Synechococcus elongatus UTEX 2973
Here we present our new chassis, Synechococcus elongatus UTEX2973 with an intensive review on its orign, recent findings and an outlook on the potential of our new power host of Synthetic Biology.
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We are proud to present our project Syntex. We established a new chassis that will revolutionize phototrophic Synthetic Biology. We tamed the wildtype cyanobacteria Synechococcus elongatus UTEX 2973 by restoring its natural competence, building a CRISPR-Cpf1 system for rapid genome engineering and built a MoClo compatible shuttle vector for rapid transformation. We integrated this shuttle vector in our Marburg Collection 2.0 by adding the Green Expansion, a set of parts dedicated to the Genome Engineering of cyanobacteria. Additionally we standardized cultivation and measurement for cyanobacteria to unite them with the ideals of Synthetic Biology. | We are proud to present our project Syntex. We established a new chassis that will revolutionize phototrophic Synthetic Biology. We tamed the wildtype cyanobacteria Synechococcus elongatus UTEX 2973 by restoring its natural competence, building a CRISPR-Cpf1 system for rapid genome engineering and built a MoClo compatible shuttle vector for rapid transformation. We integrated this shuttle vector in our Marburg Collection 2.0 by adding the Green Expansion, a set of parts dedicated to the Genome Engineering of cyanobacteria. Additionally we standardized cultivation and measurement for cyanobacteria to unite them with the ideals of Synthetic Biology. | ||
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+ | <h1>Inspiration</h1> | ||
With rising atmospheric CO2 concentrations and declining oil reserves, it is painfully obvious that the worldwide effort to change from petroleum-based industry to carbon neutral industry needs to accelerate drastically. One of the most promising key technologies right now is the use of phototrophic organisms for biotechnological applications. | With rising atmospheric CO2 concentrations and declining oil reserves, it is painfully obvious that the worldwide effort to change from petroleum-based industry to carbon neutral industry needs to accelerate drastically. One of the most promising key technologies right now is the use of phototrophic organisms for biotechnological applications. | ||
Hence, we decided quite early this year to devote ourselves to a project revolving around phototrophic organisms. During the design phase, we looked at different potential chassis like the model moss Physcomitrella patens, but soon stumbled upon many common obstacles that are characteristic for phototrophic chassis: time intensive culturing and complicated techniques to perform basic molecular biological methods. | Hence, we decided quite early this year to devote ourselves to a project revolving around phototrophic organisms. During the design phase, we looked at different potential chassis like the model moss Physcomitrella patens, but soon stumbled upon many common obstacles that are characteristic for phototrophic chassis: time intensive culturing and complicated techniques to perform basic molecular biological methods. |
Here we present our new chassis, Synechococcus elongatus UTEX2973 with an intensive review on its orign, recent findings and an outlook on the potential of our new power host of Synthetic Biology.
Introduction
Cyanobacteria have been popular in research for centuries but recently they gained a spotlight in
Synthetic Biology. The forefather of photosynthesis is interesting because of its simplicity, making
it easier to engineer the system but also because of its growth speed that surpasses that of plants.
In recent years phototrophs became the notorious revolutionizers of “green biotechnology”: as
photoautotrophic organisms, they only require CO2 and sunlight as carbon and energy sources to
generate biomass.
The following introduction serves as an overview over our new chassis
Synechococcus elongatus UTEX 2973, based on the latest research results.
The organism
The gram-negative photoautotrophic cyanobacterial strain Synechococcus elongatus UTEX 2973 is an
isolate from the 1955 described strain Anacystis nidulans. This strain was kept at the University of
Texas as Synechococcus leopoliensis UTEX 625. A colony was selected from a mixed culture of this
strain, resulting in Synechococcus elongatus UTEX 2973. The resulting organism is genetically very
close to the well studied strain Synechococcus elongatus PCC 7942. With the fastest measured
doubling time of below 90 minutes and a high tolerance to temperature and light intensity, UTEX 2973
is a chassis to keep an eye on.
Cyanobacteria have big advantages compared to other phototrophic organisms such as plants or
eukaryotic algae: next to their faster growth they also convert solar energy a lot more efficiently.
The faster generation of biomass makes cyanobacteria a potential candidate for biotechnological
application and their amenability to genetic modifications (Ungerer, Wendt, Hendry, Maranas, &
Pakrasi, 2018) make them a great platform for research. Despite these advantages, cyanobacteria have
still not arrived in Synthetic Biology quite as we want. With our highly optimized chassis
Synechococcus UTEX 2973 we want to change just that.
Genome sequencing has proven that our strain is 99.8% identical to the much better
studied strain Synechococcus elongatus PCC 7942, which is surprising since both
strains were isolated from completely different locations. While UTEX 2973 tolerates
high light intensities, PCC 7942 is photoinhibited by light intensities of less than
half of those which UTEX 2973 can withstand. In electron microscopic examinations
carboxysomes and polyphosphate bodies were found in both strains. Most conspicuous
are the spherical, 30nm sized electron-dense bodies in PCC 7942, which are not
present in UTEX 2973. It is assumed that the bodies are carbon stored in the form of
glycogen. UTEX 2973 does not generate glycogen storage and uses the carbon directly
for biomass production, resulting in faster growth.
Research has also proven that several changes in the photosynthetic apparatus cause
decreased phycobilisomes but enhancement of Photosystem I, cytochrome f and
plastocyanin contents (Ungerer et al., 2018).
The most notable advantage is UTEX’ unparalleled doubling time. PCC 7942 takes more
than twice as long, while only producing a third of its biomass. In an experiment
under the same initial conditions, the dry weight of UTEX 2973 also increased to
0.87 mg/ml, compared to only 0.33 mg/ml in PCC 7942 (Yu et al., 2015). Unlike UTEX
2973, PCC 7942 is naturally competent due to its porin-like proteins. These proteins
are encoded on the inverted region in the genome so that inversion in UTEX 2973 can
be reversed.
But what allows UTEX 2973 to have such vital advantages?
When comparing both strains, one can observe that their content of amino acids
varies greatly: the amount of amino acids in UTEX 2973 lies at 53% whereas in PCC
7942 it is 40.9% (Mueller, Ungerer, Pakrasi, & Maranas, 2017). This results in a
different composition of the biomass, which is due to the discovered single
nucleotide polymorphisms (SNP’s). Among other things, they cause an increased
translation rate through a more efficient RNA polymerase. In addition, UTEX 2973
increases.
Molecular aspect
UTEX 2973 differs from PCC 7942 in 55 single nucleotide polymorphisms and insertion-deletions,
as well as a 188.6 kb inversion and a six open reading frame deletion (Mueller, Ungerer,
Pakrasi, & Maranas, 2017). Thereby these mutations must contain the genetic determinant for
UTEX’ rapid growth rate.
Three genes have been discovered as potentially being involved in better growth. AtpA encodes
for the alpha subunit of an ATP-synthase with an apparent higher specific activity. The
difference results in a substitution of one amino acid (Cys in PCC 7942 to Tyr in UTEX 2973).
Another significant difference lies in the ppnK encoded NAD+-kinase. Glutamin acid in PCC 7942
substitutes into aspartic acid in UTEX 2973, which affects improved enzyme kinetics. Another
important gene is rpaA, which improves the circadian response regulator. These adjustments
relieve a photosynthetic bottleneck, increasing the capacity for photosynthetic electron flow.
This ensures the usage of higher light intensities while producing more ATP and NADPH to fix
CO2. All this ultimately leads to better growth of UTEX 2973. In this experiment, the SNP’s in
UTEX 2973 were inserted into PCC 7942, thereby significantly improving its growth rate (Ungerer
et al., 2018).
Thus, these minimal genetic changes cause a huge impact in growth rate and CO2 absorption, which
proves UTEX 2973 to be second to nothing. photorespiration and the synthesis of glyoxylate, a
precursor of several amino acids. Some SNPs alter kinetic parameters of metabolic enzymes and
increase the production of biomass components. Most striking is the difference in the rate of
carbon uptake and its allocation in biomass. UTEX 2973 absorbs CO2 2.06 times more efficiently
than PCC 7942.
Field of application
Synechococcus elongatus UTEX 2973 has the potential to change paradigms in Synthetic
Biology:
With its incredibly low doubling time for phototroph standards, UTEX 2973 is eligible for rapid
prototyping.
We need good innovations to accelerate feasible photosynthetic research. Phototrophic organisms
such as plants are simply too slow inhibiting innovative progress. With our organism UTEX 2973
it is possible to test different parts and plasmids quickly, inexpensively and easily.
Cloning is a time-consuming process, especially when you have hundreds of parts in a collection.
You can quickly test all the parts in UTEX 2973 and then test the working assemblies in your
actual organism, so UTEX acts as a technical prototype to accelerate the design build test
cycle.
If an assembly didn't work, it's easy to locate the error, optimize the construct design and get
the desired result quickly.
As a photoautotrophic prokaryote, UTEX 2973 is a simple organism that is easy to work with.
Plants as eukaryotes are much more complicated, so the work and research become more
complex.
So if you want to advance photosynthetic research, you can, for example, analyze and modify
pathways on this prokaryot without much effort. UTEX 2973 is perfect to design engineering and
principles in an easy chassis and afterwards apply it all to a higher organism.
UTEX 2973 can also be used to significantly enhance the process of characterization and
standardization of cyanobacteria and their biological “parts-list”.
We have already advanced this aspect and established a reproducible “parts-list” so it is
already possible to easily work with it.
In "Green biotechnology” our chassis can be used to sustainably produce carbon neutral platform
chemicals without fossil fuels. These platform chemicals can be used to produce biofuels and
bioplastics or carbohydrate feedstocks (Song, Tan, Liang, & Lu, 2016). Innovations like these
are needed to propel the development of a sustainable, fossil fuel independent industry of
tomorrow.
Many secondary metabolites have pharmaceutical benefits, such as amino acids, fatty acids,
macrolides, lipopeptides and amides (Yu et al., 2015). The cyanobacteria strain Synechocystis
sp. PCC 6803 already serves as a brilliant example for the application of cyanobacteria: it has
been genetically modified to secrete fatty acids and thus to avoid costly biomass recovery in
the production of photosynthetically produced, sustainable biofuels (Liu, Sheng, & Curtiss,
2011).
Establishing UTEX 2973 with its versatile capabilities could pave the way to making industrial
biotechnology more sustainable and thus be a solution to combating climate change, one of the
most horrific challenges humanity has ever faced. UTEX 2973 embodies progress and innovation on
the highest level.
Genetic amenability
In recent years, the CRISPR system has enabled precise gene editing. Gene editing is well
feasible in UTEX 2973 with the CRISPR technology and an alternative nuclease Cpf1 from the
organism Francisella novicida as Cas9 is toxic to cyanobacteria (Ungerer & Pakrasi, 2016).
Despite the loss of its natural competence, UTEX 2973 is a suitable candidate for genetic
engineering.
DNA can be easily introduced into UTEX 2973 trough triparental conjugation via E. coli and the
self-replicating vector pANS. Shuttle vectors are of great interest as they lead to higher gene
expression compared to genome integration. In addition, they retain large DNA inserts in the
organism even without selection pressure and are easy to transform (Chen et al., 2016).
We developed facile-transforming shuttle-vectors and thus, we were able to extend the genetic
toolbox and simplify genetic engineering.
Our engineered strain is now able to take up plasmids naturally. Thanks to this we are able to modify our chassis on genomic level thanks to CRISPR-cpf1 and on a transient level thanks to our MoClo compatible shuttle vector.
Natural Competence
One of the most important aspects when engineering an organism is the actual modification of its genetic code.
The introduction of exogenous DNA can be done in multiple ways - through electroporation, conjugation, heat shock or
via natural competence.
Electroporation is a method in which an electrical field is applied to cells, in order to increase the permeability of
the membrane, enabling DNA uptake not just in prokaryotic
(Thiel and Poo, 1989),
but also eukaryotic cells
(Potter and Heller, 2010).
Although relatively simple to perform, this technique is not ideal, as the success rate is rather low and many precautions
have to be taken: salt concentration and field strength highly effect the outcome and secreted endonucleases can degrade the
DNA beforehand (Zeaiter et al., 2018).
Conjugation is a more complicated and laborious method where cell to cell contact is needed. Pili are formed to transfer DNA
from one cell to another - but not all DNA can be transferred, as the plasmid that is to be conjugated needs to harbour a
mobilization sequence (Actor, 2012).
This method is more popular in cyanobacterial research, as it overcomes the above mentioned problems that come with
electroporation (Zeaiter et al., 2018).
And what is natural competence?
Natural competence was first discovered by Frederick Griffith in 1928 (Griffith et al., 1928) by studying different Streptococcus pneumoniae strains - a virulent and a non-virulent one. Although he did not know the biological processes behind it, he realized that genetic information can be passed on from one bacterium to another, as previously non-virulent strains could be transformed to virulent ones. In comparison to other types of competence that can e.g. be chemically induced, natural competence is the ability of cells to take up extracellular DNA from their environment under natural conditions.
Natural competence is found in different kinds of bacteria - also in cyanobacteria. Despite the fact that there is
still much to uncover about the exact mechanisms of natural DNA uptake in cyanobacteria, previous efforts by
Schuergers and Wilde, 2015,
Yoshihara et al., 2001,
Bhaya et al., 2002
and many more have led to the construction of a preliminary model of the type IV-like pilus responsible for natural
transformation in Synechocystis sp. PCC 6803
(Wendt and Pakrasi, 2019),
which can be seen in Figure 1.
In order to take up extracellular DNA several steps seem to be necessary: The double stranded DNA has to be picked up
by the type IV-like pilus and transported through an outer membrane pore comprised of PilQ subunits
(Bhaya et al., 2002)
and is then converted to single stranded DNA by a certain nuclease before being passed through an
inner membrane pore composed of ComE subunits
(Yoshihara et al., 2001).
Early studies have tried to identify cyanobacterial strains capable of natural transformation, from which just a few
species have been frequently chosen to serve as model organisms, including Synechococcus sp. PCC 7002,
Synechococcus sp. PCC 6803, Synechococcus elongatus PCC 7942
(Koksharova and Wolk, 2002).
Cyanobacterial species are known to be naturally competent, yet have been shown they have at least one complete set of the
genes identified in the above mentioned model
(Wendt and Pakrasi, 2019)
- one of them being Synechococcus elongatus PCC 7942.
This strain is closely related to Synechococcus elongatus UTEX 2973, in fact genome comparisons show just 55
single nucleotide polymorphisms and indels
(Yu et al., 2015).
Previous studies have suggested that a single point mutation in the pilN gene is responsible for the loss of
natural competence in S. elongatus UTEX 2973
(Li et al., 2018),
so in an effort to reintroduce natural competence into this strain, intact versions of the pilN gene from
S.elongatus PCC 7942 have been successfully introduced into one of the neutral sites in the genome
of UTEX 2973 via homologous recombination
(Li et al., 2018),
showing that natural competence can be achieved in this strain.
As natural competence is the easiest and often most efficient way to incorporate exogenous DNA into an organism,
this is a crucial feature that comes in handy for every chassis.
For this reason we planned to restore the natural competence of S.elongatus UTEX 2973 - what approaches we took
and how we planned all of it through can be found in our design section.
CRISPR gene editing
Clustered regularly interspaced short palindromic repeats / CRISPR associated protein (CRISPR/Cas) systems are adaptive
immune systems in bacteria and archaea that provide sequence-specific targeting of genetic sequences, in order to cut
exogenous DNA (Barrangou et al., 2007).
Simplified systems have been a rising interest for use in genetic engineering approaches, as they can be used as powerful
tools for precise genome alteration not just in prokaryotic, but also eukaryotic cells. This includes integration of whole
genes, alteration of single nucleotides, knock-outs of whole genetic regions, as well as the use of the DNA-binding property
in a multitude of applications through so called deadCas systems, where the Cas protein does not exhibit nuclease activity
(Hsu et al., 2014).
These adaptive systems incorporate invading DNA sequences, so called protospacers, into their CRISPR array, meaning
that short sequences of DNA can be stored between identical repeat sequences. This whole array is transcribed into a
long precursor CRISPR RNA (pre-crRNA) that is then processed into mature crRNAs that carry spacers which serve as guides,
leading the Cas protein to their recognition sequence, where it can then exhibit nuclease activity
(Hille and Charpentier, 2016).
Maturation of crRNAs differs in different CRISPR/Cas systems. CRISPR/Cas9 systems that are widely used in genetic
engineering approaches need an additional transactivating crRNA (tracrRNA) for crRNA maturation, while in CRISPR/Cas12a
(also called CRISPR/Cpf1) only the crRNA is necessary for precise targeting
(Gao et al., 2016).
Another crucial factor of these systems are the protospacer adjacent motifs (PAM). In order for the Cas protein to
effectively bind the targeted DNA sequence, it has to be next to a PAM sequence, proving that the PAM is an invaluable
targeting component that allows the cell to distinguish between self and non-self DNA, as the PAM sequences cannot be
found in the CRISPR array itself, preventing the Cas protein to cut inside of it
(Mali et al., 2013).
As mentioned before, different CRISPR/Cas systems are available for genetic engineering of a large number of organisms. The most commonly used system is the CRISPR/Cas9 system, but another attractive system is CRISPR/Cas12a, also called CRISPR/Cpf1. The main differences are that Cas9 introduces blunt ends when cutting DNA, while Cas12a produces sticky ends and that Cas9 requires a tracrRNA for crRNA maturation, while Cas12a only needs the crRNA as a guide. In Cas9 systems the crRNA:tracrRNA duplex can be linked to form a single guided RNA (sgRNA), which is usually ~100nt long - in comparison the Cas12a crRNA is only ~43nt long (Swarts and Jinek, 2018).
Both systems are of particular interest for genetic toolboxes, as they enable highly accurate genome engineering with a wide application range - including multiplexed alterations.
Cyanobacterial shuttle vectors
Cyanobacteria are known to contain multiple copy numbers of their chromosome, the unicellular cyanobacteria
Synechococcus elongatus reportedly contains 3-5
(Griese et al., 2011), 2-10
(Watanabe et al., 2015) or 1-10
(Chen et al., 2012) chromosomes per cell,
more recent studies have counted eight chromosomes per cell
(Yu et al., 2016).
S. elongatus PCC 7942 furthermore hosts two endogenous plasmids. The 46,4 kb pANL
(Chen et al., 2008) which is essential and the 7,8 kb pANS
(Van der Plas et al., 1992)
which is not essential for the strain and can easily be cured
(Lau & Doolittle, 1979).
In Synthetic Biology multiple approaches can be chosen to introduce exogenous DNA into an organism. Typically this
is done by integrating the DNA into the host genome or by transforming plasmids that contain the genes of interest.
As we learned above, the copy number of the chromosomes can be highly variable, which is a huge downside when trying to
engineer such organisms, as genome integrations have to be introduced into every single copy.
Shuttle vectors on the other hand can be stably maintained in the cells and are typically found in higher copy numbers,
resulting in higher gene expression rates as genomic integrations
(Chen et al., 2012).
Vectors that can be used in cyanobacteria are scarcely available, the few existing ones are mostly based on the
RSF1010 plasmid that shows a broad host range and can be maintained in multiple cyanobacterial species, although for
unknown reasons it is not present in high copy numbers
(Mermet-Bouvier et al., 1993).
The only shuttle vectors available that contain a native replication element of a cyanobacterial species are those that
have been constructed from the previously mentioned pANS plasmid
(Kuhlemeier & van Arkel, 1987;
Golden & Sherman, 1983).
Recent studies have shown that pANS-based shuttle vectors are present in a higher copy number than RSF1010- or pDU1-based
vectors in cyanobacteria, clearly indicating the advantages of native replication elements. The same study has also proven
that gene expression levels are higher when genes are expressed on the pANS-based vectors, than on pANL or the chromosome
of S. elongatus (Chen et al., 2012).
In spite of these apparent advantages, many still prefer integrating DNA into the chromosome, which is why we
incorporated parts for homologous recombination into our toolbox
and successfully identified new neutral side for integration.
providing invaluable tools for the community.
As we are certain that self replicating vectors are essential for many workflows, especially if rapid prototyping is to be done in an organism, we set out to construct the world's first MoClo compatible shuttle vector for cyanobacteria based on the modular Golden Gate Assembly method, allowing for flexible cloning into a reliable self-replicating system. With our constructs there is no need for tedious selection processes that come with genomic integrations.
Abstract?
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We present the Marburg Collection 2.0, an extensive addition to our part collection. In the green expansion we provide the first MoClo compatible shuttle vector for transient modification as well as the M.E.G.A. expansion for Modularized Engineering of Genome Areas. Next to that we provide a new assembly tool, the placeholders which we used to build standardized measurement vectors for cyanobacteria and a set of reporters we deemed absolutely useful for our chassis.
We present the Marburg Collection 2.0, an extensive addition to our part collection. In the green expansion we provide the first MoClo compatible shuttle vector for transient modification as well as the M.E.G.A. expansion for Modularized Engineering of Genome Areas. Next to that we provide a new assembly tool, the placeholders which we used to build standardized measurement vectors for cyanobacteria and a set of reporters we deemed absolutely useful for our chassis.
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...
Abstract?
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