Production of bio-xylitol from D-xylose by an engineered Pichia pastoris expressing a recombinant xylose reductase did not require any auxiliary substrate as electron donor

Abstract

Background: Xylitol is a five-carbon sugar alcohol that has numerous beneficial health properties. It has almost the same sweetness as sucrose but has lower energy value compared to the sucrose. Metabolism of xylitol is insulin independent and thus it is an ideal sweetener for diabetics. It is widely used in food products, oral and personal care, and animal nutrition as well. Here we present a two-stage strategy to produce bio-xylitol from D-xylose using a recombinant Pichia pastoris expressing a heterologous xylose reductase gene. The recombinant P. pastoris cells were first generated by a low-cost, standard procedure. The cells were then used as a catalyst to make the bio-xylitol from D-xylose.

Results: Pichia pastoris expressing XYL1 from P. stipitis and gdh from B. subtilis demonstrated that the biotransformation was very efficient with as high as 80% (w/w) conversion within two hours. The whole cells could be re-used for multiple rounds of catalysis without loss of activity. Also, the cells could directly transform D-xylose in a non-detoxified hemicelluloses hydrolysate to xylitol at 70% (w/w) yield.

Conclusions: We demonstrated here that the recombinant P. pastoris expressing xylose reductase could transform D-xylose, either in pure form or in crude hemicelluloses hydrolysate, to bio-xylitol very efficiently. This biocatalytic reaction happened without the external addition of any NAD(P)H, NAD(P)⁺, and auxiliary substrate as an electron donor. Our experimental design & findings reported here are not limited to the conversion of D-xylose to xylitol only but can be used with other many oxidoreductase reactions also, such as ketone reductases/alcohol dehydrogenases and amino acid dehydrogenases, which are widely used for the synthesis of high-value chemicals and pharmaceutical intermediates.

Keywords: And xylitol; Glucose dehydrogenase; Pichia; Whole cells; Xylose; Xylose reductase.

Fig. 1

SDS-PAGE gels of cell extracts prepared from various recombinant P. pastoris (a) expressing XR alone or (b) co-expressing XR with GDH. (a) Lanes 1 to 5: pPIC4Kx-PsXYL1 clones 1000–1, 1000–2, 4000–1, 4000–2, and 4000–3, respectively. Lanes 6–10: pPIC4Kx-NcXR clones 1000–1, 1000–3, 4000–2, 4000–3, and 4000–6, respectively. Lanes 11–15: pPIC4Kx-CpXr clones 4000–8, 4000–6, 4000–2, 1000–6, and 1000–2, respectively. (b) Lanes 1 to 5: pPIC4Kx + PsXLY1 + gdh clones 1000–7, 1000–8, 4000–4, 4000–7, and 4000–8, respectively. Lanes 6 to 10: pPIC4Kx + NcXR + gdh clones 1000–1, 1000–2, 4000–1, 4000–2, and 4000–3, respectively. Lanes 11 to 15: pPIC4Kx + CpXR + gdh clones 1000–5, 1000–6, 4000–4, 4000–5, and 4000–6, respectively. Lane C contained cell extracts of P. pastoris GS115 transformed with an empty pPIC4Kx. The red arrows indicated the over-expressed XR proteins

Fig. 2

Xylitol production by (a) NcXR + GDH 4000–1 and (b) PsXYL1 + GDH 4000–4 cells. All reactions had 10 mg/mL of cells. The weight of the cells was a dry cell weight. The cells were incubated at 30 °C with 200 mM d-xylose and 0.25 mM NAD⁺ in 50 mM KPi (pH 7.0) buffer. The reactions also had 100 mM formate (red circle), 100 mM glucose (black square) or no auxiliary substrate as electron donor (blue triangle). Solid lines represent d-xylose consumption; dashed lines represent xylitol production. The data shown in the figure is the average of 3 values. All the values are within the 5% SD

Fig. 3

Xylitol production by (a, c) PsXYL1 + GDH 4000–4 and (b) NcXR + GDH 4000–1 cells in multiple cylces of reactions. Symbols: (black diamond) 1st cycle, (open square) 2nd cycle, (red triange) 3rd cycle, (blue cross) 4th cycle, (pink square) 5th cycle, (green circle) 6th cycle. (a) 10 mg/mL of PsXYL1 + GDH 4000–4 cells in 50 mM KPi (pH 7.0) buffer were incubated at 30 °C with 200 mM d-xylose and 0.25 mM NAD⁺. At the end of each reaction cycle, the cells were collected by centrifugation and supernatants were removed. Fresh reaction mixture was added to the cells to start another cycle of reaction. (b) 10 mg/mL of NcXR + GDH 4000–1 cells in 50 mM KPi (pH 7.0) buffer were incubated at 30 °C with 200 mM d-xylose and 0.25 mM NAD⁺. Recycling was carried out as described above. During the 3rd cycle, 100 mM of formate and glucose were added as indicated. (c) 10 mg/mL of PsXYL1 + GDH 4000–4 cells in 50 mM KPi (pH 7.0) buffer were incubated at 30 °C with 200 mM d-xylose but without NAD⁺. NAD⁺ (0.25 mM) was added at the indicated time points (red and blue arrows) in the 3rd and 4th cycles. The data shown in the figure is the average of 3 values. All the values are within the 5% SD

Fig. 4

Xylitol production by PsXYL1 + GDH 4000–4 from different d-xylose concentrations. 10 mg/mL of PsXYL1 + GDH 4000–4 cells in 50 mM KPi (pH 7.0) buffer were incubated at 30 °C with 400 mM (black circle), 750 mM (red square), and 1500 mM (blue triangle) of d-xylose. NAD⁺ and auxiliary substrate were not added to the reactions. Solid lines represent xylitol production; dashed lines represent d-xylose consumption. The data shown in the figure is the average of 3 values. All the values are within the 5% SD

Fig. 5

Xylitol production by PsXYL1 + GDH 4000–4 from a hemicelluloses hydrolysate. The PsXYL1 + GDH 4000–4 cells used in this experiment was originated from a 10-L fed-batch fermentation. One L of hemicelluloses hydrolysate (pH 7.0) was incubated with 100 mg/mL PsXYL1 + GDH 4000–4 cells at 30 °C. Solid lines represent xylitol production; dashed lines represent d-xylose consumption. The data shown in the figure is the average of 3 values. All the values are within the 5% SD