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Xylitol production by Saccharomyces cerevisiae overexpressing different xylose reductases using non-detoxified hemicellulosic hydrolysate of corncob.

ABSTRACT: 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.

SUBMITTER: Kogje A

PROVIDER: S-EPMC4909029 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Xylitol production by Saccharomyces cerevisiae overexpressing different xylose reductases using non-detoxified hemicellulosic hydrolysate of corncob.

Kogje Anushree A Ghosalkar Anand A

3 Biotech 20160607 2

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 ferment ...[more]

PMID: 28330197

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Project description:BackgroundIntegrated management of hemicellulosic fraction and its economical transformation to value-added products is the key driver towards sustainable lignocellulosic biorefineries. In this aspect, microbial cell factories are harnessed for the sustainable production of commercially viable biochemicals by valorising C5 and C6 sugars generated from agro-industrial waste. However, in the terrestrial ecosystem, microbial systems can efficiently consume glucose. On the contrary, pentose sugars are less preferred carbon source as most of the microbes lack metabolic pathway for their utilization. The effective utilization of both pentose and hexose sugars is key for economical biorefinery.ResultsBioprospecting the food waste and selective enrichment on xylose-rich medium led to screening and isolation of yeast which was phylogenetically identified as Pichia fermentans. The newly isolated xylose assimilating yeast was explored for xylitol production. The wild type strain robustly grew on xylose and produced xylitol with >?40% conversion yield. Chemical mutagenesis of isolated yeast with ethyl methanesulphonate (EMS) yielded seven mutants. The mutant obtained after 15 min EMS exposure, exhibited best xylose bioconversion efficiency. This mutant under shake flask conditions produced maximum xylitol titer and yield of 34.0 g/L and 0.68 g/g, respectively. However, under the same conditions, the control wild type strain accumulated 27.0 g/L xylitol with a conversion yield of 0.45 g/g. Improved performance of the mutant was attributed to 34.6% activity enhancement in xylose reductase with simultaneous reduction of xylitol dehydrogenase activity by 22.9%. Later, the culture medium was optimized using statistical design and validated at shake flask and bioreactor level. Bioreactor studies affirmed the competence of the mutant for xylitol accumulation. The xylitol titer and yield obtained with pure xylose were 98.9 g/L and 0.67 g/g, respectively. In comparison, xylitol produced using non-detoxified xylose rich pre-hydrolysate from sugarcane bagasse was 79.0 g/L with an overall yield of 0.54 g/g.ConclusionThis study demonstrates the potential of newly isolated P. fermentans in successfully valorising the hemicellulosic fraction for the sustainable xylitol production.

Project description:BackgroundLignocellulosic biomass is one of the most abundant materials for biochemicals production. However, efficient co-utilization of glucose and xylose from the lignocellulosic biomass is a challenge due to the glucose repression in microorganisms. Kluyveromyces marxianus is a thermotolerant and efficient xylose-utilizing yeast. To realize the glucose-xylose co-utilization, analyzing the glucose repression of xylose utilization in K. marxianus is necessary. In addition, a glucose-xylose co-utilization platform strain will facilitate the construction of lignocellulosic biomass-utilizing strains.ResultsThrough gene disruption, hexokinase 1 (KmHXK1) and sucrose non-fermenting 1 (KmSNF1) were proved to be involved in the glucose repression of xylose utilization while disruption of the downstream genes of cyclic AMP-protein kinase A (cAMP-PKA) signaling pathway or sucrose non-fermenting 3 (SNF3) glucose-sensing pathway did not alleviate the repression. Furthermore, disruption of the gene of multicopy inhibitor of GAL gene expression (KmMIG1) alleviated the glucose repression on some nonglucose sugars (galactose, sucrose, and raffinose) but still kept glucose repression of xylose utilization. Real-time PCR analysis of the xylose utilization related genes transcription confirmed these results, and besides, revealed that xylitol dehydrogenase gene (KmXYL2) was the critical gene for xylose utilization and stringently regulated by glucose repression. Many other genes of candidate targets interacting with SNF1 were also evaluated by disruption, but none proved to be the key regulator in the pathway of the glucose repression on xylose utilization. Therefore, there may exist other signaling pathway(s) for glucose repression on xylose consumption. Based on these results, a thermotolerant xylose-glucose co-consumption platform strain of K. marxianus was constructed. Then, exogenous xylose reductase and xylose-specific transporter genes were overexpressed in the platform strain to obtain YHY013. The YHY013 could efficiently co-utilized the glucose and xylose from corncob hydrolysate or xylose mother liquor for xylitol production (> 100 g/L) even with inexpensive organic nitrogen sources.ConclusionsThe analysis of the glucose repression in K. marxianus laid the foundation for construction of the glucose-xylose co-utilizing platform strain. The efficient xylitol production strain further verified the potential of the platform strain in exploitation of lignocellulosic biomass.

Dataset Information

Xylitol production by Saccharomyces cerevisiae overexpressing different xylose reductases using non-detoxified hemicellulosic hydrolysate of corncob.

Publications

Xylitol production by Saccharomyces cerevisiae overexpressing different xylose reductases using non-detoxified hemicellulosic hydrolysate of corncob.

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