Alone at last! – Heterologous expression of a single gene is sufficient for establishing the five-step Weimberg pathway in Corynebacterium glutamicum

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.

A combined experimental and modelling approach for the Weimberg pathway optimisation

“…Therefore, based on the initial rate kinetics, a model was developed for the sequential conversion of D-xylose to α-ketoglutarate and was used for experimental design (choosing appropriate enzyme concentrations), to ensure that each reaction converts 5 mM substrate completely to product in 90 min, which is suitable for NMR analysis. The NMR analysis ( 1 H-NMR and 13 C-NMR, enabled by the use of D-xylose-1-13 C) allowed for a time-resolved observation (1 data point in 1 H and 13 [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27]. All of the enzymatically produced intermediates were in agreement with the proposed Weimberg pathway 13 .…”

“…The second enzyme in the pathway, XLA, seems not to be essential for the pathway, as D-xylonolactone is converted to D-xylonate in a non-enzyme catalysed reaction. In engineering projects, the XLA is often omitted, as it is not essential 19 , and its omission would also prevent accumulation of D-xylonate, which was shown to be toxic in E. coli, C. glutamicum and yeast 19,[21][22][23][24][25][26] . To test whether XLA has an effect on the pathway flux, we omitted the enzyme in the reference state incubation while keeping the other four enzymes at the reference state concentrations (Supplementary Note 1).…”

“…For whole-cell biocatalytic conversion of D-xylose to D-xylonate, the XDH and XLA were introduced into Corynebacterium glutamicum 15 , E. coli 16,17 and Saccharomyces cerevisiae 18 . The complete pathway for oxidative D-xylose degradation was introduced into E. coli 19 , Pseudomonas putida 20 , C. glutamicum [21][22][23][24] and S. cerevisiae 25,26 . However, the results obtained so far in these whole-cell bioengineering approaches have been suboptimal and indicate that intermediate accumulation (i.e., accumulation of toxic D-xylonate) and poor protein expression (e.g., XAD) might be inhibitory to host metabolism and product formation.…”

“…However, the results obtained so far in these whole-cell bioengineering approaches have been suboptimal and indicate that intermediate accumulation (i.e., accumulation of toxic D-xylonate) and poor protein expression (e.g., XAD) might be inhibitory to host metabolism and product formation. Therefore, adaptive laboratory evolution (LAE) approaches 22,26 , introduction of additional gene copies 26,27 and approaches in which the number of genes have been reduced (possible because previously unknown endogenous enzyme activities catalyse the reactions) 20,23 have been followed.…”

Heterologous expression of genes for bioconversion of xylose to xylonic acid in Corynebacterium glutamicum and optimization of the bioprocess

“…Xylose once inside the cell gets converted to xylonolactone and then into xylonic acid on the expression of two genes namely, xylB (xylose dehydrogenase) and xylC (xylonolactonase). These two enzymes are involved in both the Weimberg and Dahms pathway where xylose is metabolized to xylonic acid (Brüsseler et al 2019). In the present study, it is observed that only the xylose dehydrogenase enzyme activity is good enough for xylonic acid production.…”