Glucose Isomerase Research Articles

Highly efficient rDNA-mediated multicopy integration based on the dynamic balance of rDNA in Saccharomyces cerevisiae.

SummaryEngineered Saccharomyces cerevisiae strains are good cell factories, and developing additional genetic manipulation tools will accelerate construction of metabolically engineered strains. Highly repetitive rDNA sequence is one of two main sites typically used for multicopy integration of genes. Here, we developed a simple and high‐efficiency strategy for rDNA‐mediated multicopy gene integration based on the dynamic balance of rDNA in S. cerevisiae. rDNA copy number was decreased by pre‐treatment with hydroxyurea (HU). Then, heterologous genes were integrated into the rDNA sequence. The copy number of the integrated heterologous genes increased along with restoration of the copy number of rDNA. Our results demonstrated that HU pre‐treatment doubled the number of integrated gene copies; moreover, compared with removing HU stress during transformation, removing HU stress after selection of transformants had a higher probability of resulting in transformants with high‐copy integrated genes. Finally, we integrated 18.0 copies of the xylose isomerase gene into the S. cerevisiae genome in a single step. This novel rDNA‐mediated multicopy genome integration strategy provides a convenient and efficient tool for further metabolic engineering of S. cerevisiae.

Precipitant-Free Crystallization of Lysozyme and Glucose Isomerase by Drying

Protein crystallization is usually conducted by using precipitants, although the “salting-out” phenomenon is still unclear and complex. Moreover, the addition of precipitants sometimes results in irreversible disordered precipitation of protein molecules. Although precipitant-free lysozyme crystals obtained by centrifugal concentration showed significant changes in three-dimensional structure compared to the structure of salted-out crystals, it was rather difficult to mount crystals from a viscous dense liquid phase after centrifugal concentration, and the quality of the crystals often deteriorated during the mounting process. Here we present novel precipitant-free crystallization methods, which were effective for lysozyme and glucose isomerase. Tetragonal lysozyme crystals were successfully crystallized in a glass capillary simply by drying highly concentrated lysozyme solution in the presence of 0.01 M hydrochloric acid without using any precipitants. Glucose isomerase dissolved in ultra-pure water was also successfully crystallized in hanging drops by drying highly concentrated solution under low-humidity conditions. Oscillation images of the obtained crystals were safely collected without handling; they clearly indicated the crystals had a tetragonal form for lysozyme and an orthorhombic form for glucose isomerase, and their lattice parameters are similar to those of previously reported crystals obtained by salting-out methods.

Beef tallow injection matrix for serial crystallography

Serial crystallography (SX) enables the visualization of the time-resolved molecular dynamics of macromolecular structures at room temperature while minimizing radiation damage. In SX experiments, the delivery of a large number of crystals into an X-ray interaction point in a serial and stable manner is key. Sample delivery using viscous medium maintains the stable injection stream at low flow rates, markedly reducing sample consumption compared with that of a liquid jet injector and is widely applied in SX experiments with low repetition rates. As the sample properties and experimental environment can affect the stability of the injection stream of a viscous medium, it is important to develop sample delivery media with various characteristics to optimize the experimental environment. In this study, a beef tallow injection matrix possessing a higher melting temperature than previously reported fat-based shortening and lard media was introduced as a sample delivery medium and applied to SX. Beef tallow was prepared by heat treating fats from cattle, followed by the removal of soluble impurities from the extract by phase separation. Beef tallow exhibited a very stable injection stream at room temperature and a flow rate of < 10 nL/min. The room-temperature structures of lysozyme and glucose isomerase embedded in beef tallow were successfully determined at 1.55 and 1.60 Å, respectively. The background scattering of beef tallow was higher than that of previously reported fat-based shortening and lard media but negligible for data processing. In conclusion, the beef tallow matrix can be employed for sample delivery in SX experiments conducted at temperatures exceeding room temperature.

Glucose Isomerase: Functions, Structures, and Applications

Glucose isomerase (GI, also known as xylose isomerase) reversibly isomerizes D-glucose and D-xylose to D-fructose and D-xylulose, respectively. GI plays an important role in sugar metabolism, fulfilling nutritional requirements in bacteria. In addition, GI is an important industrial enzyme for the production of high-fructose corn syrup and bioethanol. This review introduces the functions, structure, and applications of GI, in addition to presenting updated information on the characteristics of newly discovered GIs and structural information regarding the metal-binding active site of GI and its interaction with the inhibitor xylitol. This review provides an overview of recent advancements in the characterization and engineering of GI, as well as its industrial applications, and will help to guide future research in this field.

Establishment of a carbon-efficient xylulose cleavage pathway in Escherichia coli to metabolize xylose

As the second-largest storage saccharide in the world, xylose is a promising renewable feedstock in the bio-industry. A few microorganisms can metabolize xylose naturally via the xylose isomerase pathway, Weimberg pathway, or Dahms pathway. In this study, we reported the metabolic engineering of Escherichia coli to achieve the carbon-efficient utilization of xylose through the establishment of a rationally designed xylulose cleavage pathway. With the inactivation of ribulose-phosphate 3-epimerase and expression of exogenous phosphoketolase, the engineered E. coli accumulated acetate as a major byproduct using xylose as the sole carbon source. Furtherly, acetate production pathways were successfully blocked to provide sufficient acetyl-CoA precursor for downstream biosynthesis reactions. Finally, we applied the xylulose cleavage pathway to produce poly-3-hydroxybutyrate. Our proposed metabolic engineering strategies illustrate the feasibility of efficient xylose utilization to produce commodity chemicals.

Characterization of D-xylose isomerase from Shinella zoogloeoides NN6 and its application for producing D-allulose and two D-ketopentoses in a one-pot multi-step transformation.

Researchers continue to search for efficient processes to reduce the production costs of rare sugars. In this paper, we report a novel D-xylose isomerase from Shinella zoogloeoides NN6 (SzXI) and its application for efficient rare sugar production. Purified SzXI did not show remarkable properties when compared with those of a previously reported D-xylose isomerase. However, NN6 was found to express inducible SzXI and constitutive D-allulose 3-epimerase (SzAE) when cultivated with D-xylose as the sole carbon source. These two enzymes were partially purified and immobilized onto HPA25L, an anion exchange resin. The co-immobilized SzXI and SzAE (i-XA) showed optimal activity at 65°C in sodium phosphate buffer (pH 7.5) and 90°C in sodium phosphate buffer (pH 6.5), respectively. i-XA produced D-ribulose via D-xylulose from D-xylose at a conversion ratio of D-xylose:D-xylulose:D-ribulose of 72:18:10. Furthermore, D-allulose was also produced via D-fructose using D-glucose as the substrate, with a D-allulose yield of 11.2%. This is the first report describing a bacterium expressing D-xylose isomerase and D-allulose 3-epimerase that converts readily available sugars such as D-glucose and D-xylose to rare sugars.

Nanomolar Responsiveness of Marine Phaeobacter inhibens DSM 17395 toward Carbohydrates and Amino Acids

Phaeobacter inhibens DSM 17395 is a heterotrophic member of the ubiquitous, marine Roseobacter group and specializes in the aerobic utilization of carbohydrates and amino acids via pathways widespread among roseobacters. The in vivo responsiveness of P. inhibens DSM 17395 was studied with nonadapted cells (succinate-grown), which were exposed to a single pulse (100–0.01 µM) each of N-acetylglucosamine, mannitol, xylose, leucine, phenylalanine, or tryptophan (effectors). Responsiveness was then determined by time-resolved transcript analyses (quantitative reverse transcription-PCR) of “degradation” and “uptake” genes selected based on previously reported substrate-specific proteome profiles. The transcriptional response thresholds were: 50–100 nM for nagK (N-acetylglucosamine kinase), paaA (ring 1,2-phenylacetyl-CoA epoxidase), and kynA (tryptophan 2,3-dioxygenase), 10–50 nM for xylA (xylose isomerase), and around 10 nM for mtlK (mannitol 2-dehydrogenase). A threshold for leucine could not be determined due to the elevated intrinsic presence of leucine in the exometabolome of succinate-grown cells (no effector addition). Notably, the response thresholds for presumptive carbohydrate-binding proteins of ABC-transporters were in the same range or even lower: 0.1–1 µM for c27930 (N-acetylglucosamine) and even below 10 nM for c13210 (mannitol) and xylF (xylose). These results shed new light on the sensory/regulatory sensitivity of a well-studied roseobacter for recognizing potential substrates at low ambient concentrations and on the concentration threshold below which these might escape biodegradation (“emergent recalcitrance” concept of dissolved organic matter persistence).

Relaxed control of sugar utilization in Parageobacillus thermoglucosidasius DSM 2542

Though carbon catabolite repression (CCR) has been intensively studied in some more characterised organisms, there is a lack of information of CCR in thermophiles. In this work, CCR in the thermophile, Parageobacillus thermoglucosidasius DSM 2542 has been studied during growth on pentose sugars in the presence of glucose. Physiological studies under fermentative conditions revealed a loosely controlled CCR when DSM 2542 was grown in minimal medium supplemented with a mixture of glucose and xylose. This atypical CCR pattern was also confirmed by studying xylose isomerase expression level by qRT-PCR. Fortuitously, the pheB gene, which encodes catechol 2, 3-dioxygenase was found to have a cre site highly similar to the consensus catabolite-responsive element (cre) at its 3′ end and was used to confirm that expression of pheB from a plasmid was under stringent CCR control. Bioinformatic analysis suggested that the CCR regulation of xylose metabolism in P. thermoglucosidasius DSM 2542 might occur primarily via control of expression of pentose transporter operons. Relaxed control of sugar utilization might reflect a lower affinity of the CcpA-HPr (Ser46-P) or CcpA-Crh (Ser46-P) complexes to the cre(s) in these operons.

Development and characterization of efficient xylose utilization strains of Zymomonas mobilis

BackgroundEfficient use of glucose and xylose is a key for the economic production of lignocellulosic biofuels and biochemicals, and different recombinant strains have been constructed for xylose utilization including those using Zymomonas mobilis as the host. However, the xylose utilization efficiency still needs to be improved. In this work, the strategy of combining metabolic engineering and adaptive laboratory evolution (ALE) was employed to develop recombinant Z. mobilis strains that can utilize xylose efficiently at high concentrations, and NGS-based genome resequencing and RNA-Seq transcriptomics were performed for strains evolved after serial transfers in different media to understand the impact of xylose and differences among strains with different xylose-utilization capabilities at molecular level.ResultsHeterologous genes encoding xylose isomerase and xylulokinase were evaluated, which were then introduced into xylose-utilizing strain Z. mobilis 8b to enhance its capacity of xylose utilization. The results demonstrated that the effect of three xylose isomerases on xylose utilization was different, and the increase of copy number of xylose metabolism genes can improve xylose utilization. Among various recombinant strains constructed, the xylose utilization capacity of the recombinant strain 8b-RsXI-xylB was the best, which was further improved through continuous adaption with 38 transfers over 100 days in 50 g/L xylose media. The fermentation performances of the parental strain 8b, the evolved 8b-S38 strain with the best xylose utilization capability, and the intermediate strain 8b-S8 in different media were compared, and the results showed that only 8b-S38 could completely consume xylose at 50 g/L and 100 g/L concentrations. In addition, the xylose consumption rate of 8b-S38 was faster than that of 8b at different xylose concentrations from 50 to 150 g/L, and the ethanol yield increased by 16 ~ 40%, respectively. The results of the mixed-sugar fermentation also demonstrated that 8b-S38 had a higher xylose consumption rate than 8b, and its maximum ethanol productivity was 1.2 ~ 1.4 times higher than that of 8b and 8b-S8. Whole-genome resequencing identified three common genetic changes in 8b-S38 compared with 8b and 8b-S8. RNA-Seq study demonstrated that the expression levels of genes encoding chaperone proteins, ATP-dependent proteases, phage shock proteins, ribosomal proteins, flagellar operons, and transcriptional regulators were significantly increased in xylose media in 8b-S38. The up-regulated expression of these genes may therefore contribute to the efficient xylose utilization of 8b-S38 by maintaining the normal cell metabolism and growth, repairing cellular damages, and rebalancing cellular energy to help cells resist the stressful environment.ConclusionsThis study provides gene candidates to improve xylose utilization, and the result of expressing an extra copy of xylose isomerase and xylulokinase improved xylose utilization also provides a direction for efficient xylose-utilization strain development in other microorganisms. In addition, this study demonstrated the necessity to combine metabolic engineering and ALE for industrial strain development. The recombinant strain 8b-S38 can efficiently metabolize xylose for ethanol fermentation at high xylose concentrations as well as in mixed sugars of glucose and xylose, which could be further developed as the microbial biocatalyst for the production of lignocellulosic biofuels and biochemicals.

Efficient anaerobic consumption of D-xylose by E. coli BL21(DE3) via xylR adaptive mutation

BackgroundMicroorganisms can prioritize the uptake of different sugars depending on their metabolic needs and preferences. When both D-glucose and D-xylose are present in growth media, E. coli cells typically consume D-glucose first and then D-xylose. Similarly, when E. coli BL21(DE3) is provided with both D-glucose and D-xylose under anaerobic conditions, glucose is consumed first, whereas D-xylose is consumed very slowly.ResultsWhen BL21(DE3) was adaptively evolved via subculture, the consumption rate of D-xylose increased gradually. Strains JH001 and JH019, whose D-xylose consumption rate was faster, were isolated after subculture. Genome analysis of the JH001 and JH019 strains revealed that C91A (Q31K) and C740T (A247V) missense mutations in the xylR gene (which encodes the XylR transcriptional activator), respectively, controlled the expression of the xyl operon. RT-qPCR analyses demonstrated that the XylR mutation caused a 10.9-fold and 3.5-fold increase in the expression of the xylA (xylose isomerase) and xylF (xylose transporter) genes, respectively, in the adaptively evolved JH001 and JH019 strains. A C91A adaptive mutation was introduced into a new BL21(DE3) background via single-base genome editing, resulting in immediate and efficient D-xylose consumption.ConclusionsAnaerobically-adapted BL21(DE3) cells were obtained through short-term adaptive evolution and xylR mutations responsible for faster D-xylose consumption were identified, which may aid in the improvement of microbial fermentation technology.

Protein acetylation regulates xylose metabolism during adaptation of Saccharomyces cerevisiae

BackgroundAs the second most abundant polysaccharide in nature, hemicellulose can be degraded to xylose as the feedstock for bioconversion to fuels and chemicals. To enhance xylose conversion, the engineered Saccharomyces cerevisiae with xylose metabolic pathway is usually adapted with xylose as the carbon source in the laboratory. However, the mechanism under the adaptation phenomena of the engineered strain is still unclear.ResultsIn this study, xylose-utilizing S. cerevisiae was constructed and used for the adaptation study. It was found that xylose consumption rate increased 1.24-fold in the second incubation of the yYST12 strain in synthetic complete-xylose medium compared with the first incubation. The study figured out that it was observed at the single-cell level that the stagnation time for xylose utilization was reduced after adaptation with xylose medium in the microfluidic device. Such transient memory of xylose metabolism after adaptation with xylose medium, named “xylose consumption memory”, was observed in the strains with both xylose isomerase pathway and xylose reductase and xylitol dehydrogenase pathways. In further, the proteomic acetylation of the strains before and after adaptation was investigated, and it was revealed that H4K5 was one of the most differential acetylation sites related to xylose consumption memory of engineered S. cerevisiae. We tested 8 genes encoding acetylase or deacetylase, and it was found that the knockout of the GCN5 and HPA2 encoding acetylases enhanced the xylose consumption memory.ConclusionsThe behavior of xylose consumption memory in engineered S. cerevisiae can be successfully induced with xylose in the adaptation. H4K5Ac and two genes of GCN5 and HPA2 are related to xylose consumption memory of engineered S. cerevisiae during adaptation. This study provides valuable insights into the xylose adaptation of engineered S. cerevisiae.

Directed evolution and secretory expression of xylose isomerase for improved utilisation of xylose in Saccharomyces cerevisiae

BackgroundXylose contained in lignocellulosic biomass is an attractive carbon substrate for economically viable conversion to bioethanol. Extensive research has been conducted on xylose fermentation using recombinant Saccharomyces cerevisiae expressing xylose isomerase (XI) and xylose reductase/xylitol dehydrogenase (XR/XDH) pathways along with the introduction of a xylose transporter and amplification of the downstream pathway. However, the low utilization of xylose in the presence of glucose, due to the varying preference for cellular uptake, is a lingering challenge. Studies so far have mainly focused on xylose utilization inside the cells, but there have been little trials on the conversion of xylose to xylulose by cell before uptake. We hypothesized that the extracellular conversion of xylose to xylulose before uptake would facilitate better utilization of xylose even in the presence of glucose. To verify this, XI from Piromyces sp. was engineered and hyper-secreted in S. cerevisiae for the extracellular conversion of xylose to xylulose.ResultsThe optimal pH of XI was lowered from 7.0 to 5.0 by directed evolution to ensure its high activity under the acidic conditions used for yeast fermentation, and hyper-secretion of an engineered XI-76 mutant (E56A and I252M) was accomplished by employing target protein-specific translational fusion partners. The purified XI-76 showed twofold higher activity than that of the wild type at pH 5. The secretory expression of XI-76 in the previously developed xylose utilizing yeast strain, SR8 increased xylose consumption and ethanol production by approximately 7–20% and 15–20% in xylose fermentation and glucose and xylose co-fermentation, respectively.ConclusionsIsomerisation of xylose to xylulose before uptake using extracellular XI was found to be effective in xylose fermentation or glucose/xylose co-fermentation. This suggested that glucose competed less with xylulose than with xylose for uptake by the cell. Consequently, the engineered XI secretion system constructed in this study can pave the way for simultaneous utilization of C5/C6 sugars from the sustainable lignocellulosic biomass.

Production of Glucose Isomerase from Streptomyces roseiscleroticus

This study was aimed at the isolation and characterization of a microbial strain capable of producing glucose isomerase. Microbial strain was isolated from soil using starch casein agar as a differential media. The isolated microbial strain was capable of producing glucose isomerase which was tested using 2, 3, 5 - triphenyltetrazolium solution as a chromogenic substrate. The microbial strain was identified as Streptomyces species based on its morphological and microscopic characteristics. It was further subjected to molecular characterization using 16S rRNA sequencing and was subsequently confirmed as Streptomyces roseiscleroticus. Glucose isomerase was produced from Streptomyces roseiscleroticus after 120 hr of submerged fermentation at pH 6.8 and at 37°C utilizing xylose as the sole carbon source and a compendium of peptone, beef and yeast extracts as nitrogen sources. These findings suggest that the microbial strain, Streptomyces roseiscleroticus can be a useful bacterial source for the production of glucose isomerase needed for commercial and industrial utilization.

KINETIC STUDY COMPARISON OF IMMOBILIZED GLUCOSE ISOMERASE (GENSWEET AND SWEETZYME IT) IN STIRRED TANK REACTOR AND PACKED BED REACTOR

D- Glucose/xylose isomerase catalysis the reversible isomerization of aldoses to ketoses such as D-glucose and D-xylose to D-fructose and D-xylose respectively. High fructose corn syrup (HFCS), a low calorie sugar substitute for cane sugar, utilizes Glucose isomerase enzyme for conversion of glucose to fructose. The conversion of glucose to fructose favors more at high temperature, providing an incentive to utilize thermostable and thermoactive glucose isomerase in High fructose corn syrup (HFCS) production. Present studies emphasize on enzymatic conversion and optimization using Sweetzyme IT extra &amp; Gensweet, commercially available glucose isomerases. The experiments were carried out for enzymatic conversion of glucose to fructose using Gensweet and Sweetzyme in Packed bed reactor (PBR) and Stirred tank reactor (STR). Maximum conversion was seen in Stirred tank reactor (STR) using both of these enzymes, approx 10 % more Fructose conversion comparing it to packed bed reactor (PBR). Also, Stirred tank reactor (STR) reaction conditions such as pH, buffers, cofactor (MgSO4) requirement were optimized to achieve optimum enzyme activity. Analysis of enzymatic conversion samples was done using HPLC-RID (using Zorbax Column). The importance of the divalent cation MgCl2 for optimal enzyme activity was investigated. The enzyme performed best at pH 7.5 and 60°C, using 10mM MgSO4 as a cofactor. Utilizing Gensweet in Stirred tank reactor (STR), the maximum fructose transformation was 44 %. The most activity was detected with Sodium phosphate buffers, and EPPS buffers at pH 7 and 8, accordingly, whereas the least activity was reported with TRIS HCl buffer.

Simultaneous saccharification isomerization and Co-fermentation – SSICF: A new process concept for second-generation ethanol biorefineries combining immobilized recombinant enzymes and non-GMO Saccharomyces

Integrated bioprocess strategies may facilitate ethanol production from both C6 and C5 fractions of lignocellulosic feedstocks. We propose a new process concept, SSICF, where sugarcane bagasse is hydrolyzed simultaneously with xylose isomerization and the co-fermentation of C6 and C5 sugars. A commercial cocktail was supplemented with our multi-enzymatic system composed of three recombinant enzymes immobilized in Feroxyhyte magnetic nanoparticles: β-glucosidase, β-xylosidase and xylose isomerase. SSICF was performed using non-GMO Saccharomyces at pH 6.0 and 35 °C for 72 h in a synthetic medium containing cellobiose and xylose, and another medium containing pretreated sugarcane bagasse (PSB). The results of ethanol global yields in SSICF were 77.67% and 73.24% for the synthetic medium and PSB, respectively. In a nutshell, this is the first report of a successful proof-of-concept of SSICF with four rounds of enzyme recycling and a non-GMO yeast, an innovative process with high potential for industrial use.

Enhanced catalytic efficiency and thermostability of glucose isomerase from Thermoanaerobacter ethanolicus via site-directed mutagenesis

Glucose isomerase (GI) is a key enzyme in the preparation of high fructose corn syrup (HFCS). In this study, a mutant TEGI-M-L38 M/V137 L (TEGI-M2) of glucose isomerase (TEGI-M) originated from Thermoanaerobacter ethanalicus CCSD1 was obtained by site-directed mutagenesis. The TEGI-M2 showed an optimal activity at 85 ℃ and pH 6.5 with the divalent cations Co2+ and Mg2+. The structural differences between TEGI-M and TEGI-M2 were investigated based on the homology modeling and molecular docking, to elucidate the mechanism of improvement in the enzymatic properties. Compared with the original enzyme, the TEGI-M2 showed a 2.0-fold increased enzyme activity and a decreased Km from 234.2 mM to 85.9 mM. Finally, the application of mutant TEGI-M2 in HFCS one-step biosynthesis was attempted, resulting in a d-fructose yield of 67.3 %, which was 14.3 % higher than that of TEGI-M. This improved catalytic performance of TEGI-M2 was of great importance for the industrial preparation of d-fructose in one-step process.

Catalyst Replacement Policy on Multienzymatic Systems: Theoretical Study in the One-Pot Sequential Batch Production of Lactofructose Syrup

One-pot systems are an interesting proposal to carry out multi-enzymatic reactions, though this strategy implies establishing an optimal balance between the activity and operability of the involved enzymes. This is crucial for enzymes with marked differences in their operational stability, such as one-pot production of lactofructose syrup from cheese whey permeate, which involves two enzymes—β-galactosidase (β-gal) and glucose isomerase (GI). The aim of this work was to study the behavior of one-pot sequential batch production of lactofructose syrup considering both enzymes immobilized individually, in order to evaluate and design a strategy of replacement of the catalysts according to their stabilities. To this end, the modelling and simulation of the process was carried out, considering simultaneously the kinetics of both reactions and the kinetics of inactivation of both enzymes. For the latter, it was also considered the modulating effect that sugars present in the medium may have on the stability of β-gal, which is the less stable enzyme. At the simulated reaction conditions of 40 °C, pH 7, and 0.46 (IUGI/IUβ-gal), the results showed that considering the stability of β-gal under non-reactive conditions, meaning in absence of the effect of modulation, it is necessary to carry out four replacements of β-gal for each cycle of use of GI. On the other hand, when considering the modulation caused by the sugars on the β-gal stability, the productivity increases up to 23% in the case of the highest modulation factor studied (η = 0.8). This work shows the feasibility of conducting a one-pot operation with immobilized enzymes of quite different operational stability, and that a proper strategy of biocatalyst replacement increases the productivity of the process.

Study the Behavior of Activity and Express for Enzymes

Enzymes are be defined as soluble, colloidal, organic catalysts which are produced by living cells.Enzymes are biologic catalysts to accelerate the rate of biochemical reactions.in this study Quantification of the Enzyme Activity, Where: E = enzyme concentration; S = substrateconcentration; ES = enzyme-substrate complex concentration; k1, k3= 1st order reaction rate constants(t-1); P = product concentration; k2= 2nd reaction rate constant (M-1.t-1).At first, it must be stressed thatthe formation of ES (enzyme-substrate complex) is an obligatory step to any type of enzyme-catalyzedreaction. The ES, depending on the reaction conditions, can form the product (forward reaction) ornot (reward reaction). The immobilization technique allowed a more frequent use of biomaterials inindustry, therapeutics (confection of subcutaneous capsules for controlled delivering of hormones), andlab analysis (automatic dosing equipments, enzyme electrode, biosensors and enzyme immunoassays).In industry, the use of glucose isomerase for the conversion of glucose into fructose (high fructosecorn syrup), the aminoacylase for the separation of racemic mixture of amino acids, microbial lipase intriglyceride hydrolysis, and the lactase for removing lactose from milk and/or whey are stressed.

Xylose Metabolism in Bacteria—Opportunities and Challenges towards Efficient Lignocellulosic Biomass-Based Biorefineries

In a sustainable society based on circular economy, the use of waste lignocellulosic biomass (LB) as feedstock for biorefineries is a promising solution, since LB is the world’s most abundant renewable and non-edible raw material. LB is available as a by-product from agricultural and forestry processes, and its main components are cellulose, hemicellulose, and lignin. Following suitable physical, enzymatic, and chemical steps, the different fractions can be processed and/or converted to value-added products such as fuels and biochemicals used in several branches of industry through the implementation of the biorefinery concept. Upon hydrolysis, the carbohydrate-rich fraction may comprise several simple sugars (e.g., glucose, xylose, arabinose, and mannose) that can then be fed to fermentation units. Unlike pentoses, glucose and other hexoses are readily processed by microorganisms. Some wild-type and genetically modified bacteria can metabolize xylose through three different main pathways of metabolism: xylose isomerase pathway, oxidoreductase pathway, and non-phosphorylative pathway (including Weimberg and Dahms pathways). Two of the commercially interesting intermediates of these pathways are xylitol and xylonic acid, which can accumulate in the medium either through manipulation of the culture conditions or through genetic modification of the bacteria. This paper provides a state-of-the art perspective regarding the current knowledge on xylose transport and metabolism in bacteria as well as envisaged strategies to further increase xylose conversion into valuable products.

Synthesis of aromatic amino acids from 2G lignocellulosic substrates.

SummaryPseudomonas putida is a highly solvent‐resistant microorganism and useful chassis for the production of value‐added compounds from lignocellulosic residues, in particular aromatic compounds that are made from phenylalanine. The use of these agricultural residues requires a two‐step treatment to release the components of the polysaccharides of cellulose and hemicellulose as monomeric sugars, the most abundant monomers being glucose and xylose. Pan‐genomic studies have shown that Pseudomonas putida metabolizes glucose through three convergent pathways to yield 6‐phosphogluconate and subsequently metabolizes it through the Entner–Doudoroff pathway, but the strains do not degrade xylose. The valorization of both sugars is critical from the point of view of economic viability of the process. For this reason, a P. putida strain was endowed with the ability to metabolize xylose via the xylose isomerase pathway, by incorporating heterologous catabolic genes that convert this C5 sugar into intermediates of the pentose phosphate cycle. In addition, the open reading frame T1E_2822, encoding glucose dehydrogenase, was knocked‐out to avoid the production of the dead‐end product xylonate. We generated a set of DOT‐T1E‐derived strains that metabolized glucose and xylose simultaneously in culture medium and that reached high cell density with generation times of around 100 min with glucose and around 300 min with xylose. The strains grew in 2G hydrolysates from diluted acid and steam explosion pretreated corn stover and sugarcane straw. During growth, the strains metabolized > 98% of glucose, > 96% xylose and > 85% acetic acid. In 2G hydrolysates P. putida 5PL, a DOT‐T1E derivative strain that carries up to five independent mutations to avoid phenylalanine metabolism, accumulated this amino acid in the medium. We constructed P. putida 5PLΔgcd (xylABE) that produced up to 250 mg l−1 of phenylalanine when grown in 2G pretreated corn stover or sugarcane straw. These results support as a proof of concept the potential of P. putida as a chassis for 2G processes.