Difference between revisions of "Team:Marburg/Design"

One key feature of the Marburg Collection are the connectors that provide our toolbox with the required flexibility. Our design is inspired by the “Dueber Toolbox”, a golden-gate-based cloning method designed for applications in Saccharomyces cerevisiae (Lee et al. 2015) . To our knowledge, the “Dueber Toolbox” is the first cloning system that performs assembly through all levels using restriction sites located on independent basic parts, termed connectors, instead of destination plasmids for level 1 and level 2 plasmids (Weber et al. 2011) .

Design of insulators

Natural Competence

The Flp/frt system

Our workflow

One of the central elements of synthetic biology is the Design-Build-Test-Learn cycle. It illustrates the workflow of how systems like metabolic pathways, genetic circuits or anything else are constructed and evaluated. The first step is to design the system basing on the existent knowledge about the system. Then the system is built and its functionality is tested. The results of these tests describe the ‘Learning’ part of the cycle. They are used to make an improved design. Finally, a new round in the DBTL cycle can be made and the system gets improved in each round of the cycle. For our approach we decided to establish a workflow for metabolic engineering, which bases on the DBTL cycle and combines the existing principles of metabolic engineering with the advantages of the fast-growing organism V. natriegens. First of all, we use the Marburg Toolbox to construct a library of pathways with different combinations of promoters, ribosome binding sites, coding regions, enzyme ratios and plasmid copy numbers. Next, we transform the plasmids for each pathway version together with the plasmid for the biosensor into our chassis. When the pathway is now performed, the cells produce a fluorescence or luminescence signal and its intensity correlates to the 3-hydroxypropionate ( 3HPA-) concentration. By using FACS we can select for the most efficient pathway versions. By using multiplex automated genome engineering (MAGE), refeeding 3HPA into the central metabolism and repetition of FACS selection we have a cycle of directed evolution-guided pathway optimization. The strains with the most promising pathway variants can then be isolated and their pathway composition elucidated. Thus, we get information about designing principles for our pathway and can propose an improved design for a new round in the cycle of metabolic engineering.

Our pathway

As mentioned in the description, there are many ways to produce 3HPA. We decided for a pathway, basing on the conversion of acetyl-CoA via malonyl-CoA to 3HPA. According to (Valdehuesa et al. 2013) who evaluated many pyruvate-derived production pathways from a thermodynamical point of view, this is one of the most efficient routes for 3HPA production. We decided for this route for several reasons. First it bases on glucose degradation and V. natriegens has an unbeaten glucose-uptake rate, enabling a high glucose consumption rate. Second there is no vitamin B12 necessary, the only cofactors which are involved are NADPH and biotin, both of them naturally occurring in the organism. Third there is theoretically just one further enzyme needed to complete the pathway. All enzymes necessary for the production of malonyl-CoA are still present in V. natriegens and we just have to integrate the last enzyme, malonyl-CoA reductase. Nevertheless, overexpression of acetyl-CoA carboxylase increases productivity, so we took both enzymes into consideration. (Kildegaard et al. 2016) proposed, that there are many ways to further optimize the pathway and direct the flow to a high product titer. Hence, implementing many variations of this pathway into V. natriegens and testing them in a fast manner promises a high gain of knowledge about pathway optimization and general principles of metabolic engineering.

Choice of enzymes

For the core pathway, we need to implement two enzymes, acetyl-CoA carboxylase (Acc) and malonyl-CoA reductase (Mcr). For Acc there are several candidates, which have still been tested for synthetic metabolisms ((Kildegaard et al. 2016); (Liu et al. 2013); (Vidra et al. 2017)). Our plan was to first test enzymes from different organisms and then optimize expression profiles. To benefit from the fast engineering workflow in V. natriegens, we decided to use enzymes from different organisms. We planned to use the native Acc from V. natriegens, because it is still present in the cells and we prevent unnecessary burden by overexpressing a heterologous Acc. The native Acc consists of four subunits together with an associated biotin ligase. The genetic organization is the same like in E. coli, which is shown in figure 3. Nevertheless, further looks into literature revealed, that heterologous expression of pathway enzymes could be more promising because housekeeping Accs often are strictly regulated while foreign genes have different regulation motifs. Hence, we also used the Acc from E. coli together with its associated biotin ligase (BirA). These enzymes have already been tested for 3HPA production (Liu et al. 2013). Additionaly, we decided to test the enzyme from Synechococcus elongatus. Since cyanobacteria require higher amounts of fatty acids to form thylakoid membranes, they might have more active enzymes. The S. elongatus Acc consists of four subunits, just like those from E. coli and V. natriegens. For this enzyme it was also recommended to use it with a biotin ligase, because it needs a covalently bound biotin to be activ. Unfortunately Accs consisting of four subunits form a very unstable complex and unbalanced expression levels of its subunits lead to gene repression (Abdel-Hamid et al. 2006). To circumvent this issue, we also chose the Acc from C. glutamicum. In this organism, AccB and AccC are fused to one subunit, as well as AccA and AccD. Balancing of two subunits instead of four is much easier, therefore this enzyme is beneficial for us. Gande et al., 2007 still characterized this enzyme and confirmed its applicability for synthetic biology. Since C. glutamicum is a gram-positive bacterium, we decided to codon optimize the genes for a smoother translation and coexpressed the associated biotin ligase (Accession numbers NCgl0670, NCgl0678, NCgl0679).

Rational design of plasmids

Synthetic biology proceeded significantly in recent years and we have much knowledge about rational design of circuits and pathways. Nevertheless, failures and unexpected outcomes slowdown scientific progress. To tackle this issue, we decided to follow two approaches. On the one hand we planned to model our pathway and predict a – theoretically – optimal pathway, which we can then construct and test. And on the other hand, we decided to build additionally to this particular pathway a whole library of many variations of this pathway with little differences. In this way we create many producer strains in parallel and speed up the metabolic engineering workflow. Our rational approach bases on a Michaelis-Menton-based metabolic analysis. For more details see our metabolic model. Since there are no stochiometric flux data about V. natriegens yet, we used the data from E. coli, which has a very comparable metabolome to V. natriegens (citation needed). For the model we only considered acc from C. glutamicum and mcr from C. aurantiacus, because they were from our point of view the most promising ones. The model predicted, that Acc is the rate limiting enzyme in the pathway and has to be expressed ten times stronger than Mcr. We used the promoter characterization data, obtained from our part collection team (For more details see the section about characterization of the promoters of our Marburg toolbox) and introduced them into the model. The resulting prediction was, that we have to use the promoter J23106 for acc and J23109 for mcr. Therefore, we designed the pathway plasmid according to this model. We built the plasmid based on the Marburg Toolbox with 4 transcriptional units. For the ori we chose ColEI, which has a broad host range and a high copy number in V. natriegens and E. coli (Jain et al. 2013), [Link zu qPCR von Carlos]). We decided for that one, because V. natriegens shall be a chassis for testing pathway variants, which then maybe will be transferred into other strains, therefore a broad host range vector is reasonable. For accBC and accD we chose the promoter J23106 and for mcr the promoter J23109 according to the model. For birA we also chose J23109 as promoter, because BirA is only relevant for activation of Acc and not for direct reaction catalysis. For ribosome binding sites and terminators, we used the same for each coding region, because otherwise we would have fluctuations which weren’t considered in the model. An illustration of the plasmid is shown in figure 5.

Pathway library

For the second approach of pathway design, where we build a whole library, we also used the Marburg Toolbox [Marburg Toolbox soll ein Link zur Marburg Toolbox sein] to combine different promoters, ribosome binding sites and coding regions. In theory, the toolbox enables the plasmid assembly of 21780 different part combinations per transcriptional unit (22 promoters x 5 rbs x 6 terminators x 3 oris x 11 resistance cassettes; connectors weren’t considered). For a plasmid containing accBC, accD, birA and mcr that would make 6 x 1012 possible constructs, but assembly of so many versions is irrational. The better way is to select a few parts which are combined and then for the most promising versions one can think of more combinations. Therefore, we focused on eight promoters and three ribosome binding sites with different strengths. Our idea was to build pathway plasmids containing the whole pathway, as well as plasmids containing only the acetyl-CoA carboxylase genes or only the mcr gene, which can then be cotransformed (Table 1). To enable cotransformation, we used the oris ColEI and p15a, which are compatible to each other.

The idea behind the plasmids with the whole pathway is, that the cells only have to maintain one particular plasmid. The strategy for the single-enzyme plasmids is that by cotransformation different combinations can be made to increase the number of possible pathway variants. The different plasmid types can be seen in table 1, all in all we designed the construction of 1446 plasmids for our pathway library. For plasmids of category I (see table 1), we combined eight promoters with three ribosome binding sites, a kanamycin resistance gene and a p15a ori, giving us 24 constructs. For plasmids of category II and III we used the same promoters and ribosome binding sites for both transcriptional units, leading to 576 plasmids respectively. For plasmids of category IV we restricted our selection to three promoters and three ribosome binding sites. Since a misbalance of accBC- and accD expression levels leads to occlusion bodies and decreased activity we decided to always use the same promoters and rbs for both genes. In this category we get 81 plasmid variants. For categories V, VI and VII, containing the whole pathway, the combinatorial possibilities were very high, so we decided to further restrict the selection by always coupling strong promoters to strong rbs, medium promoter to medium rbs and weak promoter to weak rbs. Therefore, we got a total of 27 plasmids for category V and 81 plasmids for category VI and VII, respectively. We also considered to integrate one or several genes, therefore we planned to search for integration sites and to characterize them. [Link zu Bauersachs Teil]

Our biosensors

The design of our biosensors was largely guided by the goal of having as little impact on the host metabolism, while simultaneously being simple and easy to implement. We chose to design all parts to be compatible with the Marburg toolbox, RFC10, RFC25, andRFC1000, to make it as easy as possible for us and future users to implement it and adapt it to new requirements.

Annotation of the location for the regulatory sequences upstream of the HdpR coding sequence was insufficient for us to precisely remove the activator binding site or the constitutive promoter driving HdpR expression. So, we included an about 200bp long region upstream of the HdpR, which should be enough to include all regulatory motifs. Also included is the HdpR coding sequence with its own ribosomal binding site (RBS). and promoter. This results in a part that includes the whole sensing function, but does not quite fit our scheme of clearly defined functional units. Since expression of any part cloned behind the regulatory region is directly controlled by it, and the part serves as both, a promoter and RBS. It is basically an inducible promoter fused to an RBS. Thus, we decided to give the part the overhangs of an RBS. on the downstream and a promoter on the upstream end.

In order to obtain the desired region from the P. putida KT2440 genome, we planned to amplify it via PCR and then isolate the fragment by gel purification of the corresponding band. Into the primer-design we factored in the overhangs we needed to make it Marburg toolbox compatible. So, we gave them non-binding overhangs which automatically added the necessary BsmBI cut sites.

For the malonyl-CoA sensor, our proceeding was very similar. Just that we knew the exact location of all functional parts, except of the location of the promoter, form the 2015 paper by Liu et al and from annotations in the published genome (Liu et al. 2015). Therefore, we could implement it fully into the Marburg toolbox, by simply treating the FapR coding sequence as coding sequence. Not that straightforwardly was the design of the fapO regulator binding site. Since we did not know the promoter strength, and the mechanism of regulation was independent of the promoter sequence, we decided to implement a promoter from the Andersen library, J23100. By placing it right in front of the fapO site, we created a construct that, in theory, should give us a FapR controlled promoter. For the amplification of the individual parts from the B. subtilis 168 genome, again, we used primers sporting non-binding regions, adding the necessary overhangs for our toolbox.

For future projects, other sensors could also be employed. In a publication from 2016, Rogers et al. propose an alternative sensor for 3HPA with a lower perception threshold (Rogerset al.2016). We choose not to implement it in our design because of its more complicated mechanism that requires the implementation of more genes. Intriguingly, it works by first converting 3HPA to acrylate via a pathway of three heterologous enzymes. The acrylate is then sensed by a transcription factor, similarly to how our sensors described above work. They also report its successful implementation and application in increasing the 3HPA titer up to 23-fold. It is conceivable that with a little more effort our system could yield comparable results. Off cause, s we have the additional benefit of having a much shorter iteration interval.

Further product sceening

The usage of biosensors for product screening is a fast and cheap method enabling high throughput analysis of pathway libraries. However, to further characterize the pathway variants and implemented enzymes we planned to measure enzyme activities. In that way we can quantitatively benchmark each enzyme and establish in vitro assays as a prototyping engineering approach. For the malonyl-CoA reductase, it is easy to measure reaction velocity, because the enzyme converts NADPH into NAD+ and both compounds have different absorbances at 345nm (De Ruyck et al. 2007). Based on that we designed an enzyme assay, where we use cell extract from Mcr producing cells or directly purified Mcr and mixed it with a defined amount of NADPH and MgCl2. By measuring the absorbance, one can determine if NADPH concentration changes. By adding malonyl-CoA to the reaction mix we then start the reaction and are able to observe a decrease in absorbance because the reaction consumes NADPH. Here the starting concentrations of malonyl-CoA and NADPH have to be high enough to ensure, that nearly each Mcr molecule in the mixture is saturated and the reaction goes proportional to enzyme concentration. By determining the slope of the linear decrease of absorbance it is possible to calculate the specific activity. The specific activity is a value which characterizes the ability of enzymes, to convert a substrate into a product. It is defined as the moles of product formed by an enzyme in a given amount of time under given conditions per milligram of total proteins, the unit is µmol min-1mg-1. To calculate the specific activity, one needs the slope of the measured absorbance. This value defines the change of absorbance per time. By using the Lambert-Beer law it is possible to calculate the amount of NADPH converted into NADP+ per time (Lambert and Anding, 1760).

Directed evolution

Metabolic Engineering offers many possibilities to create suitable pathways but it consists of more than just building them. Pathway optimization is also crucial for efficient productivity. In our workflow for metabolic engineering we want to implement directed evolution to drive metabolic fluxes towards 3HPA production. We elaborated two strategies to do that. Our first strategy was to implement genes for the conversion of 3HPA into succinate. In that way, we lead our product back into the central metabolism. By deleting all other genes responsible for succinate production, the cells are forced to use the 3HPA bypass to get succinate, which is needed in the TCA cycle. The big advantage of using V. natriegens for that purpose is that its short doubling time increases the number of cell divisions per time and thereby increases the number of mutations in a given time frame. Our second strategy is to use MAGE [Link zu project→description→metabolic engineering→Directed evolution→MAGE erklärung] together with our biosensors and fluorescence-activated cell sorting ( FACS ), to cause various genome modifications and rapidly select for cells with increased 3HPA productivity (Figure 9). We looked into literature and found 16 possible genome modifications for 3HPA production made by other groups. Table 2 shows a list of all modifications. Our plan was to order oligos for each of these modifications, leading to upregulation, downregulation, deletion or site-directed mutagenesis of the respective gene. The aimed modification for each gene is shown in the table. For the next step we decided to use natural transformation for integration of these oligos into the strain. Because each cell will take up different numbers and different combinations of oligos, we will get a population of genomic variations [Link zu Project → Design → Strainies → Mugent]. The genetically diverse population can then be put into FACS and we can select for cells with the strongest signal and thereby the best productivity. Finally, the whole cycle of MAGE and FACS can be repeated, leading to continuously improved pathways. We believe that the combination of MAGE- FACS with the fast growth of V. natriegens will speed up the process of metabolic engineering and pathway optimization significantly!

Outlook

In our project we try to accelerate the process of metabolic engineering in all of its facets and thereby find a suitable pathway variant to achieve high 3HPA productivity. Nevertheless, there will still be many possibilities to further improve the production of 3HPA in V. natriegens. Table 2 shows a list of 16 genome modifications, that were made in other organisms with the same or a similar pathway to direct the flow towards 3HPA. Some of these modifications target for instance NADPH homeostasis. During glycolysis cells produce NADH while the production of 3HPA depends on NADPH. Therefore, a misbalance between NADH and NADPH could arise, but overexpression of pntAB (nicotinamide nucleotide transhydrogenase) or gapA (NADP+-dependent glycerol-3-phosphate dehydrogenase) has been shown to stabilize cofactor balance (See table for references). Another possibility to enrich the NADPH pool is to downregulate glycolysis while upregulating pentose phosphate pathway, which produces NADPH instead of NADH. This can be achieved by deletion of NAD+-dependent gapA or deletion of pfkA (phosphofructokinase). Another critical point in the 3HPA route are side reactions. Pyruvate can be reduced to lactate or decarboxylated to acetate, what could be suppressed by deletion of ldhA (lactate dehydrogenase) or pox (pyruvate decarboxylase). The flow from pyruvate to acetyl-CoA can be increased by overexpression of pdh (pyruvate dehydrogenase). For acetyl-CoA there are also side reactions, for instance hydrolysis to acetate. This reaction can be suppressed by deletion of pta (phosphotransacetylase) and ackA (acetate kinase) or by overexpression of ald6 (aldehyde dehydrogenase) and acs (acetyl-CoA synthase) for the reverse reaction to acetyl-CoA. Malonyl-CoA can also go into side reactions for fatty acid biosynthesis instead of the reduction to 3HPA. However, downregulation of fabAB (fatty acid biosynthesis) or inducible inactivation of fabAB has been showed to increase 3HPA titer. Additionally it has also been shown that site-directed mutagenesis of acc and mcr genes increases product titer (Kildegaard et al. 2016); (Liu et. al. 2016).

We chose the production of 3HPA as application to show the functionality of the Marburg toolbox and as proof-of-principle for our metabolic engineering workflow. However, our goal is not to produce high amounts of 3HPA, but rather to show that V. natriegens can be used as test chassis for rapid testing of many pathway variations to find the best one. The next step would therefore be to evaluate, if the pathway with its genetic composition can be transmitted to other organisms to see if the route still works. Besides that, there are many other compounds of medical and economical relevance, which still can’t be produced biotechnologically. The production of the anticancer drug taxol involves for instance 19 enzymes and is therefore a very challenging pathway with lot of bottlenecks and tunable reactions (Croteau et al., 2006). We think it would be beneficial to use V. natriegens as chassis for testing this challenging pathway or parts of it. Its short doubling time and genetic accessibility will accelerate the process of enzyme engineering and finetune expression levels or gene combinations. Another crucial aspect, that has to be tackled is the universality of our workflow. We involved biosensors for product screening but for many metabolites there are still no known sensor proteins. There are three possibilities to circumvent this issue. The first is to find new biosensors, what is complicated, because many compounds, especially drugs for medical purposes don’t occur in the nature, therefore it is unlikely that there are still proteins binding specifically to these compounds. The second possibility would be to engineer proteins to become drug-specific sensors. The third possibility is to upscale LC-MS techniques to enable larger sets of measurements and faster screening. LC-MS has an unbeaten accuracy but its bottleneck is the velocity. The average time needed for one sample lies between 1 and 10 minutes, making measurements of large sets of samples impossible. Furthermore LC-MS requires high amounts of buffer. However, as mentioned in the Labautomation-section [soll ein Link sein], the company Labcyte developed a system to use acoustic waves for pipetting nanoliter droplets into microtiter plates in a very fast manner. Now they invented an acoustic mass spectrometer, which combines acoustic nanodroplet formation with mass spectrometry. The device is able to measure 3 samples per second without the need of a mobile phase. Such a device could be used instead of biosensor-based flow cytometry to broader the applicability of our metabolic engineering workflow.

References