growth performance was observed for any strain generated in this work, indicating that although the native metabolic flux towards sterols had been altered by the novel metabolite, no impact on cellular fitness could be observed in lab-scale.
Of note in this publication, was the attempt to demonstrate scalability of P. tricornutum cultivation to 550 L working volume in indoor tubular fence style photo-bioreactor cultivations (Fig. 8b). The strains exhibited growth behaviors as expected for P. tricornutum in this system. During scale-up, large variations were obtained due to the amount of culture which could be harvested. However, from 430 L of harvested culture, 98 g of an AtLUS expression strain could be collected which contained a total of 1.27 mg lupeol (at 13 µg/g dry biomass). Although yields were low, this work is an excellent first example of heterologous triterpenoid production from a diatom. Undoubtedly, future work will be able to increase yields from this host and, given the ease of its large-scale cultivation, production of high-value triterpenoids could be readily conducted with diatoms once titers are improved.
Heterologous isoprenoids from the model green microalga C. reinhardtii
C. reinhardtii has a metabolic architecture typical for Chlorophyta wherein the MEP pathway located in the chloroplast is responsible for the production of IPP, DMAPP, and all subsequent isoprenoid molecules in the cell (Lohr et al. 2012). To date, three reports of heterologous isoprenoid production have been demonstrated in C. reinhardtii, all of which were conducted by our working group (Lauersen et al. 2016, 2018; Wichmann et al. 2018). The following sections describe in detail two examples of sesquiterpenoid production and several examples of diterpenoid production from this host.
Patchoulol production from C. reinhardtii
The FPP synthase of C. reinhardtii (Uniprot ID: A8IX41) does not contain a plastid transit peptide and in vivo localization indicated that this enzyme forms a halo around the algal nucleus (Lauersen et al. 2016). The CrFPPS likely interacts with the squalene synthase that has been previously localized in the endoplasmic reticulum when expressed in onion cells (Kajikawa et al. 2015). The localization of CrFPPS indicated that FPP may be available in the cytosol; therefore, the first attempts at heterologous sesquiterpenoid production were conducted with enzymes expressed and localized there. The first demonstration of heterologous sesquiterpenoid production from C. reinhardtii was also the first transgene construct to be designed which contained repetitive spreading of the rbcS2 intron 1 (rbcS2i1) (Lauersen et al. 2016). The reasons for attempting this design strategy were two-fold; our previous trials of transgene expression using the pOptimized vector (Lauersen et al. 2015b) routinely resulted in low expression levels when using codon optimized cDNA over ~1 kb and terpene synthases (TPSs) are known to be catalytically slow enzymes (Kirby and Keasling 2009; Vickers et al. 2017). Therefore, failing to achieve robust TPS over-expression would result in heterologous isoprenoid production limitations.
The ~ 1662 bp patchoulol synthase [PcPs, (Deguerry et al. 2006)] was codon optimised for expression from the nuclear genome of C. reinhardtii and three copies of the rbcs2i1 were inserted throughout the sequence. The resultant intron containing optimized gene was 2097 bp. This strategy was employed in order to minimize exon lengths and, potentially, encourage robust transgene expression. PcPs catalyzes the conversion of FPP into patchoulol, a fragrant C15 alcohol which is the major component found in the oil of the patchouli plant Pogostemon cablin (Deguerry et al. 2006). The gene was cloned in fusion to a bright yellow fluorescent reporter (mVenus, hereafter YFP) in the pOptimized vector backbone as it was already known the PcPs could function as a fusion protein without affecting its activity (Albertsen et al. 2011) and to enable rapid as well as robust transformant identification at the agar plate level (discussed above, Fig. 6). After transformation and regeneration on selective plates, bright yellow fluorescent colonies could be detected on the primary transformant plate and a strong odor of patchouli fragrance was noted (Lauersen et al. 2016).
Successful expression of the PcPs in C. reinhardtii was significant for several reasons: the first was the successful use of introns to encourage large transgene expression, which spurred a systematic investigation in their use in numerous other transgenes (Baier et al. 2018). We have since used rbcS2i1 intron spreading as a basic tool for gene design for all transgene expression constructs from the C. reinhardtii nuclear genome. The second was that the cell had freely available FPP in the cytoplasm which could be converted into a non-native isoprenoid product via heterologous sTPS over-expression. During this work, it was determined that the non-native isoprenoid product could be produced to much higher titers if strains were cultivated with a biocompatible organic solvent overlay, the C12 alkane dodecane, as a second phase on top of the algal culture. Dodecane allowed culture growth and simultaneous capture of the heterologous isoprenoid products. It is not yet clear how excretion from the cells occurs, whether by passive diffusion or active transport. Nevertheless, patchoulol partitions more favorably into dodecane than into the algal cells or culture medium, a finding similar to production of this product in the yeast Saccharomyces cerevisiae (Gruchattka and Kayser 2015). Dodecane overlay allows straightforward capture and analysis of heterologous isoprenoid products, as the polar phase can be removed from cultures simply by centrifugation and injected directly into gas chromatography mass spectroscopy (GC–MS).
Bisabolene production from C. reinhardtii
In addition to protein overexpression, the main bottlenecks for isoprenoid flux towards farnesyl pyrophosphate (FPP), the precursor of sesquiterpenoids, were targeted for down-regulation by artificial micro RNA (amiRNA) in bisabolene producing strains. It was determined that knockdown of GGPP synthase (GGPPS) and squalene synthase (SQS) both resulted in increased production of bisabolene. GGPPS knock-down may result in increased IPP and DMAPP pools in the chloroplast; it is possible that export to the cytoplasm, where these would be converted to FPP by the FPP synthase, acts as a balance mechanism to prevent perturbed homeostasis in the chloroplast. SQS knock-down should result in a pooling of FPP. Under both scenarios, more FPP should be available for the AgBs to convert to bisabolene which was confirmed when the SQS was knocked down in a triple AgBs overexpression line (Fig. 10). SQS knockdown resulted in more than doubling of bisabolene productivity in this strain, up to 10.3 ± 0.7 mg bisabolene/g dry biomass. The results indicated that C. reinhardtii could convert up to 1% of its total biomass into a completely novel product without affecting cellular fitness or growth.
In both patchoulol and bisabolene production examples, major engineering hurdles were overcome in the green alga C. reinhardtii; the first and most important was the development of an effective transgene design strategy to enable robust expression from the nuclear genome (Baier et al. 2018), and the second was that C. reinhardtii could handle significant modifications in terms of recombinant construct expression as well as metabolic channeling to non-native products. In both cases, patchoulol and bisabolene, C. reinhardtii produced more when the cells were grown mixotrophically with light and acetate, than with CO2 as a sole carbon source. As the cytosolic FPP is a precursor also for isoprenoids which participate in respiratory metabolism in the mitochondria (ubiquinone), it is likely that flux towards FPP in the cytoplasm is higher under mixotrophic conditions. Given the levels of engineering achieved with these two projects, the capacity for reliable transgene design and expression, and a desire to access the metabolic flux of the MEP pathway towards isoprenoids in the algal chloroplast, the next metabolic engineering strategy conducted with C. reinhardtii was towards the production of C20 diterpenoids from the algal chloroplast.
Diterpenoid production from C. reinhardtii
We chose expression of heterologous constructs from the nuclear genome and consequent targeting to the algal chloroplast to mediate heterologous diterpenoid production in C. reinhardtii (Lauersen et al. 2018) as our recent gene design strategy enabled reliable expression of large transgenes from the nuclear genome and multiple transgene expression (Lauersen et al. 2016; Wichmann et al. 2018). Although algal chloroplast transformation could be used for expression of a single diTPS, a previous report found no diterpenoid product when the cis-abienol (diterpene) synthase was overexpressed from the chloroplast genome of C. reinhardtii (Zedler et al. 2014).
Heterologous expression and chloroplast targeting of taxadiene and casbene synthases as well as co-expression of both CfTPS2 and 3 could be successfully shown, all of which led to the production of the desired diterpenoid products for each respective enzyme (Lauersen et al. 2018) (Fig. 11b). Several insights were determined early in this work: very large heterologous fusion proteins could be reliably expressed from the nuclear genome of C. reinhardtii and targeted to the algal chloroplast mediated by the 36 amino acid photosystem I reaction center subunit II (PsaD) chloroplast target peptide (CTP). This CTP could be used to target multiple heterologous proteins in a single strain simultaneously to the algal chloroplast. Additionally, it was determined that diterpenoid products were not only produced in the algal chloroplast but were also found to excrete from the cells into dodecane overlays.
It was determined that although some native GGPP could be diverted to diterpenoid products by diTPSs, native mechanisms outcompeted GGPP channeling to heterologous products, limiting their productivity. Diterpenoid titers were subsequently improved by co-overexpression of MEP pathway enzymes, thereby generating more freely available GGPP for the diTPSs (Lauersen et al. 2018). The first committed step of the MEP pathway is the 1-deoxy-d-xylulose 5-phosphate synthase (DXS) (Lichtenthaler 1999); this enzyme has been found to be rate-limiting for the flux towards IPP and DMAPP as it is feedback inhibited by these compounds (Lohr et al. 2012). GGPP synthase converts these compounds into GGPP, the precursor of diterpenoids. Heterologous expression of DXS from Salvia pomifera (Trikka et al. 2015) or an engineered yeast GGPP synthase (Ignea et al. 2015) in a CfTPS2 and CfTPS3 expressing strain was found to increase 13R(+) manoyl oxide titers (Lauersen et al. 2018). The most significant increase in manoyl oxide production was observed when CfTPS2, the rate limiting enzyme for GGPP conversion to manoyl oxide, was fused to the yeast GGPPS (Fig. 11c). In this scenario, a metabolic pull was directly created on the C5 precursors IPP and DMAPP which could be converted by the heterologous GGPPS to GGPP in the vicinity of the CfTPS2, potentially increasing channeling to its active site. This step led to a strain which produced ~ 40 mg manoyl oxide/L culture in 5 days.
When cultivated in 400 mL bioreactor concepts with different carbon sources, this strain produced manoyl oxide at higher titers when CO2 was used as a sole carbon source. This is in direct contrast to observations of productivity of sesquiterpenoids produced from the cytoplasm and may indicate dynamic regulation of isoprenoid flux compartmentalization based on carbon-source in the algal cell. Of note, in 7 days, this strain produced 80 mg manoyl oxide/g dry biomass when grown on CO2 (Fig. 11d). Although not possible to quantify the carbon partitioning in this system as CO2 was flushed through the chamber continuously to saturating levels, the total 13R(+) manoyl oxide productivity was 8% of the complete biomass of the system without changes in pigmentation (Lauersen et al. 2018). The results indicate that the algal MEP pathway has an innate flexibility as well as tolerance for high flux and that the alga chassis may be a powerful future host for the production of heterologous isoprenoid products.
Diterpenoid hydroxy-functionalization by a heterologous CYP
High value diterpenoids like those mentioned above are often chemically functionalized derivatives of the C20 isoprenoid backbones produced by diTPSs. These final decorated forms are created by the concerted activity of specific cytochrome P450 enzymes (CYPs). It could be additionally shown in C. reinhardtii that co-expression of a truncated and soluble C. forskohlii microsomal P450 targeted to the algal chloroplast mediated the hydroxylation of heterologous 13R(+) manoyl oxide to 9-OH 13R(+) manoyl oxide (Lauersen et al. 2018). The heterologous CfCYP was able to take electrons from a native source in the algal chloroplast and use this energy to cause hydroxylation of the manoyl oxide backbone in absence of a dedicated CPR. Although the conversion amount in mixotrophic screening conditions was only ~ 11% of the total 13R(+) manoyl oxide produced, it is the first step towards the production of more complex decorated diterpenoid products with this host. Future investigations will be able to more systematically investigate whether electron donor fusion strategies can improve activity of these enzymes in the algal chloroplast.
Current technical limitations to engineering algal systems
For all algal strains, availability of selectable markers and low transgene expression rates are still limiting factors to complex trait stacking and eventual pathway transfer into the algal hosts. For C. reinhardtii, the pOptimized (pOpt2) vectors have 4 selectable markers for antibiotic resistance which have been used in combination with fluorescent reporters to rapidly generate transformants and select individual strains with robust expression profiles (Wichmann et al. 2018; Lauersen et al. 2018). There are also many other potential selectable markers which could be adapted to this system (Bruggeman et al. 2014; Mussgnug 2015; Scaife and Smith 2016). There is currently a movement by the greater Chlamydomonas research community to adopt the common syntax for the modular cloning (MoClo) DNA parts design strategy to facilitate construct exchange and generate more reliable expression vector constructs (Engler et al. 2008; Weber et al. 2011; Crozet et al. 2018). Using individual selectable markers, trait stacking is possible by serial transformation and screening events. However, this strategy is limited to marker availability and often results in strains carrying multiple antibiotic resistance genes, which is undesirable. Although it is also possible to combine traits from one C. reinhardtii to another via sexual crossing (Rasala et al. 2013), domesticated strains which robustly express transgenes such as the UVM4/11 and are capable of mating are not yet readily available (Barahimipour et al. 2016).
Phaeodactylum tricornutum has an innate capacity for expression of multiple transgenes with minimal adaptation to the host genomic context (discussed above). This is exemplified in the co-transformation and expression of multiple plasmids in a single transformation round conducted towards the production of the intermediate betulin in P. tricornutum (D’Adamo et al. 2018). This strategy minimized the need for multiple selection rounds; however, it still exhibited low overall efficiencies in positive expression for all transgenes simultaneously (1 out of 30 colonies). Although more amenable to transformation of multiple constructs at a time, depending on construct length, selection markers will also become an issue for reliable engineering efforts in this host.
A promising recent report may have solved this issue by designing selection markers flanked by the loxP recombinase sites and using an inducible expression strategy for the Cre recombinase in Nannochloropsis gaditana (Verruto et al. 2018). Although there are no reports in literature currently of heterologous isoprenoid production from Nannochloropsis sp., engineering in these hosts is also reaching mature levels (Ajjawi et al. 2017; Poliner et al. 2018a, b, c; Verruto et al. 2018). The Cre recombinase/loxP system was employed for serial transformation of a selectable marker and small guide RNAs for Cas9 induced knock-out trait stacking (Verruto et al. 2018). Such techniques could be equally applied to protein over-expression constructs to enable gene stacking and metabolic pathway construction. Adapting a Cre recombinase/loxP recyclable selection cassette into other algal systems may encourage higher-order gene stacking and synthetic pathway designs. This would be especially valuable for some complex isoprenoid products, for example, the highly decorated diterpenoid forskolin is built from 6 enzymatic steps: two enzymatic steps required to produce 13R(+) manoyl oxide from GGPP, followed by 3 CYPs and one acetyl transferase (Pateraki et al. 2017). Although technically possible to achieve with the current tools for both C. reinhardtii and P. tricornutum, such engineering pushes the limit of what is currently practical with these hosts. A recyclable selection cassette system would be valuable for enabling multiple transformation and selection rounds in order to facilitate complex trait stacking.
Although potentially more amenable to straightforward engineering, P. tricoronutum still exhibits slow and sensitive growth when compared to the more rapidly growing model green microalga C. reinhardtii. The desire to engineer C. reinhardtii, and other green algae strains, lies specifically with their ease of handling in the lab and rapid generation times. Through the added step of intron integration into codon-optimised transgenes, we have been able to unlock the power of nuclear gene expression in C. reinhardtii (Baier et al. 2018). This is an enabling technology towards the increased use of C. reinhardtii in biotechnological applications. However, even for over expression of single sTPSs or diTPSs, multiple rounds of transformation and selection were required to boost amounts of a single construct in the cell (Lauersen et al. 2016, 2018; Wichmann et al. 2018). Currently, transcriptional limitation is still a major hurdle to enabling mature metabolic engineering in this alga. Indeed, our understanding of the regulatory mechanisms which result in intron mediated enhancement of transgene expression is still minimal. Greater insights into the regulatory machinery in green algal hosts is certainly required to design smart transgene expression constructs which work with the inherent transcriptional machinery for improved expression rates towards enabling future mature engineering concepts (Gallegos and Rose 2015).
Although powerful, intron spreading also does not overcome some of the marked limitations of engineering in the green algal system, specifically NHEJ integration and nuclease digestion of constructs during transformation (discussed above). These issues still limit reliable engineering in this host by requiring large numbers of transformants to be screened to identify individual strains with robust expression, especially of larger genetic constructs (see Fig. 6). Novel innovations in gene delivery are needed to improve GOI expression rates. Future investigations of chemical or biological DNA stabilizing agents and reduction of inherent nuclease activities may help improve reliable integration of large DNA sequences into the nuclear genome. As DNA synthesis is rapidly becoming more reliable and less costly, it may soon be possible to design constructs which contain complex metabolic pathways on synthetic algal chromosomes. Such genetic constructs could enable customized remodeling of cellular metabolism towards desired product generation. However, this cannot be realized until DNA can be more reliably transfected to the algal host and multiple genes can be reliably expressed to tunable levels simultaneously without exhaustive screening efforts being required.
Scalability, technical limitations, and economic considerations of heterologous isoprenoid production from phototrophic algal systems
The key benefit of algal hosts for bio-production is the capacity for low-input, scalable, and sustainable production concepts using CO2 as a carbon source and growth driven by energy obtained from (sun)light. Nevertheless, algal cells still represent emerging host systems that require improvements in cultivation strategies in addition to genetic engineering techniques to economically compete with established fermentative microbial hosts or natural product extraction. To date, commercial processes with microalgal hosts are entirely focused on natural products such as carotenoids (Raja et al. 2007; Benemann 2013; Hallmann 2016), and facilities for large-scale cultivation of engineered algal cells are rare. The current market value of some exemplary isoprenoid products which can also be produced in heterologous systems are presented in Table 1.
At current rates of production, economic feasibility of heterologous isoprenoid production will not be achieved with algal systems. For example, the sesquiterpenoid cosmetic and perfume ingredient patchoulol is sold as a pure crystal for 50 cents/g and a heterologous patchouli oil, produced by microbial fermentation, is already marketed under the name Clearwood by Firmenich. Bisabolene was also shown to be produced in fermentative microbial hosts up to 900 mg/L in only several days (Peralta-Yahya et al. 2011) and is commercially available from Alfa Aesar as a natural product extract at several dollars per gram (Table 1). Maximal yields of patchoulol and bisabolene in C. reinhardtii were 1 mg/g dry biomass and 10 mg/g dry biomass in almost 1 week of cultivation, respectively (Lauersen et al. 2016; Wichmann et al. 2018). At these productivities, 100 L of algal culture would be required to produce 1 g of bisabolene if lab-scale productivities could be extrapolated, although productivities are usually lower in scale-up.
Diterpenoids and triterpenoid products have higher market value, while natural carotenoids are already sold as dietary supplements with medium–low prices. Although highly purified isoprenoid products used as analytical standards can be sold at high price points (Table 1), active isoprenoid ingredients can be acquired and used as mixtures or in extracts from their natural hosts. For diterpenoids and triterpenoids, yields from algal hosts are also still too-low to compete with natural or heterologous production in fermentative microbial systems. Additionally, current reports have only begun to demonstrate partial CYP-mediated functionalization of base terpenoid products into higher-functionalized active molecules. For example, generation of 9-OH 13R(+) manoyl oxide from C. reinhardtii is one of several functionalization steps towards forskolin and functionalization of lupeol to betulin in P. tricornutum is one functionalization step towards betulinic acid (D’Adamo et al. 2018; Lauersen et al. 2018). Both forskolin and betulinic acid can be extracted from natural plant sources and sold as pure products (Table 1).
In order for genetic engineering strategies in microalgae to extend from the laboratory into scalable biotechnological production processes, increasing communication is also needed between molecular biologists (genetic/metabolic engineers) and out-door algal cultivation specialists. This is important for the advancement of engineering concepts with photosynthetic microalgae as domesticated laboratory adapted strains, especially those lacking cell walls, often do not perform well in large-scale photobioreactor cultivation concepts. A prominent example of this is that common algal cultivation media at lab scale use ammonium as a nitrogen source for growth and buffers such as Tris to regulate pH (Gorman and Levine 1965). However, in industrial-scale algal culture medium, nitrate is favored as it does not cause pH fluctuations and different buffering systems are employed, for example by regulating CO2 inputs (Fabregas et al. 1984). The domesticated C. reinhardtii strains UVM4 and 11, which have been reliable chassis for demonstrating isoprenoid engineering concepts, are not capable of growth on nitrate, containing both nit1 and nit2 mutations from their parent strain (Neupert et al. 2009; Barahimipour et al. 2016). Although this capacity can be complemented by transformation of the Nit1 + Nit2 gene constructs (Fernández et al. 1989; Schnell and Lefebvre 1993), this is a further engineering step which must be considered. P. tricornutum strains used in lab-scale engineering do not face these same hurdles, however, may be more sensitive to light intensities in early cultivation stages than their green algal counterparts.
Increased interaction and communication between these stakeholders is important for the advancement of engineered algal concepts and their application at larger scales. It will also be prudent to shift from common model organisms and start attempting engineering in strains with already proven value to industrial-scale cultivations. Although requiring significant activation energy, including genome/transcriptome sequencing and annotation to guide genetic tool design, as well as growth characterization, and domestication steps, use of robust strains which will already tolerate large-scale cultivation for engineering concepts may facilitate transfer of engineered production hosts from the lab to industrial realisation.
Eukaryotic microalgae are interesting candidates for the development of sustainable light-driven bio-processes, especially the production of heterologous isoprenoid products. Although previous engineering efforts in these organisms had been met with limited success, the advent of appropriate transgene designs which work with host regulation machinery and DNA synthesis technologies are improving overall engineering capacities with these hosts. The diatom P. tricornutum has demonstrated favorable capacities for reliable engineering, while only recently has intron-mediated enhancement of transgene expression been shown to enable robust engineering concepts in the model green microalga C. reinhardtii. Both have now been shown to be amenable to heterologous isoprenoid production and are starting to pave the way for the next generation of sustainable algal bio-process development. Tapping into inherent metabolic flux towards heterologous isoprenoids in eukaryotic microalgae holds powerful potential and robust yields are already being reported. Despite these recent advances, engineering concepts are still at an early stage and technical limitations must be overcome in order to ensure mature engineering concepts can capture the inherent potential of algal hosts as light-driven green-cell factories. Standardization of genetic tools, increasing communication as well as collaboration between molecular biologists and process engineers will help spur developments of eukaryotic algal engineering towards practical realization of industrial-scale bioprocesses with these hosts.
Author contribution statement
K.L. wrote this review and designed the figures with original artwork or photographs. Some images were provided by collaborators as indicated in respective figure captions.
This work has been supported by the technology platform and infrastructure at the Center for Biotechnology (CeBiTec) of Bielefeld University. Sincere thanks to Dr. Thomas Baier for critical reading of this manuscript and those mentioned in the text who provided pictures.
Compliance with ethical standards
Conflict of interest
The author declares no conflict of interest.
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