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d-1,2,4-Butanetriol (BTO), a C4 platform compound, is widely used in fields such as military and pharmaceuticals. Biosynthesis of d-1,2,4-BTO from lignocellulose-derived d-xylose presents a promising production route. However, the low catalytic activity of d-xylonate dehydratase leading to the accumulation of d-xylonic acid remains a key bottleneck for the efficient production of d-1,2,4-BTO. In this study, we aimed to enhance the catalytic activity of d-xylonate dehydratase through an integrated enzyme and cofactor engineering approach. Firstly, we evolved the d-xylonate dehydratase YjhG by using both random mutagenesis and site-directed saturation mutagenesis. Among the generated variants, YjhGT325F showed an 1.82-fold increase in d-xylonic acid consumption compared to the wild-type enzyme. When introduced into the producing strain, this variant increased d-1,2,4-BTO production by 1.34-fold compared to the original strain. Further enhancement was achieved by modifying the iron-sulfur [Fe–S] cluster synthesis system, which was critical for d-xylonate dehydratase activity. We systematically evaluated three [Fe–S] assembly systems, including SUF (encoded by sufABCDSE), ISC (encoded by iscSUA-hscBA-fdx), and CSD (encoded by csdAE). Comparative analysis revealed that the overexpression of SUF system conferred the highest catalytic efficiency of YjhG. The recombinant strain of BT-YjhGT325F-SUF produced 10.36 g/L of d-1,2,4-BTO from d-xylose, achieving a molar yield of 73.6 %, which was 1.88-fold that of the original strain. This study provided a robust foundation for high-efficiency d-1,2,4-BTO production through enzyme and cofactor engineering.

We describe here the identification and characterization of two novel enzymes belonging to the IlvD/EDD protein family, the D-xylonate dehydratase from Caulobacter crescentus , Cc XyDHT, (EC 4.2.1.82), and the L-arabonate dehydratase from Rhizobium leguminosarum bv . trifolii , Rl ArDHT (EC 4.2.1.25), that produce the corresponding 2-keto-3-deoxy-sugar acids. There is only a very limited amount of characterization data available on pentonate dehydratases, even though the enzymes from these oxidative pathways have potential applications with plant biomass pentose sugars. The two bacterial enzymes share 41 % amino acid sequence identity and were expressed and purified from Escherichia coli as homotetrameric proteins. Both dehydratases were shown to accept pentonate and hexonate sugar acids as their substrates and require Mg 2+ for their activity. Cc XyDHT displayed the highest activity on D-xylonate and D-gluconate, while Rl ArDHT functioned best on D-fuconate, L-arabonate and D-galactonate. The configuration of the OH groups at C2 and C3 position of the sugar acid were shown to be critical, and the C4 configuration also contributed substantially to the substrate recognition. The two enzymes were also shown to contain an iron–sulphur [Fe–S] cluster. Our phylogenetic analysis and mutagenesis studies demonstrated that the three conserved cysteine residues in the aldonic acid dehydratase group of IlvD/EDD family members, those of C60, C128 and C201 in Cc XyDHT, and of C59, C127 and C200 in Rl ArDHT, are needed for coordination of the [Fe–S] cluster. The iron–sulphur cluster was shown to be crucial for the catalytic activity (k cat ) but not for the substrate binding (K m ) of the two pentonate dehydratases.

Improving the comprehensive utilization of sugars in lignocellulosic biomass is a major challenge for enhancing the economic viability of lignocellulose biorefinement. A robust yeast Pichia kudriavzevii N-X showed excellent performance in ethanol production under high temperature and low pH conditions and was engineered for ᴅ-xylonate production without xylitol generation. The recombinant strain P. kudriavzevii N-X/S1 was employed for sequential production of ᴅ-xylonate and ethanol from ᴅ-xylose, feeding on ᴅ-glucose without pH control in a two-stage strategy of aerobic and shifting micro-aerobic fermentation. Acid-pretreated corncob without detoxification and filtration was used for ᴅ-xylonate production, then simultaneous saccharification and ethanol fermentation was performed with cellulase added at pH 4.0 and at 40 °C. By this strategy, 33.5 g/L ᴅ-xylonate and 20.8 g/L ethanol were produced at yields of 1.10 g/g ᴅ-xylose and 84.3% of theoretical value, respectively. We propose a promising approach for the sequential production of ᴅ-xylonate and ethanol from non-detoxified corncob using a single microorganism.

In E. coli , the genes of the D-xylonate catabolic pathway are present in two distinct operons, yagEFG  and yjhIHG , which are part of two cryptic phages found in the genome of this bacterium. These operons are under the control of two transcription factors: XynR which acts as a repressor of yagEFG and yjhI which is an activator of yjhIHG . Although D-xylonate is known to induce these two operons, the true inducer has not been identified yet. Through the construction of biosensors based on xynR and yjhI , using syfp2 as reporter gene in combination with mutants of genes involved in D-xylonate catabolism, it has been demonstrated that the effector of these operons is 2-keto-3-deoxy-D-xylonate, which is formed by the dehydration of D-xylonate catalyzed by the dehydratases encoded by yagF and yjhG genes. Building on the finding that these two operons were also upregulated in E. coli challenged with high concentration of platform molecule 2,4-dihydroxybutyric acid, it is reported that both XynR- and YjhI-based biosensors were also responsive to the intermediate non-natural molecule 2-oxo-4-hydroxybutyric acid, with characteristic performances in terms of response threshold, sensitivity, cooperativity, and dynamic response comparable to those of the natural effector. Given that the bottlenecks in the production of 2,4-dihydroxybutyrate from C2 and C5/C6 carbon are D-threonate dehydratase and L-homoserine transaminase, respectively, which catalyze the formation of 2-oxo-4-hydroxybutyric acid from either D-threonate or homoserine, we showed that XynR and YjhI-based biosensors could be efficient tools for the screening and selection of more active enzymes producing this compound, thereby improving the production of 2,4-dihydroxybutyrate.

The important platform chemicals ethylene glycol and glycolic acid were produced via the oxidative D-xylose pathway in the yeast Saccharomyces cerevisiae . The expression of genes encoding D-xylose dehydrogenase (XylB) and D-xylonate dehydratase (XylD) from Caulobacter crescentus and YagE or YjhH aldolase and aldehyde dehydrogenase AldA from Escherichia coli enabled glycolic acid production from D-xylose up to 150 mg/L. In strains expressing only xylB and xylD , 29 mg/L 2-keto-3-deoxyxylonic acid [( S )-4,5-dihydroxy-2-oxopentanoic acid] (2K3DXA) was produced and D-xylonic acid accumulated to ca. 9 g/L. A significant amount of D-xylonic acid (ca. 14%) was converted to 3-deoxypentonic acid (3DPA), and also, 3,4-dihydroxybutyric acid was formed. 2K3DXA was further converted to glycolaldehyde when genes encoding by either YagE or YjhH aldolase from E. coli were expressed. Reduction of glycolaldehyde to ethylene glycol by an endogenous aldo-keto reductase activity resulted further in accumulation of ethylene glycol of 14 mg/L. The possibility of simultaneous production of lactic and glycolic acids was evaluated by expression of gene encoding lactate dehydrogenase ldhL from Lactobacillus helveticus together with aldA . Interestingly, this increased the accumulation of glycolic acid to 1 g/L. The D-xylonate dehydratase activity in yeast was notably low, possibly due to inefficient Fe–S cluster synthesis in the yeast cytosol, and leading to D-xylonic acid accumulation. The dehydratase activity was significantly improved by targeting its expression to mitochondria or by altering the Fe–S cluster metabolism of the cells with FRA2 deletion.

1,2,4-Butanetriol (BT) is a valuable chemical with extensive applications in many different fields. The traditional chemical routes to synthesize BT suffer from many drawbacks, e.g., harsh reaction conditions, multiple steps and poor selectivity, limiting its industrial production. In this study, an engineered Escherichia coli strain was constructed to produce BT from xylose, which is a major component of the lignocellulosic biomass. Through the coexpression of a xylose dehydrogenase ( CCxylB ) and a xylonolactonase ( xylC ) from Caulobacter crescentus , native E. coli xylonate dehydratase ( yjhG ), a 2-keto acid decarboxylase from Pseudomonas putida ( mdlC ) and native E. coli aldehyde reductase ( adhP ) in E. coli BL21 star(DE3), the recombinant strain could efficiently convert xylose to BT. Furthermore, the competitive pathway responsible for xylose metabolism in E. coli was blocked by disrupting two genes ( xylA and EcxylB ) encoding xylose isomerase and xyloluse kinase. Under fed-batch conditions, the engineered strain BL21ΔxylAB/pE-mdlCxylBC&pA-adhPyjhG produced up to 3.92 g/L of BT from 20 g/L of xylose, corresponding to a molar yield of 27.7%. These results suggest that the engineered E. coli has a promising prospect for the large-scale production of BT.

Glycolic acid (GA) is an ⍺-hydroxy acid used in cosmetics, packaging, and medical industries due to its excellent properties, especially in its polymeric form. In this study, Escherichia coli was engineered to produce GA from D-xylose by linking the Dahms pathway, the glyoxylate bypass, and the partial reverse glyoxylate pathway (RGP). Initially, a GA-producing strain was constructed by disrupting the xylAB and glcD genes in the E. coli genome and overexpressing the xdh(Cc) from Caulobacter crescentus. This strain was further improved through modular optimization of the Dahms pathway and the glyoxylate bypass. Results for module 1 showed that the rate-limiting step of the Dahms pathway was the xylonate dehydratase reaction, and the overexpression of yagF was sufficient to overcome this bottleneck. Furthermore, the appropriate aldolase gene for module 1 was proven to be yagE. The results also show that overexpression of the lactaldehyde dehydrogenase gene, aldA, is needed to increase the GA production while the overexpression of glyoxylate reductase gene, ycdW, was only essential when the glyoxylate bypass was active. On the other hand, the module 2 enzymes AceA and AceK were vital in activating the glyoxylate bypass, while the RGP enzymes were dispensable. The final strain (GA19) produced 4.57 g/L GA with a yield of 0.46 g/g from D-xylose. So far, this is the highest value achieved for GA production in engineered E. coli through the Dahms pathway.

Preparation of xylonic acid from xylonate fermentation broth was studied in a four-chamber bipolar membrane electrodialysis (BMED) setup. The effects of metal-ion size, current density, and xylonate concentration on BMED were evaluated principally with respect to acid yield and partially with respect to efficiency and energy consumption. Sodium xylonate was more successful than potassium xylonate because of its smaller size and easier membrane penetrability for BMDE. Efficient electrodialysis was achieved using 50 mA/cm 2 current density for 14 min; thus, we obtained 92% xylonic acid from 100 g/L sodium xylonate fermentation broth. In conclusion, BMED can be used for producing xylonic acid from fermentation broth. Moreover, this study highlights ways of improving the efficiency of BMED.

Ethylene glycol (EG) is an important platform chemical with steadily expanding global demand. Its commercial production is currently limited to fossil resources; no biosynthesis route has been delineated. Herein, a biosynthesis route for EG production from d -xylose is reported. This route consists of four steps: d -xylose →  d -xylonate → 2-dehydro-3-deoxy- d -pentonate → glycoaldehyde → EG. Respective enzymes, d -xylose dehydrogenase, d -xylonate dehydratase, 2-dehydro-3-deoxy- d -pentonate aldolase, and glycoaldehyde reductase, were assembled. The route was implemented in a metabolically engineered Escherichia coli , in which the d -xylose →  d -xylulose reaction was prevented by disrupting the d -xylose isomerase gene. The most efficient construct produced 11.7 g L −1 of EG from 40.0 g L −1 of d -xylose. Glycolate is a carbon-competing by-product during EG production in E. coli ; blockage of glycoaldehyde → glycolate reaction was also performed by disrupting the gene encoding aldehyde dehydrogenase, but from this approach, EG productivity was not improved but rather led to d -xylonate accumulation. To channel more carbon flux towards EG than the glycolate pathway, further systematic metabolic engineering and fermentation optimization studies are still required to improve EG productivity.

Corn cob is a major waste mass-produced in corn agriculture. Corn cob hydrolysate containing xylose, arabinose, and glucose is the hydrolysis product of corn cob. Herein, a recombinant Escherichia coli strain BT-10 was constructed to transform corn cob hydrolysate into 1,2,4-butanetriol, a platform substance with diversified applications. To eliminate catabolite repression and enhance NADPH supply for alcohol dehydrogenase YqhD catalyzed 1,2,4-butanetriol generation, ptsG encoding glucose transporter EIICB Glc and pgi encoding phosphoglucose isomerase were deleted. With four heterologous enzymes including xylose dehydrogenase, xylonolactonase, xylonate dehydratase, α-ketoacid decarboxylase and endogenous YqhD, E. coli BT-10 can produce 36.63 g/L 1,2,4-butanetriol with a productivity of 1.14 g/[L·h] using xylose as substrate. When corn cob hydrolysate was used as the substrate, 43.4 g/L 1,2,4-butanetriol was generated with a productivity of 1.09 g/[L·h] and a yield of 0.9 mol/mol. With its desirable characteristics, E. coli BT-10 is a promising strain for commercial 1,2,4-butanetriol production.