Team:Uppsala Universitet/Design
The plan
To ensure an effective workflow we divided the wet-lab into three main groups: cloning, yeast expression and proof of concept. These groups worked closely together with the aim of creating an enzymatic system in Pichia pastoris (P. pastoris) that can break down lignin to smaller polymers.
Cloning
The cloning groups started with designing the constructs with the coding sequence for recombinant proteins to express in P. pastoris: Aryl-alcohol oxidase (AAO), horseradish peroxidase (HRP), manganese peroxidase (MnP), lignin peroxidase (LiP), glyoxal oxidases (Glox). All these genes were codon optimized for P. pastoris. However, this species is slow growing [1], while E.coli is not. In order to take advantage of the fast growing feature of E.coli and shorten the project time, shuttle vector: pPICZαB, which can propogate in both species, was used for assembling and amplifing constructs in E. coli. Furthermore, express and secrete recombinant proteins in P. pastoris [2].
Gibson assembly was chosen as the method to assemble the constructs. Gibson is a scarless assembly method allowing multiple DNA fragments to be joined in one isothermal reaction [3]. This method was chosen since it creates no scar between the ɑ-factor signal peptide and the coding sequence for the recombinant protein. At this point, Professor Jerry Ståhlberg suggested to try to co-translate the enzymes with a readily screenable marker protein by fusing a 2A peptide between them to make expression in yeast more visible and easy to identify. It has been proven in industrial cellulase producing fungi that the 2A peptide allows transcription and translation of multiple independent genes from a single mRNA. The result is two independent gene products from one RNA strand [4].
Protein Expression
Enzyme expression is highly dependent on choosing the correct system to ensure that the proteins are correctly folded, have functional post translational modifications and show the desired activity. Due to the origin of the enzymes in this project (e.g Horseradish and white-rot fungi) a eukaryotic expression system was chosen to ensure previously mentioned factors. The most prominent options in the field of yeast expression systems are Saccharomyces cerevisiae and Pichia pastoris. Literature showed functional expression of both enzymes in both S. cerevisiae and P. pastoris [5][6][7][8][9].
It was reported that secreted proteins in S. cerevisiae are often hyperglycosylated and therefore, their activity can be affected. This process is lower in P. pastoris, which is why this organism was selected for the project [10]. The enzymes to be expressed were codon-optimised for P. pastoris.
Two different strains of P. pastoris, X-33 and KM71H, were used. X-33 is the wild type strain, while the KM71H has a mutation in the methanol-metabolising operon leading to the slow methanol utilisation (MutS). The two strains may unpredictably differ in ability to express a specific enzyme, [11] so all proteins were expressed in both strains. It has also been shown that the methanol utilization plus phenotype, Mut+ P. pastoris strains grow faster under methanol induction, but the slower growing MutS strains have almost double the amount of protein production [12].
For generating genetically modified P. pastoris cells, the pPICZɑB shuttle vector (Invitrogen Easy Select Pichia expression kit) for Escherichia coli was used. This vector contains a bacterial origin of replication (ori), and thus propagates as a self-replicating plasmid in Escherichia coli. In contrast, pPICZɑB lacks a yeast ori and replicates by recombination into the AOX1 genome locus. This leads to a stable genomic construct without the risk of plasmid-loss. Furthermore, pPICZɑB carries a Bleomycin resistance cassette, which enables selection by antibiotics, and the methanol inducible AOX1 promoter. In native conditions, the AOX1 promoter controls the first enzyme of the methanol utilisation pathway [13]. This promoter is positively regulated by methanol and negatively affected by glucose, which is the more easily accessible carbon-source for the yeast. To establish inducible expression, the gene of interest is cloned downstream of the AOX1 promoter in-frame with the N-terminal ɑ-factor secretion signal sequence. After induction with methanol, the recombinant protein will be secreted into the supernatant. This is beneficial for future potential industrial application, as the cells do not need to be lysed to obtain functional enzymes. Furthermore, P. pastoris does not secrete many proteins, leading to a supernatant composed of almost exclusively desired enzymes.
X-33 (the wild-type strain) could acquire the MutS -phenotype if the construct integrates into a region coding for methanol-utilising enzymes after transformation. This is important to know when optimising the protein expression conditions so the Mut-phenotype of X-33 colonies was determined with standardised tests for methanol utilisation.
In order to test out cultivation of P. pastoris and intended transformation and expression protocols, an already available expression vector with RV1284 was used. RV1284 is a small protein which catalyzes reversible dehydration of CO2 to form bicarbonate [14]. This protein has been expressed and secreted successfully in P. pastoris [15], which made it a suitable construct for testing transformation and expression protocols with the conditions set up in the project lab.
For gene transfer into P. pastoris, two transformation protocols were tested, chemical transformation and electroporation. Due to higher efficiency the electroporation was used for all the constructs after RV1284.
For every expression culture, both expression and secretion of our target proteins were tested. To analyze this, samples of supernatant and pelleted cells were taken every 24h for 4 days after induction of the cultures for Mut+ and 6 days for Muts. Induction was maintained by adding methanol every 24 hours. The samples were analyzed by SDS-PAGE, with uninduced culture serving as a control. The SDS-PAGE should show the overexpression of a protein of specific size and its secretion in induced cultures. The absence of these bands from controls would confirms the specificity of the expression and that the induction works.
Proof of concept
For the proof-of-concept part of the project, the enzymes that were chosen first needed to be validated for degrading lignin and to what extent. Since the expression would be accomplished at a later stage of the experiment, HRP, LiP, MnP and AAO were purchased to allow for earlier analysis, but also for comparison to the enzymes later expressed in the lab. Firstly, the enzymatic activity needed to be measured to ensure that the enzymes were active. This was done using appropriate assays for the different enzymes based on their known activity.
Earlier studies preferably used enzymatic assays based on spectrophotometry. This was taken into consideration when choosing the methods. Spectrophotometry was used in all the enzymatic assays in order to test the activity.
Horseradish peroxidase
In order to verify that the expressed HRP was active, a 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) based assay was used. ABTS is a substrate that is oxidized in the presence of peroxidase, leading to a change detectable by a spectrophotometer at 405 nm [16].
Aryl-alcohol oxidase
Since AAO releases H2O2, a ferrous oxidation-xylenol orange (FOX)-based assay was used. In this assay, hydrogen peroxide oxidizes ammonium ferrous (Fe2+) ions into ammonium ferric (Fe3+) ions. The ammonium ferrous ions were in a H2SO4 solution. The released H2O2 will oxidize the ammonium ferrous ions, which, together with xylenol orange will form a complex that can be detected by the spectrophotometer at 560 nm (590 nm in a plate-reader) [17].
Manganese peroxidase (MnP)
Two different spectrophotometric assays were used to detect MnP. The first assay used phenol red as a substrate which would be oxidized by the MnP. The absorbance would be measured at 610 nm [18]. The second assay used ABTS as a substrate, where the ABTS would be oxidized by MnP and an absorbance change would be visible at 420 nm [19].
Lignin degradation and detection
Two different types of lignin were used: Kraft Lignin (from Stora Enso), which is lignin in the form that comes out of the paper mills, and lignosulfonate (from Domsjö Fabriker), which has sulfuric groups attached to the lignin complex. The sulfur groups makes this lignin water-soluble in contrast to the Kraft Lignin, which is not.
During a visit to the associate professor Martin Lawoko at Wallenberg Wood Science Center, an advice that was given during this visit was to use size exclusion chromatography to measure the eventual degradation of Kraft Lignin. Since industrial Kraft Lignin has a high polydispersity, the lignin would preferably be fractionated into different fractions based on solubility, before degradation [20]. These fractions would be used in the degradation experiments later in the project.
The carefully picked enzyme mixtures was chosen so that the oxidase (AAO) would generate H2O2 for peroxidase (HRP). The peroxidase would then degrade parts of the lignin which would be used by the oxidase in order to fuel the production of H2O2. This enzyme mixture needed to be tested in the lab. The hopes when using enzymes is that they are able to reach into the complex structure and break the bonds that lay deeper into the structure. This would allow the use of both the Kraft Lignin and the sulfonated lignin.
Lignin was mixed with HRP and H2O2 (both from AAO and H2O2) in a water solution. The combination was expected to give enough material and fuel to accomplish lignin degradation. The samples were analysed with SDS-PAGE, Native-PAGE and TLC-plate and with the help of these methods, detection of lignin degradation could be possible.
Thin layer chromatography
When degrading lignin, there will in theory be components that have structures with different properties. These differences could be used in order to separate the lignin components using thin layer chromatography (TLC). A TLC has a stationary phase consisting of a silica gel that is polar. The mobile phase can be adjusted for the components that are being separated, in order to control the migration of the sample. The separation can therefore be based on the affinity to the stationary phase or the solubility of the structures. A small amount of samples with enzyme-treated lignin were loaded onto the TLC-plate. The samples then wander up the stationary phase, depending on the affinity to the stationary phase the samples will wander a certain distance. If degradation of lignin had occurred, the samples would migrate differently on the plate [21]. The mobile phase used consisted of methanol, acetone and water. After running the samples, the plate was observed under a UV-light which revealed dark spots and smears, caused by the lignin-compound.
PAGE
SDS-PAGE and Native-PAGE was used for the same reason as the TLC-plate, to detect whether the lignin had been degraded or not. If the enzymatically treated lignin had forms of different sized compounds, then these parts of the degraded lignin were expected to be shown on the gel [22].
Full spectrum UV-Vis spectroscopic analysis using nanodrop
Spectroscopic analysis can detect chemical groups that have an absorption maximum at certain wavelengths, this means that the changes in these groups could be detected by observing absorbance changes at these specific wavelengths. A full spectrum analysis would allow the visualization and detection of changes to the absorbance peaks [23]. This full spectrum analysis was performed using NanoDrop and the samples included fractionated lignin, sonicated lignin and enzyme-treated lignin.
Future considerations
A good project design should include future improvement, development and scale-up considerations. In this part, we discuss the possible outlooks of our project.
Experimental
Industrial
We have incorporated future scaling-up considerations into our initial project design:
Ethical
Environmental