Golden Gate Assembly is a revolutionary cloning method published 10 years ago (Engler et al., 2008). since its publication it became very popular giving rise to many assembly strategies based on this system. All systems published over the years offered improvements over previous publications. Nevertheless, although there are many systems available – often specialized for certain organisms or classes – the development by far is not at an end. We worked on a novel toolbox and developed an improved, highly flexible system focused on prokaryotes including a high number of parts characterized for the novel upcoming chassis V. natriegens.
The innovation given by Golden Gate assembly was the introduction of type IIS restriction enzymes used for DNA digestion. A current workflow at that time consisted of DNA restriction, gel purification and finally ligation of the single parts. For restriction different type II restriction enzymes were taken making it important to check for compatible buffers and getting a six base pair scar after ligation. By introducing type IIS restriction enzymes into this workflow the system was highly improved. This new method offered many advantages like I) an independent enzyme recognition site that is removed after restriction, II) restriction and ligation in one pot III) directional assembly by conscious design of the cleavage sequences IV) no buffer incompatibilities due to the usage of one enzyme cutting at several spots and still producing different cleavage sequences (Engler et al., 2008) (Figure will be added…).
Based on this, many new systems arose offering the possibility of building complex constructs out of single genetic parts in a fast, simple and flexible way. Full libraries of standardized genetic parts were developed enabling the user to build full combinatorial libraries. The first system developed was the modular cloning system (MoClo) offering a new hierarchical cloning method of defined biological parts (Weber et al., 2011) and consequently setting a new standard over the years.
Here we summarize some Golden Gate based systems and compare their advantages and disadvantages.
Image description
The modular cloning system was the first one proposing a standard for Golden Gate based assembly. This toolbox offers 5 types of modules designed mainly for eukaryotes. The modules are stored in level 0 acceptor plasmids derived from the pUC19 backbone with a spectinomycin resistance and a LacZα cassette as dropout for blue/white screening. Custom level 0 plasmids are assembled by flanking the sequences with BpiI recognition sites and setting a single restriction-ligation-reaction with the correspondent plasmid. Up to 5 level 0 modules are assembled in an acceptor plasmid with ampicillin resistance and a LacZα-dropout by restriction with BsaI to transcription units. 6 transcription units can be assembled using BpiI into level 2 multigene constructs containing a kanamycin resistance and a Cred-dropout. Alternatively Esp3I can be use to transfer the constructs to intermediary levels to reach higher levels for assembly of bigger constructs.
Our Goal
Our goal is to accelerate metabolic engineering by establishing a workflow for fast pathway construction, product screening and pathway optimization. Metabolic engineering is a broad field in synthetic biology, where existing pathways are divided into modular parts and then used and combined to build new pathways ( (Stephanopoulos et al.2012) ). One of the best examples for the impact of metabolic engineering on solving global problems is the engineered production pathway for artemisinic acid, the precursor for a drug against malaria (Ro et al.2006) . In the old days the drug had to be isolated out of the plant Artemisia annua. This was very inefficient because of the limitations of plant growth and low yields of artemisinic acid. Therefore, the group of Jay Keasling engineered a metabolic pathway to produce the drug in yeast cells, which grow faster and produce a lot more artemisinic acid. As in this example, metabolic engineering enables the production of drugs and other valuable products with an increased yield when compared to product isolation from natural sources. Furthermore, the large variety of usable enzymes, pathways and chassis enables more flexibility in pathway design compared to chemical approaches.
Why should we accelerate metabolic engineering?
Although much progress in metabolic engineering was made in the last 20 years, there are still many drawbacks and limitations.
For instance, it is crucial to have a big set of genes as combinable parts to find the optimal pathway and they have to be predictable.
With many of them just poorly characterized, rational pathway design remains difficult.
Our understanding of the working and interactions of enzymes at the level of the metabolism is still rudimentary. Consequently, metabolic engineering consists of much trial-and-error and researchers have to endure series of failures.
One strategy to face these issues is modeling of enzyme behavior but currently limitations in computing power prevent this from being used in high-throughput. Another strategy is to use chassis as workhorses to test a lot of enzyme combinations and pathway variants. Here, organisms like E. coli, S. cerevisiae or C. glutamicum are often used but product formation is limited by growth rate and nutrient uptake
(Stephanopoulos et al.2007).
When a pathway is constructed and to be tested, one has to wait until the production strain has grown and then product formation is still limited by substrate uptake. Another big drawback is that screening for product often is expensive, time consuming and complicated.
Conventional methods like LC-MS need a lot of time and resources and it is hardly possible to use it in high-throughput, for instance for a whole pathway library.
To overcome these limitations and accelerate metabolic engineering, we established V. natriegens as chassis for metabolic engineering.
Its doubling time of under 10 minutes makes it the perfect organism to quickly test many pathway variants or enzyme versions.
Additionally, it has a glucose uptake rate of 3.90 g g-1h-1 while E. coli, S. cerevisiae and C. glutamicum have much lower rates (1.90, 3.52 and 0.37 g g-1h-1, respectively).
Coupled with the short doubling time, V. natriegens is a powerful chassis not only for research purposes but also for industrial usage, where high productivity is crucial.
How are we accelerating metabolic engineering?
For optimal execution of metabolic engineering we established a workflow, basing on the DBTL-cycle. We use our Marburg Toolbox to build whole pathway libraries, which can then be transformed into our producer strain VibriXpress. By using biosensors for product screening, we circumvent the expensive and time-intensive usage of LC-MS and learn which pathway variants are the most promising. These findings can then be fed into our pathway-design, thereby closing the DBTL-cycle. To further optimize these pathways, we planned to use directed evolution for more adaptations to that route.
3-Hydroxypropionic acid 3HPA
As a proof of concept for our engineering workflow we chose the biological production of 3-Hydroxypropionic acid ( 3HPA ). 3HPA is a compound of high industrial value. In 2004 the U.S. Department of Energy recommended 3HPA as alternative to fossil oils for the r chemical industry (Werpy et al.2004). 3HPA as a platform chemical can be converted into many other compounds like acrylic acid and acrylamide, which both are also precursors for further compounds. According to an estimate of 2014, acrylates alone have an annual market value of USD12 billion, making its precursor 3HPA an optimal target product for our metabolic engineering approach. By finding the optimal pathway to produce 3HPA we could show the efficiency of metabolic engineering in V. natriegens as well as getting a big step closer to an alternative to fossil oils by biological, renewable resources. [Abbildung: T--Marburg--Metabolic_Applications.svg --- noch nicht fertig!]
Our pathway
In recent years many people concentrated on producing 3HPA via many different metabolic pathways
(Valdehuesa et al.2013)
;
(Vidra et al.2017).
Most of these pathways use glycerol or glucose as starting substrate, but there are also publications where acrylic acid, CO2 or uracil were converted to 3HPA
(Vidra et al.2017).
One promising route starts with glycerol which is dehydrated and oxidized.
For E. coli, a productivity of 6.6 mmol g-1 cdw h-1 (oder Lieber andere units? 6.5mmol l-1 oder 0.48 mol mol-1 glycerol) has been shown
(Raj et al.2008).
However, this route depends on vitamin B12, which often can’t be taken up or produced by the chassis.
Many other routes start with glucose, which gets metabolized via propionate, lactate, β-alanine or malonyl-CoA
(Vidra et al.2017).
We decided to use a pathway, based on the conversion of acetyl-CoA into malonyl-CoA and finally 3HPA . According to
(Valdehuesa et al.2013)
who evaluated many pyruvate-derived production pathways from a thermodynamic point of view, this is one of the most efficient routes for 3HPA production.
This pathway was first established in E. coli, but it was also tried in many other chassis like S. cerevisiae and S. elongatus
(Kildegaard et al.2016);
(Liu et al.2017);
(Rathnasingh et al.2012).
We chose this route for several reasons.
Firstly, it is based on glucose degradation and V. natriegens has an unbeaten glucose-uptake rate, enabling high glucose consumption. Secondly, vitamin B12 is not necessary, the only cofactors involved are NADPH and biotin, both of which occur naturally in the organism.
Thirdly, theoretically just one further enzyme is needed to complete the pathway. All enzymes necessary for the production of malonyl-CoA are already present in V. natriegens and we just have to integrate the last enzyme, malonyl-CoA reductase.
Nevertheless, overexpression of acetyl-CoA carboxylase increases production, so we took both enzymes into consideration.
(Liu et al.2017)
proposed, that there are many ways to further optimize the pathway and direct the flow towards a high product titer.
Hence, implementing many variations of this pathway into V. natriegens and testing them in a fast manner promises a wealth of knowledge about pathway optimization and general principles of metabolic engineering.
First isolated from salt marsh mud on Sapelo island off the coast of Georgia in 1958 (Payne et al. 1958), the gram-negative bacterium was first named Pseudomonas natriegens (Payne et al. 1960). These early studies also revealed a broad range of tolerated pH conditions with an optimum at a pH of 7.5 (Payne et al. 1960). One feature of this newly discovered organism became immediately apparent: The incredible doubling rate, first determined to be around 9.8 minutes (Eagon 1962). We found that doubling rates of around 7 minutes were possible (own Data).
Only much later, studies found several possible reasons for this. Firstly, as most Vibrio species, V. natriegens genome is split and distributed on two chromosomes. This means that replication can start at two origins of replication (Ori), resulting in more in parallel replication.
Additionally, it was shown that the gamma-proteobacterium has an increased rRNA activity. This is because of a greater number of rRNA operons which are additionally controlled by stronger promoters (Aiyar et al. 2002) when compared to E. coli . More rRNA means more ribosomes since rRNA synthesis has been shown to be the rate-limiting step in their assembly (Miura et al. 1981). Estimates for the number of ribosomes in V. natriegens in the exponential phase suggest around 115,000 per cell, while E. coli is estimated to have between 70,000 and 90,000 (Failmezger et al. 2018).
This gives V. natriegens higher protein expression and lets it create biomass more quickly. Could this also be exploited for the production of high-value proteins? Most likely.
It was soon discovered that V. natriegens would make a perfect example by which to effectively and harmlessly demonstrate the basic techniques of microbiology to students, taking full advantage of the immense doubling rate (Mullenger 1973; Delpech 2001). Experiments regarding population structure, UV stress, and simply bare growth can be observed in a much shorter timeframe.
V. natriegens has a single stage lifecycle and does not form spores. The rod shaped bacterium does however possess a single polar flagella (Austin et al. 1978).
The rod-shaped bacterium occupies a special ecological nice in estuarine and salt marsh regions, where strongly fluctuating salt concentrations and nutrient availability create a challenging environment. In order to adapt to these challenges, V. natriegens can utilize a wide range of substrates to outgrow competitors, as well as remain in a low metabolic state for extended periods of time when the nutrient pool is depleted (Nazly 1980). Furthermore, it has the ability to quickly draw available phosphorous from its environs and store them in polyphosphate bodies (Nissen et al. 1987) for later utilization. Yet another way in which V. natriegens is adapted to its variable environment is the storage of carbon as polyhydroxyalkanoates (PHAs), biodegradable polyesters that are accumulated in intracellular granules that can be broken down under starvation conditions (Chien et al. 2007).
From the well oxygenated upper layer of mud down to the anoxic layers, V. natriegens is able to grow in many surroundings. These extremes are challenges to which V. natriegens found solutions. At the surface of the mud, as well as in open waters, the bacteria are exposed to strong UV radiation. As a response to this, the DNA damage repair systems are highly active (Simons et al. 2010), with significantly elevated expression levels of some parts of the system compared to E. coli , thus increasing DNA sequence integrity. By switching to fermentation, growth can be maintained under anaerobic conditions, allowing the colonization of the lower strata in the mud.
V. natriegens was shown to be a vital part of the marine ecosystem because it has the rare ability to fix atmospheric nitrogen under anaerobic conditions, thereby enriching its habitat and playing a crucial role as a provider of essential, bioavailable nitrogen (Coyer et al. 1996) under anaerobic conditions.Unsurprisingly, V. natriegens has its own predators in its native habitat: Bacteriophages (Zachary 1974). Studies in the 70ies found a link between different phages and the prevailing environmental conditions (Zachary 1978) . This suggests the phages employ a divide and conquer strategy, specializing, thereby limiting competition while improving reproductive success. One phage may have adapted to be effective at lower temperatures, while others prefer higher temperatures. The same applies to other conditions like for instance salt concentration (Zachary 1976). Intriguingly, that opens the possibility of creating genetic parts derived from these phages, applicable to future work with V. natriegens, analogous to the T7 system in E. coli. On the other hand, one study could show, that the phage carrying the toxins associated with V. cholerae could not replicate in V. natriegens (Lee et al. 2016), further showing its harmlessness towards humans.
In contrast to that, some strains of Vibrio natriegens seem to be predators themselves. Confirmed as being pathogenic in several marine crustacea, most notably in the swimming crab Portunus trituberculatus, in which V. natriegens can lead to mortalities up to 85% (Bi et al. 2016). This is especially problematic since P. trituberculatus is farmed commercially in aquaculture in south-east China. The mechanism of that infection is poorly studied and it is not clear whether all strains of V. natriegens are pathogenic to crustaceans, but it is well known that many Vibrio species cause opportunistic infections in crustacea.
On a more positive note, a recent study could show that the sponge Aplysilla rosea develops a more diverse microbiome when exposed to V. natriegens (Mehbub et al. 2018). It can be speculated that this helps to protect the sponge from pathogenic microbes that then find it harder to colonize its surface.
A diverse, well-balanced metabolism allows a bacterium to grow in many conditions, while simultaneously making it interesting for biotechnological applications. V. natriegens can utilize a wide range of substrates (Hoffart et al. 2017), as well as grow in the absence of oxygen while being faster than other bacteria under the same conditions.
For many applications in which a modern laboratory work horse is expected to be useful, a high-resolution map of its metabolism is a prerequisite. One recent study generated just that (Long et al. 2017). By tracking 13C labeled carbon through the intricate network that constitutes V. natriegens metabolism, they were able to gain insight. Fore one, they found the it to be very similar to the well-studied E. coli metabolism. It has al the mayor catabolic and anabolic pathways, the same canonical amino acids and building blocks. Some notable differences between the two were the RNA content being 29% in V. natriegens compared to only 21% in E. coli, the presence of an enzyme for the decarboxylation of oxalate (Long et al 2017).
The specific uptake of many carbon sources per gram dry weight per time was sown to be significantly higher than in other comparable species of bacteria (Hoffart et al. 2017). Many commercially interesting compounds are already produced by unmodified V. natriegens. In the case of alanine production, only four deletions resulted in a strain that outperformed highly specialized E. coli and C. glutamicum production-strains by a factor of 9 to 13 times (Hoffart et al. 2017). Bioreactor based production on a large scale is expensive, therefore a strain generating similar or even higher yield per gram carbon source is highly desirable, improving temporal yield per fermentation unit.
When growing anaerobically, acids are produced that lead to an acidification of the medium but when grown under strong aeration, the pH can, depending on the medium, rise. This is most likely due to formation of ammonia (Eagon 1962).
The dependence on Na+ ions for its metabolic activity conveys a form of natural biocontainment, inhibiting growth in accidentally released V. natriegens (Webb and Payne 1971). The high salt content of the LBv2 media also presents possible contaminants with a barrier, reducing the likelihood of airborne microbes to take hold.
Recently, a study revealed that V. natriegens, surprisingly, is the most effective producer of selenium nanoparticles yet described (Fern`ndez-Llamosas et al. 2017). These particles with a diameter from 100-400nm have applications in diverse fields, such as medicine, microelectronics and more. Producing the nanoparticles in living systems has benefits over other methods, for instance, the low energy consumption as well as a coat of proteins preventing agglomeration of the particles. Also, V. natriegens exceptionally high resistance to selenite suggests possible applications in bioremediation of contaminated water and soils.
Working with V. natriegens
ATCC 14048 is the most commonly used strain of V. natriegens. Most results from earlier studies were generated from this strain. We recommend using ATCC 14048 in order to ensure that the findings will be applicable to your work and to adopt it as the standard strain. It is also the fastest strain known (Weinstock et al. 2016) so you won´t miss out on its incredible doubling time.
Stemming from subtropical regions, V. natriegens has not the same tolerance to low temperatures that we are used to from E. coli, but it is still much more resilient to cold than B. subtilis. At the root of that lies the low catalytic activity of V. natriegens native catalase at low temperatures, which is then unable to detoxify reactive oxygen species. To address that problem, scientists from SGI introduced homologous catalases, thereby creating a strain with comparable cold tolerance. With this adaptation, storage on plates can now be extended beyond the four weeks at room temperature by placing the plates in the fridge (Weinstock et al. 2016) In storage at -80°C in a standard 20% glycerol stock the cells stay viable almost indefinitely. And even when working with the wildtype, your cells won´t die overnight when placed at 4°C. Only after a week they will notably start decreasing in viability.
When working with V. natriegens ATCC 14048, no special precautions have to be taken since it has been shown that no known pathogenicity associated genes were present (Weinstock et al. 2016) Also, there is continued record of research since 1958 with not a single documented incident of a human infection with V. natriegens.
Lately, a new branch of microbiologists developed an interest in V. natriegens. Their main focus lies in establishing state of the art methods and bringing V. natriegens to its full potential. We see our project as part of this movement and wish to make V. natriegens easily accessible to researchers. One groundbreaking paper (Weinstock et al. 2016) established several methods to make V. natriegens genetically accessible, as well as characterizing some central genetic parts. This paved the way for more research into applying its potential.
If you want to introduce genetic material into V. natriegens, you have several tried and tested options to choose from. You could use electroporation, a quick and simple method and the one with the highest transformation efficiency. Electroporation can also be used on wildtype cells. Or you could use chemically competent cells, the transformation works very similar to E. coli heat shock transformation. But in order to achieve good efficiencies using chemically competent cells, a nuclease deletion strain is needed. And if you want to insert especially large constructs, you can also use conjugation with very good success ( (Lee et al. 2016; Weinstock et al. 2016).
A fully sequenced genome is also a prerequisite for many studies. Currently, several sequences are available (Lee et al. 2016; Maida et al. 2013), one of them generated by us. The genome has a size of approximately 5.17 Mb. Automated annotation was performed, revealing more of the genomic structure, as well as enabling the reconstruction of the codon usage profile (Lee et al. 2016).
The genome of V. natriegens is, like in many species of the genus, split into two chromosomes. Chromosome one contains 3.24 Mb, chromosome two 1.92 Mb.
Plasmids, Promoter, resistance
Usually, a big hurdle when changing your chassis organism is a very basic question: Will my constructs still work? Do I need different ori? Do I have to redesign everything from scratch? Fortunately, the most commonly used origins of replication, ColE1, p15A and pMB1 are maintained just fine in V. natriegens (Weinstock et al. 2016) We could observe some differences in copy number, which we quantified. You can find the corresponding data on our Part collection page. To retrieve these plasmids from the cells, we have tested a wide variety of commercially available kits and found that all of them worked, returning plasmid of the same purity and yield as for E. coli. Promoter commonly used in E. coli also lead to expression in V. natriegens. Expression levels are slightly different, but we also quantified these and you can find the results on our toolbox page. The same goes for other basic genomic parts like terminators and ribosomal binding sites.
Commonly, when very high gene expression is needed, for instance in protein overproduction, the phage derived T7 system is used. The T7 phage infects E. coli but it was also successfully inserted into V. natriegens and shown to drive protein expression (Weinstock et al. 2016) One way of generating even stronger, phage derived promoters could be using V. natriegens derived phage expression systems.
Every organism has its unique set of resistances to certain antimicrobial molecules. In order to successfully use a new organism, awareness of its individual tolerances is required to use antibiotics successfully for selection. We could show that chloramphenicol, carbenicillin, tetracycline and kanamycin worked, but at different concentrations from E. coli. We measured the V. natriegens specific concentrations and made them available on our strain engineering page.
Plasmids in absence of a selective pressure were only retained for a short time (Own Data). Curing of plasmids that have no longer a purpose is therefore quick and fairly effortless.
MuGENT - Recombineering
Investigations into alternative genetic tools were also undertaken, most notably MuGENT and Recombineering.
MuGENT takes advantage of V. natriegens natural competence by inducing the TfoX system. The TfoX system, which is known from many Vibrio species, enables uptake and genomic integration of DNA parts via homologous recombination (Pollack-Berti 2010). In most cases, growth on chitin was identified as the trigger which activated the system. Regrettably, the trigger is not known for V. natriegens. Using this system, a group of researchers characterized this system in V. natriegens, making it usable by placing it under the control of an inducible promoter. Additionally, they demonstrated its potential by increasing the natural poly-hydroxybutyrate (PHB) production 100-fold (Dalia et al. 2017).
Recombineering takes advantage of the fact that short DNA oligonucleotides, that can easily be delivered into cells by a plethora of ways, can be used to generate point mutations, knockouts and small modifications in the genome, as well as on plasmids. The homologous flanks required are relatively short, allowing for constructs under 100 bp. Having this system available in V. natriegens means that generating new strains and gene variants is easy and quickly implemented (Lee et al. 2017).
Cell Free Protein-expression Systems (CFPS)
Very recently, several groups have published their work about cell free protein expression systems (CFPS) in V. natriegens (Failmezger et al. 2018; Des Soye et al. 2018; Wiegand et al. 2018). Cell free systems offer the advantage of direct access to many cell components as well as fine control over the conditions in the reaction (Carlson et al. 2012). While these are true for all CFPSs, the advantage of creating such a system from V. natriegens is the much higher ribosome count (Failmezger et al. 2018).
Although the system works well when expressing plasmids from E. coli, it could be shown that expression was much higher when plasmids prepared from V. natriegens were used. Possible explanations are differences in methylation patterns, resulting in degradation of the plasmid by the system (Failmezger et al. 2018). All this could be done by using protocols very similar to well established methods for other bacteria.
Cre- lox
The Cre- lox system was also successfully implemented and shown to work consistently with 600bp homology flanks (Weinstock et al. 2016) Recycling of selection markers, like antibiotic resistances, as well as the removal of incorporated genes becomes easy and reliable.
Also interesting for future protein related applications is, that a putative secretion signal was identified (Weinstock et al. 2016)
Fun facts:
The epithet natriegens was chosen after the realization that salt is needed for it to grow, natrie stemming from the Latin word natrium meaning sodium and the Latin verb egere, meaning to need.
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