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Biotechnological perspectives on algae: a viable option for next generation biofuels
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ScienceDirect Biotechnological perspectives on algae: a viable option for next generation biofuels Sikandar Khan1,2 and Pengcheng Fu1 Because of their biofuel producing capabilities, algae (including microalgae and cyanobacteria) are effective and sustainable tools to attain energy security with a growing world population and for reduction of our current reliance on fossil fuels. Algal metabolic and genetic engineering could provide substantial advancements in producing novel and promising strains for the production of alternative biofuels. In this review, we have highlighted biotechnological strategies for microalgae and cyanobacteria that target the improvement of: (1) biosynthesis of biofuel precursors (fatty acid, TAGs, and lipids etc.), (2) carbon-capture ability to accumulate more lipids, and (3) engineering hydrogenases for augmented production of biohydrogen. Other strategies for improving quality and quantity of algal biofuels are also explored. Addresses 1 State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou, 570228 China 2 Department of Biotechnology, Shaheed Benazir Bhutto University, Sheringal, KPK, Pakistan Corresponding author: Fu, Pengcheng ([email protected])
Current Opinion in Biotechnology 2020, 62:146–152 This review comes from a themed issue on Energy biotechnology Edited by Joe Shaw and Kirsten Benjamin
https://doi.org/10.1016/j.copbio.2019.09.020 0958-1669/ã 2019 Elsevier Ltd. All rights reserved.
Introduction Microalgae and cyanobacteria have received considerable attention as promising biological tools to grapple with the pressing problem of climate change caused by the reliance on fossil fuels, the emission of anthropogenic CO2, and a growing world population [1,2]. In accordance with their efficient photosynthetic CO2 fixation and ability to utilize industrial CO2 waste streams, use of non-arable land for cultivation of algal biomass and their higher productivity, algae are aimed at by the biotechnology research community to enable biofuels to gain economic competitiveness [2,3]. Algae biotechnology continues to attract interest in sustainable bioenergy production and emission reduction Current Opinion in Biotechnology 2020, 62:146–152
[4]. Many biotechnological studies regarding microalgal strain improvement for biofuels (biodiesel, bioethanol, biogas, and biohydrogen) production have focused on either mutagenesis of single target gene or engineering of multiple genes including complete pathways involved in lipid, starch or pigment biosynthesis [5]. This can be followed by genetic engineering strategies to modify microalgae and cyanobacteria for better ability to produce fatty acids and lipids (which upon transesterification generate biodiesel) including both knocking-out and overexpression of genes involved in lipid biosynthesis [1]. As illustrative examples, microalgae such as Chlorella vulgaris and the cyanobacterium Synechocystis PCC-6803 are frequently used as host microorganisms for genetic transformation or overexpression of native fermentation-related genes (for example, the PDC gene coding pyruvate decarboxylase and the ADH gene coding for alcohol dehydrogenase) in the production of bioethanol [6]. In addition, biogas (methane) produced through anaerobic digestion of algal biomass is a well-studied bioprocess configuration [7], biohydrogen is an attractive fuel alternative because its combustion produces no carbon byproducts and it is superior for bio-electricity production via fuel cells [8]. Therefore, genetic manipulation of hydrogenases in algae has improved the yield of biohydrogen production recently [9]. The successful genetic engineering of microalgae and cyanobacteria is not limited to the effective gene transformation and its expression, but it must also include other aspects, such as effect of heterologous DNA on the normal algal growth and physiology, the kind of strategy used, overall productivity, cost effectiveness and type of biofuel produced. The relevant information and tools required for engineering optimization of these photosynthetic microorganisms are diverse and not unidirectional. Therefore, we attempt to provide a brief but comprehensive review of the most frequently used biotechnological strategies for the genetic manipulation of cyanobacterial and microalgal strains that produce biofuels.
Framework for biofuel producing algae Because of their innate power of significant biomolecular synthesis for carbohydrate, protein, lipid and other biomolecules, algae are being considered as a viable renewable host system for biofuel production [10]. Sustainable production of algal bioproducts has a great potential to become a next-generation biofuel because of their higher productivity, ability to grow on non-arable land and high solar energy conversion efficiency [8]. In the last www.sciencedirect.com
Impact of biotechnology on algal biofuels Khan and Fu 147
decades, there has been an increasing interest in exploiting microalgae and cyanobacteria for the production of biofuel precursors, such as lipids in the form of triacylglycerol (TAG) and starch, which can be transformed into biodiesel and bioethanol, respectively [11,12]. In addition, some green algal species are capable of photolysismediated biohydrogen (H2) production, which is an attractive, and clean fuel that helps in carbon emission reduction [8,13]. A schematic presentation of the basic steps in eco-friendly algal biofuel production is summarized in Figure 1. The figure shows a typical algal cell presenting a closed loop of carbon cycle from photosynthetic carbon-capture by green algae to biosynthesis of starch, fatty acids, TAGs and derived lipids that upon transesterification create FAMEs (fatty acid methyl esters), which are the major components of biodiesel.
Biotechnological strategies for improvement of algal biofuels As the cell size, lipid composition and starch content differ among microalgae and cyanobacterial species, therefore, not all of them are useful for the production of biofuels. Some species are better-suited for biofuel
production as they are able to naturally biosynthesize and accumulate considerable amounts of intracellular lipids and starch which can be further improved upon genetic modifications [1]. For example, Chlamydomonas reinhardtii, Synechocystis sp. PCC 6803, Phaeodactylum tricornutum, and various Chlorella species are among the extensively studied engineered algal strains for their biofuel producing potentials [9,14,15]. A detailed description of various genetically engineered algal species is given in the upcoming section which will provide a solution to increase strain productivity and facilitate the development of economically feasible production of algal biofuels. Both unicellular microalgae and cyanobacteria occasionally produce excessive amounts of commercially important bio-products like biofuels [16]. Therefore, the genetic modification of them needs to be introduced to augment the production of precursor molecules for biofuels [17–22]. To date, commercially achievable production of these biofuel precursors has been limited by their high cost and by the fact that these compounds are usually accumulated under stressed conditions which
Figure 1
MICROALGAE BIOFUELS
1. Enhancing carbon capture ability & biomass of algae
2. G.E. for enhanced biosynthesis of fatty acids & lipid (Carboxylases)
3. Genetic manipulation of algae for enganced bioethanol production
4. Genetic engineering for Biohydrogen synthesis (Hydrogenases) Current Opinion in Biotechnology
A typical algal cell depicting biotechnological engineering for boosting algal biofuel (biodiesel, bioethanol, and biohydrogen) production. Photosynthetic carbon-capture by green algae leads to biosynthesis of starch, fatty acids, TAGs and lipids which upon transesterification making fatty acid methyl esters (FAMEs-major components of biodiesel). The rectangles (1–4) displayed at the bottom emphasizing the main and selfexplanatory biotechnological strategies for improvement of algal biofuels. The abbreviations used are TAGs (Triacylglycerols), and FAMEs (Fatty Acid Methyl Esters). www.sciencedirect.com
Current Opinion in Biotechnology 2020, 62:146–152
Biotechnological modifications of microalgae and cyanobacteria for enhanced algal biofuel production Algal strain
Type of modification
Targeted gene
Outcomes a
Type of biofuel produced
Ref.
Chlamydomonas reinhardtii (strain 704) Chlamydomonas reinhardtii + Pseudomonas sp. (strain D) Synechocystis sp. PCC 6803
Nuclear transformation
Acetyl-CoA synthetase (ACS2)
Biodiesel
[11]
Algae-bacteria consortium
–
Starch content = " 2-fold Triacylglycerol = " 2.4-fold H2 = " 26-fold (154 mL L1)
Biohydrogen
[8]
Knocking out (KO) and Triparental conjugation (TC)
KO = Acyl-ACP synthetase (aas), TC = Thioesterase (tes3), Carboxylic acid reductase (car), Phosphopanteth-einyl transferase (sfp) Carbonic anhydrase (CA)
1-octanol and 1-decanol = " 26-fold (<100 mg L1)
Bio-alcohol
[12]
Lipid accumulation = " 2.2-fold (1.1 g L1) Single mutation = " 7-fold, Double mutation = " 30-fold H2
Biodiesel
[43]
Biohydrogen
[9]
Augmented hydrogenase activity = " H2 Lipid contents = " 28.5% Productivity = 34.9 mg L1 day1 Neutral lipids = " 94% Total lipids = " 56% Intracellular TAGs = " 2.4-fold
Biohydrogen
[10]
Biodiesel
[25]
Biodiesel
[17]
Biojet/biodiesel
[18]
Lipid contents = " 2.7-fold, 55.7% of dry weight Biomass and FAME = " 2-fold
Biodiesel
[14]
Biodiesel
[19]
Hydrocarbon accumulation (free fatty acids, fatty alcohols, alkanes and alkenes) in UVM4 = " 8-fold, and in PCC 6803 = " 19-fold
Hydrocarbon fuel (biodiesel, bioethanol)
[15]
Lipid contents = " 67% Productivity = 91 mg L1 day1
Biodiesel
[23]
Lipid contents in AG125 deficient Obi = " 65.4%, Biomass = # 26.2%
Biodiesel
[37]
Chlorella sorokiniana (CS) and Chlorella vulgaris (CV) Chlorella sp. (strain DT)
Genetic transformation by electroporation Site directed mutagenesis and homologous transformation
Chlorococcum minutum
Environmental and biochemical stresses CRISPRi based transcriptional silencing Genetic transformation (heterologous expression) CmFAX1 overexpression and knockout
Chlamydomonas reinhardtii (strain CC-400) Chlamydomonas reinhardtii (strain CC-424) Cyanidioschyzon merolae (strain 10D) Phaeodactylum tricornutum Chlamydomonas reinhardtii (strain CC-124) Chlamydomonas reinhardtii (strain UVM4), and Synechocystis sp. PCC 6803
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Chlorella vulgaris mutant (UV715) Coccomyxa sp. strain Obi
Genetic transformation by electroporation (GenePulser) Polyploidization Knocking out, Triparental conjugation and heterologous expression
2nd round of mutagenesis creating (UV715-EMS25) Mutagenesis by a transcription activator-like effector nuclease (TALEN)
Hydrogenase (hydA)- mutatedCshydAc.DT (modify A.A. residues A105I, V265W, G113I, and V273I around the gas tunnel) Hydrogenase Phosphoenolpyruvate carboxylase (PEPC1) Dunaliella tertiolecta fatty acyl-ACP thioesterase (DtTE) CmFAX1(chloroplast inner membrane protein—export of fatty acids) Glucose-6-phosphate dehydrogenase (G6PD) Duplication of genome (Diploid) Five synthetic metabolic pathways, 1. TesA (thioesterase) and Daas (acyl ACP synthase), 2. CAR (carboxylic acid reductase), 3. UndA and UndB (responsible for 1-undecene biosynthesis in Pseudomonas, 4. OleT (responsible for olefin biosynthesis in Jeotgalicoccus), 5. FAP (fatty acid photodecarboxylase) Random mutagenesis using ethyl methanesulfonate (EMS) ATP:a-D-glucose-1-phosphate adenylyltransferase (AGPL) (starchless mutant AG125)
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Table 1
The outcomes or fold changes mentioned in this column are with respect to control or wild type of the algal/cyanobacterial strains. " indicate enhanced/improved, while # show decreased/reduced. a
[46] Strategy to improve biodiesel production rbcL and accD Chlorella sorokiniana
Genetic transformation, overexpression and transcriptomics Over expression and Transcriptomics
Transcriptomics
Lipid productivity = " 2.3-fold, Biomass = " 1.6-fold
[45] Strategy for improvement (Enhancing tolerance to biofuel)
[33]
[44]
Strategy to improve quality and quantity of biofuel Strategy to improve productivity
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Acetyl-CoA carboxylase (acc), and Acyl-ACP synthetase (aas) Triacylglycerol lipases (TAG lipases) Response regulator genes slr1037 and sll0039 Transcription profile analysis
Synechococcus sp. (strain HS01) Fistulifera solaris JPCC (strain DA0580) Synechocystis sp. PCC 6803
[26] Strategy to improve quality and quantity of biofuel
Degree of unsaturation in transcripts DGAT2A:2C:2D for SFA:MUFA: PUFA in TAG were "1.3-, "3.7-, and "11.2-fold, respectively Lipid productivity = " 2.8-fold, role of acc and aas in lipid 16 of the 42 lipase genes were upregulated during lipid degradation Tolerance to 1-butanol = "133%
[32]
Type-2 Diacylglycerol Acyltransferases (DGAT2s)
Ref.
Biodiesel Lipids content = " 64.25% Phospholipase A2 (PLA2)
CRISPR-Cas9 based targetspecific knockout Overexpression and Knockdown of DGAT2A, 2C, and 2D Chlamydomonas reinhardtii (strains CC-4349) Nannochloropsis oceanica (strain IMET1)
Table 1 (Continued )
Outcomes a Targeted gene Type of modification Algal strain
Type of biofuel produced
Impact of biotechnology on algal biofuels Khan and Fu 149
may limit algal growth and their ultimate yield [11,23]. Nevertheless, these organisms, particularly cyanobacteria are excellent biotechnological platforms to accumulate valuable compounds, with the advantages of comparatively simple genomes, easy transformation, diversity of suitable strains and availability of fully sequenced genomes of some of the model strains [12,24]. The biotechnological strategies used for enhancing algal biofuel production through genetic manipulation of these microorganisms are diverse, but the main themes are; enhancing lipids and starch biosynthesis, increasing carbon-capture ability and lipids accumulation, and genetic engineering for biohydrogen production. Genetic engineering for enhancing algal fatty acids, lipids and starch biosynthesis
Engineering attempts of lipid production in microalgae include overexpression of enzymes involved in the biosynthesis of fatty acids and lipids [12,17,18,25], TAG biosynthesis and assembly [26–30], or enhancing fatty alcohols [12,31], as well as targeted knocking down or blocking competitive pathways, such as starch biosynthesis or catabolism of lipids and so on [25,32,33–35]. Several approaches have also focused on transcription factors, which regulate lipid biosynthetic pathways [36], or enhancing availability of the reducing agent NADPH, by overexpression of the relative genes, which has been used to increase the fatty acid content in P. tricornutum [14]. The degree of success of these genetic approaches is largely variable; therefore, in many cases the results obtained are not those expected. Table 1 highlights some key features of biotechnological engineering in various algal strains for enhanced algal biofuel production. The outcomes after genetic modifications are presented in terms of fold change in starch and fatty acid content, lipid and fatty alcohol productivity as well as biohydrogen production with respect to control or wild type strains (Table 1). Chlorella vulgaris mutant (UV715), Coccomyxa sp. strain Obi and C. reinhardtii (strains CC-4349) are at the top three among the genetically modified algal strains mentioned in this review. In terms of improvement, their percent lipid content are " 67%, " 65% and " 64% for the above strains, respectively [23,32,37]. The modification strategies used for this improvement were two rounds of mutagenesis, mutagenesis by a transcription activator-like effector nuclease (TALEN) and a CRISPR-Cas9 based targetspecific knockout of PLA2, respectively (Table 1). In this case, the TALEN-based mutagenesis was conducted on Coccomyxa sp. strain Obi to create a starchless mutant (AG125) that improved lipid productivity at the cost of a 26.2% reduction in biomass. Therefore, suppression of starch biosynthesis to increase lipid productivity in microalgae seems to be a risky strategy [37]. Additionally, five synthetic metabolic pathways, such as 1. TesA (thioesterase) and Daas (acyl ACP synthase), 2. CAR (carboxylic acid Current Opinion in Biotechnology 2020, 62:146–152
150 Energy biotechnology
reductase), 3. UndA and UndB (responsible for 1-undecene biosynthesis in Pseudomonas, 4. OleT (responsible for olefin biosynthesis in Jeotgalicoccus), 5. FAP (fatty acid photodecarboxylase) were introduced in to two green cell factories (C. reinhardtii strain UVM4, and Synechocystis sp. PCC 6803) [15]. This engineering strategy (Knocking out, Triparental conjugation and heterologous expression) resulted in enhanced hydrocarbon accumulation (free fatty acids, fatty alcohols, alkanes and alkenes), or eightfold increase for UVM4, and 19-fold increase for PCC 6803 (Table 1). Enhancing carbon-capture ability and lipid accumulation
Overexpression of key genes related to carbon fixation pathways in microalgae was found to be a promising strategy to capture excess CO2 for enhanced lipid accumulation [38–40]. Carbonic anhydrase (CA) is an efficient and wellstudied enzyme existing in the majority of the algal strains for catalyzing the CO2 and bicarbonate interconversion. Various studies upon genetic engineering of CA has proved its active role in CO2-sequestration. It is thus considered an essential component of the carbon concentrating mechanism in Nannochloropsis oceanica [41,42]. Additionally, an exogenous CA gene was successfully overexpressed in C. vulgaris and Chlorella sorokiniana [43]. As a result, the transgenic algae had improved biomass production, accelerated carbon fixation rates as well as increased lipid accumulation (2.2-fold increased lipid content compared to the wild types, with up to 1.1 g/L titer) (Table 1).
and more diverse biofuel molecules for this societally important technology. Therefore, a shift of research paradigms is needed to introduce cutting edge biotechnologies, such as synthetic biology, gene editing and artificial intelligence to promote the industrial scale production of algal biofuels.
Conflict of interest statement Nothing declared.
Acknowledgements SK and PF were financially supported by the Research Start-Up Funds from Hainan University in China (KYQD_ZR2017212).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest Urtubia HO, Betanzo LB, Va´squez M: Microalgae and cyanobacteria as green molecular factories: tools and perspectives. Algae-organisms for Imminent Biotechnology. IntechOpen; 2016 A comprehensive review chapter, highlighting the current strategies in microalgae biotechnology and presenting analysis of the challenges to genetically manipulate microalgae and cyanobacteria, including their transformation and selection methodologies.
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Genetic modification for biohydrogen production
The key enzymes responsible for bio-hydrogen production, upon reduction of protons (H+) to H2, in algae are hydrogenases and nitrogenases [47]. Some important activities on genetic engineering of enzymes and metabolic pathways for enhanced hydrogen production in microalgae and cyanobacteria are described in a recent review by Khetkorn et al. [48]. The algal [FeFe] hydrogenase is the most efficient H2-producing enzyme, although it is highly O2 sensitive. This sensitivity is the main obstruction for algal photolysis based hydrogen production [49]. Many solutions have been devised to reduce the O2 level by algal-bacterial consortiums or to increase hydrogenase resistance to oxidative damage and overcome this problem [8,10,50]. In one such attempt of genetic engineering the Chlorella sp. (strain DT) hydrogenase A amino acid residues A105I, V265W, G113I, or V273I around the H2 gas tunnel were modified. As a result, O2 accessing the active site of enzyme was prevented, which enhanced H2 production up to 30-fold [9].
Conclusions So far, successful industrial-scale applications of algal-based biofuel production are rare, highlighting the scientific and applied challenges that must still be overcome before largescale, economically competitive bio-manufacturing is achieved. The recent examples of biotechnology-driven catalyst improvement offer an exciting path forward towards more effective CO2 capture, higher biofuel productivities, Current Opinion in Biotechnology 2020, 62:146–152
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42. Tan S-I, Han Y-L, Yu Y-J, Chiu C-Y, Chang Y-K, Ouyang S, Fan KC, Lo K-H, Ng I-S: Efficient carbon dioxide sequestration by using recombinant carbonic anhydrase. Process Biochem 2018, 73:38-46.
47. Bolatkhan K, Kossalbayev BD, Zayadan BK, Tomo T, Veziroglu TN, Allakhverdiev SI: Hydrogen production from phototrophic microorganisms: reality and perspectives. Int J Hydrogen Energy 2019, 44:5799-5811.
43. Lin W-R, Lai Y-C, Sung P-K, Tan S-I, Chang C-H, Chen C-Y, Chang J-S, Ng I-S: Enhancing carbon capture and lipid accumulation by genetic carbonic anhydrase in microalgae. J Taiwan Inst Chem E 2018, 93:131-141 In this paper, an exogenous carbonic anhydrase gene was successfully overexpressed in both Chlorella sorokiniana and Chlorella vulgaris. The transgenic algae exhibit improved biomass productivity and lipid accumulation as a result of accelerating the carbon capture and fixation.
48. Khetkorn W, Rastogi RP, Incharoensakdi A, Lindblad P, Madamwar D, Pandey A, Larroche C: Microalgal hydrogen production–a review. Bioresource Technol 2017, 243:1194-1206.
44. Modiri S, Zahiri HS, Vali H, Noghabi KA: Evaluation of transcription profile of acetyl-CoA carboxylase (ACCase) and acyl-ACP synthetase (AAS) to reveal their roles in induced lipid accumulation of Synechococcus sp. HS01. Renew Energy 2018, 129:347-356.
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49. Weiner I, Shahar N, Feldman Y, Landman S, Milrad Y, Ben-Zvi O, Avitan M, Dafni E, Schweitzer S, Eilenberg H: Overcoming the expression barrier of the ferredoxin-hydrogenase chimera in Chlamydomonas reinhardtii supports a linear increment in photosynthetic hydrogen output. Algal Res 2018, 33:310-315. 50. Kubas A, Orain C, De Sancho D, Saujet L, Sensi M, Gauquelin C, Meynial-Salles I, Soucaille P, Bottin H, Baffert C: Mechanism of O2 diffusion and reduction in FeFe hydrogenases. Nat Chem 2017, 9:88.
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ScienceDirect Biotechnological perspectives on algae: a viable option for next generation biofuels Sikandar Khan1,2 and Pengcheng Fu1 Because of their biofuel producing capabilities, algae (including microalgae and cyanobacteria) are effective and sustainable tools to attain energy security with a growing world population and for reduction of our current reliance on fossil fuels. Algal metabolic and genetic engineering could provide substantial advancements in producing novel and promising strains for the production of alternative biofuels. In this review, we have highlighted biotechnological strategies for microalgae and cyanobacteria that target the improvement of: (1) biosynthesis of biofuel precursors (fatty acid, TAGs, and lipids etc.), (2) carbon-capture ability to accumulate more lipids, and (3) engineering hydrogenases for augmented production of biohydrogen. Other strategies for improving quality and quantity of algal biofuels are also explored. Addresses 1 State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou, 570228 China 2 Department of Biotechnology, Shaheed Benazir Bhutto University, Sheringal, KPK, Pakistan Corresponding author: Fu, Pengcheng ([email protected])
Current Opinion in Biotechnology 2020, 62:146–152 This review comes from a themed issue on Energy biotechnology Edited by Joe Shaw and Kirsten Benjamin
https://doi.org/10.1016/j.copbio.2019.09.020 0958-1669/ã 2019 Elsevier Ltd. All rights reserved.
Introduction Microalgae and cyanobacteria have received considerable attention as promising biological tools to grapple with the pressing problem of climate change caused by the reliance on fossil fuels, the emission of anthropogenic CO2, and a growing world population [1,2]. In accordance with their efficient photosynthetic CO2 fixation and ability to utilize industrial CO2 waste streams, use of non-arable land for cultivation of algal biomass and their higher productivity, algae are aimed at by the biotechnology research community to enable biofuels to gain economic competitiveness [2,3]. Algae biotechnology continues to attract interest in sustainable bioenergy production and emission reduction Current Opinion in Biotechnology 2020, 62:146–152
[4]. Many biotechnological studies regarding microalgal strain improvement for biofuels (biodiesel, bioethanol, biogas, and biohydrogen) production have focused on either mutagenesis of single target gene or engineering of multiple genes including complete pathways involved in lipid, starch or pigment biosynthesis [5]. This can be followed by genetic engineering strategies to modify microalgae and cyanobacteria for better ability to produce fatty acids and lipids (which upon transesterification generate biodiesel) including both knocking-out and overexpression of genes involved in lipid biosynthesis [1]. As illustrative examples, microalgae such as Chlorella vulgaris and the cyanobacterium Synechocystis PCC-6803 are frequently used as host microorganisms for genetic transformation or overexpression of native fermentation-related genes (for example, the PDC gene coding pyruvate decarboxylase and the ADH gene coding for alcohol dehydrogenase) in the production of bioethanol [6]. In addition, biogas (methane) produced through anaerobic digestion of algal biomass is a well-studied bioprocess configuration [7], biohydrogen is an attractive fuel alternative because its combustion produces no carbon byproducts and it is superior for bio-electricity production via fuel cells [8]. Therefore, genetic manipulation of hydrogenases in algae has improved the yield of biohydrogen production recently [9]. The successful genetic engineering of microalgae and cyanobacteria is not limited to the effective gene transformation and its expression, but it must also include other aspects, such as effect of heterologous DNA on the normal algal growth and physiology, the kind of strategy used, overall productivity, cost effectiveness and type of biofuel produced. The relevant information and tools required for engineering optimization of these photosynthetic microorganisms are diverse and not unidirectional. Therefore, we attempt to provide a brief but comprehensive review of the most frequently used biotechnological strategies for the genetic manipulation of cyanobacterial and microalgal strains that produce biofuels.
Framework for biofuel producing algae Because of their innate power of significant biomolecular synthesis for carbohydrate, protein, lipid and other biomolecules, algae are being considered as a viable renewable host system for biofuel production [10]. Sustainable production of algal bioproducts has a great potential to become a next-generation biofuel because of their higher productivity, ability to grow on non-arable land and high solar energy conversion efficiency [8]. In the last www.sciencedirect.com
Impact of biotechnology on algal biofuels Khan and Fu 147
decades, there has been an increasing interest in exploiting microalgae and cyanobacteria for the production of biofuel precursors, such as lipids in the form of triacylglycerol (TAG) and starch, which can be transformed into biodiesel and bioethanol, respectively [11,12]. In addition, some green algal species are capable of photolysismediated biohydrogen (H2) production, which is an attractive, and clean fuel that helps in carbon emission reduction [8,13]. A schematic presentation of the basic steps in eco-friendly algal biofuel production is summarized in Figure 1. The figure shows a typical algal cell presenting a closed loop of carbon cycle from photosynthetic carbon-capture by green algae to biosynthesis of starch, fatty acids, TAGs and derived lipids that upon transesterification create FAMEs (fatty acid methyl esters), which are the major components of biodiesel.
Biotechnological strategies for improvement of algal biofuels As the cell size, lipid composition and starch content differ among microalgae and cyanobacterial species, therefore, not all of them are useful for the production of biofuels. Some species are better-suited for biofuel
production as they are able to naturally biosynthesize and accumulate considerable amounts of intracellular lipids and starch which can be further improved upon genetic modifications [1]. For example, Chlamydomonas reinhardtii, Synechocystis sp. PCC 6803, Phaeodactylum tricornutum, and various Chlorella species are among the extensively studied engineered algal strains for their biofuel producing potentials [9,14,15]. A detailed description of various genetically engineered algal species is given in the upcoming section which will provide a solution to increase strain productivity and facilitate the development of economically feasible production of algal biofuels. Both unicellular microalgae and cyanobacteria occasionally produce excessive amounts of commercially important bio-products like biofuels [16]. Therefore, the genetic modification of them needs to be introduced to augment the production of precursor molecules for biofuels [17–22]. To date, commercially achievable production of these biofuel precursors has been limited by their high cost and by the fact that these compounds are usually accumulated under stressed conditions which
Figure 1
MICROALGAE BIOFUELS
1. Enhancing carbon capture ability & biomass of algae
2. G.E. for enhanced biosynthesis of fatty acids & lipid (Carboxylases)
3. Genetic manipulation of algae for enganced bioethanol production
4. Genetic engineering for Biohydrogen synthesis (Hydrogenases) Current Opinion in Biotechnology
A typical algal cell depicting biotechnological engineering for boosting algal biofuel (biodiesel, bioethanol, and biohydrogen) production. Photosynthetic carbon-capture by green algae leads to biosynthesis of starch, fatty acids, TAGs and lipids which upon transesterification making fatty acid methyl esters (FAMEs-major components of biodiesel). The rectangles (1–4) displayed at the bottom emphasizing the main and selfexplanatory biotechnological strategies for improvement of algal biofuels. The abbreviations used are TAGs (Triacylglycerols), and FAMEs (Fatty Acid Methyl Esters). www.sciencedirect.com
Current Opinion in Biotechnology 2020, 62:146–152
Biotechnological modifications of microalgae and cyanobacteria for enhanced algal biofuel production Algal strain
Type of modification
Targeted gene
Outcomes a
Type of biofuel produced
Ref.
Chlamydomonas reinhardtii (strain 704) Chlamydomonas reinhardtii + Pseudomonas sp. (strain D) Synechocystis sp. PCC 6803
Nuclear transformation
Acetyl-CoA synthetase (ACS2)
Biodiesel
[11]
Algae-bacteria consortium
–
Starch content = " 2-fold Triacylglycerol = " 2.4-fold H2 = " 26-fold (154 mL L1)
Biohydrogen
[8]
Knocking out (KO) and Triparental conjugation (TC)
KO = Acyl-ACP synthetase (aas), TC = Thioesterase (tes3), Carboxylic acid reductase (car), Phosphopanteth-einyl transferase (sfp) Carbonic anhydrase (CA)
1-octanol and 1-decanol = " 26-fold (<100 mg L1)
Bio-alcohol
[12]
Lipid accumulation = " 2.2-fold (1.1 g L1) Single mutation = " 7-fold, Double mutation = " 30-fold H2
Biodiesel
[43]
Biohydrogen
[9]
Augmented hydrogenase activity = " H2 Lipid contents = " 28.5% Productivity = 34.9 mg L1 day1 Neutral lipids = " 94% Total lipids = " 56% Intracellular TAGs = " 2.4-fold
Biohydrogen
[10]
Biodiesel
[25]
Biodiesel
[17]
Biojet/biodiesel
[18]
Lipid contents = " 2.7-fold, 55.7% of dry weight Biomass and FAME = " 2-fold
Biodiesel
[14]
Biodiesel
[19]
Hydrocarbon accumulation (free fatty acids, fatty alcohols, alkanes and alkenes) in UVM4 = " 8-fold, and in PCC 6803 = " 19-fold
Hydrocarbon fuel (biodiesel, bioethanol)
[15]
Lipid contents = " 67% Productivity = 91 mg L1 day1
Biodiesel
[23]
Lipid contents in AG125 deficient Obi = " 65.4%, Biomass = # 26.2%
Biodiesel
[37]
Chlorella sorokiniana (CS) and Chlorella vulgaris (CV) Chlorella sp. (strain DT)
Genetic transformation by electroporation Site directed mutagenesis and homologous transformation
Chlorococcum minutum
Environmental and biochemical stresses CRISPRi based transcriptional silencing Genetic transformation (heterologous expression) CmFAX1 overexpression and knockout
Chlamydomonas reinhardtii (strain CC-400) Chlamydomonas reinhardtii (strain CC-424) Cyanidioschyzon merolae (strain 10D) Phaeodactylum tricornutum Chlamydomonas reinhardtii (strain CC-124) Chlamydomonas reinhardtii (strain UVM4), and Synechocystis sp. PCC 6803
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Chlorella vulgaris mutant (UV715) Coccomyxa sp. strain Obi
Genetic transformation by electroporation (GenePulser) Polyploidization Knocking out, Triparental conjugation and heterologous expression
2nd round of mutagenesis creating (UV715-EMS25) Mutagenesis by a transcription activator-like effector nuclease (TALEN)
Hydrogenase (hydA)- mutatedCshydAc.DT (modify A.A. residues A105I, V265W, G113I, and V273I around the gas tunnel) Hydrogenase Phosphoenolpyruvate carboxylase (PEPC1) Dunaliella tertiolecta fatty acyl-ACP thioesterase (DtTE) CmFAX1(chloroplast inner membrane protein—export of fatty acids) Glucose-6-phosphate dehydrogenase (G6PD) Duplication of genome (Diploid) Five synthetic metabolic pathways, 1. TesA (thioesterase) and Daas (acyl ACP synthase), 2. CAR (carboxylic acid reductase), 3. UndA and UndB (responsible for 1-undecene biosynthesis in Pseudomonas, 4. OleT (responsible for olefin biosynthesis in Jeotgalicoccus), 5. FAP (fatty acid photodecarboxylase) Random mutagenesis using ethyl methanesulfonate (EMS) ATP:a-D-glucose-1-phosphate adenylyltransferase (AGPL) (starchless mutant AG125)
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Table 1
The outcomes or fold changes mentioned in this column are with respect to control or wild type of the algal/cyanobacterial strains. " indicate enhanced/improved, while # show decreased/reduced. a
[46] Strategy to improve biodiesel production rbcL and accD Chlorella sorokiniana
Genetic transformation, overexpression and transcriptomics Over expression and Transcriptomics
Transcriptomics
Lipid productivity = " 2.3-fold, Biomass = " 1.6-fold
[45] Strategy for improvement (Enhancing tolerance to biofuel)
[33]
[44]
Strategy to improve quality and quantity of biofuel Strategy to improve productivity
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Acetyl-CoA carboxylase (acc), and Acyl-ACP synthetase (aas) Triacylglycerol lipases (TAG lipases) Response regulator genes slr1037 and sll0039 Transcription profile analysis
Synechococcus sp. (strain HS01) Fistulifera solaris JPCC (strain DA0580) Synechocystis sp. PCC 6803
[26] Strategy to improve quality and quantity of biofuel
Degree of unsaturation in transcripts DGAT2A:2C:2D for SFA:MUFA: PUFA in TAG were "1.3-, "3.7-, and "11.2-fold, respectively Lipid productivity = " 2.8-fold, role of acc and aas in lipid 16 of the 42 lipase genes were upregulated during lipid degradation Tolerance to 1-butanol = "133%
[32]
Type-2 Diacylglycerol Acyltransferases (DGAT2s)
Ref.
Biodiesel Lipids content = " 64.25% Phospholipase A2 (PLA2)
CRISPR-Cas9 based targetspecific knockout Overexpression and Knockdown of DGAT2A, 2C, and 2D Chlamydomonas reinhardtii (strains CC-4349) Nannochloropsis oceanica (strain IMET1)
Table 1 (Continued )
Outcomes a Targeted gene Type of modification Algal strain
Type of biofuel produced
Impact of biotechnology on algal biofuels Khan and Fu 149
may limit algal growth and their ultimate yield [11,23]. Nevertheless, these organisms, particularly cyanobacteria are excellent biotechnological platforms to accumulate valuable compounds, with the advantages of comparatively simple genomes, easy transformation, diversity of suitable strains and availability of fully sequenced genomes of some of the model strains [12,24]. The biotechnological strategies used for enhancing algal biofuel production through genetic manipulation of these microorganisms are diverse, but the main themes are; enhancing lipids and starch biosynthesis, increasing carbon-capture ability and lipids accumulation, and genetic engineering for biohydrogen production. Genetic engineering for enhancing algal fatty acids, lipids and starch biosynthesis
Engineering attempts of lipid production in microalgae include overexpression of enzymes involved in the biosynthesis of fatty acids and lipids [12,17,18,25], TAG biosynthesis and assembly [26–30], or enhancing fatty alcohols [12,31], as well as targeted knocking down or blocking competitive pathways, such as starch biosynthesis or catabolism of lipids and so on [25,32,33–35]. Several approaches have also focused on transcription factors, which regulate lipid biosynthetic pathways [36], or enhancing availability of the reducing agent NADPH, by overexpression of the relative genes, which has been used to increase the fatty acid content in P. tricornutum [14]. The degree of success of these genetic approaches is largely variable; therefore, in many cases the results obtained are not those expected. Table 1 highlights some key features of biotechnological engineering in various algal strains for enhanced algal biofuel production. The outcomes after genetic modifications are presented in terms of fold change in starch and fatty acid content, lipid and fatty alcohol productivity as well as biohydrogen production with respect to control or wild type strains (Table 1). Chlorella vulgaris mutant (UV715), Coccomyxa sp. strain Obi and C. reinhardtii (strains CC-4349) are at the top three among the genetically modified algal strains mentioned in this review. In terms of improvement, their percent lipid content are " 67%, " 65% and " 64% for the above strains, respectively [23,32,37]. The modification strategies used for this improvement were two rounds of mutagenesis, mutagenesis by a transcription activator-like effector nuclease (TALEN) and a CRISPR-Cas9 based targetspecific knockout of PLA2, respectively (Table 1). In this case, the TALEN-based mutagenesis was conducted on Coccomyxa sp. strain Obi to create a starchless mutant (AG125) that improved lipid productivity at the cost of a 26.2% reduction in biomass. Therefore, suppression of starch biosynthesis to increase lipid productivity in microalgae seems to be a risky strategy [37]. Additionally, five synthetic metabolic pathways, such as 1. TesA (thioesterase) and Daas (acyl ACP synthase), 2. CAR (carboxylic acid Current Opinion in Biotechnology 2020, 62:146–152
150 Energy biotechnology
reductase), 3. UndA and UndB (responsible for 1-undecene biosynthesis in Pseudomonas, 4. OleT (responsible for olefin biosynthesis in Jeotgalicoccus), 5. FAP (fatty acid photodecarboxylase) were introduced in to two green cell factories (C. reinhardtii strain UVM4, and Synechocystis sp. PCC 6803) [15]. This engineering strategy (Knocking out, Triparental conjugation and heterologous expression) resulted in enhanced hydrocarbon accumulation (free fatty acids, fatty alcohols, alkanes and alkenes), or eightfold increase for UVM4, and 19-fold increase for PCC 6803 (Table 1). Enhancing carbon-capture ability and lipid accumulation
Overexpression of key genes related to carbon fixation pathways in microalgae was found to be a promising strategy to capture excess CO2 for enhanced lipid accumulation [38–40]. Carbonic anhydrase (CA) is an efficient and wellstudied enzyme existing in the majority of the algal strains for catalyzing the CO2 and bicarbonate interconversion. Various studies upon genetic engineering of CA has proved its active role in CO2-sequestration. It is thus considered an essential component of the carbon concentrating mechanism in Nannochloropsis oceanica [41,42]. Additionally, an exogenous CA gene was successfully overexpressed in C. vulgaris and Chlorella sorokiniana [43]. As a result, the transgenic algae had improved biomass production, accelerated carbon fixation rates as well as increased lipid accumulation (2.2-fold increased lipid content compared to the wild types, with up to 1.1 g/L titer) (Table 1).
and more diverse biofuel molecules for this societally important technology. Therefore, a shift of research paradigms is needed to introduce cutting edge biotechnologies, such as synthetic biology, gene editing and artificial intelligence to promote the industrial scale production of algal biofuels.
Conflict of interest statement Nothing declared.
Acknowledgements SK and PF were financially supported by the Research Start-Up Funds from Hainan University in China (KYQD_ZR2017212).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest Urtubia HO, Betanzo LB, Va´squez M: Microalgae and cyanobacteria as green molecular factories: tools and perspectives. Algae-organisms for Imminent Biotechnology. IntechOpen; 2016 A comprehensive review chapter, highlighting the current strategies in microalgae biotechnology and presenting analysis of the challenges to genetically manipulate microalgae and cyanobacteria, including their transformation and selection methodologies.
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Genetic modification for biohydrogen production
The key enzymes responsible for bio-hydrogen production, upon reduction of protons (H+) to H2, in algae are hydrogenases and nitrogenases [47]. Some important activities on genetic engineering of enzymes and metabolic pathways for enhanced hydrogen production in microalgae and cyanobacteria are described in a recent review by Khetkorn et al. [48]. The algal [FeFe] hydrogenase is the most efficient H2-producing enzyme, although it is highly O2 sensitive. This sensitivity is the main obstruction for algal photolysis based hydrogen production [49]. Many solutions have been devised to reduce the O2 level by algal-bacterial consortiums or to increase hydrogenase resistance to oxidative damage and overcome this problem [8,10,50]. In one such attempt of genetic engineering the Chlorella sp. (strain DT) hydrogenase A amino acid residues A105I, V265W, G113I, or V273I around the H2 gas tunnel were modified. As a result, O2 accessing the active site of enzyme was prevented, which enhanced H2 production up to 30-fold [9].
Conclusions So far, successful industrial-scale applications of algal-based biofuel production are rare, highlighting the scientific and applied challenges that must still be overcome before largescale, economically competitive bio-manufacturing is achieved. The recent examples of biotechnology-driven catalyst improvement offer an exciting path forward towards more effective CO2 capture, higher biofuel productivities, Current Opinion in Biotechnology 2020, 62:146–152
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25. Kao P-H, Ng I-S: CRISPRi mediated phosphoenolpyruvate carboxylase regulation to enhance the production of lipid in Chlamydomonas reinhardtii. Bioresource Technol 2017, 245:1527-1537 This study used CRISPRi to regulate expression of an exogenously expressed rfp gene as a proof-of-concept, and of the endogenous PEPC1 gene as a proof-of-function in Chlamydomonas reinhardtii for higher biomass and lipid accumulation in microalgae. 26. Xin Y, Lu Y, Lee Y-Y, Wei L, Jia J, Wang Q, Wang D, Bai F, Hu H, Hu Q: Producing designer oils in industrial microalgae by rational modulation of co-evolving type-2 diacylglycerol acyltransferases. Mol Plant 2017, 10:1523-1539. 27. Fukuda S, Hirasawa E, Takemura T, Takahashi S, Chokshi K, Pancha I, Tanaka K, Imamura S: Accelerated triacylglycerol production without growth inhibition by overexpression of a glycerol-3-phosphate acyltransferase in the unicellular red alga Cyanidioschyzon merolae. Sci Rep 2018, 8:12410. 28. Balamurugan S, Wang X, Wang H-L, An C-J, Li H, Li D-W, Yang WD, Liu J-S, Li H-Y: Occurrence of plastidial triacylglycerol synthesis and the potential regulatory role of AGPAT in the model diatom Phaeodactylum tricornutum. Biotechnol Biofuels 2017, 10:97. 29. Klaitong P, Fa-Aroonsawat S, Chungjatupornchai W: Accelerated triacylglycerol production and altered fatty acid composition in oleaginous microalga Neochloris oleoabundans by overexpression of diacylglycerol acyltransferase 2. Microb Cell Fact 2017, 16:61. 30. Wei H, Shi Y, Ma X, Pan Y, Hu H, Li Y, Luo M, Gerken H, Liu J: A type-I diacylglycerol acyltransferase modulates triacylglycerol biosynthesis and fatty acid composition in the oleaginous microalga, Nannochloropsis oceanica. Biotechnol Biofuels 2017, 10:174. 31. Kaczmarzyk D, Cengic I, Yao L, Hudson EP: Diversion of the long-chain acyl-ACP pool in Synechocystis to fatty alcohols through CRISPRi repression of the essential phosphate acyltransferase PlsX. Metab Eng 2018, 45:59-66. 32. Shin YS, Jeong J, Nguyen THT, Kim JYH, Jin E, Sim SJ: Targeted knockout of phospholipase A2 to increase lipid productivity in Chlamydomonas reinhardtii for biodiesel production. Bioresource Technol 2019, 271:368-374 The CRISPR-Cas9 system was applied for a target-specific knockout of the phospholipase A2 gene in Chlamydomonas reinhardtii. The authors obtained a mutant that showed an increased DAGs pool and a higher accumulation of TAGs without a reduction in cell growth. 33. Nomaguchi T, Maeda Y, Liang Y, Yoshino T, Asahi T, Tanaka T: Comprehensive analysis of triacylglycerol lipases in the oleaginous diatom Fistulifera solaris JPCC DA0580 with transcriptomics under lipid degradation. J Biosci Bioeng 2018, 126:258-265. 34. Huang C-H, Shen CR, Li H, Sung L-Y, Wu M-Y, Hu Y-C: CRISPR interference (CRISPRi) for gene regulation and succinate production in cyanobacterium S. elongatus PCC 7942. Microb Cell Fact 2016, 15:196. 35. Wei L, Xin Y, Wang Q, Yang J, Hu H, Xu J: RNA i-based targeted gene knockdown in the model oleaginous microalgae Nannochloropsis oceanica. Plant J 2017, 89:1236-1250. 36. Ajjawi I, Verruto J, Aqui M, Soriaga LB, Coppersmith J, Kwok K, Peach L, Orchard E, Kalb R, Xu W: Lipid production in Nannochloropsis gaditana is doubled by decreasing expression of a single transcriptional regulator. Nat Biotechnol 2017, 35:647. 37. Takahashi K, Ide Y, Hayakawa J, Yoshimitsu Y, Fukuhara I, Abe J, Kasai Y, Harayama S: Lipid productivity in TALEN-induced starchless mutants of the unicellular green alga Coccomyxa sp. strain Obi. Algal Res 2018, 32:300-307. 38. Oh Y-K, Hwang K-R, Kim C, Kim JR, J-S Lee: Recent developments and key barriers to advanced biofuels: a short review. Bioresource Technol 2018, 257:320-333. 39. Dinamarca J, Levitan O, Kumaraswamy GK, Lun DS, Falkowski PG: Overexpression of a diacylglycerol acyltransferase gene in Phaeodactylum tricornutum directs carbon towards lipid biosynthesis. J Phycol 2017, 53:405-414. Current Opinion in Biotechnology 2020, 62:146–152
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40. Huang Y, Cheng J, Lu H, He Y, Zhou J, Cen K: Transcriptome and key genes expression related to carbon fixation pathways in Chlorella PY-ZU1 cells and their growth under high concentrations of CO2. Biotechnol Biofuels 2017, 10:181.
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41. Gee CW, Niyogi KK: The carbonic anhydrase CAH1 is an essential component of the carbon-concentrating mechanism in Nannochloropsis oceanica. Proc Natl Acad Sci U S A 2017, 114:4537-4542.
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43. Lin W-R, Lai Y-C, Sung P-K, Tan S-I, Chang C-H, Chen C-Y, Chang J-S, Ng I-S: Enhancing carbon capture and lipid accumulation by genetic carbonic anhydrase in microalgae. J Taiwan Inst Chem E 2018, 93:131-141 In this paper, an exogenous carbonic anhydrase gene was successfully overexpressed in both Chlorella sorokiniana and Chlorella vulgaris. The transgenic algae exhibit improved biomass productivity and lipid accumulation as a result of accelerating the carbon capture and fixation.
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49. Weiner I, Shahar N, Feldman Y, Landman S, Milrad Y, Ben-Zvi O, Avitan M, Dafni E, Schweitzer S, Eilenberg H: Overcoming the expression barrier of the ferredoxin-hydrogenase chimera in Chlamydomonas reinhardtii supports a linear increment in photosynthetic hydrogen output. Algal Res 2018, 33:310-315. 50. Kubas A, Orain C, De Sancho D, Saujet L, Sensi M, Gauquelin C, Meynial-Salles I, Soucaille P, Bottin H, Baffert C: Mechanism of O2 diffusion and reduction in FeFe hydrogenases. Nat Chem 2017, 9:88.
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