Difference between revisions of "Team:TU Darmstadt/Project/Glycolic acid/S cerevisiae"

Glycolic Acid Production in S. cerevisiae

Introduction

By modifying the natural glyoxylate cycle, we aim to generate a biosynthetic pathway to produce glycolic acid in S. cerevisiae using the strains CEN.PK-1C and H3847^[1]

Figure 1: Reaction mechanism of ICL1 and AtGLYR1.

GOR1 is the natural occurring gene in S. cerevisiae, which encodes the enzyme converting glyoxylate into glycolic acid by reducing the aldehyde group in a NADPH-dependent reaction. Since the gene is not expressed^[2] under normal growth conditions and the encoded glyoxylate reductase shows moderate affinity to the substrate glyoxylate, we used GLYR1 from Arabidopsis thaliana instead. Previous studies reported an enhanced affinity of AtGLYR1 to glyoxylate ^[3] and also enhanced activity ^[1]. The enzyme has a molecular weight of 30,7 kDa ^[4].

[Bild/Video Glyr1]

Figure 2: Structure of the NADPH-dependent glyoxylate reductase (GLYR1) with a molecular weight of 30,7 kDa.

Accumulating glyoxylate benefits the yield of glycolic acid. The isocitrate lyase 1, ICL1, catalyzes the reaction from isocitrate to glyoxylate. In the formation of glyoxylate, succinate occurs as a byproduct of the reaction. By overexpressing ICL1, a high substrate concentration is ensured. ICL1 has a molecular weight of 62.4 kDa^[5].

[Bild ICL1]

Figure 3: Structure of the isocitrate lyase 1 (ICL1) with a molecular weight of 62,4 kDa.

The accumulation of glyoxylate is supported by the deletion of MLS1 and IDP2, since these enzymes degrade glyoxylate and isocitrate in unwanted side reactions. Consequently, previous deletions of both enzymes in S. cerevisiae significantly increased glycolic acid production titers^[1]. MLS1, a malate synthase, catalyzes the conversion from glyoxylate to malate, and IDP2, an isocitrate dehydrogenase, catalyzes the oxidation of isocitrate to α-ketoglutarate.

As a result of our metabolic engineering approach, we were able to prove the activity of the glyoxylate reductase AtGLYR1. Due to the lack of time we were not able to detect the glycolic acid production in S. cerevisiae.

Methods

Figure 4: Schematic representation of work flow.

Cloning

Firstly, the sequence for AtGLYR1 was modified with a Strep-tag on the 3´end and 20 base pairs overhangs t the two ends of the gene, which are complementary with the alcohol dehydrogenase promotor (ADH) of the pAT423 plasmid. The sequence was ordered from Integrated DNA Technologies (IDT) and inserted into the pAT423 plasmid using the Gibson assembly method. E. coli TOP10 were transformed with generated plasmids and positive colonies were identified via colony PCR and DNA sequencing.

[[->2 Plasmidkarten pro Teamseite]]

Figure 5: pAT423 plasmid including expression cassette of AtGLYR1

Deletion

MLS1 and IDP2 were deleted from the genome of the S. cerevisiae using deletion cassettes, which were integrated into the genome by homologous recombination. Both deletion cassettes (KanMX-cassette and Leucin-cassette) were first amplified via PCR. The primers were binding to the loxP-sites and had a 40 bp homologous region to the gene. After that, S. cerevisiae cells were transformed with the linearized amplifed deletion cassettes, with the result that MLS1 was deleted using the KanMX- and IDP2 using the Leucin-cassette.

SDS-PAGE and western blot

To verify that AtGLYR1 was expressed and the corresponding protein was translated, a SDS-PAGE was performed, followed by a western blot. The resulting bands were compared to the expected protein size of 32.9 kDa.

Purification

After expression of At GLYR1in S. cerevisiae, a GE Healthcare ÄKTA Pure machine was used to purify the desired Strep-tagged enzyme.

Enzyme assays

The assay for the glyoxylate reductase AtGLYR1 is based on the different absorption maxima of NADPH (absorption maximum at 340 nm) and NADP⁺ (maximum at 260 nm). During the reaction, the enzyme uses NADPH as a cofactor. NADPH is converted into NADP⁺, which leads to a decrease of absorption at 340 nm. By measuring the absorption at 340 nm over time, it is possible to quantify the enzyme activity and infer glycolic acid production. To calculate the NADPH conversion rate, a calibration curve was created by measuring the absorption of different NADPH concentrations.

Figure 6: Reaction mechanism of NADPH-dependent conversion of glyoxylate into glycolic acid performed by AtGLYR1

HPLC analysis

To detect the produced monomer (glycolic acid), as well as its precursors, high-performance liquid chromatography (HPLC) was utilized. An organic acid separation column as stationary phase, and sulfuric acid as mobile phase was used. This allowed the separation of glycolic acid, isocitrate and glyoxylate. Signals were recorded by a refractive index detector.

Results and Discussion

Deletions

To increase the yield of glycolic acid in S. cerevisiae, the genes MLS1 (malate synthase) and IDP2 (isocitrate dehydrogenase) were deleted. MLS1 converts glyoxylate to malate. Therefore, the amount of substrate for GLYR1 is minimized. IDP2 converts isocitrate to α-ketoglutarate. Isocitrate is also used by ICL1 as a substrate to produce glyoxylate. Sine glyoxylate is the precursor of glycolic acid, as much isocitrate as possible should be converted to glyoxylate instead of α-ketoglutarate. To bypass these problems, we deleted the genes encoding MLS1 and ICL1. The deletions were performed by transformation of CEN.PK-1C cells with a deletion cassette containing homologous regions to the flanking areas of the genes of interest (for a more detailed explanation see Methods). To determine whether or not the deletion of MLS1 and IDP2 worked, a PCR with two sets of primers was carried out. The primer sets contained the same forward primer but different reverse primers. If the deletion was not successful, only the reverse primer within the gene of interest would bind. If the deletion was successful, the reverse primer would bind within deletion cassette. Therefore, two PCRs per colony were performed. To see if the gene of interest or the deletion cassette was part of the genome, the PCR product was analyzed via agarose gel electrophoresis (see Fig. 7).

Figure 7: Gel electrophoretic separation for control of deletions. KanMX= primers bind in the KanMX cassette, MLS1 = primers bind in the MLS1 sequence. Leu= primers bind in the Leucin cassette, IDP2 = primers bind in the IDP2 sequence. 2-log ladder from NEB was used.

Figure 7 shows 3 different colonies which were screened. The expected fragment sizes are 1650 bp for the KanMX cassette and 1012 bp for MLS1. Colony 1 shows a fragment for KanMX as well as MLS1. If the deletion of MLS1 had worked, only a fragment for KanMX should be visible. Therefore, colony 1 is a mixed culture. Colony 2 and 5 only show a fragment for KanMX but not for MLS1. This means that the deletion was performed successfully. IDP2 was deleted using a Leucin cassette instead of KanMX. The expected fragment sizes for the cassette and IDP2 are 1156 bp and 1498 bp. All three tested colonies show a clear fragment for Leucin. The deletion of IDP2 was therefore successful. Colony 1 was not used for further experimental procedures.

Cloning and Expression

To produce glycolic acid in S. cerevisiae, a glyoxylate reductase from Arabidopsis thaliana needs to be expressed. The native glyoxylate reductase GOR1 does not have an affinity to glyoxylate as high as GLYR1 from A. thaliana. A S. cerevisiae codon optimized version of the sequence coding for AtGLYR1 containing a Strep-tag was ordered from IDT. The sequence was inserted into a shuttle vector (pAT423) which can be used for gene expression in S. cerevisiae, as well as in in E. coli. The cloning was done using the Gibson Assembly method. The accuracy of the cloning was confirmed by sequencing.

To test whether AtGLYR1 is produced in S. cerevisiae, we performed a western blot. We tested three colonies which were grown on selective medium after transformation with pAT423xGLYR1. As a negative control, untransformed CEN.PK-1C cells, were used. The resulting western blot is shown in Figure 8. AtGLYR1 was designed to contain a Strep-tag. Therefore, an anti-Strep antibody was used to detect the tagged AtGLYR1. The expected protein size was 31.9 kDa.

Figure 8: Western blot for AtGLYR1. Three colonies were screened for the translation of AtGLYR1 in S. cerevisiae. Thermo Scientific PageRuler Prestained Protein Ladder was used as protein standart. 1-3 = CEN.PK-1C was transformed using pAT423xAtGLYR1; - = Negative control of untransformed CEN.PK-1C.

The western blot shows three colonies which were screened for the production of GLYR1. Colony 1 and 3 produced GLYR1. However, Colony 2 shows no band at the expected height. A corresponding positive colony was used for further experimental procedures. After gene expression for up to 22h, the protein was purified via StrepTactin columns using an ÄKTA system.

Enzyme assay

The purified enzyme was tested for its activity. The assay is based in the oxidization of NADPH. While the purified AtGLYR1 oxidizes NADPH glyoxylate is being reduced to glycolic acid. NADPH has its absorption maximum at 340 nm. Therefore, the conversion of NADPH can be recognized through a decrease in absorption at 340 nm. Glyoxylate is included as the substrate. Since the conversion of NADPH results in the production of glycolic acid, this assay can proof the ability of the enzyme to produce glycolic acid. Figure 9 shows the calibration curve for the enzyme assay. For the calibration curve the absorption of different concentrations of NADPH was measured. The curve can be used to calculate the substrate turnover rate. Therefore, the results of the assay (Figure 10) must be compared to the calibration curve.

Figure 9: Calibration curve for AtGLYR1 enzyme assay. Different NADPH concentrations (1mM, 0.5 mM, 0.25 mM, 0.125 mM and 0 mM) were measured and plotted against the average absorption. Figure 10: NADPH dependent enzyme assay of AtGLYR1. The graph shows the average absorption at 340 nm in correlation to the time (min). The graph shows the turnover rate of 2.7 ng/µL enzyme in the total reaction mix, positive (NADP⁺) and negative control (NADPH) were included.

Figure 10 clearly shows that AtGLYR1 converts NADPH. The average absorption, which reflects the NADPH concentration, decreases. As the positive and negative controls stay constant, it can be assumed that the decrease of the average absorption in the enzyme containing sample is due to the functionality and activity of AtGLYR1. After around 3.5 minutes the average absorption is at 0.5 and thus half of the NADPH has been used to convert glyoxylate to glycolic acid. Furthermore, the curve almost reaches the positive control. This shows that presumably all of the NADPH has been used and highlights the activity of the purified enzyme. The high activity is also recognizable by the start of the average absorption at 0.9. The concentration of NADPH at the start of the assay was the same as for the negative control. Even though the enzyme is added at last, the enzyme starts to work before the measurement starts. Therefore, quite a bit of NADPH has already been converted before. Next, a Student’s was performed to see whether or not these results are significant. For this purpose five data sets (0, 5, 10, 20 and 30 minutes) were chosen. This is shown in figure 11.

Figure 11: Enzyme assay absorption for 0, 5, 10, 20 and 30 minutes with standard error. n=3.

Table 1 includes the values for figure 11. It also shows the NADPH-absorption at chosen times as well as the associated standard error.

Table 1: Calculated significance of AtGLYR1 data sets for 0, 5, 10, 20 and 30 minutes at 30 °C. The table also shows the NADPH-absorption as well as the particular standard error. A= Absorption, *= Significance (p-value < 0.05), n= 3.

Both figure 11 and table 1 show the significance of the enzymes activity. The calculated substrate conversion rate of AtGLYR1 at 30 °C is 3,9 µM/s.

Outlook

Due to metabolic engineering of the glyoxylate cycle in S. cerevisiae, we were able to produce glycolic acid as one of our monomers for PLGA- and PGLC-synthesis. It has been shown that yeast acts as a suitable organism for genetic modifications and production of small organic acids. Yet, an emerging number of different yeast species have established themselves as microbial cell factories with at present unknown potential to further increase the yield of metabolic products. Recent work with Kluveromyces lactis ^[1] suggests that a higher titer of glycolic acid can be received by implementation of similar biosynthetic approaches on different organisms. Obtaining a quantitative analysis of the so produced monomers would therefore be of particular relevance for the comparison of the chosen production strains. In this regard, the currently applied method ^[6] including high-performance liquid chromatography with use of a refractive index detector for signal recording can be complemented with external standards in order to create a quantifying calibration curve.

Furthermore, the construction of a BioBrick-compliant shuttle vector for overexpression, protein purification with Strep-tag affinity chromatography and subsequent activity assays, similar to those performed on AceA in E. coli, is the next step in the process of glycolic acid production. For the construction of a stable GLYR1-encoding strain, the glyoxylate reductase gene could be integrated into the genome via homologous recombination, as transformed populations can suffer from plasmid instability ^[7]. Since the reaction catalyzed by GLYR1 is depending on NADPH, the effect of cofactor limitation and regenerating systems, like we used for the production of ε-caprolactone, could also be investigated. Coordinating the two-gene expression ratio of heterologous GLYR1 and overexpressed ICL1 is one potential future outlook.

1. ↑ ^{1.0 1.1 1.2 1.3 1.4} Koivistoinen, O. M., Kuivanen, J., Barth, D., Turkia, H., Pitkänen, J.-P., Penttilä, M., & Richard, P. (2013). Glycolic acid production in the engineered yeasts Saccharomyces cerevisiae and Kluyveromyces lactis. Microbial Cell Factories, 12, 82. [1].
2. ↑ Rintala, E., Pitkänen, J., Vehkomäki, M., Penttilä, M., & Ruohonen, L. (2007). The ORF YNL274c (GOR1) codes for glyoxylate reductase in Saccharomyces cerevisiae. Yeast, 24, 129-136.[2].
3. ↑ Hoover, G. J., Van Cauwenberghe, O. R., Breitkreuz, K. E., Clark, S. M., Merrill, A. R., & Shelp, B. J. (2007). Characteristics of an Arabidopsis glyoxylate reductase: general biochemical properties and substrate specificity for the recombinant protein, and developmental expression and implications for glyoxylate and succinic semialdehyde metabolism in planta. Canadian Journal of Botany, 85, 883-895. [3].
4. ↑ UniProtKB - Q9LSV0 (GLYR1_ARATH) [4].
5. ↑ ICL1 / YER065C Overview [5].
6. ↑ Zahoor, A., Otten, A., & Wendisch, V. F. (2014). Metabolic engineering of Corynebacterium glutamicum for glycolate production. Journal of Biotechnology, 192, 366-375 [6].
7. ↑ Zhang, Z., Moo-Young, M., & Christi, Y. (1996). Plasmid stability in recombinant Saccharomyces cerevisiae. Biotechnology Advances, 14(4), 401-435. [7].