Abstract
Corynebacterium glutamicum can grow on d-xylose as sole carbon and energy source via the five-step Weimberg pathway when the pentacistronic xylXABCD operon from Caulobacter crescentus is heterologously expressed. More recently, it could be demonstrated that the C. glutamicum wild type accumulates the Weimberg pathway intermediate d-xylonate when cultivated in the presence of d-xylose. Reason for this is the activity of the endogenous dehydrogenase IolG, which can also oxidize d-xylose. This raised the question whether additional endogenous enzymes in C. glutamicum contribute to the catabolization of d-xylose via the Weimberg pathway. In this study, analysis of the C. glutamicum genome in combination with systematic reduction of the heterologous xylXABCD operon revealed that the hitherto unknown and endogenous dehydrogenase KsaD (Cg0535) can also oxidize α-ketoglutarate semialdehyde to the tricarboxylic acid cycle intermediate α-ketoglutarate, the final enzymatic step of the Weimberg pathway. Furthermore, heterologous expression of either xylX or xylD, encoding for the two dehydratases of the Weimberg pathway in C. crescentus, is sufficient for enabling C. glutamicum to grow on d-xylose as sole carbon and energy source. Finally, several variants for the carbon-efficient microbial production of α-ketoglutarate from d-xylose were constructed. In comparison to cultivation solely on d-glucose, the best strain accumulated up to 1.5-fold more α-ketoglutarate in d-xylose/d-glucose mixtures.
Highlights
The Gram-positive bacterium Corynebacterium glutamicum has a long history in the industrial production of proteinogenic amino acids
Presence of the endogenous dehydrogenase IolG oxidizing D-xylose to 1,4-D-xylonolactone and the observation that hydrolyzation of this lactone can occur spontaneously, indicates that heterologous expression of the xylose dehydrogenase and the D-xylonolactonase from C. crescentus might not be required for establishing the Weimberg pathway in C. glutamicum
Since reduction of the Weimberg pathway encoding operon has not been tried yet, a synthetic operon comprised of codon-optimized genes for 2-keto-3-desoxyxylonate dehydratase, xylonate dehydratase and the α-ketoglutarate semialdehyde dehydrogenase, all originating from C. crescentus, was constructed
Summary
The Gram-positive bacterium Corynebacterium glutamicum has a long history in the industrial production of proteinogenic amino acids. Several C. glutamicum strains, capable of utilizing D-xylose via the Isomerase pathway have been engineered for the production of succinate, ethanol, lysine, glutamate, ornithine, putrescine and 1,5-diaminopentane (Buschke et al, 2011; Jo et al, 2017; Meiswinkel et al, 2013). Functional introduction of the xylXABCD operon from Caulobacter crescentus enabled C. glutamicum to grow on D-xylose as sole carbon and energy source via the five-step Weimberg pathway (Radek et al, 2014). In this pathway, D-xylose is initially oxidized to 1,4-D-xylonolactone via a xylose dehydrogenase (XylB) and subsequently hydrolyzed by a D-xylonolactonase (XylC) yielding D-xylonate (Fig. 1). Two subsequent dehydration reactions, catalyzed by a D-xylonate dehydratase (XylD) and a 2-keto-3-deoxyxylonate dehydratase (XylX), lead to license (http://creativecommons.org/licenses/by/4.0/)
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