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.

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.

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.

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.

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.

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.

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.

Draft Genome Sequence of Newly Isolated Agarolytic Bacteria Cellulophaga omnivescoria sp. nov. W5C Carrying Several Gene Loci for Marine Polysaccharide Degradation.

The continued research in the isolation of novel bacterial strains is inspired by the fact that native microorganisms possess certain desired phenotypes necessary for recombinant microorganisms in the biotech industry. Most studies have focused on the isolation and characterization of strains from marine ecosystems as they present a higher microbial diversity than other sources. In this study, a marine bacterium, W5C, was isolated from red seaweed collected from Yeosu, South Korea. The isolate can utilize several natural polysaccharides such as agar, alginate, carrageenan, and chitin. Genome sequence and comparative genomics analyses suggest that strain W5C belongs to a novel species of the Cellulophaga genus, from which the name Cellulophaga omnivescoria sp. nov. is proposed. Its genome harbors 3,083 coding sequences and 146 carbohydrate-active enzymes (CAZymes). Compared to other reported Cellulophaga species, the genome of W5C contained a higher proportion of CAZymes (4.7%). Polysaccharide utilization loci (PUL) for agar, alginate, and carrageenan were identified in the genome, along with other several putative PULs. These PULs are excellent sources for discovering novel hydrolytic enzymes and pathways with unique characteristics required for biorefinery applications, particularly in the utilization of marine renewable biomass. The type strain is JCM 32108T (= KCTC 13157BPT).

Performance evaluation of poly-urethane foam packed-bed chemical scrubber for the oxidative absorption of NH3 and H2S gases.

The feasibility of open-pore polyurethane (PU) foam as packing material for wet chemical scrubber was tested for NH3 and H2S removals. The foam is inexpensive, light-weight, highly porous (low pressure drop) and provides large surface area per unit volume, which are desirable properties for enhanced gas/liquid mass transfer. Conventional HCl/HOCl (for NH3) and NaOH/NaOCl (for H2S) scrubbing solutions were used to absorb and oxidize the gases. Assessment of the wet chemical scrubbers reveals that pH and ORP levels are important to maintain the gas removal efficiencies >95%. A higher re-circulation rate of scrubbing solutions also proved to enhance the performance of the NH3 and H2S columns. Accumulation of salts was confirmed by the gradual increase in total dissolved solids and conductivity values of scrubbing solutions. The critical elimination capacities at >95% gas removals were found to be 5.24 g NH3-N/m3-h and 17.2 g H2S-S/m3-h at an empty bed gas residence time of 23.6 s. Negligible pressure drops (< 4 mm H2O) after continuous operation demonstrate the suitability of PU as a practical packing material in wet chemical scrubbers for NH3 and H2S removals from high-volume dilute emissions.

Removal of odorous compounds emitted from a food-waste composting facility in Korea using a pilot-scale scrubber.

Monitoring and control of odorous compound emissions have been enforced by the Korean government since 2005. One of the point sources for these emissions was from food waste composting facilities. In this study, a pilot-scale scrubber installed in a composting facility was evaluated for its performance in the removal of malodorous compounds. The exhaust stream contained ammonia and methylamine as the major odorants detected by the threshold odor test and various instrumental techniques (GC-FID, FPD, MS and HPLC/UV). For the scrubber operation, the column was randomly packed with polypropylene Hi-Rex 200, while aqueous sulfuric acid was selected as the scrubbing solution. To achieve 95% removal, the scrubber must be operated by using H2SO4 solution with pH at < 6.5, liquid to gas ratio > 4.5, gas loading rate < 1750 m3/m3-hr and contact time < 0.94 s. The scrubber performance was further evaluated by determining the mass transfer coefficients and then monitoring for 355 days of operation. The pilot-scale scrubber maintained > 95% ammonia and methylamine removal efficiencies despite the fluctuations in the inlet (from composting facility exhaust stream) concentration. The optimum operating conditions and scrubber performance indicators determined in this study provides a basis for the design of a plant-scale scrubber for treatment of composting facility gas emissions.

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.

Metabolic engineering of Escherichia coli for biosynthesis of D-galactonate.

D-galactose is an attractive substrate for bioconversion. Herein, Escherichia coli was metabolically engineered to convert D-galactose into D-galactonate, a valuable compound in the polymer and cosmetic industries. D-galactonate productions by engineered E. coli strains were observed in shake flask cultivations containing 2 g L(-1) D-galactose. Engineered E. coli expressing gld coding for galactose dehydrogenase from Pseudomonas syringae was able to produce 0.17 g L(-1) D-galactonate. Inherent metabolic pathways for assimilating both D-galactose and D-galactonate were blocked to enhance the production of D-galactonate. This approach finally led to a 7.3-fold increase with D-galactonate concentration of 1.24 g L(-1) and yield of 62.0 %. Batch fermentation in 20 g L(-1) D-galactose of E. coli ∆galK∆dgoK mutant expressing the gld resulted in 17.6 g L(-1) of D-galactonate accumulation and highest yield of 88.1 %. Metabolic engineering strategy developed in this study could be useful for industrial production of D-galactonate.

Combining De Ley-Doudoroff and methylerythritol phosphate pathways for enhanced isoprene biosynthesis from D-galactose.

An engineered Escherichia coli strain was developed for enhanced isoprene production using D-galactose as substrate. Isoprene is a valuable compound that can be biosynthetically produced from pyruvate and glyceraldehyde-3-phosphate (G3P) through the methylerythritol phosphate pathway (MEP). The Leloir and De Ley-Doudoroff (DD) pathways are known existing routes in E. coli that can supply the MEP precursors from D-galactose. The DD pathway was selected as it is capable of supplying equimolar amounts of pyruvate and G3P simultaneously. To exclusively direct D-galactose toward the DD pathway, an E. coli ΔgalK strain with blocked Leloir pathway was used as the host. To obtain a fully functional DD pathway, a dehydrogenase encoding gene (gld) was recruited from Pseudomonas syringae to catalyze D-galactose conversion to D-galactonate. Overexpressions of endogenous genes known as MEP bottlenecks, and a heterologous gene, were conducted to enhance and enable isoprene production, respectively. Growth test confirmed a functional DD pathway concomitant with equimolar generation of pyruvate and G3P, in contrast to the wild-type strain where G3P was limiting. Finally, the engineered strain with combined DD-MEP pathway exhibited the highest isoprene production. This suggests that the equimolar pyruvate and G3P pools resulted in a more efficient carbon flux toward isoprene production. This strategy provides a new platform for developing improved isoprenoid producing strains through the combined DD-MEP pathway.

Recent advances in the metabolic engineering of microorganisms for the production of 3-hydroxypropionic acid as C3 platform chemical.

Development of sustainable technologies for the production of 3-hydroxypropionic acid (3HP) as a platform chemical has recently been gaining much attention owing to its versatility in applications for the synthesis of other specialty chemicals. Several proposed biological synthesis routes and strategies for producing 3HP from glucose and glycerol are reviewed presently. Ten proposed routes for 3HP production from glucose are described and one of which was recently constructed successfully in Escherichia coli with malonyl-Coenzyme A as a precursor. This resulted in a yield still far from the required level for industrial application. On the other hand, strategies employing engineered E. coli and Klebsiella pneumoniae capable of producing 3HP from glycerol are also evaluated. The titers produced by these recombinant strains reached around 3 %. At its current state, it is evident that a bulk of engineering works is yet to be done to acquire a biosynthesis route for 3HP that is acceptable for industrial-scale production.

Biosynthesis of ethylene glycol in Escherichia coli.

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.

Intermittent trickling bed filter for the removal of methyl ethyl ketone and methyl isobutyl ketone.

Biodegradations of methyl ethyl ketone and methyl isobutyl ketone were performed in intermittent biotrickling filter beds (ITBF) operated at two different trickling periods: 12 h/day (ITBF-12) and 30 min/day (ITBF-0.5). Ralstonia sp. MG1 was able to degrade both ketones as evidenced by growth kinetic experiments. Results show that trickling period is an important parameter to achieve high removal performance and to maintain the robustness of Ralstonia sp. MG1. Overall, ITBF-12 outperformed ITBF-0.5 regardless of the target compound. ITBF-12 had high performance recovery at various inlet gas concentrations. The higher carbon dioxide production rates in ITBF-12 suggest higher microbial activity than in ITBF-0.5. Additionally, lower concentrations of absorbed volatile organic compound (VOC) in trickling solutions of ITBF-12 systems also indicate VOC removal through biodegradation. Pressure drop levels in ITBF-12 were relatively higher than in ITBF-0.5 systems, which can be attributed to the decrease in packed bed porosity as Ralstonia sp. MG1 grew well in ITBF-12. Nonetheless, the obtained pressure drop levels did not have any adverse effect on the performance of ITBF-12. Biokinetic constants were also obtained which indicated that ITBF-12 performed better than ITBF-0.5 and other conventional biotrickling filter systems.

Hydroxypicolinic acid tethered on magnetite core-silica shell (HPCA@SiO2@Fe3O4) as an effective and reusable adsorbent for practical Co(II) recovery.

The soaring demand and future supply risk for cobalt (Co) necessitate more efficient adsorbents for its recycling from electronic wastes, as a cheaper and less hazardous option for its production. Herein, a magnetic adsorbent covalently tethered with 5-hydroxypicolinic acid (HPCA) as Co(II) ligand was developed. The magnetic component (Fe3O4) was protected with silica (SiO2), then silanized with chloroalkyl linker and subsequently functionalized with HPCA via SN2 nucleophilic substitution (HPCA@SiO2@Fe3O4). Results from FTIR, TGA, EA, and XPS confirm the successful adsorbent preparation with high HPCA loading of 2.62 mmol g-1. TEM-EDS reveal its imperfect spherical morphology with ligands well-distributed on its surface. HPCA@SiO2@Fe3O4 is hydrophilic, water-dispersible and magnetically retrievable, which is highly convenient for its recovery. The Co(II) capture on HPCA@SiO2@Fe3O4 involves monodentate coordination with carboxylate (COO-) and lone pair acceptance from pyridine (aromatic -N = ) moiety of HPCA, with minor interaction from acidic silanols (Si-O-). The binding occurs at 2 HPCA: 1 Co(II) ratio, that follows the Sips isotherm model with competitive Qmax = 92.35 mg g-1 and pseudo-second order kinetics (k2 = 0.0042 g mg-1 min-1). In a simulated LIB liquid waste, HPCA@SiO2@Fe3O4 preferentially captures Co(II) over Li(I) with αLi(I)Co(II)=166 and Mn(II) with αMn(II)Co(II)=55, which highlights the importance of HPCA for Co(II) recovery. Silica protection of Fe3O4 rendered the adsorbent chemically stable in acidic thiourea solution for its regeneration by preventing the deterioration of the magnetic component. Covalent functionalization averted ligand loss, which allowed HPCA@SiO2@Fe3O4 to deliver consistent and reversible adsorption/desorption performance. Overall results demonstrate the potential of HPCA@SiO2@Fe3O4 as a competitive and practical adsorbent for Co(II) recovery in liquid waste sources.