Xylitol production is increased by expression of codon-optimized Neurospora crassa xylose reductase gene in Candida tropicalis

Abstract

Xylose reductase (XR) is the first enzyme in d-xylose metabolism, catalyzing the reduction of d-xylose to xylitol. Formation of XR in the yeast Candida tropicalis is significantly repressed in cells grown on medium that contains glucose as carbon and energy source, because of the repressive effect of glucose. This is one reason why glucose is not a suitable co-substrate for cell growth in industrial xylitol production. XR from the ascomycete Neurospora crassa (NcXR) has high catalytic efficiency; however, NcXR is not expressed in C. tropicalis because of difference in codon usage between the two species. In this study, NcXR codons were changed to those preferred in C. tropicalis. This codon-optimized NcXR gene (termed NXRG) was placed under control of a constitutive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter derived from C. tropicalis, and integrated into the genome of xylitol dehydrogenase gene (XYL2)-disrupted C. tropicalis. High expression level of NXRG was confirmed by determining XR activity in cells grown on glucose medium. The resulting recombinant strain, LNG2, showed high XR activity (2.86 U (mg of protein)−1), whereas parent strain BSXDH-3 showed no activity. In xylitol fermentation using glucose as a co-substrate with xylose, LNG2 showed xylitol production rate 1.44 g L−1 h−1 and xylitol yield of 96% at 44 h, which were 73 and 62%, respectively, higher than corresponding values for BSXDH-3 (rate 0.83 g L−1 h−1; yield 59%).

Furfural tolerance and detoxification mechanism in Candida tropicalis

“…Gene disruption of C. tropicalis was performed by homologous recombination [31, 32]. The URA3 gene (with promoter and terminator, GenBank accession number AB006207.1) was amplified by PCR from the genomic DNA of wild-type C. tropicalis with primers URA3-F and URA3-R [31].…”

Translation elongation can control translation initiation on eukaryotic mRNAs

“…The use of codons that are overrepresented in naturally highly expressed proteins in recombinant sequences usually improves expression levels compared to random codon usage, particularly in eukaryotic hosts (Kotula & Curtis, 1991;Nagata et al, 1999;Outchkourov et al, 2002;Sinclair & Choy, 2002;Slimko & Lester, 2003;Yadava & Ockenhouse, 2003;Mossadegh et al, 2004;Hu et al, 2006;Lombardi et al, 2009;Mirzaei et al, 2010;Jeon et al, 2012). The situation may be distinct in prokaryotes, where codon usage was found to have only minor effects on gene expression levels (Kudla et al, 2009).…”

Rapid ethanol production at elevated temperatures by engineered thermotolerant Kluyveromyces marxianus via the NADP(H)-preferring xylose reductase-xylitol dehydrogenase pathway

“…Although SsXR were used widely in engineering xylose fermentation strains, the activity of SsXR is not strong enough (the kcat/Km value of SsXR for NADPH is only 167 μM/min) and much weaker than XR from N. crassa, which prefers NADPH to NADH (kcat/Km for NADPH and NADH were 2000 and 19 μM/min, respectively) (Woodyer et al, 2005). Additionally, NcXR has been found to be more thermotolerant with a half-life of 94 min at 40 1C and has been proven to be effectively expressed in K. marxianus for xylitol production at elevated temperatures (Jeon et al, 2011;Woodyer et al, 2005;Zhang et al, 2014;. In this study, NcXR was proven more efficient during ethanol production with xylose in a XR-XDH pathway at elevated temperatures.…”