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

Tai Man Louie,Kailin Louie,Mani Subramanian,Samuel Denhartog,Shuvendu Das,Mark Arnold,Sridhar Gopishetty

doi:10.1186/s12934-021-01534-1

Abstract

BackgroundXylitol 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.ResultsPichia 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.ConclusionsWe 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.

Highlights

Xylitol is a five-carbon sugar alcohol that has numerous beneficial health properties
A Xylose reductase (XR) gene was previously identified in C. parapsilosis KFCC-18075 and the gene product was characterized as an NADHpreferring XR [22]
We failed to amplify any PCR product after numerous attempts. Using another pair of PCR primers designed from an uncharacterized XR gene identified within the genome of C. parapsilosis isolate 317 [24], a XR gene was amplified from ATCC 22019 and was successfully expressed in P. pastoris

Summary

Introduction

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 it is an ideal sweetener for diabetics It is widely used in food products, oral and personal care, and animal nutrition as well. Xylitol is a five-carbon sugar alcohol that has numerous beneficial health properties [1] It has almost the same sweetness as sucrose, but with an energy value of 3 cal/g compared to the 4 cal/g of sucrose [2]. Cirino et al [11] engineered an E. coli that produced 250 mM xylitol from d-xylose with glucose as a co-substrate. Detoxification of the hydrolysates prior to being used as fermentation substrates is necessary, which will increase xylitol production cost and time [19]

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Conclusion