Recent Developments on Genetic Engineering of Microalgae for Biofuels and Bio-Based Chemicals
I-Son Ng,* Shih-I Tan, Pei-Hsun Kao, Yu-Kaung Chang, and Jo-Shu Chang
Microalgae serve as a promising source for the production of biofuels and bio-based chemicals. They are superior to terrestrial plants as feedstock in many aspects and their biomass is naturally rich in lipids, carbohydrates, proteins, pigments, and other valuable compounds. Due to the relatively slow growth rate and high cultivation cost of microalgae, to screen efficient and robust microalgal strains as well as genetic modifications of the available strains for further improvement are of urgent demand in the development of microalgae-based biorefinery. In genetic engineering of microalgae, transfor- mation and selection methods are the key steps to accomplish the target gene modification. However, determination of the preferable type and dosage of antibiotics used for transformant selection is usually time-consuming and microalgal-strain-dependent. Therefore, more powerful and efficient techni- ques should be developed to meet this need. In this review, the conventional and emerging genome-editing tools (e.g., CRISPR-Cas9, TALEN, and ZFN) used in editing the genomes of nuclear, mitochondria, and chloroplast of microalgae are thoroughly surveyed. Although all the techniques mentioned above demonstrate their abilities to perform gene editing and desired
phenotype screening, there still need to overcome higher production cost and lower biomass productivity, to achieve efficient production of the desired products in microalgal biorefineries.
1. Introduction
The global population is expected to increase from 6.3 billion at 2015 to more than 9 billion at 2050. The humanity’s top ten problems for next 50 years will be energy, water, food, environment, poverty, terrorism and war, disease, education, democracy, and population issues. To solve such kinds of problems, many policies have been developed focusing on
creating alternative energy, exploring sustainable agriculture, supporting food security, preventing disease transmis- sion, and providing new concept in education.[1] However, some conflict exists between continuous economic growth and sustainable development.
Therefore, the crucial problem is green- house effect caused by carbon dioxide from combustion of fossil oil, which had led to the anthropogenic climate change over the past 20 years. Inaction under such circumstances will lead to severe consequences, including the abrupt rise of CO2 by 15%.[2] In order to decrease carbon dioxide emissions and reduce fossil fuels consumption, many efforts have been devoted to the search of alternative energy.[3]Among the existing fossil fuel alternatives, biomass energy (in a recognized life cycle assessment, LCA) seems to have superior advantages from the perspective of environmental protection as biomass absorbs carbon dioxide during growth, thereby reducing the atmospheric concentration of carbon dioxide.[4,5]In addition, biofuel is porta- ble and can be used in the transportation sector, which is not possible with other renewable energies like solar energy and wind power.[6] Microalgal biomass and the energy rich compounds derived from microalgae, such as carbohydrates and lipids have emerged as the most popular feedstock for the production of biofuels.[7] Compared with other biofuels feedstock, microalgae do not compete with food crops for arable land or water resources, and they can grow in seawater or industrial/domestic wastewaters with a relatively fast growth rate than other plants.[8,9]Therefore, microalgae as the non-food biofuel feedstocks are environment friendly resour- ces of biomass energy and they could possible reduce greenhouse gas effect as they account for about 40% of global carbon fixation. Furthermore, compared to terrestrial plants, microalgae are more appropriate for biofuel produc- tion due to their relatively higher growth rate and higher lipid content compared to terrestrial plants. The oil content of microalgae by dry weight is typically in the range of 20–50%, while it can reach up to 70% in some microalgal strains.[10]It is also known that an enormous proportion of the crude oil is of microalgal origin, especially from the diatoms.[11]Several startup companies have been developed in the past few years trying to commercialize microalgae-derived fuels.[12]
Dr. I-S. Ng, S.-I Tan, P.-H. Kao, Dr. J.-S. Chang Department of Chemical Engineering
National Cheng Kung University, Tainan 70101, Taiwan
E-mail: [email protected] Dr. I-S. Ng, Dr. J.-S. Chang
Research Center for Energy Technology and Strategy
National Cheng Kung University, Tainan 70101, Taiwan
Prof. Y.-K. Chang
Graduate School of Biochemical Engineering Ming Chi University of Technology, New Taipei City 24301, Taiwan DOI: 10.1002/biot.201600644
Moreover, microalgae have metabolic pathways for the production of high value-added compounds, such as carote- noids,[13] polyunsaturated fatty acid (PUFA),[14] astaxanthin and lutein,[15]which can be applied in a biorefinery concept to make the production economically feasible.
Despite plenty of advantages for microalgae to be an excellent resource for biorefinery, there are still some technical difficulties that need to be overcome before commercializing biofuels and biologically active compound(s) from microalgae.[16] Several constraints for microalgae biorefinery are as follows: 1) cell stoichiometry or mass balance to shift composition toward the desired product; 2) higher cost of cultivation and conditions; and 3) suitable marketing and sale price.[17]Till now, it is estimated that the cost of a barrel of algae-based fuel using current technology is greater than US$300, compared with petroleum which is available at US$40 to 60 per barrel. The most cost incurring part in microalgal biofuel production is cultivation and the energy intensive harvesting of microalgae, and the strains used for biofuel production must have high biomass productivity and high carbohydrate/lipid productivity. One of the solutions is to cultivate microalgae in heterotrophic mode which offers a promising approach to obtain higher biomass and economically useful metabolites or high-value compounds.[18]On the other hand, increasing the biomass productivity by process engineer- ing strategies led to the design of expensive photobioreactors for stable outdoor cultivation.[19] Flotation systems for harvesting and new reactor designs for high biomass productivity have been considered to overcome these barriers[20] while different mediums were tested and optimized for the production of the target products.[21,22]However, the intrinsic metabolic capability of microalgae to accumulate lipids could not be improved by such strategies and lipid productivity is still limited in many microalgal strains.[14]Lipid accumulation is triggered in micro- algae when cell division is blocked by the depletion of certain nutrients like sulfur or nitrogen, whereas carbon is continuously fixed by cells leading to lipid accumulation. As a result, high lipid accumulation usually cannot be achieved during rapid micro- algal growth, thereby strongly limiting the lipid productivity of microalgae. It was also observed that some microalgal species with fast growth rate possess very lower lipid content.[23]Thus, it seems very difficult to achieve high cell growth rate and high cellular lipid content simultaneously. To overcome the intrinsic limitations associated with production of lipids or other function compounds from microalgae or cyanobacteria, a series of studies have been focused on genetic modification of microalgae to enhance carbon fixation,[24] lipid accumulation[25–27]and high value-added chemical formation.[28,29]
Genetic engineering recently gained more attentions because new and powerful genetic tools are increasingly available, and the genome can be edited to our need more accurately than before.[30] The metabolic approach or synthetic biology has received the most attention, as the design of a metabolic pathway in a system where it does not exist opens up new possibilities for industrialization of microalgae.[31]In synthetic biology, the basic gene manipulation process remains the same, including host selection, gene target, plasmid construction, transformation tools, selection system, and DNA editing tools.
In microalgae genetic engineering, the transformation and selection methods are key steps to accomplish the target gene
modification.[31–33]There are four main methods being used for transformation of microalgae: 1) agitation with glass beads; 2) electroporation; 3) particle bombardment; and 4) Agrobacte- rium-construction. Each method has its own advantages and disadvantages based on efficiency, integration, or stability of the transgene. Alternatively, the selection system can be based on antibiotic resistance or reporter gene selection. Different host and promoter strength would affect the selection efficiency.[34]
Besides, in metabolic engineering, genome-editing and gene- interfering tools are important for efficiently targeting the gene.
Recently, Clustered Regularly Interspaced Short Palindromic Repeats – CRISPR associated protein 9 (CRISPR-Cas9) and Transcription Activator-Like (TAL) Effector Nucleases (TALEN) which are the new genome-editing tools, as well as Zinc-Finger Nucleases (ZFN) are used in gene modification.[35–37]Desired microalgae phenotypes without gene modification could be achieved by gene-interfering tools, such as CRISPR-dCas9, micro RNA (miRNA), and silence RNA (siRNA), to repress or activate the gene expression.[38–40]The Omics approach is also an integrative technology for microalgal research in sustainable development.[41]
I-Son Ngis an assistant professor in the Department of Chemical Engineering at National Cheng Kung University (NCKU), Taiwan since 2014. She received her Ph.D. from NCKU in 2005 and then worked for Academia Sinica in 2006; and Xiamen University in 2010. She is devoted to the study of synthetic biology, genetic and enzymatic engineering, biorefinery, and bioremediation. She has developed a robust and efficient platform to explore the novel and functional enzymes by genetic and proteomics approach. She has published more than 40 SCI papers and also created the first iGEM team in NCKU.
Jo-Shu Changis a university chair professor in the Department of Chemical Engineering at National Cheng Kung University (NCKU), Taiwan. He received his Ph.D.
degree from University of California, Irvine in 1993. His research interests cover biochemical engineering, environmental biotechnology, and applied microbiology focus on microalgae-based CO2reutilization, biofuels production, and biorefineries. He plays an important role in Taiwan’s biomass energy R&D and policy making. He also serves as executive committee member of Asia Federation of Biotechnology (AFOB). He recently became fellow of American Institute of Medical and Biological Engineering (AIMBE) in 2015 and has received numerous prestigious domestic and international academic awards.
In this review, we summarize the basic gene manipulation tools in microalgal genetic engineering, including the transfor- mation methods, selection systems, and tools for genome engineering as well as gene expression levels. In particular, the CRISPR-Cas9 gene manipulation in microalgae will be discussed in detail. Moreover, we will delineate the metabolic engineering of microalgae with an emphasis on biofuel production, carbon fixation, and lycopene production, where the targeted function of cells could be enhanced by single gene enhancement, metabolic flux redirection, or new pathway construction. Finally, future perspectives on genetic engineering of microalgae are also discussed.
2. Microalgal Genome Editing
Metabolic engineering has generally become a central strategy for optimizing genetic and biosynthetic pathways within cells to increase the yield and rate of any metabolite. More than 30 years ago, the earliest successful DNA modification ofC. reinhardtii was accomplished by Rochaix and van Dillewijn.[42]From then on, a number of genetic techniques for improving efficiency[43]
and approaches have been developed forChlamydomonas.[24]But very few has been developed for other microalgae, where genetic tools are not available or not yet developed. Most successful biosynthetic results are reported for the model eukaryotic algal systemsChlamydomonas andChlorella[44]or prokaryotic micro- algae, cyanobacteria,[45] which produced high value biocompound(s) or proteins through the expression of certain
genes in algal cells, or by using genetic knockout and knockdown strategies to change the metabolicflow to a specific path. In the genetic engineering of microalgae, two important steps are involved: 1) the genetic delivery tools and 2) selectable and screenable markers. Some successful examples have been compiled in the following sections.
2.1. Transformation Techniques
Gene delivery by transformation to host is an important technique for molecular biotechnological applications, especially in the production of foreign proteins or modifications of specific metabolic pathway. A prerequisite for genetic engineering of any algal species is the formation of reliable and reproducible transformation systems, ideal for both nuclear and chloroplast genomes. Nuclear transformation usually occurs as a random insertion of the transgene in the nuclear genome (Figure 1A) by agitation with glass beads, where the algal cells and the DNA are agitated in the presence of 0.5 mm beads and this is the oldest method reported for microalgal transformation.[46,47] The transgenes would be inserted at random in the nuclear genome and selected by antibiotic resistance or phenotypic variation.
This method is quite simple and a high ratio of transformed cells can be achieved, but using a cell wall-less strain is required.
Electroporation requires the use of electrical impulses to deliver exogenous DNA into cells. It involves specific instrumentation and optimization steps, but has the highest rate of nuclear transformation for microalgae compared with the other
Figure 1.Genetic transformation strategies of microalgae. A) Nuclear transformation by glass beads agitation, electroporation and agrobacterium transfection, and random insertion. B) Chloroplasts transformation by biolistic and homologous recombination.
transformation systems.[48–52]In addition, other strategies that have been deemed suitable for nuclear transformation of microalgae involve agitation with silicon carbide whiskers,[53,54]
sonication in the presence of polyethylene glycol[55] or using Agrobacterium tumefaciens-mediated transfection to accomplish transformation.[56,57] Currently, the most efficient strategy for delivering foreign DNA fragments into chloroplast genome of algal cells, where the DNA must traverse multiple membranes, is microparticle bombardment with a biolistic particle gun.[58,59]
The chloroplast genome is altered by homologous recombina- tion between the chloroplast DNA (cpDNA) and the foreign DNA delivered by transformation (Figure 1B). Therefore, the transgene would integrate into the chloroplast chromosome within left border (LB) and right border (RB).
The four major transformation techniques, including bom- bardment, glass beads, electroporation, and Agrobacterium transfection for microalgae are used differently and summarized inTable 1. Bombardment technique is the only successful method to transform chloroplast DNA,[60] but the numbers of trans- formants obtained are relatively low (i.e., 100 clones per 106cells).
Bombardment, as a choice for chloroplast transformation, requires a biolistic device for DNA delivery like PDS-1000/He apparatus (Biorad, CA, USA) to accomplish the foreign gene transformation in chloroplast, where pressure control is the critical point.[59]Glass beads method is accomplished by vortexing the DNA and bead, which is the simplest method among four of them but the cell wall must be removed or cell wall less strain is required.[61] The efficiency is similar to bombardment and operates in very low cost for C. reinhardtii. However, it is not directly applied in many kinds of microalgae (i.e., Chlorella, Nannochloropsis, orPhaeodactylum) because their cell-walls are rigid.Agrobacteriumassisted transfection is widely used in many plants but it is still technically challenging for usage in microalgae, and only have been reported forC. reinhardtii,[56]Haematococcus pluvialis,[57] Chlorella vulgaris,[62] and oil-bearing marine algae Parachlorella kessleri.[63]Except for eukaryotic microalgae, prokary- otic microalgae such as cyanobacteria are amenable to genetic manipulation by conjugation or electroporation.[64]It produced important natural products, such as protein and vitamin or chemical compounds of 3-hydroxypropionic acid[65]and succinate acid[42] through electroporation. The electroporation, as an operation friendly equipment and well established protocol, is widely, effectively and dominantly applied for nuclear
transformation of microalgae.[59,66]The benefit of electroporation transformation is well-documented for different electric field intensity on different alga. For example, voltage ranging from 1000 to 2000 (cm 1) for pulse duration around 5 ms is acceptable for ChlamydomonasandChlorella, while voltage at 6000 (cm 1) for pulse 50 ms is preferred for Dunaliella salina. All the genetic modification was random insertion or deletion. Therefore, the long-term stability of transformants is a great concern and is discussed in the next section.
2.2. Transgenic Microalgal Strains, Stability, and Antibiotic Resistant
Following the successful and diversified genetic transformation of numerous algal species, the genomes of microalgae could be more easily manipulated. However, the genetic stability after transformation and the effect of continuation were a great concern. As shown in Table S1, Supporting Information, Chlamydomonas, Dunaliella, Chlorella, and Nannochloropsis, which are demonstrative microalgal species, show high stability after transformation.[46,67–70]However, other special algae, such as Thalassiosira weissflogii, Ulva lactuca, Poryhyra miniata, Kappaphycus alvarezii, andGracilaria changiiare unstable after nuclear transformation.[71–75] The transgenic DNA in chloro- plast of Chlamydomonas,[58] Chlorella,[76] Porphyridium,[77] and Euglena gracilis[78] are quite stable. Till now, the stability is highly uncertain in genetic engineering of microalgae. But more stable transformants have been reported in nuclear transformation.
Two mechanisms are primarily used to screen microalgal nuclear transformants: 1) generating auxotrophic defective mutants and then transforming them with the wild-type authentic gene,[79]or 2) integrating a gene that induces resistance to an antibiotic or herbicide. Antibiotic screening is the most frequently used approach. For a powerful genetic selection, the resistance genes require high efficiency and stability. However, the type and the concentration of selectable marker or antibiotic in different species plays a crucial role, thus these parts of conditional test are usually time consuming. The success of antibiotic marker for genetic transformation in various species of microalgae and specific concentration of antibiotics used are summarized in Table S2, Supporting Information. For commercial vectors (i.e.,
Table 1. Comparison of bombardment, glass beads, electroporation, andAgrobacteriumtransfection for transforming microalgae.
Criteria Bombardment
Glass
beads Electroporation Agrobacterium
Required equipment Complex PDS-1000/He apparatus (Biorad)
Simple Complex Gene Pulser Xcell (Biorad) or Gemini Systems (BTX)
Complex
Equipment cost High Low High High
Difficulty of usage Need of specialized Quite easy Easy Technically
challenging
Predominant type of transformation Chloroplast Nucleus Nucleus Nucleus
Removal of cell wall required No Yes No No
Demonstrated presence of exogenous DNA
Yes Yes Yes Yes
pChlamy), hygromycin is the commonly used antibiotic for selection. Zeocin, chloramphenicol, erythromycin, spectinomy- cin, and paromomycin are also used successfully for selection in Chlamydomonas reinhardtii, Chlorella sp., Synechococcus, and Nannochloropsissp. As shown in Table S2, Supporting Informa- tion, the concentration of hygromycin and zeocin resistance ranging from 10 to 20 mg L 1 in different kinds of algae.
Kanamycin, spectiomycin, and chloramphenicol are usually used at 100 mg L 1. It is worth mentioning that the model diatom P. tricornutum, is resistant to higher concentration of antibiotics like zeocin at 100 mg L 1[80] and chloramphenicol at 300 mg L 1,[81]respectively. The promoters CAMV35S and RbcS2 are used frequently for all species. Table S2, Supporting Information, provided an index for screening of genetically modified microalgae from the proper antibiotics with referred promoter at the beginning.
2.3. CRISPR Technology for Genome Editing in Microalgae
Over the past decade, genetic engineering of microalgae developed from delivery of DNA, focusing on transformation, selection, and currently till the most recent CRISPR/Cas9 technique. The timeline of genetic roadmap is shown in Figure 2. Since 1988, genetic engineering of microalgae has been developing from a variety of gene delivery techniques, including the chloroplast genome and the nuclear genome, even some improved methods are used to increase the efficiency of transformation. A breakthrough came in the 2000s, the regulation of microalgae metabolism began to be controlled by the developing gene editing strategies such as RNA interference, ZFNs, and TALENs. However ZFNs is more challenging to be programmed, as thefinger domain is 3–6 nucleotide triplets and the nucleases to which they are attached function only as dimers, thus the pairs of ZFNs are required to target any specific locus. TALENs is similar to ZFNs, but instead of recognizing DNA triplets, each domain recognizes a
single nucleotide would be much easier for design and operation.[82]
Over the past 20 years, the dominant genetic editing tools were zinc finger nucleases (ZFNs)[83] and transcription activator-like effector nucleases (TALENs).[84]They are artificial enzymes made by fusing an engineered DNA-binding domain to the FokI DNA-cleavage domain for targeting specific DNA sequences. Zincfinger domains or transcription activator-like effectors (TALEs) can be engineered to bind any desired DNA sequence that then ZFNs or TALENs perform specific cleavage at specific locations (Figure S1A, Supporting Information).
Both ZFNs and TALENs have lots of successful academic reports on genome editing in plants, insects and mam- mals[85,86] but seldom in the diatom P. tricornutum.[87]
Discovered in 2013, the CRISPR-Cas9 system belonging to the bacterial adaptive immune system is receiving extensive attention. A simplified variant of the type-II CRISPR-Cas9 system from Streptococcus pyogenes rely on CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) or single synthetic guide RNA (sgRNA) before the protospacer adjacent motif (PAM) to lead the Cas9 nuclease for triggering double- strand breaks (DSBs) in genomic DNA[88] (Figure S1A, Supporting Information). The schematic diagram and time- scale of development and applicability of the three gene editing tools including ZFNs, TALENs, and CRISPR/Cas9 system are summarized in Figure S1, Supporting Information. When compared with ZFNs and TALEN, CRISPR/Cas9 showed higher applicability but need more whole genomic data to prevent off-target sgRNA design. The CRISPR interference (CRISPRi) system uses the same design of guide RNA but with nuclease-deficient Cas9 (or dead Cas9) which lack the ability to cleave DNA and only function as a DNA binding complex for gene interference instead of gene modification for gene regulation.[89]The CRISPRi is a new approach, but the concept is similar to traditional RNA interference (RNAi) such as siRNA and miRNA (Figure S1B, Supporting Information). RNA interference is initiated by the enzyme Dicer, which cleaves
Figure 2.Timeline of genetic engineering development in microalgae.
molecules into short double-stranded fragments called siRNAs and split out miRNA. Inside the cell, protein Argonaute 2 (Ago) would bind with RNA-induced silencing complex (RISC) and induce RNA regulation, causing interference. As RNAi in microalgae is challenged by low efficiency, non-specific targeting and silencing of the RNA constraints, the new technology CRISPRi has great potential as it is much controllable. From 2014, demonstrations of CRISPR/Cas9- mediated genome editing in C. reinhardtii cells marked the beginning of a new age of genome editing in microalgae. Of course, the challenge of CRISPR/Cas9 genetic editing for microalgae is the toxicity of Cas9 nuclease, thus the mutation rate is within 10%.[89] To use Cas9 protein-gRNA ribonucleo- proteins (RNPs) is an alternative approach to overcome the toxicity of Cas9.[90]The successful clones are quite low and need further optimization in the future.
Thefirst assays to demonstrate the CRISPR-Cas9 based gene modification in Chlamydomonas reinhardtii, the model micro- algae, showed clear evidence that Cas9 and sgRNA can successfully express functions in algae, but lack efficiency and have low surviving ratio due to the toxicity of vector-driven Cas9.[89]This extreme problem was solved by directly delivering Cas9 protein-gRNA ribonucleoproteins (RNPs) into C. rein- hardtiito induce mutations at three loci and improved up to 100- fold compared to the earlier study.[35]In addition, some simple features of the application have been reported using the same method for the knockout ofCpFTSYandZEPtwo-gene.[90]There are still other demonstrations of CRISPR/Cas9-based genome editing of microalgae species. The genome of the marine diatom Phaeodactylum tricornutum can be efficiently edited by using optimized CRISPR/Cas9 vector.[91]CRISPR technology has also been successfully implemented onNannochloropsisspp., which became new model microalgae of carbon sequestration and oil- producing variety.[92] However, the practical use of this technology for the production of metabolites from microalgae is not yet demonstrated. Till 2017, CRISPRi isfirst applied for C. reinhardtii CC400 to enhance lipid production through repression of CrPEPC1 gene effectively.[93] For the study of photosynthetic mechanism, cyanobacteria are the evolutionary ancestors of plastids and serve as the conceptual model.
Important chemical compounds, such as 1-butanol, ethylene, and limonene are reported to be produced by genetically modified prokaryotic microalgae,Synechococcus sp.[94]In recent years, very few studies reported application of a CRISPR/Cas9 genome editing system in the fast-growing cyanobacterium S. elongatusUTEX 2973, as a proof of concept for the ability to produce a marker-free deletion mutant to target thenblAgene.[95]
The CRISPRi applied inSynechocystissp. PCC 6803 has been reported for multiple genes repression recently.[38] Moreover, several studies employed both CRISPR and CRISPRi system not only to trigger the gene regulation, but also achieved significantly improved succinate production inS. elongatusPCC 7942 for the first time.[39,96] For enhancement of lactate production, glutamine synthetase (glnA) repression strains were obtained from cyanobacterium Synechococcus sp. PCC 7002 via CRISPRi technology without reducing autotrophic growth rates and mutation on chromosome.[97]The successful applications of CRISPR/Cas9 gene editing in algae are summarized in Table 2.
3. Case Studies for Metabolic Engineering of Microalgae
3.1. Biofuels Production
Biofuels can be produced via thermal conversion, chemical conversion, and biochemical conversion of the biomass or its metabolic products. In the case of microalgae, the entire biomass and extracts could be converted into different forms of biofuels, such as biogasoline, bioethanol, biodiesel, and even jet fuel by the biorefinery processes.[98]In addition, with higher growth rate and lipid productivity, microalgae are more appropriate to serve as the biodiesel feedstock with a genetically modified lipid pathway, including enhancement of single or multiple genes involved in the production pathway or blocking the competing pathway.[99]There are three main steps in the lipid production pathway: namely, malonyl-CoA synthesis, acyl chain elongation, and triacylglycerol (TAG) formation in the sequential order.[100]
Also, three existing competitive pathway to lipid production are the b-oxidation,[101] phospholipid biosynthesis,[102] and the conversion of phosphoenolpyruvate to oxaloacetate.
In the first step of the lipid biosynthesis, acetyl-CoA carboxylase (ACC) plays a crucial role in metabolicflux to lipid biosynthesis since ACC catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, which is thefirst intermediate product to the fatty acid elongation pathway.[103,104]ACC possesses three activity subunits, including biotin carboxylase activity, biotin carboxyl carrier protein, and carboxyl transferase activity, which are composed of several polypeptides encoded by distinct genes.[105]A fully functional ACC is assembled by these three activity subunits with some specific difference amongst organisms.[105,106] So far, many reports on the overexpression of ACC from different species in diverse organisms, such as bacteria, microalgae, plants, etc., has concluded that single ACC overexpression indeed increased the activity of ACC and fatty acid biosynthesis rate because of the increased malonyl-CoA pool, while the lipid content was not enhanced signifi- cantly.[103,107] It is suggested that the committed step of lipid production catalyzed by ACC is not the rate-determining step in some special species or there are secondary rate-determining steps in the lipid production pathway as the expression of ACC exceeded in some level.[108]
After malonyl-CoA is synthesized by acetyl-CoA carboxylase from acetyl-CoA, a series of reactions for fatty acid production is catalyzed by fatty acid synthase (FAS).[109]The FAS is a cascade protein and classified into two types. Type I FAS existing in fungi, mammalian, and CMN group of bacteria is a multi- subunit protein, while Type II FAS composed of independent polypeptides encoded by separate genes are found in the archaea, bacteria, and plants.[110,111] Regardless of the classification of FAS, it belongs to the multi-enzymatic family, which including malonyl/acetyltransferase (MAT), acyl carrier protein (ACP), ketoacyl synthase (KS), ketoacyl reductase (KR), dehydrase (DH), enoyl reductase (ER), and thioesterase (TE).[112]First, malonyl- CoA:ACP transacetylase catalyzes the reaction of adding acryl carrier protein (ACP) to malonyl-CoA and produces the intermediate product, which the malonyl-CoA-ACP would influx into the fatty acid elongation cycle.[109] In the fatty acid elongation cycle, a series of enzyme-based Claisen condensation
will be taken place by KS, KR, DH, and ER to form thefinal product palmitic-ACP or stearic-ACP.[113,114] Then, palmitic- ACP or stearic-ACP thioester bonds are hydrolyzed by the TE, which produces palmitic acid or stearic acid.[115] In order to increase the accumulation of fatty acid, KS was stimulated resulting in changes in cell physiology and lipid profile, decreases in cell growth rate and lipid synthesis rate.[111] On the other hand, it is difficult to enhance the fatty acid production by modification of the pathway owing to the intrinsic properties of fatty acid synthase (FAS), as the enzyme is composed of many subunits, thus the modification on any subunit would affect the activity of whole FAS.[99]Besides, for the purpose of obtaining longer or unsaturated fatty acid, desaturases and elongases were introduced into the fatty acid synthesis, which uses palmitic acid or stearic acid as substrates.[116–121]
Final step of lipid production in microalgae is the triacylglyceride (TAG) formation. In glycerol phosphate-based pathway, glycerol-3-phosphate would be transformed into phosphatidate (PA) by glycerol phosphate acyltransferase (GPAT) and acylglycerolphosphate acyltransferase (AGPAT) sequentially. GPAT catalyzes the conversion of glycerol-3- phosphate into lysophosphatidate (LPA) which would be
condensed into phosphatidate by AGPAT afterward.[102,122,123]
Subsequently, dephosphorylation of PA would be catalyzed by phosphatidic acid phosphatase (PAP) to produce diacylglycerol.
Finally, the diacylglycerol would be esterified into triacyl- glycerol by diacylglycerol acyl-transferase (DGAT) which is regarded as one of the most important enzyme in TAG synthesis pathway.[124–126] Due to the importance of DGAT, overexpression of DGAT has been reported in many plants and microorganisms, which reveals that it could increase TAG accumulation.[114,127–130]The successful expression of diacylgly- cerol acyl-transferase gene in Scenedesmus obliquus were enhanced 128% of lipid content.[131]Therefore, the esterification of diacylglycerol catalyzed by DGAT is the rate-determining step in the lipid synthesis pathway.
Aside from the overexpression of gene in the lipid synthesis pathway, another approach is to block the competitive pathways for lipid synthesis. As mentioned earlier, there are three competitive pathways. One is the b-oxidation pathway which is the most straightforward competitive pathway and breaks down the fatty acid in cytosol or mitochondria and peroxisome for prokaryotes or eukaryotes separately.[101,132] Studies show that direct knock out ofb-oxidation pathway related genes or Table 2.Overview of published studies using CRISPR technology in microalgae.
Microalgae
species Editing method Target gene
Methods for verifying
the editing Positive contributions Ref.
C. reinhardtii (CC-530)
Vector-driven CRISPR/Cas9 system (C. reinhardtiicodon- optimized Cas9)
Three exogenous gene:mGFP, mGluc,Hygror
PCR/restriction enzyme analysis
First study of successful expression CRISPR/Cas9 system in microalgae
[89]
A endogenous gene:FKB12 C. reinhardtii
(CC-124)
Cas9 ribonucleoproteins (RNPs) MAA7,CpSRP43,ChlM Phenotypic characterization Improve the toxicity problem of producing Cas9 in algal cells
[90]
C. reinhardtii (CC-4349)
Cas9 ribonucleoproteins (RNPs) CpFTSY,ZEP Phenotypic characterization Knockout two-gene to constitutively produce zeaxanthin and improve the photosynthetic productivity
[35]
P. tricornutum (CCMP2561)
Vector-driven CRISPR/Cas9 system (P. tricornutumcodon- optimized Cas9)
CpSRP54 Phenotypic characterization Showed a demonstration of CRISPR editing in the diatom
[91]
N. oceanica (IMET1)
Vector-driven CRISPR/Cas9 system (codon-optimized Cas9)
NR(nitrate reductase) PCR/restriction enzyme analysis and phenotypic characterization
Showed a demonstration of CRISPR editing in the emerging oil-producing model microalgae
[92]
C. reinhardtii (CC-400)
Vector-driven CRISPR/Cas9 system (C. reinhardtiicodon- optimized Cas9)
CrPEPC1 PCR/restriction enzyme
analysis and phenotypic characterization
First successful enhanced lipid by CRISPRi/
dCas9 in microalgae
[93]
S. elongatus UTEX 2973
Vector-driven CRISPR/Cas9 system
nblA deletion Conjugation efficiency Toxicity effect in Cyanobacteria, markless demonstration
[95]
Synechcocystic sp. PCC6803
Vector-driven CRISPR/dCas9 system
phaE,glgC PCR/restriction enzyme
analysis and phenotypic characterization
Showed a multiple genes repression by CRISPRi
[38]
S. elongatus PCC 7942
Vector-driven CRISPR/Cas9 system
ppc,glgc,gltA PCR/restriction enzyme analysis and phenotypic characterization
Succinate production enhanced by passages [96]
S. elongatus PCC 7942
Vector-driven CRISPRi/dCas9 system
glycogenaccumulation (glgc), succinate dehydrogenase (sdhA and sdhB)
PCR/restriction enzyme analysis and phenotypic characterization
Succinate production in cyanobacterium [39]
Synechococcus sp. PCC 7002
Vector-driven CRISPRi/dCas9 system
Yfp,cpc cluster,ccm,glnA PCR/restriction enzyme analysis and fluorescent
Regulation of carboxysome to fix CO2and produced chemical
[97]
indirect inhibition of b-oxidation pathway by decreasing acyl- CoA transportation system could also efficiently enhance the lipid production pathway.[122,133]Another one is the phospho- lipid biosynthesis pathway where the phosphatidate would be converted into CDP-diacylglycerol, and influxes into the phospholipid biosynthesis pathway to form the cell membrane instead of TAG formation.[102,134] However, the inhibition of phospholipid biosynthesis pathway leads to abnormally long fatty acid production because the inhibition of this pathway affects the cell physiology by lack of phospholipids for cell membrane formation.[135] Another case is the reaction that converts phosphoenolpyruvate to oxaloacetate. In bio-lipid synthesis pathway, phosphoenolpyruvate (PEP) is the important metabolite that would be converted into pyruvate or oxaloacetate.
As a general rule, PEP is converted into pyruvate and the metabolic flux follows TCA cycle or lipid production pathway;
however, through genetic engineering for the phosphoenolpyr- uvate to be converted into oxaloacetate, catalyzed by phospho- enolpyruvate carboxylase (PEPC), the metabolicflux would only flow into the TCA cycle.[136] Consequently, many reports have exhibited that inhibition of the PEPC activity would enhance the lipid content by knock down of PEPC gene.[137–141] CRISPRi enhanced lipid production up to 94% by successful knock-down of PEPC inC. reinhardtii.[93]On the other hand, expression of transgenic malic enzyme in P. tricornutum enhanced lipid productivity by 2.5-fold compared to wild-type, while retaining the same growth rate.[142]
3.2. Carbon Fixation
Carbon fixation is the process by which the autotrophic organisms convert inorganic carbon available from the atmo- sphere to organic compounds. The metabolic pathway for carbon fixation in carbon-fixing organisms is crucial, because the atmospheric carbon dioxide must be mobilized as energy-rich organic forms for life on earth. Till now, six autotrophic carbon fixation pathways have been reported. The photoautotrophic organisms, such as the plants, algae, and cyanobacteria, absorb sunlight to convert water and carbon dioxide into organic carbon like glucose in chlorophyll by Calvin cycle.[143,144]Other special photoautotrophic organisms such as purple sulfur bacteria, convert hydrogen sulfide, instead of water, and carbon dioxide into organic carbon while releasing solid sulfurs.[145,146]Except for the Calvin cycle, the otherfive of six carbonfixation pathways are the reductive citric acid cycle, reductive acetyl CoA pathway and three relative cycles of 3-hydroxypropionate production discovered in some kind of bacterium,Chlorobium,Clostridium, Chloroflexus, enabling survival in harsh environment.[147–150]
RuBisCo (ribulose-1,5-bisphosphate carboxylase/oxygenase) is involved in thefirst step of carbonfixation in Calvin cycle and directs the carbon dioxide into Calvin cycle.[151]RuBisCo usually consists of two subunits. One of the subunit is encoded byrbcL gene or large-chain gene present in chloroplast DNA. Another subunit is encoded by small-chain genes, including several related genes in the nuclear DNA. A fully functional RuBisCo is assembled by eight large-chains and eight small-chains into a complex of about 54 000 kDa.[152]RuBisCo catalyzes not only the conversion of ribilose-1,5-bisphosphate and carbon dioxide to
3-phosphoglycerate, but also a side reaction of ribilose-1,5- bisphosphate and oxygen into 2-phosphoglycolate, a toxic metabolite for cell growth.[153] Consequently, the phosphogly- colate related metabolic product would be recycled by photo- respiratory pathway in mitochondria and peroxisomes and carbon dioxide would be released in the process of photorespi- ration.[154] In order to decrease the futile side reaction and enhance RuBisCo activity, a series of research on RuBisCo was focused on genetic modification of RuBisCo to enhance its selectivity and velocity.[155] However, limited success was reported owing to the intrinsic problems of RuBisCo as selectivity and velocity could not be enhanced simulta- neously.[156] Another approach to enhance carbon fixation is to heterologously overexpress some natural variants of RuBisCo, especially from red-algae, as it is slightly more effective than others.[157]To identify an efficient RuBisCo from the diverse pool is more effective than mutation of RuBisCo gene.[156,158]
Additionally, assembling different RuBisCo with high selectivity or catalytic velocity has been reported to enhance carbon fixation.[159]Overall, genetic modification of RuBisCo to increase catalytic velocity is preferable than that of improving selectivity, since the selectivity problem could be overcome by bioreactor design with high concentration of carbon dioxide.[160]
Actually, RuBisCo is not the sole enzyme to enhance carbon fixation. Metabolic flux control and regulation of metabolic pathway from Calvin cycle are also important to enhance carbon fixation.[153] As for the neighboring pathway of Calvin cycle, photorespiratory pathway could be bypassed by introducing the phosphoglycolate rerouting enzyme, which results in a higher carbon fixation rate and biomass accumulation owing to less energy consumption in the process of rerouting phosphoglyco- late.[161]As for the metabolicflux control of Calvin cycle, some of Calvin cycle enzymes have been tested, such as sedoheptulose- 1,7-bisphosphatase, transketolase, aldolase and so on, in order to enhance carbonfixation. A successful metabolicflux control for enhancing carbonfixation by overexpression of sedoheptulose- 1,7-bisphosphatase indeed increased photosynthetic effi- ciency.[144,162,163] However, it is not always the enhancement in carbonfixation by strengthening all the enzymes involved in Calvin cycle.[162]Therefore, theflux balance in Calvin cycle plays a critical role in carbon fixation. Through computer-based metabolic pathway construction/analysis by modeling the photosynthesis in algae and cyanobacteria or dynamic tracking of isotopes, Calvin cycle regulation could be more precisely controlled to enhance carbonfixation.[144,164]On the other hand, except RuBisCo, thioredoxin regulated enzymes such as sedoheptulose-bisphosphatase (SBPase), fructose-1,6-bisphos- phatase (FBPase), and ribulose-5-phosphate kinase (PRKase) are key enzymes in the Calvin cycle which involved in plant growth.[165] These enzymes are also supposed to accelerate carbonfixation.
Abiotic factors for carbonfixation also affect the efficiency of carbonfixation. For example, photoautotrophic organisms use light as energy to perform photosynthesis. However, excess light would induce photoinhibition, which results in inefficient utilization of light and decreased photosynthetic efficiency.[160]
To reduce photoinhibition, Beckmann et al.,[166] Mussgnug et al.,[167] and Masuda et al.,[168]have reported that shrinking chlorophyll antenna size allows more light transmission and
higher absorptive capacity of light resulting in higher biomass productivity. Another abiotic factor is the concentration of inorganic carbon. As mentioned above, the concentration of carbon dioxides affects the efficiency of Calvin cycle. Photoauto- trophic organisms have developed their own carbon concentrat- ing strategies. In algae, carboxysomes and pyrenoids are used to concentrate carbon dioxide by diverse carbonic anhy- drase.[169,170]Enhancement of carbonic anhydrase (CA) or other enzymes involved in carbon concentrating pathway may increase the carbon fixation rate.[171] One of the example is genetic S. elongatesPCC7942 with CA which accelerates the CO2fixation as well as biomass productivity.[172]Many environmental factors significantly influence the efficiency of Calvin cycle, especially metal ions and temperature.[173–176]Therefore, the abiotic factors are also critical for carbonfixation.
3.3. Other Biocompond(s) Production
First at all, carotenoids are a vast group of pigments widely found and synthesized in higher plants and green algae. In photosynthesis, carotenoids serve as light energy absorber and also protect chlorophyll from photodamage, which means that
carotenoids play a crucial role in effective photosynthesis.[177]As for humans, carotenoids have been reported to prevent some diseases and lung cancer.[178–180] Therefore, carotenoids are becoming attractive in the field of health-promoting foods.
Nowadays, carotenoids are produced in large quantities and extracted especially from microalgae due to their high productivity and high growth rate.[33,181] In order to enhance the mass production of carotenoids, aside from screening a high carotenoid producing microalgae, metabolic engineering approaches have been also applied because the pathway of the carotenoids biosynthesis has been extensively studied and most of the genes have been identified.[182–184]
In the carotenoid synthesis pathway, phytoene synthase (PSY) catalyzes thefirst step of carbonflux toward the production of phytoene, which is considered as the rate determining step in this pathway.[185,186]Afterward, the phytoene would be converted into lycopene by a cascade of enzyme, phytoene desaturase (PDS), z-carotene desaturase (ZDS), and carotene cis-trans isomerase (CRISCO).[187–189] Lycopene is the most significant intermediate which would turn on the production of high-value carotenoids. Therefore, enhancement of the expression of specific genes upstream of lycopene synthesis would indeed increase carotenoids production. The PSY and PDS were
Table 3.Summary of the bio-chemicals production by transgenic microalgae.
Host Target gene Genetic method
Transformation
method Result Ref.
Biofuel production
Navicula saprophila Acc1gene fromCyclotella cryptica Over-expression Bombardment 2–3ACC activity, no change in lipid content [107]
C. reinhardtii DGAT2gene fromBrassica napus Over-expression Electroporation 1.5 times increase in the lipid content and change the lipid profile
[127]
P. tricornutum DGAT2gene fromPhaeodactylum tricornutum
Over-expression Electroporation Increase the neutral lipid content by 35% [130]
C. reinhardtii PEPC1 gene RNA interfere Glass bead Increase the TAG level by 20% [139]
P. tricornutum PEPCK gene RNA interfere Electroporation 1.5 times increase in the lipid content and increase the TAG accumulation about 1.1 times
[141]
Carbon fixation
C. reinhardtii ModifiedNAB1gene from
Chlamydomonas reinhardtii(T541A, T676A)
Over-expression Glass bead Reduction of LHC antenna size by 10–17%
and about 50% increase of photosynthetic efficiency
[166]
S. elongates PCC7942
Carbonic anhydrase (CA) Over-expression Electroporation Carbon dioxide fixation increased 41% [172]
Other chemical compounds
C. reinhardtii PSYgene fromChlorella zofingiensis Over-expression Glass bead Content of the carotenoids were 2.0- and 2.2-fold increase
[185]
C. reinhardtii PSYgene fromD. salina Over-expression Glass bead Increase content of carotenoids by 25–160% [191]
H. pluvialis Modified PDSgene from Haematococcus pluvialis (L504R)
Over-expression Bombardment 43-fold higher resistance to the bleaching herbicide norflurazon and increase the astaxanthin production by about 35%
[194]
C. reinhardtii Modified PDSgene from Chlamydomonas reinhardtii (L505F)
Over-expression Glass bead 27.7-fold higher resistance to the herbicide norflurazon and increase the carotenoids production by about 20%
[195]
C. zofingiensis ModifiedPDSgene fromChlorella zofingiensis
Over-expression Bombardment Produce 32.1% more total carotenoids (TCs) and 54.1% more astaxanthin
[198]
C. vulgaris Human growth hormone Over-expression PEG-mediated 200–600 ng mL 1of protein in the culture [208]
highlighted to be the candidate genes for carotenoids produc- tion.[190]Regulation of the PSY gene was investigated in some microalgae, such as C. reinhardtii, H. pluvialis, and P. tricornutum, and the results indeed show an increase in carotenoids production on PSY gene over-expression.[185,191–193]
On the other hand, manipulation of PDS gene expression has also been reported to enhance carotenoids production in C. reinhardtii,H. pluvialis, andC. zofingiensis.[194–197]
High-value carotenoids will be synthesized in vivo just after the production of lycopene. Lycopene isfirst cyclized to forma- orb- type carotene by different lycopene cyclase, which would influence the carotenoid to be produced. Next, hydrolysis of carotene by different carotene hydrolase would produce lutein and zeaxanthin from a- and b-type carotene, respectively.[198–200] It has been reported that the deprivation of nitrogen also enhanced expression of cyclase.[198]However, reports regarding the regulation of cyclase and hydrolase by genetic engineering for microalgae are very few.
Additionally,b-carotene and zeaxanthin could be oxidized to form more high-value biocompound(s), such as canthaxanthin, viola- xanthin, or astaxanthin in specific microalgae,H. pluvialisand C. zofingiensis.[201–204]In this pathway,b-carotene oxygenase (BKT) is the key enzyme to be enhanced or introduced into other model microbes to produce astaxanthin. BKTwas successfully introduced inC. reinhardtiiand astaxanthin was produced as a result.[205]In thisfield, most researchers focus on enhancement of carotenoids production by changing culture conditions, by introducing some nutritional or abiotic stress. Enhancement of carotenoid produc- tion by genetic engineering is still limited and needs more attention. Moreover, the codon optimization for all genes expressed in different microalgae remains significant.
Fast growth rate, high protein content, and FDA-approval for human use are the advantage ofChlorellaspecies. For example, lutein content is up to 42.0 mg L 1 in C. sorokiniana.[206] C.
pyrenoidosaandC. vulgarisconsisted 57 and 58% of protein while the average of protein content inChlorellawas near 50%.[17,207]
Most successful genetic modification ofC. zofingiensishas used in stimulating carotenoids,[197–199] other cases are dominating in heterologous expressed protein in Chlorella species.[44] For example, the human growth hormone was the first report for genetically modified C. vulgaris in pharmaceutical use.[208]
However, the yield was quite low and genetic Chlorellaare still difficult to adapt in industry at the current stage. Some scientific reports on transgenic microalgae to produce versatile biocom- pound(s) are summarized inTable 3.
4. Conclusion and Prospects
Currently, microalgae are becoming an ideal biomass feedstock due to their ability of increased CO2sequestration, rapid growth rate, enhanced lipid content, and production of other high-value compounds like fatty acids and pigments. To reduce the production cost of high-value biocompounds from microalgae, new extraction methods, expansion of the use of microalgae and aquaculture, and the utilization of whole cells need to be explored further. Development of high performance microalgal strains by metabolic engineering is significant for making microalgae-derived products economically competitive.
However, the use of genetically modified microalga is still
challenged as non-GMO products are more favorable for human use, although over 100 Nobel Prize winners claimed that the uses of GMO products have no security issues in 2016. This review describes the essential strategies of genetic modification of microalgae, the tools used for gene editing, along with detailed discussion of genetically modified microalgae used for various purposes. In particular, the development of using CRISPR technology for targeted genome editing in certain species of microalgae and cyanobacteria are summarized. More successful and efficient studies need more whole genomic data of microalgae.
Omics approaches are also in high priority among all the techniques provided. As CRISPR technology can further evolve to seamless or scarless cloning without antibiotic marker, it is expected that the CRISPR-Cas9 system holds the potential to revolutionize future solutions for efficient and precise genetic engineering to produce biofuels or other valuable products from microalgae in a more efficient and commercially viable way.
Abbreviations
ACC, acetyl-CoA carboxylase; ACP, acyl carrier protein; AGPAT, acylglycerolphosphate acyltransferase; BKT, b-carotene oxygenase;
CRILSO, carotene cis-trans isomerase; CRISPR, clustered regularly interspaced short palindromic repeats; Cas9, CRISPR associated nuclease 9; CRISPRi, CRISPR interference; dCas9, dead Cas9; DH, dehydrase;
DGAT, diacylglycerol acyl-transferase; ER, enoyl reductase; FAS, fatty acid synthase; GPAT, glycerol phosphate acyltransferase; KR, ketoacyl reductase; KS, ketoacyl synthase; LPA, lysophosphatidate; MAT, malonyl/acetyltransferase; miRNA, micro RNA; PAP, phosphatase; PA, phosphatidate; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; PDS, phytoene desaturase; PSY, phytoene synthase; PUFA, polyunsaturated fatty acid; siRNA, small interfering RNA; TE, thioester- ase; TALENs, transcription activator-like effector nucleases; TAG, triacylglyceride; ZFNs, zinc finger nucleases; ZDS,z-carotene desaturase.
Supporting Information
Supporting Information is available from the Wiley Online or from the author.
Acknowledgement
The authors are grateful to the financial support for this study provided by the Ministry of Science and Technology (MOST 105-2221-E-006-225-MY3, MOST-105-2621-M-006-012-MY3 and MOST-105-2218-E-006-021) in Taiwan.
Conflict of Interest
The authors declare no commercial or financial conflict of interest.
Keywords
CRISPR-Cas9, genetic engineering, microalgae, selection, transformation
Received: February 23, 2017 Revised: July 24, 2017 Published online: September 18, 2017