Enhanced glycolic acid yield through xylose and cellobiose utilization by metabolically engineered Escherichia coli.

Microbial biorefinery is a promising route toward sustainable production of glycolic acid (GA), a valuable raw material for various industries. However, inherent microbial GA production has limited substrate consumption using either D-xylose or D-glucose as carbon catabolite repression (CCR) averts their co-utilization. To bypass CCR, a GA-producing strain using D-xylose via Dahms pathway was engineered to allow cellobiose uptake. Unlike glucose, cellobiose was assimilated and intracellularly degraded without repressing D-xylose uptake. The final GA-producing E. coli strain (CLGA8) has an overexpressed cellobiose phosphorylase (cep94A) from Saccharophagus degradans 2-40 and an activated glyoxylate shunt pathway. Expression of cep94A improved GA production reaching the maximum theoretical yield (0.51 g GA g-1 xylose), whereas activation of glyoxylate shunt pathway enabled GA production from cellobiose, which further increased the GA titer (2.25 g GA L-1). To date, this is the highest reported GA yield from D-xylose through Dahms pathway in an engineered E. coli with cellobiose as co-substrate.

Improved cell growth and biosynthesis of glycolic acid by overexpression of membrane-bound pyridine nucleotide transhydrogenase.

The non-conventional D-xylose metabolism called the Dahms pathway which only requires the expression of at least three enzymes to produce pyruvate and glycolaldehyde has been previously engineered in Escherichia coli. Strains that rely on this pathway exhibit lower growth rates which were initially attributed to the perturbed redox homeostasis as evidenced by the lower intracellular NADPH concentrations during exponential growth phase. NADPH-regenerating systems were then tested to restore the redox homeostasis. The membrane-bound pyridine nucleotide transhydrogenase, PntAB, was overexpressed and resulted to a significant increase in biomass and glycolic acid titer and yield. Furthermore, expression of PntAB in an optimized glycolic acid-producing strain improved the growth and product titer significantly. This work demonstrated that compensating for the NADPH demand can be achieved by overexpression of PntAB in E. coli strains assimilating D-xylose through the Dahms pathway. Consequently, increase in biomass accumulation and product concentration was also observed.

Everyone loves an underdog: metabolic engineering of the xylose oxidative pathway in recombinant microorganisms.

The D-xylose oxidative pathway (XOP) has recently been employed in several recombinant microorganisms for growth or for the production of several valuable compounds. The XOP is initiated by D-xylose oxidation to D-xylonolactone, which is then hydrolyzed into D-xylonic acid. D-Xylonic acid is then dehydrated to form 2-keto-3-deoxy-D-xylonic acid, which may be further dehydrated then oxidized into α-ketoglutarate or undergo aldol cleavage to form pyruvate and glycolaldehyde. This review introduces a brief discussion about XOP and its discovery in bacteria and archaea, such as Caulobacter crescentus and Haloferax volcanii. Furthermore, the current advances in the metabolic engineering of recombinant strains employing the XOP are discussed. This includes utilization of XOP for the production of diols, triols, and short-chain organic acids in Escherichia coli, Saccharomyces cerevisiae, and Corynebacterium glutamicum. Improving the D-xylose uptake, growth yields, and product titer through several metabolic engineering techniques bring some of these recombinant strains close to industrial viability. However, more developments are still needed to optimize the XOP pathway in the host strains, particularly in the minimization of by-product formation.

Engineering Escherichia coli for glycolic acid production from D-xylose through the Dahms pathway and glyoxylate bypass.

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.

Identification of aldehyde reductase catalyzing the terminal step for conversion of xylose to butanetriol in engineered Escherichia coli.

Biosynthetic pathways for the production of biofuels often rely on inherent aldehyde reductases (ALRs) of the microbial host. These native ALRs play vital roles in the success of the microbial production of 1,3-propanediol, 1,4-butanediol, and isobutanol. In the present study, the main ALR for 1,2,4-butanetriol (BT) production in Escherichia coli was identified. Results of real-time PCR analysis for ALRs in EWBT305 revealed the increased expression of adhP, fucO, adhE, and yqhD genes during BT production. The highest increase of expression was observed up to four times in yqhD. Singular deletion of adhP, fucO, or adhE gene showed marginal differences in BT production compared to that of the parent strain, EWBT305. Remarkably, yqhD gene deletion (KBTA4 strain) almost completely abolished BT production while its re-introduction (wild-type gene with its native promoter) on a low copy plasmid restored 75 % of BT production (KBTA4-2 strain). This suggests that yqhD gene is the main ALR of the BT pathway. In addition, KBTA4 showed almost no NADPH-dependent ALR activity, but was also restored upon re-introduction of the yqhD gene (KBTA4-2 strain). Therefore, the required ALR activity to complete the BT pathway was mainly contributed by YqhD. Increased gene expression and promiscuity of YqhD were both found essential factors to render YqhD as the key ALR for the BT pathway.

Enhanced yield of ethylene glycol production from d-xylose by pathway optimization in Escherichia coli.

The microbial production of renewable ethylene glycol (EG) has been gaining attention recently due to its growing importance in chemical and polymer industries. EG has been successfully produced biosynthetically from d-xylose through several novel pathways. The first report on EG biosynthesis employed the Dahms pathway in Escherichia coli wherein 71% of the theoretical yield was achieved. This report further improved the EG yield by implementing metabolic engineering strategies. First, d-xylonic acid accumulation was reduced by employing a weak promoter which provided a tighter control over Xdh expression. Second, EG yield was further improved by expressing the YjgB, which was identified as the most suitable aldehyde reductase endogenous to E. coli. Finally, cellular growth, d-xylose consumption, and EG yield were further increased by blocking a competing reaction. The final strain (WTXB) was able to reach up to 98% of the theoretical yield (25% higher as compared to the first study), the highest reported value for EG production from d-xylose.

Overexpression and secretion of AgaA7 from Pseudoalteromonas hodoensis sp. nov in Bacillus subtilis for the depolymerization of agarose.

Interest in agar or agarose-based pharmaceutical products has driven the search for potent agarolytic enzymes. An extracellular ß-agarase (AgaA7) recently isolated from Pseudoalteromonas hodoensis sp. nov was expressed in Bacillus subtilis, which was chosen due to its capability to overproduce and secrete functional enzymes. Phenotypic analysis showed that the engineered B. subtilis secreted a functional AgaA7 when fused with the aprE signal peptide (SP) at the amino-terminus. The maximum agarolytic activity was observed during the late logarithmic phase. To further improve the secretion of AgaA7, an expression library of AgaA7 fused to different naturally occurring B. subtilis SPs was created. The amount of AgaA7 secreted by the clones was compared through activity assay, immuno-blot, and purification via affinity chromatography. Although the aprE SP can readily facilitate the secretion of AgaA7, other SPs such as yqgA, pel, and lipA were relatively more efficient. Among these SPs, lipA was the most efficient in improving the secretion of AgaA7.The use of B. subtilis as host for the expression and secretion of agarolytic and other hydrolytic enzymes can be a useful tool in the field of white biotechnology.