Achieving arabitol-free xylitol production through modified xylose reductase in a Candida tropicalis isolate

To maximise the environmental, economic sustainability of the second generation lignocellulosic sugar platform and drive its widespread commercial uptake, it is crucial to devise holistic approaches for generating value added products from the hemicellulose sugar fraction, particularly xylose. With an estimated market of 537 M US$ per annum, growing at an annual rate of 15%, the artificial sweetener xylitol is an attractive candidate to derive value from hemicellulosic xylose. Despite considerable research aimed at exploring the bioconversion of xylose to xylitol using a number of different xylose-utilising microorganisms, including the fungi Candida tropicalis, commercial production of the sugar alcohol is largely achieved via chemical routes. One major drawback of xylitol synthesis through fungal fermentation is the concomitant production of arabitol, an impurity that significantly confounds and adds to downstream processing costs. Arabitol is predominantly generated due to the promiscuous nature of xylose reductase (XR), the enzyme which can non-specifically bind and convert both xylose and its epimer arabinose to the corresponding sugar alcohol. This work aims to present efforts directed at rendering XR derived from a C. tropicalis isolate (Y4) more xylose specific via in silico modelling and site directed mutagenesis followed by evaluating enzyme kinetics. Apart from enhancing XR’s substrate specificity, any impact of altering the enzyme's cofactor specificity (from NADPH to NADH) on arabitol production will be discussed. The impact of enzyme modifications in wild type Y4 and that adapted to dilute acid hydrolysates (generated from willow, wheat straw, corn stover and miscanthus) would be demonstrated by assessing the microorganism’s fermentation potential.