Part:BBa_K4324000

lacPO-RBS-XR-T1

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).

Figure 1: Protein structure of xylose reductase from AlphaFold

Sequence and Features

Assembly Compatibility:

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  Illegal SapI.rc site found at 827
  

Usage and Biology

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:

D-xylose + NAD(P)H + H⁺ ⇌ xylitol + NAD(P)⁺

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.

Improvement of an Existing Part (BBa_K1602004)

This part improves upon BBa_K1602004, which expresses aldose reductase from the GRE3 gene from S. cerevisiae.

Figure 4: Aldose reductase (GRE3) Michaelis constants, from Purification and partial characterization of an aldo-keto reductase from Saccharomyces cerevisiae by Kuhn A., van Zyl C., van Tonder A., Prior B.A.

Inspecting the enzyme kinetics of aldose reductase (GRE3), the K_m value of D-xylose is 27.90mM whilst for D-glucose, it is only 9.34mM. This reveals that aldose reductase has a higher affinity for glucose than it does for xylose. However, as our project sought to increase the uptake of xylose for E. coli, it was necessary that the enzyme have a stronger affinity towards xylose.

By instead utilising xylose reductase (XYL1) from S. stipitis, which has a higher affinity for xylose (K_m = 42mM) than glucose (K_m = 420mM) as shown in the Usage and Biology section, its ability to increase the efficiency of the xylose metabolism pathway (XR-XDH pathway) has been improved.

Furthermore, this part is from the GRE3 gene and can only be expressed in S. cerevisiae. Our improved part took the XYL1 gene from S. stipitis and codon-optimised its sequence for expression in E. coli. Furthermore, we added an appropriate lac promoter, RBS and T1 terminator to enable its expression in E. coli through IPTG induction in this composite part.

References

1. https://www.uniprot.org/uniprotkb/P31867/entry
2. https://biotechnologyforbiofuels.biomedcentral.com/articles/10.1186/s13068-020-1662-x
3. https://pubmed.ncbi.nlm.nih.gov/3921014/
4. https://journals.asm.org/doi/epdf/10.1128/aem.61.4.1580-1585.1995
5. https://www.cmbe.engr.uga.edu/assays/xylosereductase.pdf