Date Published: June 7, 2016
Publisher: Springer Berlin Heidelberg
Author(s): Anushree Kogje, Anand Ghosalkar.
Xylitol production was compared in fed batch fermentation by Saccharomyces cerevisiae strains overexpressing xylose reductase (XR) genes from Candida tropicalis, Pichia stipitis, Neurospora crassa, and an endogenous gene GRE3. The gene encoding a xylose specific transporter (SUT1) from P. stipitis was cloned to improve xylose transport and fed batch fermentation was used with glucose as a cosubstrate to regenerate NADPH. Xylitol yield was near theoretical for all the strains in fed batch fermentation. The highest volumetric (0.28 gL−1 h−1) and specific (34 mgg−1 h−1) xylitol productivities were obtained by the strain overexpressing GRE3 gene, while the control strain showed 7.2 mgg−1 h−1 specific productivity. The recombinant strains carrying XR from C. tropicalis, P. stipitis, and N. crassa produced xylitol with lower specific productivity of 14.3, 6.8, and 6.3 mgg−1 h−1, respectively, than GRE3 overexpressing strain. The glucose fed as cosubstrate was converted to biomass and ethanol, while xylose was only converted to xylitol. The efficiency of ethanol production was in the range of 38–45 % of the theoretical maximum for all the strains. Xylitol production from the non-detoxified corncob hemicellulosic hydrolysate by recombinant S. cerevisiae was reported for the first time. Xylitol productivity was found to be equivalent in the synthetic xylose as well as hemicellulosic hydrolysate-based media showing no inhibition on the S. cerevisiae due to the inhibitors present in the hydrolysate. A systematic evaluation of heterologous XRs and endogenous GRE3 genes was performed, and the strain overexpressing the endogenous GRE3 gene showed the best xylitol productivity.
Xylitol, the sugar alcohol is a promising polyol due to its application as a low calorie sweetener. It can be used for its antimicrobial properties to prevent dental and other infections (Nevoigt 2008). Xylitol is conventionally produced by hydrolysis and hydrogenation of xylan to xylitol under high temperature and pressure conditions. In spite of very high conversion efficiency, this technology has certain constrains viz. requirements of high pressure, temperature, expensive catalyst, and extensive downstream operations (Leathers 2003). Microorganisms convert xylose to xylitol by the cofactor-dependent xylose reductase (XR or Xyl1) enzyme (Saha 2003; Cocotle-Ronzon et al. 2012). The majority of essential cofactor NADPH/NADH for the XR activity is regenerated through pentose phosphate pathway (PPP) (Bengtsson et al. 2009; Schwartz et al. 2012). Several microorganisms have been screened and studied with respect to their ability for assimilation of xylose as carbon source and conversion to xylitol. Extensive research has been conducted to isolate and identify the suitable yeasts for xylitol production (Barbosa et al. 1988; Parajo et al. 1998a). Xylitol producing yeasts, such as P. stipitis, C. tropicalis, C. guilliermondii, D. hansenii (Mohamad et al. 2015), and K. marxianus (Zhang et al. 2013), have been studied for the optimization of the fermentation parameters, utilization of various nutrients, and fermentation of xylose rich hemicellulosic hydrolysate obtained from different sources of pretreated biomass (Parajo et al. 1998a, b; Dominguez et al. 1996; Ping et al. 2013). These organisms were preferred, as they exhibited efficient conversion of xylose to xylitol. Several reports on Candida species involved studies on the fermentation of pretreated biomass hydrolysates. Ghindea et al. (2010) have provided a review on various microorganisms studied for xylose transport and xylitol production.
This is the first report on xylitol production using non-detoxified hemicellulosic hydrolysate of corn cob by recombinant S. cerevisiae. As a GRAS organism, S. cerevisiae is a preferable biocatalyst with high tolerance to inhibitory compounds present in hemicellulosic hydrolysates. It converts xylose to xylitol with near theoretical yield, whereas Candida sp. also utilizes it for cell growth. In the present study, a systematic evaluation of different reductase genes was done in recombinant S. cerevisiae containing heterologous xylose transporter. The strain overexpressing the endogenous GRE3 gene along with the xylose transporter gene SUT1 showed the highest xylitol productivity over control as compared to the strains carrying C. tropicalis, P. stipitis, and N. crassa xylose reductases. The fed batch process involved feeding of cosubstrate glucose, which could be available in the bio-refineries based on cellulosic substrates. Ethanol produced from the glucose fed during process can be recovered from the fermentation broth to improve the overall process economics. It can also help in the prevention of contamination during fermentation in the hydrolysate-based media. Fermentation was conducted under microaerobic conditions which was lesser than the aeration required for xylitol production by other naturally xylose fermenting yeast species, such as Candida.