Part:BBa_K1602004

Aldose reductase - GRE3

GRE3 is a gene, coding for an aldose reductase. The reductase catalyzes the conversion from xylose to xylitol in dependance of NADPH.
	Figure 1 aldose reductase (coded by GRE3)

Characteristics

Molecular Weight	37118.78
Residues	327
Charge	3.5
Isoelectric Point	7.0925
A280 Molar Extinction Coefficients	45380 (reduced) 45755 (cystine bridges)
Improbability of expression in inclusion bodies	0.605
[Data taken from PEPSTATS]

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology: TheKingsSchool_AU_HS

Aldose reductase is also notated as NAD(P)H-dependent D-xylose reductase, or xylose reductase (XR).

Figure 1: 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) 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. cerevisiae and S. stipitis yeast cells, xylose reductase forms the first process in the XR-XDH pathway, as shown in Figure 1, which converts xylose into xylulose via xylitol. Xylulose is then converted into xylulose-5-phosphate (X5P) for further metabolism in the pentose phosphate pathway. This XR reaction is reversible.

Figure 2: 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. 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. This presents opportunities for expressing this enzyme in microorganisms that struggle to metabolise xylose in the presence of glucose (e.g. E. coli) for improved xylose uptake.

In such case, as the reaction is reversible from xylitol to D-xylose, expressing 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.

Improvements: TheKingsSchool_AU_HS

The improved part for the coding sequence is BBa_K4324100, and a functional composite part with a promoter, RBS and terminator is at BBa_K4324000.

Figure 3: 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 Km 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 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.