Recent Toolboxes for Synthetic Biology in
Cyanobacteria
Due to limitations in the molecular biology tools available for
cyanobacteria in comparison to the other bacteria (E. coli ),
there is a need to design these tools using a synthetic biology
approach. Synthetic biology manipulates the already available tools
native or foreign and recombining them using different combinations for
a better output. In this view, several tools like promoters,
riboswitches, ribosome binding sites, CRISPR/Cas system, etc. were
developed, which are briefly reviewed here. Strains likeSynechococcus elongatus PCC 7942, Synechocystis sp . PCC
6803, etc. are used as a host to test these genetic tools. One of the
major demerits of these strains is the longer doubling time. Recently Yu
et al. (2015) discovered Synechococcus elongatus UTEX 2973, which
is the fastest-growing strain reported to date. Table 1 summarizes the
commonly used cyanobacterial strains as hosts. <Table
1 >
2.1 Promoters
There are number of native and foreign promoters that are used in
cyanobacteria and are summarized in Table 2. In bacteria, promoters are
recognised by the σ factor of the RNA polymerase (RNA P) enzyme and aids
in the transcription of the gene of interest. The promoters can be
constitutive or inducible. Constitutive promoters transcribe the genes
continually in an unregulated way. Whereas inducible promoters are
specific to the signals like light, dark, heavy metals, nitrate/nitrite,
etc. and are helpful when the intermediate/end products are toxic to the
host cells. Pcpc560 considered as the super-strong
promoter, was discovered by Zhou et al. (2014). It has the same
expression rate as that of the E. coli producing functional
proteins at a level of up to 15 % of total soluble proteins. It has two
promoters from the cpcB gene and 14 transcription factor binding sites,
which are assumed to be the crucial factor for its strength. In one of
the studies done by Liu and Pakrasi (2018), promoter cpcB showed the
highest expression (sequence identical to cpc560). They compared 13
different promoters by checking the expression of enhanced yellow
fluorescence protein. Out of the thirteen promoters, twelve were native,
and one was E. coli originated. Wang et al. (2018) constructed
promoters’ library, compared 17 different promoters, and concluded that
Ptrc gives two times better expression than the promoter
PpsbA (from chloroplast of the flowering plantAmaranthus hybridus ) and seven times expression than
PcpcB (Pcpc560) and its variants. The expression level
was checked by expressing the ethylene forming enzyme inSynechocystis sp. PCC 6803. Ptrc2O and
Ptrc1O (Huang et al., 2010 and Camsund et al., 2014) is
the promoter derived from Ptrc (Brosius et al., 1985).
Ptrc1O has a
strong lac operator than Ptrc, whereas
Ptrc2O has two lac operator site showing efficient
repression. Markley et al. (2014) constructed a promoter which performs
better than trc promoter, giving 48±7 fold expression of YFP. They
constructed two orthogonal promoter libraries with the IPTG induction
system, which were tried and tested in Synechococcus sp. strain
PCC 7002. Promoter MB1, MB2, and MB3 were obtained by a change in
sequences in the Biobrick promoter J23119. Similarly, 20 different
synthetic promoters were assembled by modifying J23119,
Ptrc1O, Ptic10, and
Ptac10 promoters, and the intensity of fluorescence was
checked in Synechocystis and Synechococcus elongatus UTEX
2973 (Vasudevan et al., 2019). In both the cases, J23119 showed the
highest fluorescence levels. Werner et al. (2018) discovered and
characterized nineteen native promoters in Synechocystis sp. PCC
6803, which are induced by 12:12 hour light and dark cycles (LD cycles).
Out of these nineteen promoters, four of the promoters
PhliC, Prbp1, Pslr0006l,and PsigA shows a strong correlation with 12:12 LD
cycles when characterized using bacterial luciferase bioluminescent
gene. In the same species, metal ions (nickel, cobalt, and zinc) induced
nrsB, nrsD, nrsS, coaT, and ziaA promoters were compared with endogenous
constitutive promoters (Englund et al., 2016). PnrsB was
found to be the most efficient promoter, which can be regulated and
tuned with the help of nickel ion. Kelly et al. (2018) inserted a
rhamnose-inducible rhaBAD promoter from E. coli toSynechocystis sp. PCC 6803 which showed a controlled expression
system. PsynDIF, a short 48 nucleotides long synthetic
promoter for heterocyst-specific expression in filamentous cyanobacteria
(Wegelius et al., 2018) can be used well for the production of oxygen
intolerant enzymes as the promoter gives 10 times more expression after
the heterocyst formation. Expression of the promoter changes when it is
relocated from the native location to the new one (Albers and Peebles
2016). PpsbAII within the native genomic location ofSynechocystis sp. PCC 6803 showed 15.8 times increase in the
transcript in comparison to only 1.6 times when the promoter is moved to
neutral region slr0168. Promoter psbA was used to control the expression
of mannitol encoding genes (mtlD and mlp ) inSynechococcus sp. PCC 7002, giving a yield of 1.1 g mannitol
L−1 with a production rate of 0.15 g mannitol
L−1 day−1 (Jacobsen and Frigaard,
2014). Huang and Lindblad (2013) constructed non-inducible promoters and
showed that altering a few base pairs can change the strength of the
promoters. R40 promoter was used as a template for promoter designing.
Specific base pairs changes were done in the R40 promoter at TATAAT site
(L12 promoter created whose strength is less than R40) and between -10
element and transcription start site (L12 promoter having strength more
than R40). Three line of modifications were done between -10 element and
transcription start site creating a total of 19 promoters, L01 to L016,
L21 and L22, and L31. The study shows that L21 promoter has 110 (±1)
times strength than L22, opening the possibilities to change the region
between −27 and +3 at TATAAT sequence. Bioinformatics tools play an
important role in the prediction of promoters in cyanobacteria. Btss
finder is one of the means for bacterial promoter prediction, which
includes E. coli and cyanobacteria. Being novel, it can identify
the promoters of different sigma classes of two different phyla. Various
native and foreign promoters are shown in table 2.
<Table 2 >
2.2 Ribosome Binding Sites
As the promoters regulate the initiation of transcription, in the same
way, ribosome binding sites (RBS) regulate translation initiation rate
of downstream target genes (Kierzek et al., 2001). Upon translation
initiation, with the help of complementary base pairing of the
nucleotides, the 3-terminal sequence of the 16S rRNA interacts with the
core Shine-Dalgarno (SD) sequence of RBS. Ma et al. (2002) showed this
in Synechocystis 6803 that the 3 terminal sequence of the 16S
rRNA is AUCACCUCCUUU and its complementary SD sequence is AAAGGAGGUGAU
(core SD sequence underlined). To enhance the production of
2,3-butanediol in Synechococcus 7942, expression levels of the
three genes (alsS, alsD and adh) are coordinated by utilizing four
different RBS from E. coli (Oliver et al., 2014). Wang et al. 2016
increased limonene synthesis by RBS engineering in Synechococcus
elongatus PCC 7942. Strain L1113 showed limonene production of 32.8
μg/L/OD/d by changing the original RBS of the trc promoter. Similarly, a
synthetic RBS introduced in psbA promoter increased the limonene
production to 885.1 μg/L/OD/d. RBS library for cyanobacteria was created
by Englund et al. (2016) by utilizing 8 RBS sequences from BioBrick
Registry of standard biological parts and two from Synechocystis
sp . PCC 6803 and predicted by “RBS library calculator”. The mentioned
library was used to express an enhanced yellow fluorescent protein
(eYFP) with the help of PnrsB, PnrsD, PnrsS, PcoaT and PziaA promoters
which are induced by nickel, cobalt and zinc metal efflux pumps. More
recently, Liu et al. (2018) evaluated 20 native RBS, which were 22 base
pair long. Ptrc1O was selected as the promoter to check the strength of
the RBS sequences by the EYFP gene. In the same manner, Thiel et al.
(2018) also assessed 13 RBS out of which 7 were native ofSynechocystis 6803 , and 6 were from E. coli .
Codon-optimized GFPmut3, sYFP2, and ethylene forming enzyme were used as
the reporter proteins for checking translation efficiency. These studies
help in the selection of suitable RBSs for overexpression of the end
product. According to Reeve et al. (2014), the same RBS can have
inconstant translational efficiencies in different microorganisms or
different genes in the same organisms. RBS calculating tools plays an
important role which is based on the thermodynamic model to predict the
changes in the start codon and 5 untranslated regions in an mRNA
transcript. RBS calculator, UTS design and RBS designer are the majorly
used tools to determine translation rates. Each calculator is used
efficiently for reverse and forward engineering. RBS calculator
prognosticates TIR by enumerating the strength of 30S complex and mRNA
transcript interaction (Salis et al., 2009 and Salis et al., 2011). RBS
designer (Na and Lee, 2010) works by designing RBS sites synthetically
on the RNA transcript, while the UTR designer (Seo et al., 2013) focuses
on changing 5-UTR to alter protein expression and predicts translation
efficiency. These tools serve an importance purpose of generating RBS
libraries, but efficiency of them can vary. Wang et al. (2017) reported
the low efficiency of RBS library created by RBS calculator and
established a rational RBS design strategy. Likewise, Thiel et al.
(2018) also stated that the data predicted by UTR designer and RBS
calculator shows different translation efficiency than the experimental
one. In another study numerous RBS calculated for bisabolene synthase
gene gives 7.8 mg/L titer (Sebesta and Peebles, 2020).
2.3 Riboswitches
In comparison with the inducible promoters, riboswitches do not require
additional protein factors (RNA P) and are cis-acting regulatory element
which changes the conformation on binding with its ligand controlling
TIR (Henkin 2008; Domin et al., 2017). This makes riboswitches an ideal
tool for gene regulation. Nakahira et al. (2013) illustrated that
modified theophylline-responsive riboswitches regulate gene (luciferase)
expression more efficiently than inducible promoters. Further, this
riboswitch was used in many studies in Synechococcus elongatusPCC 7942, Leptolyngbya sp . strain BL0902, Anabaena sp .
strain PCC 7120, and Synechocystis sp . Strain WHSyn andSynechocystis 6803 to check the expression regulation of yellow
and green fluorescent protein (Ma et al., 2014; Ohbayashi et al., 2016).
The theophylline-responsive riboswitches used were earlier screened and
characterized in the past in Gram-negative alpha- and
gamma-proteobacteria and Gram-positive bacteria (Lynch and Gallivan,
2009; Topp et al., 2010). It is also used to regulate intracellular
glycogen content (40 to 300% of wild type) in Synechococcus
elongatus PCC7942 by controlling ADP-glucose pyrophosphorylase (GlgC).
Optimised level of glycogen increases cellular robustness (Chi et al.,
2019). Apart from theophylline-responsive riboswitches,
cobalamin-dependent riboswitch work well in Synechococcus 7002 as
the strain cannot synthesize the cobalamin itself (Perez et al., 2016).
But this riboswitch cannot work in the strain which synthesize cobalamin
such as Synechococcus 7942, Synechocystis 6803,Crocosphaerawatsonii WH8501 and Synechococcus sp. WH7803
(Helliwell et al., 2016). Other riboswitches used in cyanobacteria
includes S-box (SAM), SAM-II (a-proteobacteria) and SAMI/IV-variant
riboswitch, thiamine pyrophosphate (TPP)-riboswitch, Glycine riboswitch,
SMK box translational riboswitch, Purine riboswitch, FMN riboswitch (RFN
element), Lysine riboswitch, SAH (S -adenosyl-l-homocysteine)
riboswitch, THF (Tetrahydrofolate) riboswitch Moco (molybdenum cofactor)
riboswitch, (Sun et al., 2013; Zhang and Gladyshev, 2008; Singh et al.,
2018). Some of the riboswitches inducer are toxic to the host organism
and are key metabolic intermediates, therefore, only theophylline
dependent riboswitch is widely used in cyanobacterial systems.
2.4 CRISPR Based Technique
The most recent synthetic biology tool is the CRISPR/Cas system, which
is marker less. CRISPR/Cas stands for Clustered regularly interspaced
short palindromic repeats/ CRISPR associated proteins. Its targetability
is provided by the single-guide RNA (sgRNA), which is specific to the
target genomic site. SgRNA directs the Cas protein to the target site
which cleaves both the strand of the genome. Cyanobacteria being
oligoploid and polyploid in nature shows difficulty in producing
homozygous mutants (Watanabe et al., 2015 and Zerulla et al., 2016).
CRISPR based technique improve this editing efficiency. Wendt et al
(2016) used CRISPR/Cas 9 system to produce nonbleaching protein A (nblA)
mutants of Synechococcus elongatus UTEX 2973. Once all the copies
of the genes are deleted the mutants show visible results (within 1
week) as the nblA serves as visual reporter gene. These results were
verified by Li et al (2016) by increasing the succinate concentration inSynechococcus elongatus PCC 7942 through glgc knock-out glta/ppc
(citrate synthase/phosphoenol pyruvate carboxylase) knock in by
CRISPR-Cas 9 editing. Cas 9 at higher concentrations showed toxicity inS. elongatus UTEX 2973 and PCC 7942 cells (Wendt et al., 2016 and
Li et al., 2016). The quick fix to the problem was the transient
expression of Cas 9 through temperature-controlled plasmid. This gave
inkling to Ungerer & Pakrasi (2016) to prospect CRISPR/Cas 12a (also
known as Cpf1). Markerless point mutation, a knock-out mutation or a
knock-in mutation were generated S. elongatus UTEX 2973,Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120. Cas
12 have several merits over Cas 9 which includes, no requirement of
tracrRNA to activate crRNA, cost efficient as it needs only 42
nucleotides RNA which is cheaper to produce, and PAM (Protospacer
adjacent motif) sequence is more specific to the cleavage site. A recent
application of Cas 12a was seen in developing high-throughput
prototyping tool for promoter characteristics (Choi et al., 2021). This
gives opportunity to rapidly characterize promoters by cell free
transcription. A newer version of CRISPR/Cas system is
CRISPR-interference (CRISPRi) which utilizes the dead Cas9 (dCas9) which
binds to the target DNA but has lost the ability to cleave it. This type
of system is essential for the genes which are crucial for the cell
viability and can only be downregulated rather deleted. Yao et al.
(2016) first reposted the application of CRISPRi in Synechocystis
sp . PCC 6803. The sgRNAs were placed in the neutral sites
(slr2030-slr2031) due to this 94 % of repression was seen in the GFP
protein. CRISPRi can be used for producing carbon-based products as
shown by Huang et al. (2016). They effectively repressed extrinsic
(EYFP) and intrinsic genes (glgc, sdhA and sdhB) to redirect the carbon
flow. This laid the foundation for the metabolic pathways in
cyanobacteria. Similarly, CRISPRi is being also used to redirect fatty
acid flux (Kaczmarzyk et al., 2018). Regulating plsx (phosphate
acyltransferase) gene by CRISPRi system fatty alcohol production
enhanced to a great extent at 10.3 mg/g dry weight. Modulation of
glutamine sythetase (glnA helps in nitrogen assimilation) inAnabaena sp . PCC 7120 was shown by Higo et al. (2018). The
process is regulated in such a way that ammonium is produced only when
dCas inducer is present in the system. Lately, dCas12a-mediated CRISPR
interference system (CRISPRi-dCas12a) was developed in cyanobacteria for
repressing genes which are not needed to produce value added chemicals
(repression upto 53-94%). The technique was implemented in PCC 7942 to
increase squalene production by repressing aconitase (Choi and Woo,
2020).
2.5 Vectors
After successfully finalizing the genetic elements like promoters, RBS,
riboswitches, etc. a platform is required for taking the gene of
interest into the host cell (here, cyanobacteria). For this purpose,
vectors come into role, which is a plasmid with properties having
antibiotic resistance gene for selection, mobilization elements for
transfer and unique restriction sites for cloning. The heterologous gene
can be inserted into the genome of the cyanobacteria or replicate
autonomously. The former is done with the help of integrative and latter
with replicative vectors. Replicative vectors are easy to use to insert
gene of interest in cyanobacteria for the bioproduction and other
purposes (Heidorn et al., 2011). Autonomous expression of gene without
getting inserted in the genome gives higher expression (Xia et al.,
2019). Shuttle vectors, commonly used are replicative plasmids as they
can express in two hosts. Jin et al. (2018) constructed shuttle vector
for PCC 6803 utilizing its own plasmid. PCC 6803’s plasmid PCC 5.2
consists a replicon and combining it with pMB1 (origin of replication ofE. coli ) leads to the formation of shuttle vector pSCB-YFP.
Replicative vectors require antibiotic selection/stress to stably
maintain them. Table 3 shows the list of replicative vectors available
for research purposes. There are number of neutral sites detected in the
genome of cyanobacteria like NS I, NS II, etc. (Ng et al., 2015).
Replacing these neutral sites with the gene of interest with the help of
homologous recombination is the widely used method as shown in figure 1
(Lee et al., 2017). Another strategy includes integrating the
heterologous gene in place of the genes which do have function in
cyanobacteria or does not affect the robustness of the strain. In this
case the heterologous gene utilizes the promoter, RBS, and terminator
sequences of the source gene. High ploidy level in cyanobacteria prove
to be the major drawback in integration of gene of interest as it is to
be ensured that each and every copy has the gene (Heidorn et al., 2011).
This is epitomized by Griese et al. (2011) stating PCC 7942 having 3 to
4 genomic copies per cell, and PCC 6803 having 218 and 58 genomic copies
in exponential and stationary phase respectively. Earlier there was no
modular cloning (MoClo) (Engler et al., 2014) system for cyanobacteria,
however Vasudevan et al. (2019) combined plant MoClo with cyanobacteria
making CyanoGate system kit. The kit consists of 96 parts and these can
be combined with each other from level 0 to level T to form replicative
or integrative vectors. All vectors are submitted at addgene for
research purposes (Addgene Kit #1000000146). Commercially available
replicative plasmids are listed in table 3 and integrative plasmids have
been listed in table 4.
<Figure 1 > <Table
3 > <Table 4 >