Optimization of ethylene glycol production from (D)-xylose via a synthetic pathway implemented in Escherichia coli

Abstract

Background: Ethylene glycol (EG) is a bulk chemical that is mainly used as an anti-freezing agent and a raw material in the synthesis of plastics. Production of commercial EG currently exclusively relies on chemical synthesis using fossil resources. Biochemical production of ethylene glycol from renewable resources may be more sustainable.

Results: Herein, a synthetic pathway is described that produces EG in Escherichia coli through the action of (D)-xylose isomerase, (D)-xylulose-1-kinase, (D)-xylulose-1-phosphate aldolase, and glycolaldehyde reductase. These reactions were successively catalyzed by the endogenous xylose isomerase (XylA), the heterologously expressed human hexokinase (Khk-C) and aldolase (Aldo-B), and an endogenous glycolaldehyde reductase activity, respectively, which we showed to be encoded by yqhD. The production strain was optimized by deleting the genes encoding for (D)-xylulose-5 kinase (xylB) and glycolaldehyde dehydrogenase (aldA), and by overexpressing the candidate glycolaldehyde reductases YqhD, GldA, and FucO. The strain overproducing FucO was the best EG producer reaching a molar yield of 0.94 in shake flasks, and accumulating 20 g/L EG with a molar yield and productivity of 0.91 and 0.37 g/(L.h), respectively, in a controlled bioreactor under aerobic conditions.

Conclusions: We have demonstrated the feasibility to produce EG from (D)-xylose via a synthetic pathway in E. coli at approximately 90 % of the theoretical yield.

Fig. 1

Synthetic (blue) and natural (black) d-xylose assimilation pathways. In the synthetic pathway (d)-xylose is transformed to (d)-xylulose by endogenous xylose isomerase (XylA). (d)-xylulose-1-kinase (Khk-C) phosphorylates (d)-xylulose to obtain (d)-xylulose-1P, and (d)-xylulose-1-phosphate aldolase (Aldo-B) cleaves (d)-xylulose-1P into glycolaldehyde and DHAP. Ethylene glycol is produced via the action of an unknown endogenous aldehyde reductase.

Fig. 2

Identification of YqhD as the major glycolaldehyde reductase in E. coli. a Production of ethylene glycol by E. coli strains depending on the deletion of candidate glycolaldehyde reductases. Cells were cultivated on mineral (d)-xylose medium and exposed to 10 mM glycolaldehyde. Production of ethylene glycol was estimated after 10 h of incubation. b Log2 transformed expression levels of candidate glycolaldehyde reductases in wild-type cells (C1), strain Pen205 (ΔxylB expressing pEXT20-khk-C-aldoB) (C2), and wild-type cells exposed to 10 mM glycolaldehyde (C3). Genes were clustered according to the Euclidean distance between their expression levels using complete-linkage clustering [30]. Red and blue correspond to high and low expression levels, respectively, using arbitrary units.

Fig. 3

Growth and product formation kinetics depending on the presence and absence of YqhD. a Pen205 (ΔxylB expressing pEXT20-khk-C-aldoB) and b Pen334 (ΔxylB ΔyqhD pEXT20-khk-C-aldoB) were cultivated on mineral medium containing (d)-xylose at 70 mmol/L initial concentration. Experiments were performed in 250 mL shake flask containing 50 mL medium.

Fig. 4

Growth and product formation kinetics of strain Pen641 in controlled bioreactors. Cells were cultivated under fully aerobic conditions (a), or under micro-aerobic conditions (b) on xylose mineral medium enriched with 1 g/L tryptone and 0.5 g/L yeast extract. Initial (d)-xylose concentration was 55 g/L. Aerobic and micro-aerobic conditions were imposed by maintaining the dissolved oxygen tension above 40 and 2 %, respectively.