Understanding D-xylonic acid accumulation: a cornerstone for better metabolic engineering approaches.

The xylose oxidative pathway (XOP) has been engineered in microorganisms for the production of a wide range of industrially relevant compounds. However, the performance of metabolically engineered XOP-utilizing microorganisms is typically hindered by D-xylonic acid accumulation. It acidifies the media and perturbs cell growth due to toxicity, thus curtailing enzymatic activity and target product formation. Fortunately, from the growing portfolio of genetic tools, several strategies that can be adapted for the generation of efficient microbial cell factories have been implemented to address D-xylonic acid accumulation. This review centers its discussion on the causes of D-xylonic acid accumulation and how to address it through different engineering and synthetic biology techniques with emphasis given on bacterial strains. In the first part of this review, the ability of certain microorganisms to produce and tolerate D-xylonic acid is also tackled as an important aspect in developing efficient microbial cell factories. Overall, this review could shed some insights and clarity to those working on XOP in bacteria and its engineering for the development of industrially applicable product-specialist strains. KEY POINTS: D-Xylonic acid accumulation is attributed to the overexpression of xylose dehydrogenase concomitant with basal or inefficient expression of enzymes involved in D-xylonic acid assimilation. Redox imbalance and insufficient cofactors contribute to D-xylonic acid accumulation. Overcoming D-xylonic acid accumulation can increase product formation among engineered strains. Engineering strategies involving enzyme engineering, evolutionary engineering, coutilization of different sugar substrates, and synergy of different pathways could potentially address D-xylonic acid accumulation.

Engineering of xylose metabolism in Escherichia coli for the production of valuable compounds.

The lignocellulosic sugar d-xylose has recently gained prominence as an inexpensive alternative substrate for the production of value-added compounds using genetically modified organisms. Among the prokaryotes, Escherichia coli has become the de facto host for the development of engineered microbial cell factories. The favored status of E. coli resulted from a century of scientific explorations leading to a deep understanding of its systems. However, there are limited literature reviews that discuss engineered E. coli as a platform for the conversion of d-xylose to any target compounds. Additionally, available critical review articles tend to focus on products rather than the host itself. This review aims to provide relevant and current information about significant advances in the metabolic engineering of d-xylose metabolism in E. coli. This focusses on unconventional and synthetic d-xylose metabolic pathways as several review articles have already discussed the engineering of native d-xylose metabolism. This paper, in particular, is essential to those who are working on engineering of d-xylose metabolism using E. coli as the host.

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.

Overexpression and characterization of a novel GH16 ß-agarase (Aga1) from Cellulophaga omnivescoria W5C.

OBJECTIVE: To identify and characterize a new ß-agarase from Cellulophaga omnivescoria W5C capable of producing biologically-active neoagarooligosaccharides from agar. RESULTS: The ß-agarase, Aga1, has signal peptides on both N- and C-terminals, which are involved in the type IX secretion system. It shares 75% protein sequence identity with AgaD from Zobellia galactanivorans and has a molecular weight of 54 kDa. Biochemical characterization reveals optimum agarolytic activities at pH 7-8 and temperature 30-45 °C. Aga1 retains at least 33% activity at temperatures lower than the sol-gel transition state of agarose. Metal ions are generally not essential, but calcium and potassium enhance its activity whereas iron and zinc are inhibitory. Finally, hydrolysis of agarose with Aga1 yields neoagarotetraose, neoagarohexaose, and neoagarooctaose. CONCLUSIONS: Aga1 displays unique traits such as moderate psychrophilicity, stability, and synergy with other agarases, which makes it an excellent candidate for biosynthetic production of neoagarooligosaccharides from agar.

Correction to: A pH-responsive genetic sensor for the dynamic regulation of D-xylonic acid accumulation in Escherichia coli.

In the published version, the y-axis data of Fig. 3c was incorrectly inserted (OD600 instead of D-xylonate (g L-1) and the x-axes of Figs. 3b, 3d, 3e and 3f ended at 48 h instead of 72 h. See the correct Fig. 3 below.

A pH-responsive genetic sensor for the dynamic regulation of D-xylonic acid accumulation in Escherichia coli.

The xylose oxidative pathway (XOP) is continuously gaining prominence as an alternative for the traditional pentose assimilative pathways in prokaryotes. It begins with the oxidation of D-xylose to D-xylonic acid, which is further converted to α-ketoglutarate or pyruvate + glycolaldehyde through a series of enzyme reactions. The persistent drawback of XOP is the accumulation of D-xylonic acid intermediate that causes culture media acidification. This study addresses this issue through the development of a novel pH-responsive synthetic genetic controller that uses a modified transmembrane transcription factor called CadCΔ. This genetic circuit was tested for its ability to detect extracellular pH and to control the buildup of D-xylonic acid in the culture media. Results showed that the pH-responsive genetic sensor confers dynamic regulation of D-xylonic acid accumulation, which adjusts with the perturbation of culture media pH. This is the first report demonstrating the use of a pH-responsive transmembrane transcription factor as a transducer in a synthetic genetic circuit that was designed for XOP. This may serve as a benchmark for the development of other genetic controllers for similar pathways that involve acidic intermediates.

Discovering a novel D-xylonate-responsive promoter: the PyjhI-driven genetic switch towards better 1,2,4-butanetriol production.

The capability of Escherichia coli to catabolize D-xylonate is a crucial component for building and optimizing the Dahms pathway. It relies on the inherent dehydratase and keto-acid aldolase activities of E. coli. Although the biochemical characteristics of these enzymes are known, their inherent expression regulation remains unclear. This knowledge is vital for the optimization of D-xylonate assimilation, especially in addressing the problem of D-xylonate accumulation, which hampers both cell growth and target product formation. In this report, molecular biology techniques and synthetic biology tools were combined to build a simple genetic switch controller for D-xylonate. First, quantitative and relative expression analysis of the gene clusters involved in D-xylonate catabolism were performed, revealing two D-xylonate-inducible operons, yagEF and yjhIHG. The 5'-flanking DNA sequence of these operons were then subjected to reporter gene assays which showed PyjhI to have low background activity and wide response range to D-xylonate. A PyjhI-driven synthetic genetic switch was then constructed containing feedback control to autoregulate D-xylonate accumulation and to activate the expression of the genes for 1,2,4-butanetriol (BTO) production. The genetic switch effectively reduced D-xylonate accumulation, which led to 31% BTO molar yield, the highest for direct microbial fermentation systems thus far. This genetic switch can be further modified and employed in the production of other compounds from D-xylose through the xylose oxidative pathway.

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.