Metabolic Engineering of E. coli for Xylose Production from Glucose as the Sole Carbon Source.

Xylose is the raw material for the synthesis of many important platform compounds. At present, xylose is commercially produced by chemical extraction. However, there are still some bottlenecks in the extraction of xylose, including complicated operation processes and the chemical substances introduced, leading to the high cost of xylose and of synthesizing the downstream compounds of xylose. The current market price of xylose is 8× that of glucose, so using low-cost glucose as the substrate to produce the downstream compounds of xylose can theoretically reduce the cost by 70%. Here, we designed a pathway for the biosynthesis of xylose from glucose in Escherichia coli. This biosynthetic pathway was achieved by overexpressing five genes, namely, zwf, pgl, gnd, rpe, and xylA, while replacing the native xylulose kinase gene xylB with araL from B. subtilis, which displays phosphatase activity toward d-xylulose 5-phosphate. The yield of xylose was increased to 3.3 g/L by optimizing the metabolic pathway. Furthermore, xylitol was successfully synthesized by introducing the xyl1 gene, which suggested that the biosynthetic pathway of xylose from glucose is universally applicable for the synthesis of xylose downstream compounds. This is the first study to synthesize xylose and its downstream compounds by using glucose as a substrate, which not only reduces the cost of raw materials, but also alleviates carbon catabolite repression (CCR), providing a new idea for the synthesis of downstream compounds of xylose.

Characterization of hemicelluloses in sugarcane (Saccharum spp. hybrids) culm during xylogenesis.

Hemicelluloses are effective renewable biopolymers that can be used in many different industrial processes and preparations. In plants, the content of hemicellulose might change with different developmental stages and/or tissues. Thus, in here chemical and structural differences in hemicellulose isolated from the apical, middle and basal segments of sugarcane stem were characterized using chemical techniques. Further, difference in expression levels of genes related to synthesis of hemicelluloses from these three segments were studied by RNA-seq and qRT-PCR etc. The sugarcane hemicellulose backbone was xylose residues connected via ß-1,4 glycosidic linkages which was further substituted with arabinose, acetyl and glucuronic acid side chains. Hemicellulose content was higher in the middle and basal segments with less backbone substitutions compared to apical segments. In terms of gene expression, hemicellulose synthesis and modification genes were intensely expressed in middle and basal segments. Taken together, our research describes differences in hemicellulose content and substitutions in sugarcane during xylogenesis, which will increase our knowledge for finding more refined use of sugarcane bagasse.

Enzymatically-synthesized xylo-oligosaccharides laurate esters as surfactants of interest.

Lipase-catalyzed synthesis of xylo-oligosaccharides esters from pure xylobiose, xylotriose and xylotetraose in the presence of vinyl laurate was investigated. The influence of different experimental parameters such as the loading of lipase, the reaction duration or the use of a co-solvent was studied and the reaction conditions were optimized with xylobiose. Under the best conditions, a regioselective esterification occurred to yield a monoester with the acyl chain at the OH-4 of the xylose unit at the non-reducing end. Surface-active properties of these pure xylo-oligosaccharides fatty esters have been evaluated. They display interesting surfactant activities that differ according to the degree of polymerization (DP) of the glycone moiety.

Exploiting the NADPH pool for xylitol production using recombinant Saccharomyces cerevisiae.

Xylitol is a five-carbon sugar alcohol that has a variety of uses in the food and pharmaceutical industries. In xylose assimilating yeasts, NAD(P)H-dependent xylose reductase (XR) catalyzes the reduction of xylose to xylitol. In the present study, XR with varying cofactor specificities was overexpressed in Saccharomyces cerevisiae to screen for efficient xylitol production. Xylose consumption and xylitol yields were higher when NADPH-dependent enzymes (Candida tropicalis XR and S. cerevisiae Gre3p aldose reductase) were expressed, indicating that heterologous enzymes can utilize the intracellular NADPH pool more efficiently than the NADH pool, where they may face competition from native enzymes. This was confirmed by overexpression of a NADH-preferring C. tropicalis XR mutant, which led to decreased xylose consumption and lower xylitol yield. To increase intracellular NADPH availability for xylitol production, the promoter of the ZWF1 gene, coding for the first enzyme of the NADPH-generating pentose phosphate pathway, was replaced with the constitutive GPD promoter in a strain expressing C. tropicalis XR. This change led to a ~12% increase in xylitol yield. Deletion of XYL2 and SOR1, whose gene products can use xylitol as substrate, did not further increase xylitol yield. Using wheat stalk hydrolysate as source of xylose, the constructed strain efficiently produced xylitol, demonstrating practical relevance of this approach.

The pentose phosphate pathway of cellulolytic clostridia relies on 6-phosphofructokinase instead of transaldolase.

The genomes of most cellulolytic clostridia do not contain genes annotated as transaldolase. Therefore, for assimilating pentose sugars or for generating C5 precursors (such as ribose) during growth on other (non-C5) substrates, they must possess a pathway that connects pentose metabolism with the rest of metabolism. Here we provide evidence that for this connection cellulolytic clostridia rely on the sedoheptulose 1,7-bisphosphate (SBP) pathway, using pyrophosphate-dependent phosphofructokinase (PPi-PFK) instead of transaldolase. In this reversible pathway, PFK converts sedoheptulose 7-phosphate (S7P) to SBP, after which fructose-bisphosphate aldolase cleaves SBP into dihydroxyacetone phosphate and erythrose 4-phosphate. We show that PPi-PFKs of Clostridium thermosuccinogenes and Clostridium thermocellum indeed can convert S7P to SBP, and have similar affinities for S7P and the canonical substrate fructose 6-phosphate (F6P). By contrast, (ATP-dependent) PfkA of Escherichia coli, which does rely on transaldolase, had a very poor affinity for S7P. This indicates that the PPi-PFK of cellulolytic clostridia has evolved the use of S7P. We further show that C. thermosuccinogenes contains a significant SBP pool, an unusual metabolite that is elevated during growth on xylose, demonstrating its relevance for pentose assimilation. Last, we demonstrate that a second PFK of C. thermosuccinogenes that operates with ATP and GTP exhibits unusual kinetics toward F6P, as it appears to have an extremely high degree of cooperative binding, resulting in a virtual on/off switch for substrate concentrations near its K½ value. In summary, our results confirm the existence of an SBP pathway for pentose assimilation in cellulolytic clostridia.

Enhancing delignification and subsequent enzymatic hydrolysis of corn stover by magnesium oxide-ethanol pretreatment.

Corn stover pretreatment by MgO-ethanol was investigated to improve sugar recovery by reducing sugar degradation and enhance enzymatic hydrolysis by improving delignification and reducing inhibitor formation. Results showed MgO as an effective additive and Lewis base, functioned to neutralize the acids released from hemicellulose during pretreatment, reduce monosaccharide degradation and inhibitor formation, and enhance delignification. The optimal pretreatment conditions were 50% ethanol, 0.08 mol/L MgO, and 10% solid loading at 190 °C for 40 min. Under optimal conditions, 98% glucose and 92% xylose were recovered with 89% glucan and 71% xylan recoveries and 60% lignin removal. A total sugar yield of 63% on a received biomass basis after enzymatic hydrolysis was obtained with 78% glucose and 41% xylose yields. The resulting biomass slurry was near-neutral and free of furfural and 5-hydroxymethylfurfural. Thus, the process to isolate high-purity value-added lignin and recover sugars from biomass liquor can be largely simplified.

Enzymatic hydrolysis of brewer's spent grain to obtain fermentable sugars.

Lignocellulosic biomass is a feedstock with the potential to be converted into value-added bioproducts. The use of enzymatic hydrolysis allows the cleavage of lignocellulose into their monomeric units, but there are some drawbacks that make its use in industrial biocatalysis unfeasible. In the present study, we describe the hydrolysis of brewer's spent grain (BSG) with an enzymatic cocktail produced by Aspergillus niger CECT 2700 and its comparison with commercial enzymes. In addition, it was determined whether pretreating the BSG (non-pressurized alkaline hydrolysis or treatment with cholinium glycinate ionic liquid) is necessary. Results show that both pretreatments enhanced xylose release (10.55 ±â€¯0.07 g/L and 8.14 ±â€¯0.13 g/L respectively), meanwhile the hydrolysis of raw BSG with the enzymatic cocktail produced solutions containing high levels of glucose (18.45 ±â€¯1.66 g/L) and xylose (6.38 ±â€¯0.26 g/L).

Miscanthus straw as substrate for biosuccinic acid production: Focusing on pretreatment and downstream processing.

The main aim of this study was to optimize pretreatment strategies of Miscanthus × giganteus for biosuccinic acid production. A successful pretreatment with organosolv method (80% w/w of glycerol, 1.25% of H2SO4), prevented sugars conversion to furfurals and organic acids, and thereby resulted in high sugar recovery (glucan > 98%, xylan > 91%) and biomass delignification (60%). Pretreated biomass was subjected to hydrolysis with various cellulolytic enzyme cocktails (Viscozyme® L, Carezyme 1000L®, ß-Glucanase, Cellic® CTec2, Cellic® HTec2). The most effective enzymes mixture composed of Cellic® CTec2 (10% w/w), ß-Glucanase (5% w/w) and Cellic® HTec2 (1% w/w) resulted in high glucose (93.1%) and xylose (69.2%) yields after glycerol-based pretreatment. Succinic acid yield of 75-82% was obtained after hydrolysates fermentation, using Actinobacillus succinogenes 130Z. Finally a successful downstream concept for succinic acid purification was proposed. The succinic acid recovery with high purity (>98%) was developed.

Mild reaction conditions induce high sugar yields during the pretreatment of Agave tequilana bagasse with 1-ethyl-3-methylimidazolium acetate.

Sequential 2k factorial and central composite designs were used to optimize Agave tequilana bagasse (ATB) pretreatment by using 1-ethyl-3-methylimidazolium acetate ([Emim][OAc]). Reaction time, temperature and solids loading were the studied factors while sugar yield was the response variable. Results indicated that optimal conditions (119 °C, 142 min) using high solids loading (30%) were achieved at lower temperatures and reaction times than those previously reported in the literature. It was also revealed that solid recovery after pretreatment with [Emim][OAc] is a key factor. The increase in enzymatic digestibility of pretreated ATB was correlated to a decrease in crystallinity and lower lignin content as observed using microscopy techniques and weaken chemical bonds by Fourier transform infrared spectroscopy. Yields of glucose and xylose in the hydrolysate were 41.3, and 13.0 kg per 100 kg of untreated ATB, which are equivalent to glucan and xylan conversions of 75.9% and 82.9%, respectively.

GH-10 and GH-11 Endo-1,4-ß-xylanase enzymes from Kitasatospora sp. produce xylose and xylooligosaccharides from sugarcane bagasse with no xylose inhibition.

A novel strategy for the low-cost, high-yield co-production of xylose and xylooligosaccharides together with no xylose inhibition was developed using a novel heterologous expression of XYN10Ks_480 endo-1,4-ß-xylanase with a ricin-type ß-trefoil type of domain and XYN11Ks_480 endo-1,4-ß-xylanase with a CBM 2 superfamily from the Kitasatospora sp in an actinomycetes expression system. Xylose is the main building block for hemicellulose xylan. Our findings demonstrated high levels of expression and catalytic activity for XYN10Ks_480 during hydrolysis of the extracted xylan of bagasse, and three types of xylan-based substrates were used to produce xylose and xylooligosaccharides. However, hydrolysis by XYN11Ks_480 produced xylooligosaccharides without xylose formation. This study demonstrated how integrating sodium hypochlorite-extracted xylan and enzymatic hydrolysis could provide an alternative strategy for the generation of XOS from lignocellulosic material.

d-Xylose and l-arabinose laurate esters: Enzymatic synthesis, characterization and physico-chemical properties.

Efficient enzymatic synthesis of d-xylose and l-arabinose lauryl mono- and diesters has been achieved by transesterification reactions catalysed by immobilized Candida antarctica lipase B as biocatalyst, in organic medium in the presence of d-xylose or l-arabinose and vinyllaurate at 50 °C. In case of l-arabinose, one monoester and one diester were obtained in a 57% overall yield. A more complex mixture was produced for d-xylose as two monoesters and two diesters were synthesized in a 74.9% global yield. The structures of all these pentose laurate esters was solved. Results demonstrated that the esterification first occurred regioselectively onto the primary hydroxyl groups. Pentose laurate esters exhibited interesting features such as low critical aggregation concentrations values all inferior to 25 µM. Our study demonstrates that the enzymatic production of l-arabinose and d-xylose-based esters represents an interesting approach for the production of green surfactants from lignocellulosic biomass-derived pentoses.

[Fermentations of xylose and arabinose by Kluyveromyces marxianus].

Kluyveromyces marxianus, as unconventional yeast, attracts more and more attention in the biofuel fermentation. Although this sort of yeasts can ferment pentose sugars, the fermentation capacity differs largely. Xylose and arabinose fermentation by three K. marxianus strains (K. m 9009, K. m 1911 and K. m 1727) were compared at different temperatures. The results showed that the fermentation performance of the three strains had significant difference under different fermentation temperatures. Especially, the sugar consumption rate and alcohol yield of K. m 9009 and K. m 1727 at 40 ℃ were better than 30 ℃. This results fully reflect the fermentation advantages of K. marxianus yeast under high-temperature. On this basis, five genes (XR, XDH, XK, AR and LAD) coding key metabolic enzymes in three different yeasts were amplified by PCR, and the sequence were compared by Clustalx 2.1. The results showed that the amino acid sequences coding key enzymes have similarity of over 98% with the reference sequences reported in the literature. Furthermore, the difference of amino acid was not at the key site of its enzyme, so the differences between three stains were not caused by the gene level, but by transcribed or translation regulation level. By real-time PCR experiment, we determined the gene expression levels of four key enzymes (XR, XDH, XK and ADH) in the xylose metabolism pathway of K. m 1727 and K. m 1911 at different fermentation time points. The results showed that, for thermotolerant yeast K. m 1727, the low expression level of XDH and XK genes was the main factors leading to accumulation of xylitol. In addition, according to the pathway of Zygosaccharomyces bailii, which have been reported in NCBI and KEGG, the xylose and arabinose metabolic pathways of K. marxianus were identified, which laid foundation for further improving the pentose fermentation ability by metabolic engineering.

Production and Characteristics of a Novel Xylose- and Alkali-tolerant GH 43 ß-xylosidase from Penicillium oxalicum for Promoting Hemicellulose Degradation.

ß-xylosidase is a pivotal enzyme for complete degradation of xylan in hemicelluloses of lignocelluloses, and the xylose- and alkali-tolerant ß-xylosidase with high catalytic activity is very attractive for promoting enzymatic hydrolysis of alkaline-pretreated lignocellulose. In this study, a novel intracellular glycoside hydrolase family 43 ß-xylosidase gene (xyl43) from Penicillium oxalicum 114-2 was successfully high-level overexpressed in Pichia pastoris, and the secreted enzyme was characterized. The ß-xylosidase Xyl43 exhibited great pH stability and high catalytic activity in the range of pH 6.0 to 8.0, and high tolerance to xylose with the Ki value of 28.09 mM. The Xyl43 could effectively promote enzymatic degradation of different source of xylan and hemicellulose contained in alkaline-pretreated corn stover, and high conversion of xylan to xylose could be obtained.

Direct Ethanol Production from Ionic Liquid-Pretreated Lignocellulosic Biomass by Cellulase-Displaying Yeasts.

Among the many types of lignocellulosic biomass pretreatment methods, the use of ionic liquids (ILs) is regarded as one of the most promising strategies. In this study, the effects of four kinds of ILs for pretreatment of lignocellulosic biomass such as bagasse, eucalyptus, and cedar were evaluated. In direct ethanol fermentation from biomass incorporated with ILs by cellulase-displaying yeast, 1-butyl-3-methylimidazolium acetate ([Bmim][OAc]) was the most effective IL. The ethanol production and yield from [Bmim][OAc]-pretreated bagasse reached 0.81 g/L and 73.4% of the theoretical yield after fermentation for 96 h. The results prove the initial concept, in which the direct fermentation from lignocellulosic biomass effectively promoted by the pretreatment with IL.

The Influence of Sugar Cane Bagasse Type and Its Particle Size on Xylose Production and Xylose-to-Xylitol Bioconversion with the Yeast Debaryomyces hansenii.

In the present study, the effect of the type of sugar cane bagasse (non-depithed or depithed) and its particle size on the production of xylose and its subsequent fermentation to xylitol by Debaryomyces hansenii CBS767 was investigated using a full factorial experimental design. It was found that the particle size range and whether bagasse was depithed or not had a significant effect on the concentration and yield of xylose in the resulting hemicellulose hydrolysate. Depithed bagasse resulted in higher xylose concentrations compared to non-depithed bagasse. The corresponding detoxified hemicellulose hydrolysates were used as fermentation media for the production of xylitol. The hemicellulose hydrolysate prepared from depithed bagasse also yielded meaningfully higher xylitol fermentation rates compared to non-depithed bagasse. However, in the case of non-depithed bagasse, the hemicellulose hydrolysate prepared from larger particle size range resulted in higher xylitol fermentation rates, whereas the effect in the case of non-depithed bagasse was not pronounced. Therefore, depithing of bagasse is an advantageous pretreatment when it is to be employed in bioconversion processes.

Molecular Characterization of Xylobiose- and Xylopentaose-Producing ß-1,4-Endoxylanase SCO5931 from Streptomyces coelicolor A3(2).

Streptomyces coelicolor A3(2) sco5931 gene was predicted to encode a putative xylanase A, a 477 amino acid protein belonging to glycoside hydrolase family 10. The entire sco5931 coding region was cloned and overexpressed in Streptomyces lividans TK24. Mature SCO5931 protein comprising 436 amino acids (47 kDa) was purified by single-step gel filtration chromatography from culture broth after ammonium sulfate precipitation, with 25.8-fold purification and yield of 30.6 %. The purified protein displayed a pronounced activity toward beechwood xylan as a substrate, but no activity was detected toward carboxymethylcellulose, Avicel, galactan, barley ß-glucan, and xyloglucan, demonstrating that SCO5931 is a substrate-specific xylanase. Optimal xylanase activity was observed at 60 °C and pH 6.0. The addition of metal ions or EDTA did not affect the xylanase activity, while 4 mM MnCl2 severely inhibited the enzyme, reducing its activity by 87 %. Kinetic parameters of SCO5931 toward beechwood xylan were determined (K m = 0.24 mg/mL, V max = 6.86 µM/min). Thin layer chromatography and mass spectrometry analyses of the beechwood xylan SCO5931 hydrolysis products were conducted. Product masses corresponded to sodium adducts of xylobiose (m/z 305.24) and xylopentaose (m/z 701.59), indicating that SCO5931 specifically cleaves the ß-1,4 linkage of xylan to yield xylobiose and xylopentaose.

Pretreatment of Dried Distiller Grains with Solubles by Soaking in Aqueous Ammonia and Subsequent Enzymatic/Dilute Acid Hydrolysis to Produce Fermentable Sugars.

Dried distillers grains with solubles (DDGS), a co-product of corn ethanol production in the dry-grind process, was pretreated by soaking in aqueous ammonia (SAA) using a 15 % w/w NH4OH solution at a solid/liquid ratio of 1:10. The effect of pretreatment on subsequent enzymatic hydrolysis was studied at two temperatures (40 and 60 °C) and four reaction times (6, 12, 24, and 48 h). Highest glucose yield of 91 % theoretical was obtained for the DDGS pretreated at 60 °C and 24 h. The solubilized hemicellulose in the liquid fraction was further hydrolyzed with dilute H2SO4 to generate fermentable monomeric sugars. The conditions of acid hydrolysis included 1 and 4 wt% acid, 60 and 120 °C, and 0.5 and 1 h. Highest yields of xylose and arabinose were obtained at 4 wt% acid, 120 °C, and 1 h. The fermentability of the hydrolysate obtained by enzymatic hydrolysis of the SAA-pretreated DDGS was demonstrated in ethanol fermentation by Saccharomyces cerevisiae. The fermentability of the hydrolysate obtained by consecutive enzymatic and dilute acid hydrolysis was demonstrated using a succinic acid-producing microorganism, strain Escherichia coli AFP184. Under the fermentation conditions, complete utilization of glucose and arabinose was observed, whereas only 47 % of xylose was used. The succinic acid yield was 0.60 g/g total sugar consumed.

Synthetic pathway optimization for improved 1,2,4-butanetriol production.

1,2,4-Butanetriol (BT) is an important non-natural chemical with a variety of industrial applications. Identifying the bottlenecks for BT biosynthesis is expected to contribute to improving the efficiency of this process. In this work, we first constructed a prototype strain for BT production by assembling a four-step synthetic pathway and disrupting the competing pathways for xylose in Escherichia coli BW25113. Using this prototype strain, we conducted systematic fine-tuning of the pathway enzyme expression level to identify the potential bottlenecks and optimize the BT biosynthesis. Production conditions were also optimized by exploring the effects of temperature, pH and cell density on BT titer. BT production was increased by 4.3-fold from the prototype strain, achieved a final titer of 1.58 g/L with a yield of 7.9% after 72-h biotransformation.

Dilute oxalic acid pretreatment for high total sugar recovery in pretreatment and subsequent enzymatic hydrolysis.

Oxalic acid was evaluated as an alternative reagent to mineral inorganic acid in pretreatment of corncob to achieve high xylose yield in addition to highly digestible solid residue. A quadratic polynomial model of xylose formation was developed for optimization of pretreatment process by the response surface methodology based on the impact factors of pretreatment temperature, reaction time, acid concentration, and solid-to-liquid ratio. The highest xylose yield was 94.3 % that was obtained under the pretreatment condition of 140 °C for 40 min with 0.5 wt% oxalic acid at a solid loading of 7.5 %. Under these conditions, the xylose yield results of verification experiments were very close to the model prediction, which indicated that the model was applicable. The solid residue generated under this condition also demonstrated a satisfactory enzymatic digestibility and fermentability.

Xylose production from corn stover biomass by steam explosion combined with enzymatic digestibility.

A novel conversion process using steam explosion combined with enzymatic digestibility was exploited to increase sugar yield. Results showed that glucan and xylan recovery decreased with the increase of holding temperature and residence time in SE, respectively, while glucan and xylan conversion exhibited an opposite trend. The optimal conditions of steam explosion were 160 °C and 48 min, under which glucan and xylan recovery was 93.4% and 71.6%, respectively. Glucan and xylan conversion at 18% solid loading by periodic peristalsis increased by 3.4-5.8% and 4.5-6.2%, respectively, compared with that by water baths shaker. In the whole process, glucose, xylose and total sugar yield reached to 77.3%, 62.8% and 72.3%, respectively. The yield of hydroxymethyl furfural, furfural and lignin-derived products was 6.3 × 10(-2), 7.5 × 10(-2) and less than 3.7 × 10(-2) g/100 g feedstock, respectively. This novel conversion process increased sugar recovery, reduced degradation products formation, improved digestibility efficiency, and hence increased sugar yield.