Difference between revisions of "Part:BBa K4324300"

Latest revision as of 03:18, 14 October 2022

XR-XDH-XK-PK for E. coli

This part is a collection of all composite parts for xylose reductase (BBa_K4324000), xylitol dehydrogenase (BBa_K4324001), xylulose kinase (BBa_K4324002), and phosphoketolase (BBa_K4324003). This part is a collection of all composite parts within TheKingsSchool_AU_HS's project.

This part enables E. coli to express the XR-XDH pathway, further express XK, and express phosphoketolase (links xylulose-5-phosphate to glycolysis).

Sequence and Features

Xylose Reductase (BBa_K4324000)

This part is the composite part of the XYL1 gene from S. stipitis that induces xylose reductase, and has been codon-optimised for expression in E. coli. It has a lac promoter (BBa_K4324201), RBS (BBa_K4324200), and T1 terminator from E. coli's rrnB gene (BBa_B0010).

Our project focused on the improvement of xylose utilisation in E. coli, such that it is able to grow more efficiently on organic bio-waste matter. One part of this process was to incorporate a yeast-derived XR-XDH pathway.

A significant portion of organic biomass contains plant dry matter, or lignocellulose, which is comprised of three substances: cellulose, hemicellulose, and lignin.

Figure 2: Composition of various lignocellulosic biomass, from Production of Bioethanol from Waste Newspaper by Byadgi et al.

Cellulose ([1] KEGG C00760) is a chain of many β-1,4-linked glucose units with a chemical formula of (C₆H₁₀O₅)_n, usually found in plant cell walls. Lignin is comprised of various oxygenated phenylpropane units, usually found between cell walls, such as plant tissues. Hemicellulose is primarily comprised of D-xylose, which is the second most abundant sugar in lignocellulosic biomass, after glucose.

D-xylose is a aldopentose sugar with a chemical formula of C₅H₁₀O₅. It can serve as a sole carbon source for E. coli with its XI pathway and pentose phosphate pathway that is explained further below. E. coli has two transporter systems for xylose - XylE and XylFGH - both of which are inhibited by catabolite repression which is in favour of glucose.

Figure 2: Xylose metabolism pathways of various microorganisms, from Biochemical routes for uptake and conversion of xylose by microorganisms by Zhao, Z., Xian, M., Liu, M. et al.

Xylose reductase (EC 1.1.1.307), an aldose reductase, is an enzyme that serves as a catalyst for the conversion of xylose into xylitol, and vice versa, according to the following chemical equation:

In S. stipitis yeast cells, xylose reductase forms the first process in the XR-XDH pathway, as shown in Figure 2, which converts xylose into xylulose via xylitol. Xylulose is then converted into xylulose-5-phosphate (X5P) for further metabolism in the pentose phosphate pathway.

Figure 3: Xylulose-5-phosphate within the pentose phosphate pathway, from Fermentation of Glucose and Xylose to Hydrogen in the Presence of Long Chain Fatty Acids by Stephen Reaume

E. coli do not exhibit the XR-XDH pathway, instead having an XI pathway that directly isomerises xylose into xylulose using the xylose isomerase enzyme. Hence, together with xylitol dehydrogenase (BBa_K4324101) which can convert xylitol to xylulose, xylose reductase presents an alternate xylose metabolism pathway for E. coli.

Furthermore, as the reaction from xylose to xylitol is reversible, xylose reductase enables E. coli to utilise xylitol as an energy source through its conversion to xylose, which then follows the XI pathway.

Figure 3: Xylose reductase kinetic parameters, from Properties of the NAD(P)H-dependent xylose reductase from the xylose-fermenting yeast Pichia stipitis by C Verduyn, R Van Kleef, J Frank, H Schreuder, J P Van Dijken, W A Scheffers

Analysing the kinetic parameters of xylose reductase, we see that xylose reductase has a K_m value of 42mM for D-xylose, and 420mM for D-glucose. This demonstrates that xylose reductase in S. stipitis has a much higher affinity for xylose than glucose, and hence we chose it for expression in E. coli with the expectation that it will efficiently catabolise xylose despite E. coli's carbon catabolite repression (CCR) and diauxic growth. Furthermore, we can see higher V_max values on both NADH (16.7 vs 11.8) and NADPH (23.2 vs 17.5) as a coenzyme.

The reaction is reversible, from xylitol to D-xylose, and on expression within E.coli, would allow utilization of xylitol as the sole carbon source. This will occur first by this reverse reaction to xylose, then by direct isomerisation through xylose isomerase (XI pathway) which exists natively within E. coli. However, it must be noted that the reverse reaction incurs a reaction rate which is 4-5% that of the forward reaction, and so it is hardly useful.

Characterisation

To functionally characterise XR, we suggest demonstrating improved growth on defined media with xylose as a carbon source. Alternatively, successfully screened cell extracts of XR can undergo in vitro xylose reduction assay to confirm catalytic activity, using NADPH as a cofactor. To do this:

Cell extract should be prepared by centrifuging enough cells to produce an optical density of 20 to 30 when diluted to 5mL. Then, pelletise and resuspend twice in a potassium phosphate buffer. Using a spectrophotometer at 340nm, cuvettes of control and experimental cocktails should be placed inside, then the cell extract added simultaneously and mixed. The spectrophotometer will record the rates of change in absorbance in the cocktail (should increase). From this, the xylose reductase activity can be calculated as the amount of enzyme needed to produce 1.0 micromole of xylitol in a minute.

Then the enzymatic activity of xylose reductase can be calculated through the below equation:

Calculation for enzymatic activity, from S.-I. Yokoyama, T. Suzuki, K. Kawai, H. Horitsu, K. Takamizawa (1995) Purification, Characterization and Structure Analysis of NADPH-Dependent D-Xylose Reductase from Candida tropicalis. J. Ferm. Bioeng. 79 (3) 217-223.
N.B.: CF is the dilution factor of the cell extract.

The specific activity of xylose reductase on xylose can be further calculated using the concentration of xylose.

Calculation for specific activity, from S.-I. Yokoyama, T. Suzuki, K. Kawai, H. Horitsu, K. Takamizawa (1995) Purification, Characterization and Structure Analysis of NADPH-Dependent D-Xylose Reductase from Candida tropicalis. J. Ferm. Bioeng. 79 (3) 217-223.

Xylitol Dehydrogenase (BBa_K4324001)

This part is the composite part of the XYL2 gene from S. cerevisiae that induces xylitol dehydrogenase, and has been codon-optimised for expression in E. coli. It has a lac promoter (BBa_K4324201), RBS (BBa_K4324200), and T1 terminator from E. coli's rrnB gene (BBa_B0010).

Our project focused on the improvement of xylose utilisation in E. coli. One part of this process was to incorporate a yeast-derived XR-XDH pathway

Figure 2: Xylose metabolism pathways of various microorganisms, from Biochemical routes for uptake and conversion of xylose by microorganisms by Zhao, Z., Xian, M., Liu, M. et al.

Xylitol dehydrogenase (EC 1.1.1.9), an oxidoreductase, is an enzyme that serves as a catalyst for the conversion of xylitol into xylulose, and vice versa, according to the following chemical equation:

In S. cerevisiae yeast cells, xylitol dehydrogenase forms the second process in the XR-XDH pathway, as shown in Figure 2, which converts xylose into xylulose via xylitol. Xylulose is then converted into xylulose-5-phosphate (X5P) for further metabolism in the pentose phosphate pathway.

E. coli do not exhibit the XR-XDH pathway, instead having an XI pathway that directly converts xylose into xylulose. Hence, together with xylose reductase (BBa_K4324100) which can convert xylose to xylitol, xylitol dehydrogenase presents an alternate xylose metabolism pathway for E. coli.

Furthermore, xylitol dehydrogenase enables E. coli to utilise xylitol as an energy source through its direct conversion to xylulose, which then follows the pentose phosphate pathway.

Xylulose Kinase (BBa_K4324002)

This part is the composite part of the xylB gene from E. coli that induces xylulose kinase, and has been codon-optimised for expression in E. coli. It has a lac promoter (BBa_K4324201), RBS (BBa_K4324200), and T1 terminator from E. coli's rrnB gene (BBa_B0010).

Our project focused on the improvement of xylose utilisation in E. coli, such that it is able to grow more efficiently on organic bio-waste matter. One part of this process was to induce an over-expression of xylulose kinase in E. coli.

A significant portion of organic biomass contains plant dry matter, or lignocellulose, which is comprised of three substances: cellulose, hemicellulose, and lignin.

Figure 2: Composition of various lignocellulosic biomass, from Production of Bioethanol from Waste Newspaper by Byadgi et al.

Cellulose ([2] KEGG C00760) is a chain of many β-1,4-linked glucose units with a chemical formula of (C₆H₁₀O₅)_n, usually found in plant cell walls. Lignin is comprised of various oxygenated phenylpropane units, usually found between cell walls, such as plant tissues. Hemicellulose is primarily comprised of D-xylose, which is the second most abundant sugar in lignocellulosic biomass, after glucose.

In E. coli, D-xylose is directly isomerised by xylose isomerase into D-xylulose.

D-xylulose is a sugar with a chemical formula of C₅H₁₀O₅. E. coli has two transporter systems for xylose - XylE and XylFGH - both of which are inhibited by catabolite repression which is in favour of glucose.

Figure 2: Xylose metabolism pathways of various microorganisms, from Biochemical routes for uptake and conversion of xylose by microorganisms by Zhao, Z., Xian, M., Liu, M. et al.

Xylulose kinase (EC 2.7.1.17) is an enzyme that serves as a catalyst for the phosphorylation of xylulose into xylulose-5-phosphate, according to the following chemical equation:

E. coli natively expresses xylulose kinase through its xylB gene. In both yeast and E. coli cells, xylulose kinase forms a process that converts xylulose into X5P, for it to then be processed through the pentose phosphate pathway, as shown in Figure 3. Xylulose kinase also serves as a catalyst for the phosphorylation of 1-deoxy-D-xylulose to 1-deoxy-D-xylulose 5-phosphate, albeit with a lower efficiency (Wungsintaweekul et al.).

Figure 3: Xylulose-5-phosphate within the pentose phosphate pathway, from Fermentation of Glucose and Xylose to Hydrogen in the Presence of Long Chain Fatty Acids by Stephen Reaume

Figure 4: Xylulose kinase kinetic parameters, from Structural and kinetic studies of induced fit in xylulose kinase from Escherichia coli by Di Luccio E, Petschacher B, Voegtli J, Chou HT, Stahlberg H, Nidetzky B, Wilson DK.

Xylulose kinase can also utilise D-ribulose, xylitol and D-arabitol as substrates. However, analysing the kinetic parameters of xylulose kinase in Figure 4, we see that it has a K_m value of 0.29mM for D-xylulose, whilst the K_m values for the other substrates are comparatively high, from 14mM (D-ribulose) to 127mM (xylitol) and 141 (D-arabitol). This demonstrates that xylulose kinase in E. coli has a significantly higher affinity for xylulose of any other substrates. This is further confirmed through comparing the k_cat values of each substrate, with D-xylulose inducing the highest turnover.

Optical Density Growth Curve

We measured the growth rate of E. coli on various types of media by measuring the optical density through a biophotometer.

Figure A: Growth rate of XK on M9 media with KAN (glucose, xylose, xylitol)

E. coli containing XK were grown in the M9 media with KAN antibiotics, containing different carbon sources (glucose, xylose and Xylitol) over a period of 26 hours, with and without IPTG induction. OD600 were taken every 3 hours.

Analysing the results, IPTG helped double the growth rate in glucose and significantly increase growth rate in xylose. They grew well in these two carbon sources, with glucose still the most preferable, due to E. coli's inherent characteristics. Interestingly, this engineered strain of E. coli grew well in xylitol, with the growth rate as fast as xylose. More tests and analysis needs to be done to understand these unusual characteristics.

Spot Growth

Plasmids for XDH, XK, and phosphoketolase were transformed into E. coli K12, which were grown on M9 media (with KAN) with their respective parts induced for a few days to check growth on glycerol as a sole carbon source.

Figure B: Spot growth on M9 media of glycerol (with KAN) of control, XR, XDH and phosphoketolase

On the M9 media with glycerol and KAN, we observed minimal growth of the controls, no growth of XDH and XK, but a large growth of XK. Through literature research, we discovered that xylulose kinase could actually phosphorylate glucose to some extent, which we believed was causing it to display prominent growth on the glycerol medium (Luccio et al., Structural and Kinetic Studies of Induced Fit in Xylulose Kinase from Escherichia coli, Journal of Molecular Biology, Volume 365, Issue 3, 2007, Pages 783-798, https://doi.org/10.1016/j.jmb.2006.10.068).

Phosphoketolase (BBa_K4324003)

This part is the composite part of the XFP gene from B. lactis that induces phosphoketolase, and has been codon-optimised for expression in E. coli. It has a lac promoter (BBa_K4324201), RBS (BBa_K4324200), and T1 terminator from E. coli's rrnB gene (BBa_B0010).

Our project focused on the improvement of xylose utilisation in E. coli, such that it is able to grow more efficiently on organic bio-waste matter. One part of this process was to incorporate phosphoketolase to induce a part of the PK pathway.

A significant portion of organic biomass contains plant dry matter, or lignocellulose, which is comprised of three substances: cellulose, hemicellulose, and lignin.

Figure 2: Composition of various lignocellulosic biomass, from Production of Bioethanol from Waste Newspaper by Byadgi et al.

Cellulose ([3] KEGG C00760) is a chain of many β-1,4-linked glucose units with a chemical formula of (C₆H₁₀O₅)_n, usually found in plant cell walls. Lignin is comprised of various oxygenated phenylpropane units, usually found between cell walls, such as plant tissues. Hemicellulose is primarily comprised of D-xylose, which is the second most abundant sugar in lignocellulosic biomass, after glucose.

D-xylulose-5-phosphate is a phosphorylated sugar with a chemical formula of C₅H₁₁O₈P. In xylose metabolism, it generally occurs as a result of the phosphorylation of xylulose by xylulose kinase.

Figure 2: Xylose metabolism pathways of various microorganisms, from Biochemical routes for uptake and conversion of xylose by microorganisms by Zhao, Z., Xian, M., Liu, M. et al.

Phosphoketolase (EC 4.1.2.9) is an enzyme that serves as a catalyst for the conversion of xylulose-5-phosphate to glyceraldehyde-3-phosphate, according to the following chemical equation:

In E. coli cells, xylulose-5-phosphate generally leads into the pentose phosphate pathway, as shown in Figure 3. Phosphoketolase allows X5P to also be broken down through glycolysis through its conversion to G3P. Thiamine diphosphate is a cofactor of phosphoketolase.

Figure 3: Xylulose-5-phosphate within the pentose phosphate pathway, from Fermentation of Glucose and Xylose to Hydrogen in the Presence of Long Chain Fatty Acids by Stephen Reaume

E. coli do not exhibit phosphoketolase natively, but we have implemented it into our project to alleviate the flux of X5P through another method of metabolism.

Phosphoketolase can also utilise fructose-6-phosphate as a substrate, and in fact, the K_m value for F6P is lower (10mM) than it is for X5P (45mM), meaning it has a higher affinity for F6P.

Optical Density Growth Curve

We measured the growth rate of E. coli on various types of media by measuring the optical density through a biophotometer.

Figure A: Growth rate of PK on M9 media with CAM (glucose, xylose, xylitol)

E. coli containing PK were grown in the M9 media with CAM antibiotics, containing different carbon sources (glucose, xylose and Xylitol) over a period of 26 hours, with and without IPTG induction. OD600 were taken every 3 hours.

Analysing the results, cells grew slightly better without IPTG induction. As expected, Glucose is the most preferred carbon source, with the cell growth rate more than 3 times faster than in xylose. Also as expected, PK cells could not grow in xylitol, as E. coli does not natively have a xylose metabolism pathway. Interestingly, the induction of PK reduced the growth rate slightly on xylose. As the main intention of adding phosphoketolase was to alleviate the flux of X5P, there may not have been enough flux generated by the XI pathway alone for there to be a benefit in adding phosphoketolase, and hence random deviations in growth produced the slightly differing results.