Enhancement of Xylitol Production from Hemicellulose biomass hydrolyzate in Candida tropicalis

Abstract

Xylitol is a five-carbon sugar alcohol that has same order of sweetness level but calorie is lower compared to sucrose. It is used as an alternative natural sweetener in the food and confectionary industry. As it does not require insulin for metabolic regulation, it can be used as an insulin-independent sugar substitute for diabetics. In Candida tropicalis, xylitol is produced by an enzymatic reaction in which xylose reductase (XR) catalyzes the reduction of D-xylose to xylitol with accompanying NADPH oxidation. The biomass used for xylitol production is hydrolyzed by a mineral acid to produce a hydrolysate that consists primarily of xylose, arabinose and glucose. The glucose in the hydrolysate can be used directly as a co-substrate. This approach has economic and convenience advantages However, to use biomass hydrolyzate directly, several obstacles should be overcome. First of all, suppression of XR activity and xylose uptake by glucose should be overcome. To solve above problems, XR from Neurospora crassa and xylose transporter(XT1164) from C.tropicalis were cloned and over-expressed in C. tropicalis. Resulting strain, XT1164 showed shorten delayed initial xylitol production caused by glucose repression. And both xylitol volumetric productivity and specific productivity of XTSTP2 were 67% and 69% higher than LXU, the control strain. Another critical problem to be overcome is the reduction of arabitol formation as a byproduct in the biological xylitol production which reduces the xylitol crystallization in downstream processing. Arabitol, an epimer of xylitol, is difficult to remove during xylitol production and interferes with xylitol crystallization in the downstream processing used for purification. To solved the above problem bacterial arabinose assimilation pathway(araBAD) and arabinose specific transporter(STP2) from Arabidopsis thaliana were induced C. tropicalis strain for efficient uptake of L-arabinose. Resultinf strain JYSTP2 completely consumed, arabitol was not formed. Finally, PFK and ICL were knock out for recycling process which occur between PPP and EMP pathway and TCA cycle for efficient regeneration of NADPH. PFK, ICL double mutant(JYBI) showed 1.7 fold higher intracellular NADPH concentration and 1.5 fold enhanced xylitol volumetric productivity compared to control strain (LXU).