The synthetic biology toolkit for photosynthetic ... · 111 commercial significance. In 1999, the...
Transcript of The synthetic biology toolkit for photosynthetic ... · 111 commercial significance. In 1999, the...
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Short title: Synthetic biology in photosynthetic microorganisms 1
The synthetic biology toolkit for photosynthetic microorganisms 2
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Konstantinos Vavitsas1,2,5, Pierre Crozet3, Marcos Hamborg Vinde1,2, Fiona Davies4, Stéphane D. 4
Lemaire3, Claudia E. Vickers1,2* 5
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1 Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St 7
Lucia, Queensland 4072 8
2 CSIRO Synthetic Biology Future Science Platform, CSIRO Land & Water, GPO Box 2583, Brisbane, 9
Qld, 4001 10
3 Institut de Biologie Physico-Chimique, UMR 8226, CNRS, Sorbonne Université, Paris, France 11
4 Department of Chemistry, Colorado School of Mines, Golden, CO 80401, USA 12
5current address: Department of Biology, National and Kapodistrian University of Athens, 13
Panepistimioupolis, Athens 15784, Greece 14
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*Corresponding Author: [email protected] / [email protected] 16
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One-sentence summary: Photosynthetic microorganisms offer novel characteristics as synthetic 18
biology chassis, and the toolbox of componentry and techniques for cyanobacteria and algae is 19
rapidly increasing. 20
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Author contributions: All authors contributed to the writing of the paper 22
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Abstract 24
Cyanobacteria and algae are photosynthetic microorganisms that are emerging as attractive hosts 25
for synthetic biology applications, ranging from metabolic engineering for the production of 26
industrial biochemicals to microbial energy storage. Photosynthetic microbes have a unique 27
combination of characteristics that drives their consideration for synthetic biology, including 28
relatively simple physiology, fast photosynthetic growth, an availability of model systems, and useful 29
subcellular structures. However, their development as synthetic biology hosts/chassis has been 30
relatively slow compared to that of model heterotrophs. Here we review current synthetic biology 31
tools and applications in photosynthetic microbes and identify opportunities and challenges moving 32
into the future. We focus on cyanobacteria as model photosynthetic prokaryotes, specifically the 33
freshwater unicellular species Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942, 34
Plant Physiology Preview. Published on July 1, 2019, as DOI:10.1104/pp.19.00345
Copyright 2019 by the American Society of Plant Biologists
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the marine unicellular species Synechococcus sp. PCC 7002, and the nitrogen fixing filamentous 35
species Nostoc sp. PCC 7120. Moreover, we present algae as model photosynthetic eukaryotes, 36
specifically the unicellular green alga Chlamydomonas reinhardtii and the diatom Phaeodactylum 37
tricornutum. Finally, we include an examination of some emerging systems. For each organism, the 38
toolbox of genetic parts, high-throughput assembly systems, and other methods that are key 39
components to facilitate synthetic biology are described. Challenges and future directions for 40
synthetic biology and its applications in photosynthetic microorganisms are also discussed. 41
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Photosynthetic micro-organisms: attractive targets for synthetic biology 42
The appearance of microorganisms capable of oxygenic photosynthesis was a turning point in the 43
history of life: they shaped evolution by oxygenating the oceans and the atmosphere (Catling et al., 44
2005; Buick, 2008). A few billion years after the first cyanobacterial-like microbe started splitting 45
water into oxygen and hydrogen, photosynthetic microbes were found in almost every terrestrial 46
ecosystem, from hot springs to arctic ice. Moreover, a cyanobacterium was the likely ancestor of 47
chloroplasts (Jensen and Leister, 2014), an endosymbiosis which was deemed crucial for the 48
colonization of land by plants (Cavalier-Smith, 2010). 49
Photosynthetic microorganisms—specifically, cyanobacteria and algae—offer novel characteristics 50
as synthetic biology hosts. Their unique combination of characteristics, including relatively fast 51
photosynthetic growth, availability of redox power, a plethora of internal membranes, and 52
subcellular microcompartments, opens up a completely new palette of synthetic biology strategies 53
that is not possible in other well-studied heterotrophic host organisms. The photosynthetic 54
machinery is full of interesting targets for synthetic biology strategies (Leister, 2019), such as 55
enhancing photosynthesis for increased biomass production and yield (Zhu et al., 2010), direct 56
coupling of metabolic pathways to photosynthetic reducing power (Mellor et al., 2017), and creation 57
of bio-nano hybrids where photosynthetic modules are used as a source of electrons for non-58
biological processes by linking them to abiotic catalysts or electrode nanomaterials (Saar et al., 2018) 59
or by derivatization of photosynthetic electrons by redox mediators (Longatte et al., 2015; Fu et al., 60
2017). Although in principle such applications can be hosted in plants, photosynthetic 61
microorganisms offer distinct advantages: the combination of photosynthesis with simple unicellular 62
organization, facile genetic manipulation strategies, quick growth in liquid cultures, relative ease of 63
scale-up, and, if grown in contained facilities, potentially fewer regulatory challenges. 64
However, synthetic biology applications in photosynthetic microbes lags behind when compared to 65
model heterotrophs (e.g., Escherichia coli and yeast). One reason is the lack of synthetic biology 66
tools and enabling technologies, which are crucial to achieve their full potential. Fortunately, in 67
recent years, significant advances have rendered synthetic biology applications in algae and 68
cyanobacteria using classical design-build-test cycles more feasible (Figure 1). In its most advanced 69
form, synthetic biology relies on standardization of genetic parts and high-throughput assembly and 70
transformation, the latter of which is far more achievable in photosynthetic microbes than in higher 71
photosynthetic organisms. This perspective provides an overview of recent developments in these 72
areas and identifies some of the challenges facing synthetic biology applications in photosynthetic 73
microbes. 74
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Synthetic biology in cyanobacteria 76
Cyanobacteria are photoautotrophic prokaryotes with fossil records dating back 3.5 billion years, 77
making them the oldest known microbes (Giordano et al., 2005). The cyanobacterial clade is quite 78
diverse; despite the apparent morphological and physiological similarity between different species, 79
extant cyanobacteria may have branched evolutionarily from their last common ancestor more than 80
a billion years ago (Tomitani et al., 2006). Now, in the era of synthetic biology, cyanobacteria have 81
been identified as excellent chassis for microbial cell factories due to their fast growth, small 82
genome size, genetic amenability, and natural transformability. The recently identified 83
Synechococcus elongatus UTEX 2973 strain has one of the fastest growth rates reported for any 84
photosynthetic microbe (Yu et al., 2015; Ungerer et al., 2018); high rates of photosynthesis to 85
produce photosynthates and biomass are required to support this growth. Cyanobacteria are also 86
known for their metabolic flexibility (Xiong et al., 2017), meaning they can thrive in conditions where 87
dramatic fluctuations in nutrient availability, salinity, pH, irradiance, temperature and moisture 88
occur. Thus, cyanobacteria can rapidly acclimate to environmental fluctuations, which is an 89
important characteristic of a robust industrial strain. They can also grow at environmental extremes. 90
For example, Athrospira sp. (‘Spirulina’) are grown successfully in large-scale open ponds at high pH 91
and salt concentrations, conditions that resist invasion from predating and contaminating species 92
(Jiménez et al., 2003). 93
Cyanobacteria have carbon fixation and photosynthesis characteristics that may lead to unique 94
synthetic biology applications. Firstly, carbon fixation takes place in dedicated organelles called 95
carboxysomes. Carboxysomes can be manipulated to host heterologous enzymes, thus generating 96
artificial metabolic microcompartments with a variety of uses (Gonzalez-Esquer et al., 2016). 97
Secondly, photosynthesis does not take place in specialized photosynthetic organelles. This means 98
that their photosynthetic membranes and photosystems therein are more accessible to 99
heterologous proteins. This has enabled the direct linkage of photosynthesis with heterologous 100
metabolic reactions (direct light-driven synthesis) by expression of electron-consuming enzymes 101
(cytochromes P450) in the thylakoid membranes (Berepiki et al., 2016; Wlodarczyk et al., 2016; 102
Mellor et al., 2017). A drawback of the extensive membrane system in cyanobacteria is that our 103
understanding of metabolic regulation through their intermingling respiratory and photosynthetic 104
electron transport chains is incomplete. 105
The most commonly utilized cyanobacteria for basic and applied research are the freshwater 106
unicellular species Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942, the marine 107
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unicellular species Synechococcus sp. PCC 7002, and the nitrogen-fixing filamentous species Nostoc 108
sp. PCC 7120. Synthetic biology applications in cyanobacteria have principally been directed towards 109
metabolic engineering to redistribute photosynthetically fixed carbon towards chemicals of 110
commercial significance. In 1999, the first synthetic metabolic pathway was constructed in 111
Synechococcus elongatus PCC 7942 by introducing Zymomonas mobilis pyruvate decarboxylase (pdc) 112
and alcohol dehydrogenase II (adh) genes to allow ethanol production in this species (Deng and 113
Coleman, 1999). A wealth of subsequent literature describes synthetic biology applications in 114
cyanobacterial metabolic engineering (Nielsen et al., 2016; Matson and Atsumi, 2018; Sengupta et 115
al., 2018). Synthetic biology is also being used to deliver applications other than industrial 116
biochemical production in cyanobacteria. For example, it was recently shown that 3-D printed 117
cyanobacteria could generate and store electricity from light and subsequently power small devices 118
for a limited time (McCormick et al., 2015; Sawa et al., 2017). 119
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Genetic parts 121
Many cyanobacteria (including all of the model species) are naturally transformable and can 122
integrate transgenes into their chromosomes via homologous recombination. Whereas current 123
resources are not as extensive as in heterotrophic model microbes, numerous synthetic components 124
have been characterized for use in cyanobacteria. Vectors and genome editing tools are described in 125
subsequent sections. 126
Promoters and other control elements. Promoters are key synthetic biology components in 127
cyanobacteria (Camsund and Lindblad, 2014; Stensjö et al., 2018); a large selection is available, 128
including constitutive, inducible, and repressible promoters and riboswitches (Ma et al., 2014; 129
Ohbayashi et al., 2016; Videau et al., 2016; Qiao et al., 2017; Immethun and Moon, 2018; Wegelius 130
et al., 2018).A detailed description of the promoter landscape is provided in Box 1, and promoter 131
characteristics summarized in Table 1 for ease of reference. In addition, ribosome binding sites 132
(Englund et al., 2016; Thiel et al., 2018) and riboregulators, which are RNA sequences that respond 133
to signal nucleic acids to modify expression (Abe et al., 2014b), have been characterized. 134
Selectable Markers. Genetic engineering of cyanobacteria is facilitated by antibiotic resistance 135
cassettes. Thus far, resistance to spectinomycin, kanamycin, chloramphenicol, gentamycin, and 136
erythromycin has been used successfully (Eaton-Rye, 2010; Heidorn et al., 2011). Another selection 137
technique that has been applied results in unmarked mutants, and it is based in two consecutive 138
recombination events: the first introduces an antibiotic resistance marker and a sucrose-sensitivity 139
gene (selection on antibiotic), the second replaces the selection markers with the native sequence, 140
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maintaining the transgene (selection on sucrose) (Lea-Smith et al., 2016). This technique is 141
applicable only to cyanobacteria that can grow mixotrophically. For example, Synechocystis sp. PCC 142
6803 and Synechocystis sp. PCC 7002 are naturally mixotrophic, but Synechococcus elongatus UTEX 143
2973 and Synechococcus elongatus PCC 7942 are not. However, mixotrophy can also be engineered, 144
as has been demonstrated in Synechcystis elongatus PCC 7942 (Kanno and Atsumi, 2017). 145
Reporter Genes. Reporter genes are used to illustrate the behavior of genetic parts in a variety of 146
different contexts and are essential to characterize synthetic biology circuits. Bacterial luciferase and 147
fluorescent proteins are most commonly used [reviewed in (Heidorn et al., 2011; Berla et al., 2013)]. 148
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Promoters, characterized using reporter genes, are used for transcriptional regulation. Numerous 149
studies have identified and characterized promoters in cyanobacteria. Cyanobacteria have been 150
classically studied as model photosynthetic organisms and, as a result, several key parts of the 151
synthetic biology toolbox are sourced from photosynthesis genes. The light-inducible promoter 152
driving expression of the D1 subunit of Photosystem II (psbA2) is commonly used, due to strong 153
expression in the light and repression under limited UV (Máté et al., 1998). 154
Heterologous promoters, broadly LacI-repressible and metal-inducible promoters, have also been 155
characterized in cyanobacteria. LacI-inducible promoters, such as ptac and ptrc, are well characterized 156
in eubacteria. However, cyanobacteria have fundamental differences in their transcription 157
machinery (Stensjö et al., 2018); consequently, the same elements behave differently than in other 158
eubacteria [cyanobacteria are part of the eubacteria (Cavalier-Smith, 2010)]. The main distinction is 159
reduced efficiency of repression by LacI, rendering the construction of tightly-controlled on/off 160
transcriptional circuits practically impossible in cyanobacteria (Guerrero et al., 2012; Huang and 161
Lindblad, 2013; Oliver et al., 2013; Camsund and Lindblad, 2014). 162
Inducible promoters are widely used in synthetic biology for intermittent controlled expression. 163
These include promoters activated by increased metal concentration, such as copper, iron, zinc, and 164
nickel, in the growth medium (Briggs et al., 1990; Peca et al., 2008). These promoters show good 165
induction ratios and are useful for experimentation, but are not suitable for large-scale applications 166
due to the cost and the toxicity of the metal ions. A rhamnose-inducible expression system has been 167
developed by Kelly et al. (2018), which combines strong expression and good induction, suitable for 168
synthetic biology applications. Several riboswitches also provide tightly regulated, highly tunable 169
expression systems in cyanobacteria (Ma et al., 2014; Ohbayashi et al., 2016; Immethun and Moon, 170
2018). 171
A very strong, constitutive, synthetic promoter (cpc560) was constructed by combining the high-172
level expression phycocyanin C promoter with 14 transcription factor recognition sequences (Zhou 173
et al., 2014). 174
For expression characteristics of promoters in specific species, see the following references: 175
Synechocystis sp. PCC 6803 (Huang et al., 2010; Huang and Lindblad, 2013; Englund et al., 176
2016; Liu and Pakrasi, 2018; Ferreira et al., 2018) 177
Synechococcus sp. PCC 7002 (Markley et al., 2015; Ruffing et al., 2016; Zess et al., 2016) 178
Synechococcus elongatus PCC 7942 (Ma et al., 2014; Qiao et al., 2017) 179
Synechococcus elongatus UTEX 2973 (Li et al., 2018) 180
Nostoc sp. PCC 712mix0 (Videau et al., 2016; Wegelius et al., 2018) 181
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Vectors and standardized assembly 183
The first shuttle vector that enabled assembly of genetic constructs and expression in several 184
cyanobacteria [using the then-popular BioBricks standard (Shetty et al., 2008)] was reported in 2010 185
(Huang et al., 2010). Standardized genetic assembly paved the way for large-scale development and 186
characterization of parts. The first broad-host cyanobacterial shuttle vector pPMQAK1 (Huang et al., 187
2010), as well as the pDF-trc vector (Guerrero et al., 2012), enabled more streamlined construct 188
generation and strain characterization using restriction-based and isothermal cloning techniques. 189
There are several advantages to using extrachromosomal (ectopic) self-replicating vectors: (1) more 190
efficient transformation using conjugation and electroporation (compared to homologous 191
recombination); (2) avoids the need for time-consuming segregation to achieve incorporation of the 192
transgenic sequence in all copies of the genome; and (3) increased orthogonality of the heterologous 193
sequences, meaning no insertion in genomic loci which may not be neutral. A hybrid strategy that 194
combines the advantages of using a shuttle vector and the stability of genomic insertion involves 195
using native cyanobacterial plasmids, either as insertion sites (Ng et al., 2015) or as true shuttle 196
vectors after isolation and modification (Jin et al., 2018). 197
BioBricks and isothermal assembly techniques are adequate for the generation of a limited number 198
of constructs. However, the automated generation of very large numbers of plasmids and the 199
subsequent streamlined strain generation and characterization requires an alternative approach for 200
genetic assembly. Golden Gate-based techniques, which rely on Type II restriction enzymes (Engler 201
et al., 2008), are more compatible with high-throughput applications. So far, there are two reported 202
Golden Gate toolboxes for cyanobacteria. The first is a streamlined approach for generating 203
constructs either for insertion in the genome or to be incorporated into self-replicating vectors 204
(Taton et al., 2014). This toolkit works in the frequently used model species as well as the Californian 205
species Leptolyngbya sp. BL0902, showing that tools can function across the cyanobacterial phylum. 206
A more recent Golden Gate kit is based on the modular cloning (MoClo; Weber et al., 2011) 207
assembly standard (Vasudevan et al., 2019). This kit contains more than 100 parts for transforming 208
four model cyanobacterial species, including a broad selection of native, heterologous, and synthetic 209
promoters, terminators, linkers, fluorescent makers, antibiotic resistance genes, and more. It also 210
uses the same assembly syntax as the plant and algal MoClo kits, thus making the exchange of parts 211
straightforward. To display the suitability of this kit for high-throughput applications and to 212
characterize promoter/terminator combinations, Vasudevan et al. (2019) used automated assembly 213
to generate more than two hundred constructs. 214
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Genome editing tools 215
A significant development was the implementation of CRISPR-mediated modification systems, 216
including transformation using CRISPR/Cas9 and Cpf1 (Li et al., 2016a; Ungerer and Pakrasi, 2016), 217
and CRISPRi, the latter of which has the potential to provide simultaneous knock-down of multiple 218
genes (Gordon et al., 2016; Wendt et al., 2016; Yao et al., 2016). 219
Challenges and future directions 220
Despite the recent advances, there are several challenges that still limit the synthetic biology 221
potential of cyanobacteria. Cyanobacteria harbour multiple genome copies per cell (Watanabe et al., 222
2015), making the generation of stable transformant lines a slow process that requires extensive 223
segregation to achieve genomic homogeneity. Although homologous recombination is very efficient 224
[requiring as little as 100 bp of overlap for successful transformation (Vogel et al., 2017)], it is 225
difficult to ensure that multiple genes have been edited on the same genome copy. This makes the 226
use of elaborate synthetic biology methods challenging, and sometimes impossible. For example, 227
multiplex automated genome engineering (MAGE; Wang et al., 2009) and its counterpart yeast 228
oligo-mediated genome engineering (DiCarlo et al., 2013), which are very useful in E. coli and yeast 229
for genomic editing; and Synthetic Chromosome Recombination and Modification by LoxP-mediated 230
Evolution (SCRaMbLE), used for accelerated evolution in yeast artificial chromosomes (Dymond and 231
Boeke, 2012), face significant challenges in cyanobacteria due to their genomic redundancy. 232
Whereas CRISPR systems are available (see above), CRISPR-mediated marker-free and multiplex 233
transformation have not yet been achieved in cyanobacteria. A marker-free transformation system 234
has been reported, based on SacB toxicity in the presence of sucrose; however, this system relies on 235
two rounds of segregation—thus obtaining unmarked colonies after a couple of months—and works 236
only on cyanobacteria that can grow mixotrophically (Lea-Smith et al., 2016). 237
Success in synthetic biology applications often requires reasonably solid understanding of the target 238
biological system. However, the metabolism of cyanobacteria remains only partly elucidated. This is 239
particularly problematic for modelling, as it interferes with the predictive capability of models. More 240
reactions and major metabolic routes are being discovered even in model species (Xiong et al., 2015; 241
Chen et al., 2016; Zhang et al., 2018), whereas key regulation mechanisms remain more or less 242
unknown (Vavitsas et al., 2017; Stensjö et al., 2018). Pioneering work in understanding 243
cyanobacterial metabolism, such as the use of transposon libraries to determine essential and 244
beneficial genes (Rubin et al., 2015), as well as advances in metabolic modelling (Nogales et al., 245
2012; Knoop et al., 2013; Broddrick et al., 2016; Abernathy et al., 2017) provide essential tools for 246
studying metabolism. Good knowledge and careful manipulation of metabolism have been proven 247
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essential for metabolic engineering applications, as displayed in the production of isoprene (titer: 248
1.26 g/L) and 2,3-butanediol (titer: 12.6 g/L) in Synechococcus elongatus PCC 7942 (Gao et al., 2016; 249
Kanno et al., 2017). 250
Synthetic biology in eukaryotic algae 251
Eukaryotes present distinct advantages compared to prokaryotes for synthetic biology. This includes 252
the existence of membrane-enclosed organelles that allow exploitation of separated cellular 253
compartments (Polka et al., 2016), thus permitting the engineering of channeling and 254
compartmentalization of metabolism to enhance overall production rates and limit side reactions for 255
metabolic engineering applications. The existence of multiple genetic systems in eukaryotes 256
(nucleus, mitochondria, and chloroplasts) can also offer new design possibilities and allows 257
exploration of the coordination between the compartments, which plays a major role in numerous 258
fundamental cellular processes and is especially crucial for photosynthesis and respiration. The main 259
eukaryotic chassis are baker’s yeast and mammalian cells (Adams, 2016). In the case of 260
photosynthetic eukaryotes, synthetic biology tools have been developed for Arabidopsis thaliana 261
and Marchantia polymorpha (Boehm et al., 2017); however, unicellular chassis have only emerged 262
recently. Many eukaryotic algal genomes have been sequenced (data from NCBI Genome: 64 Green 263
Algae, 9 Red algae, 9 Diatoms, 5 Dinoflagellates, 3 Brown algae). Several industrial hosts of different 264
phyla (Brodie et al., 2017) are used for biotechnology or metabolic engineering applications, 265
including Nannochloropsis sp. (Poliner et al., 2018) and diverse green algae (Scaife and Smith, 2016), 266
such as Dunaliella salina (Feng et al., 2014) and Chlorella sp. (Yuan et al., 2018). Nevertheless, most 267
of these organisms are not yet amenable to advanced synthetic biology approaches, which require 268
robust genetic tools, reliable parts, standardized high-throughput construction, and advanced 269
molecular biology techniques (Hlavova et al., 2015). Two model organisms are therefore rising as 270
interesting chassis: the unicellular green alga Chlamydomonas reinhardtii (herein referred to as 271
Chlamydomonas) and the diatom Phaeodactylum tricornutum (Nguyen et al., 2016). Both are easily 272
cultivated even at moderate to large scale (Posten, 2009) and can be transformed efficiently, with 273
known physiology and available systems-wide datasets (genome, transcriptomics, proteomics, 274
metabolomics; Scaife and Smith, 2016). Synthetic biology may allow these two model organisms to 275
become industrial hosts (Scaife et al., 2015), and the knowledge and tools developed might also be 276
applicable to other algal species (Doron et al., 2016). 277
Genetic parts 278
Many genetic elements have been characterized in, or adapted to, microalgae. Several recent 279
reviews have been published on algal synthetic biology and chloroplast genome modification (Doron 280
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et al., 2016; Scaife and Smith, 2016; Dyo and Purton, 2018; Poliner et al., 2018). Therefore, we will 281
focus here on new developments and on nuclear genetic elements. 282
Promoters and terminators. Numerous genetic parts are available for the control of gene expression 283
including diverse inducible, repressible and constitutive promoters and multiple 3′UTR/terminators 284
(Ohresser et al., 1997; Schroda et al., 2000; Fischer and Rochaix, 2001; Ferrante et al., 2008; Helliwell 285
et al., 2014; Sawyer et al., 2015; Baek et al., 2016; Lee et al., 2018; Watanabe et al., 2018; Beltran-286
Aguilar et al., 2019). The promoters are detailed in Box 2. Available nuclear 3′UTR/terminators are 287
derived from various genes such as PSAD, RBCS2, RPS29, CA1, TUB2, or RPL23 (Crozet et al., 2018). 288
Interestingly, in the context of a small transgene expressing the nanoluciferase reporter, the 289
3′UTR/terminator was found to have a stronger impact than the promoter on global expression, 290
when comparing PSAD and AR/RBCS2 promoter and terminator elements (Crozet et al., 2018). 291
Further studies will be required to precisely assess the impact of multiple combinations of genetic 292
elements on gene expression. 293
Post-transcriptional control tools. Coordinated transcription of multiple genes can be achieved 294
through use of the foot-and-mouth-disease-virus 2A peptide, which allows cistron-like gene 295
expression in eukaryotes and was found to be functional in Chlamydomonas (Rasala et al., 2012; 296
Plucinak et al., 2015; Lopez-Paz et al., 2017). To control gene expression post-transcriptionally, two 297
other versatile genetic parts were characterized: riboswitches and artificial microRNA. Riboswitches 298
are RNA sensors that bind small molecules and in turn regulate gene expression (Nguyen et al., 299
2016). These regulatory elements are particularly interesting for synthetic biology as they are 300
modular with an aptamer part that recognizes the ligand and an expression platform that controls 301
the expression. This modularity and the knowledge of the design rules of riboswitches make them 302
primed for engineering (Hallberg et al., 2017). The THI4 riboswitch identified in Chlamydomonas 303
responds to vitamin B1 and can be used to control the expression of any gene (Croft et al., 2007; 304
Moulin et al., 2013; Crozet et al., 2018). Artificial microRNA (amiRNA) is based on the endogenous 305
microRNA machinery that controls gene expression post-transcriptionally (Rogers and Chen, 2013). 306
This complex machinery was identified in Chlamydomonas (Molnar et al., 2007) and now it is 307
possible to design amiRNA that can repress the expression of any gene in a sequence-dependent 308
manner (Molnar et al., 2009). 309
Selection systems. Several antibiotic resistance genes are available in Chlamydomonas, including the 310
newly developed sulfadiazine resistance gene (Tabatabaei et al., 2019) and the well-established 311
selectable marker genes conferring resistance to paromomycin, hygromycin, spectinomycin, 312
kanamycin, and zeocin. Most of these genes are functional in numerous other algal species (Doron 313
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et al., 2016; Poliner et al., 2018). In addition, the Nourseothricin resistance gene (Nat) is routinely 314
used in P. tricornutum (Zaslavskaia et al., 2001), and very recently a novel selection marker, namely 315
the Blasticidin-S resistance gene (encoding Blasticidin-S-deaminase), was added to the diatom 316
toolbox (Buck et al., 2018). 317
Reporter genes. A set of reporter genes are also available, including fluorescent proteins and diverse 318
luciferases (Lauersen et al., 2015; Huang and Daboussi, 2017), which can be targeted in 319
Chlamydomonas to most compartments thanks to a collection of targeting peptides (Lauersen et al., 320
2015). Recently, the very sensitive and highly stable Nanoluciferase (Hall et al., 2012) was adapted to 321
Chlamydomonas (Crozet et al., 2018). 322
Parts for other eukaryotic microalgae. Although less diverse, genetic parts including promoters, 323
reporters and targeting peptides have been developed for other diatoms (Apt et al., 1996; 324
Rosenwasser et al., 2014; Chu et al., 2016; Doron et al., 2016; Huang and Daboussi, 2017) and for the 325
Nannochloropsis genus (Poliner et al., 2018). 326
Vectors and standardized assembly 327
The pOptimized vector collection provided the first step towards standardized genetic tools in 328
Chlamydomonas, including several reporter genes and a collection of targeting peptides with each 329
gene part flanked by unique restriction sites (Lauersen et al., 2015). This toolkit is standardized but 330
still lacks the modularity of modern cloning methodologies. More recently, we participated in the 331
development of the first Modular Cloning (MoClo) toolkit for Chlamydomonas (Crozet et al., 2018). It 332
is based on standardized golden gate cloning (Engler et al., 2008) and allows rapid and standardized 333
assembly of genes and multigenic constructs (Weber et al., 2011). Most of the genetic resources 334
cited above were standardized, validated and are openly distributed to the community (Crozet et al., 335
2018). The kit comprises 119 genetic parts, including 7 promoters, 7 5′UTRs, the CrTHI4 riboswitch, 8 336
immunological or purification tags, 9 signal/targeting peptides, 12 reporter genes, 5 antibiotic 337
resistance genes, the 2A peptide, 2 microRNA (miRNA) backbones and associated controls, and 6 3′ 338
UTR/terminators. This toolkit enables rapid building of engineered cells for both fundamental 339
research and algal biotechnology. To make Chlamydomonas and other algae easier to engineer, we 340
invite our readers to follow the proposed standard (Patron et al., 2015; Crozet et al., 2018) and to 341
share their parts openly. A molecular toolbox was created for diatoms using the Gateway standard 342
(Heijde et al., 2007). More recently, D’Adamo and colleagues used the MoClo (Weber et al., 2011) 343
and its Plant standard (Patron et al., 2015) to build plasmids later successfully used to transform P. 344
tricornutum (D’Adamo et al., 2019). 345
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A recent breakthrough in diatoms was the design of an artificial circular chromosome delivered via 346
bacterial conjugation to P. tricornutum and Thalassiosira pseudonana (Karas et al., 2015; Diner et al., 347
2017). 348
Genome editing tools 349
A recent breakthrough was the development of genome editing through Zn-Finger Nucleases, 350
Transcription Activator-Like Effector Nuclease (TALEN) and CRISPR/Cas9 for different eukaryotic 351
algae. TALENs were successfully used for P. tricornutum (Weyman et al., 2015; Serif et al., 2018), 352
whereas Zn-finger nucleases were developed for Chlamydomonas (Sizova et al., 2013; Greiner et al., 353
2017). CRISPR/Cas9 genome editing was achieved in Chlamydomonas (Shin et al., 2016; Greiner et 354
al., 2017), P. tricornutum (Nymark et al., 2016; Serif et al., 2018; Slattery et al., 2018), and T. 355
pseudonana (Hopes et al., 2016). In Chlamydomonas, efficient genome editing was also achieved 356
through the Cpf1 single-stranded DNA-dependent nuclease (Ferenczi et al., 2017). In the case of 357
Chlamydomonas, a library of mapped mutants generated through insertional mutagenesis is a useful 358
resource for reverse genetics (Li et al., 2016b). A very recent study reported that DNA integration 359
through homologous recombination was enabled through a knock-down of a LigIV homolog, thus 360
decreasing the NHEJ in profit of Homologous Recombination (HR) (Angstenberger et al., 2019). 361
Challenges and future directions 362
The development of all the tools described above is nourishing synthetic biology in photosynthetic 363
eukaryotes, and is particularly useful for metabolic engineering applications. Several successes were 364
achieved in recent years, such as the production of terpenes in Chlamydomonas (Lauersen et al., 365
2016; Wichmann et al., 2018) and P. tricornutum (D’Adamo et al., 2019), and other compounds such 366
as alkanes in Chlamydomonas (Sorigue et al., 2016; Yunus et al., 2018) and astaxanthin in D. salina 367
(Anila et al., 2016). Synthetic biology of microalgae is not exclusively restricted to metabolic 368
engineering. For example, the possibility to harness photosynthesis to generate electrical power is 369
currently being considered. Extraction of photocurrents from photosystem II using exogenous 370
quinone as a redox mediator was investigated (Longatte et al., 2015; Longatte et al., 2016). The 371
redesign of the QA binding site of Chlamydomonas allowed reduction of exogenous quinones (Fu et 372
al., 2017) and this strategy provided, 10–60 µA/cm-2 for 1 hour on a high surface electrode (Longatte 373
et al., 2018). 374
Despite all the recent advances enabling synthetic biology in eukaryotic algae, some challenges 375
remain. Some of these challenges relate to the relatively recent development of photosynthetic 376
microorganisms as model chassis for engineering. Relative to more established heterotrophic 377
microbes, there are fewer genetic components and they are not as well characterized. In particular, 378
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14
whereas transcriptional characteristics in cyanobacteria that are relevant for metabolic engineering 379
applications have recently been reviewed (Stensjö et al., 2018), much more work is needed to 380
characterize promoters, especially the commonly used light-responsive promoters. Moreover, non-381
modularity in expression (commonly observed when promoters are coupled with different gene 382
sequences) is not well understood. These are important considerations for true standardization of 383
components, required for predictability and reproducibility of synthetic biology approaches in 384
photosynthetic microbes. More extensive characterization of componentry is required to improve 385
their utility and application in photosynthetic microbes for synthetic biology projects. 386
Expression of transgenes is often problematic since they are randomly integrated in the genome, 387
leading to multiple insertions, gene knockout, position effects and long-term silencing. It should be 388
possible in the future to solve some of these issues using, as in mammals, a landing pad for 389
transgene insertion through site-specific recombination flanked by insulators (Raab and Kamakaka, 390
2010; Duportet et al., 2014). Another solution might be to use the recently published strategy 391
demonstrated in the model diatom P. tricornutum in other algae, which allows a shift in the balance 392
of DNA repair from NHEJ to HR (Angstenberger et al, ACS Synth Biol, 2018 DOI 393
10.1021/acssynbio.8b00234). Low transgene expression, which is often problematic in 394
Chlamydomonas, was recently circumvented using introns (Baier et al., 2018; Wichmann et al., 395
2018). It could also be improved by targeting specific loci known to be stable and highly expressed. 396
The recently-developed diatom artificial circular chromosome (Karas et al., 2015; Diner et al., 2017) 397
could allow genome engineering without random integration in native chromosomes. This paves the 398
way for the next frontier in algal engineering, which is certainly the (re)design of natural 399
chromosomes or entire genomes as in yeast (Richardson et al., 2017). 400
Beyond molecular tools, algal synthetic biology will require the development of single cell 401
approaches and automation based on microfluidics (Best et al., 2016; Kim et al., 2016) and robotized 402
platforms for DNA assembly, strain handling and phenotyping to allow high-throughput building and 403
testing of advanced designs, possibly improved through directed evolution. 404
Concluding remarks 405
Photosynthetic microorganisms represent attractive chassis for synthetic biology applications, and 406
significant recent advances demonstrate their potential. However, there are some challenges that 407
remain to be overcome and opportunities yet to be exploited in order to realize this potential (see 408
Outstanding Questions). 409
Current research is focused on only a few model species; there is enormous diversity yet to be 410
tapped in photosynthetic microorganism clades. Even in model species, metabolic processes are not 411
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15
fully elucidated and therefore cannot be fully exploited. Other valuable chassis organisms and 412
component sources are waiting to be discovered. Cyanobacteria are clearly the front runner in terms 413
of model prokaryotic photosynthetic microorganisms, but their multiple genome copies slows the 414
production of stable transformants and creates barriers for the development and application of 415
some valuable synthetic biology tools. For both prokaryotes and eukaryotes, random integration 416
presents problems in control and predictability of transgene behavior; mechanisms for more precise 417
control of insertion and expression are required. 418
Going forwards, further development of standardized parts, high-throughput assembly systems, and 419
other synthetic biology methods will be required. Toolboxes remain limited in eukaryotic systems, 420
particularly for the diatom P. tricornutum, which has emerged even more recently as a model. An 421
improved understanding of the biology and physiology of photosynthetic microorganisms will 422
improve our ability to develop and apply synthetic biology tools. 423
For microbial cell factory/metabolic engineering applications, production rate, yield, and titer are 424
critical, especially for high volume, low value products (Vickers et al., 2010). Photosynthesis is not an 425
efficient process, although recent impressive advances in engineering to improve photosynthetic 426
efficiency can deliver significant photosynthetic yield benefits (South et al., 2019). Converting this 427
fixed carbon into improved yields, and increasing rates and titers, remain challenges for many 428
products, including fuels and commodity biochemicals, which are particularly attractive for 429
photosynthetic systems compared to high value/low volume products where the technoeconomic 430
evaluation may return more positively in systems that are easier to engineer. Solving these 431
challenges will require significant engineering and application of clever synthetic biology tools. For 432
practical application, the potential to use non-arable land for production and to deploy engineered 433
organisms in extreme environments are both attractive. Photoautotrophic production may be 434
required for applications in desert and extra-planetary (i.e. beyond Earth) environments (Llorente et 435
al., 2018). Achieving sufficient light penetration into cultures without loss of the growth medium 436
(especially in high-density cultures required for high titers) also represents a challenge in bulk 437
production systems. Mixotrophic growth, while failing to fully exploit the free carbon and energy 438
available through photosynthesis, improves growth rates and yields in cyanobacteria (Kanno and 439
Atsumi, 2017; Matson and Atsumi, 2018) and may be required to reach maximum potential. An 440
under-developed opportunity for metabolic engineering applications is the use of consortia, where a 441
photosynthetic microbe provides the sugar or other carbon source for a heterotroph that acts as the 442
production platform (Ducat et al., 2012; Fedeson and Ducat, 2017; Ramos-Martinez et al., 2017). 443
This provides the benefits of both photoautotrophy and engineerability. 444
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16
More broadly, expanding applications where photosynthetic microorganisms provide key 445
advantages beyond microbial cell factories is an attractive option. Microbial fuel cells for energy 446
storage, exploiting paired autotrophy and extremophile capabilities for bespoke applications such as 447
environmental remediation, and engineered symbiosis for applications such as nitrogen fixation, are 448
likely future targets. 449
450
Acknowledgements 451
KV was supported by a CSIRO Synthetic Biology Future Science Fellowship and by the European 452
Union and Greek national funds through the Operational Program "Competitiveness, 453
Entrepreneurship and Innovation", under the call “RESEARCH-CREATE-INNOVATE” (project 454
code:15282). MHV was supported by a CSIRO PhD Top-Up Scholarship. CEV was supported by the 455
CSIRO-UQ Synthetic Biology Alliance. This work was supported in part by Agence Nationale de la 456
Recherche Grant ANR-17-CE05-0008 and LABEX DYNAMO ANR-LABX-011. 457
458
Tables 459
460
461
Table 1. Overview of cyanobacterial promoters
Characteristics Example References
Constitutive Pcpc560 (Huang et al., 2010; Zhou et al., 2014; Englund et al., 2016;
Ruffing et al., 2016; Ferreira et al., 2018; Li et al., 2018; Liu
and Pakrasi, 2018)
Inducible
CO2 PcpcB (Sengupta et al., 2019)
Cobalt PcoaT (Peca et al., 2008; Guerrero et al., 2012; Englund et al., 2016)
Copper PpetE (Briggs et al., 1990; Guerrero et al., 2012; Englund et al., 2016)
Green-light PcpcG2 (Abe et al., 2014a)
Heavy metals Psmt (Guerrero et al., 2012)
Light PpsbA2 (Englund et al., 2016; Li et al., 2018)
Nickel PnrsB (Peca et al., 2008; Englund et al., 2016)
Nitrite PnirA (Qi et al., 2005)
Rhamnose PrhaBAD (Kelly et al., 2018)
UV-B PpsbA3 (Máté et al., 1998)
Zinc PziaA (Peca et al., 2008; Englund et al., 2016)
Repressible
CO2 Prbc (Sengupta et al., 2019)
High light intensity PcpcB (Sengupta et al., 2019)
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LacI
(IPTG Inducible)
Ptrc (Huang et al., 2010; Guerrero et al., 2012; Oliver et al., 2013;
Markley et al., 2015; Ferreira et al., 2018; Li et al., 2018)
TetR
(aTc Inducible)
L03 (Huang and Lindblad, 2013; Zess et al., 2016; Ferreira et al.,
2018)
462
463
Figure legends 464
465
Figure 1: Design-Build-Test cycle using photosynthetic microorganisms. Recent technologies have 466
expanded the capabilities and throughput of this cycle by providing improved tools and design 467
options, greater build capacity, and more efficient and informative strain characterization. Overview 468
of modular cloning system based on that in Weber et al. (2011). 469
470
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ADVANCES
• New synthetic biology advances in photosynthetic microorganisms are aimed at making genetic engineering more facile and offer more options for applications. A key factor for the successful establishment of photosynthetic microbes as synthetic biology hosts is the development of standardized circuits and techniques.
• New tools such as artificial circular chromosomes for more controlled assembly and expression, modular cloning kits, component libraries, and genome editing tools are accelerating development of applications in photosynthetic microorganisms
• The increasing availability of genome sequences and genome scale models can be exploited for identification and harvesting of useful genetic components
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OUTSTANDING QUESTIONS
• What genetic diversity are we missing by focusing on relatively few model photosynthetic microorganisms, and how do we most easily access it for new chassis organisms and circuits? Will expansion into new organisms come at the expense of needed development in current chassis?
• Can the problems resulting from multiple genome copies in cyanobacteria be overcome using novel technologies?
• Can new mechanisms for precise control of transgene insertion and expression be developed?
• For metabolic engineering applications, which engineered processes and products will provide the rates, yields and titers required for economic viability? Which outcomes can be delivered in a short time frame, and which will require sustained long-term investment?
• For other applications, where else will photosynthetic microorganisms shine compared to more easily-engineered model systems?
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BOX 1. Promoters for cyanobacterial synthetic
biology
Promoters, characterized using reporter genes, are used for transcriptional regulation. Numerous studies have identified and characterized promoters in cyanobacteria. Cyanobacteria have been classically studied as model photosynthetic organisms and, as a result, several key parts of the synthetic biology toolbox are sourced from photosynthesis genes. The light-inducible promoter driving expression of the D1 subunit of Photosystem II (psbA2) is commonly used, due to strong expression in the light and repression under limited UV (Máté et al., 1998)
Heterologous promoters, broadly LacI-repressible and metal-inducible promoters, have also been characterized in cyanobacteria. LacI-inducible promoters, such as ptac and ptrc, are well characterized in eubacteria. However, cyanobacteria have fundamental differences in their transcription machinery (Stensjö et al., 2018); consequently, the same elements behave differently than in other eubacteria [cyanobacteria are part of the eubacteria (Cavalier-Smith, 2010)]. The main distinction is reduced efficiency of repression by LacI, rendering the construction of tightly-controlled on/off transcriptional circuits practically impossible in cyanobacteria (Guerrero et al., 2012; Huang and Lindblad, 2013; Oliver et al., 2013; Camsund and Lindblad, 2014).
Inducible promoters are widely used in synthetic biology for intermittent controlled expression. These include promoters activated by increased metal concentration, such as copper, iron, zinc, and nickel, in the growth medium (Briggs et al., 1990; Peca et al., 2008).
These promoters show good induction ratios and are useful for experimentation, but are not suitable for large-scale applications due to the cost and the toxicity of the metal ions. A rhamnose-inducible expression system has been developed by Kelly et al. (2018), which combines strong expression and good induction, suitable for synthetic biology applications. Several riboswitches also provide tightly regulated, highly tunable expression systems in cyanobacteria (Ma et al., 2014; Ohbayashi et al., 2016; Immethun and Moon, 2018).
A very strong, constitutive, synthetic promoter (cpc560) was constructed by combining the high-level expression phycocyanin C promoter with 14 transcription factor recognition sequences (Zhou et al., 2014).
For expression characteristics of promoters in specific species, see the following references:
• Synechocystis sp. PCC 6803 (Huang et al., 2010; Huang and Lindblad, 2013; Englund et al., 2016; Liu and Pakrasi, 2018; Ferreira et al., 2018)
• Synechococcus sp. PCC 7002 (Markley et al., 2015; Ruffing et al., 2016; Zess et al., 2016)
• Synechococcus elongatus PCC 7942 (Ma et al., 2014; Qiao et al., 2017)
• Synechococcus elongatus UTEX 2973 (Li et al., 2018)
• Nostoc sp. PCC 712mix0 (Videau et al., 2016; Wegelius et al., 2018)
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BOX 2. Promoters for eukaryotic algae synthetic
biology
Promoters are usually seen as the key genetic element
controlling gene expression. Despite the absolute
requirement of a promoter to recruit RNA polymerase,
other genes parts, including the untranslated regions
(UTRs) and terminators, also contribute to the
regulation of gene expression, ultimately leading to
fine-tuning of protein abundance in vivo.
In Chlamydomonas, these other gene parts used to
influence expression include regulatory sequences from
the PSAD gene (Fischer and Rochaix, 2001) and the
chimeric promoter HSP70A-RBCS2 and the RBCS2
terminator (Schroda et al., 2000) for strong constitutive
expression, as well as the NIT1 (Ohresser et al., 1997) or
METE (Helliwell et al., 2014) promoters, which are
repressible by ammonium and vitamin B12, respectively.
Three inducible promoters are known: the CYC6
promoter induced by nickel and also repressed by
copper (Ferrante et al., 2008), the sulfur starvation-
induced promoter of LHCBM9 (Sawyer et al., 2015), and
the Dunaliella LIP promoter that is light-inducible and
functional in Chlamydomonas (Baek et al., 2016).
Recent reports added two new inducible promoters to
the toolbox: a salt-inducible promoter from CrGPDH3
(Beltran-Aguilar et al., 2019) and the alcohol-inducible
promoter recently adapted from the fungus Aspergillus
nidulans (Lee et al., 2018). The latter requires the
specific transcription factor alcR and may be coupled to
another input to generate a logic gate, which should
enable more complex design of genetic circuits. In
diatoms, several promoters were developed including
constitutive or inducible ones (Huang and Daboussi,
2017). Very recently, a new promoter, namely the
endogenous V-ATPase C promoter, was isolated for
driving high level expression of transgenes in
Phaeodactylum tricornutum (Watanabe et al., 2018).
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