Exploring d-xylose oxidation in Saccharomyces cerevisiae through the Weimberg pathway

Abstract

Engineering of the yeast Saccharomyces cerevisiae towards efficient d-xylose assimilation has been a major focus over the last decades since d-xylose is the second most abundant sugar in nature, and its conversion into products could significantly improve process economy in biomass-based processes. Up to now, two different metabolic routes have been introduced via genetic engineering, consisting of either the isomerization or the oxido-reduction of d-xylose to d-xylulose that is further connected to the pentose phosphate pathway and glycolysis. In the present study, cytosolic d-xylose oxidation was investigated instead, through the introduction of the Weimberg pathway from Caulobacter crescentus in S. cerevisiae. This pathway consists of five reaction steps that connect d-xylose to the TCA cycle intermediate α-ketoglutarate. The corresponding genes could be expressed in S. cerevisiae, but no growth was observed on d-xylose indicating that not all the enzymes were functionally active. The accumulation of the Weimberg intermediate d-xylonate suggested that the dehydration step(s) might be limiting, blocking further conversion into α-ketoglutarate. Although four alternative dehydratases both of bacterial and archaeon origins were evaluated, d-xylonate accumulation still occurred. A better understanding of the mechanisms associated with the activity of dehydratases, both at a bacterial and yeast level, appears essential to obtain a fully functional Weimberg pathway in S. cerevisiae.Electronic supplementary materialThe online version of this article (10.1186/s13568-018-0564-9) contains supplementary material, which is available to authorized users.

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.…”

The renaissance of yeasts as microbial factories in the modern age of biomanufacturing

“…Synthetic biology is a discipline that combines biology, nanotechnology, and engineering to design and build novel organisms and systems. The ability to integrate heterologous genes and to delete, upregulate, or downregulate native genes made it possible to engineer pathways that reshape the metabolism and extend the substrate (e.g., D-xylose; Wasserstrom et al, 2018) and product ranges of yeast strains, (cannabinoids and opioids; Galanie, Thodey, Trenchard, Filsinger Interrante, & Smolke, 2015, Luo et al, 2019.…”

Deciphering bacterial xylose metabolism and metabolic engineering of industrial microorganisms for use as efficient microbial cell factories

“…The engineered strain of the xylAB gene-deficient expressing heterologous xylose dehydrogenase (XylB), 2-keto-3deoxyxylonate dehydratase (XylX), and α-ketoglutarate semialdehyde dehydrogenase (XylA) from Caulobacter crescentus demonstrated a 53.5% increased biomass over the wild type although the growth rate was decreased (Rossoni et al 2018). Instead of the XI or XR-XDH pathway, the WMB pathway has been introduced to xylose-negative S. cerevisiae to attempt Dxylose assimilation (Wasserstrom et al 2018). However, no growth was observed due to possible inactive dehydratase reactions.…”