The metal cofactor: stationary or mobile?

Metal cofactors are essential for catalysis and enable countless conversions in nature. Interestingly, the metal cofactor is not always static but mobile with movements of more than 4 Å. These movements of the metal can have different functions. In the case of the xylose isomerase and medium-chain dehydrogenases, it clearly serves a catalytic purpose. The metal cofactor moves during substrate activation and even during the catalytic turnover. On the other hand, in class II aldolases, the enzymes display resting states and active states depending on the movement of the catalytic metal cofactor. This movement is caused by substrate docking, causing the metal cofactor to take the position essential for catalysis. As these metal movements are found in structurally and mechanistically unrelated enzymes, it has to be expected that this metal movement is more common than currently perceived. KEY POINTS: • Metal ions are essential cofactors that can move during catalysis. • In class II aldolases, the metal cofactors can reside in a resting state and an active state. • In MDR, the movement of the metal cofactor is essential for substrate docking.

The immune response to RNA suppresses nucleic acid synthesis by limiting ribose 5-phosphate.

During infection viruses hijack host cell metabolism to promote their replication. Here, analysis of metabolite alterations in macrophages exposed to poly I:C recognises that the antiviral effector Protein Kinase RNA-activated (PKR) suppresses glucose breakdown within the pentose phosphate pathway (PPP). This pathway runs parallel to central glycolysis and is critical to producing NADPH and pentose precursors for nucleotides. Changes in metabolite levels between wild-type and PKR-ablated macrophages show that PKR controls the generation of ribose 5-phosphate, in a manner distinct from its established function in gene expression but dependent on its kinase activity. PKR phosphorylates and inhibits the Ribose 5-Phosphate Isomerase A (RPIA), thereby preventing interconversion of ribulose- to ribose 5-phosphate. This activity preserves redox control but decreases production of ribose 5-phosphate for nucleotide biosynthesis. Accordingly, the PKR-mediated immune response to RNA suppresses nucleic acid production. In line, pharmacological targeting of the PPP during infection decreases the replication of the Herpes simplex virus. These results identify an immune response-mediated control of host cell metabolism and suggest targeting the RPIA as a potential innovative antiviral treatment.

Involvement of Glucosamine 6 Phosphate Isomerase 2 (GNPDA2) Overproduction in ß-Amyloid- and Tau P301L-Driven Pathomechanisms.

Alzheimer's disease (AD) is a neurodegenerative olfactory disorder affecting millions of people worldwide. Alterations in the hexosamine- or glucose-related pathways have been described through AD progression. Specifically, an alteration in glucosamine 6 phosphate isomerase 2 (GNPDA2) protein levels has been observed in olfactory areas of AD subjects. However, the biological role of GNPDA2 in neurodegeneration remains unknown. Using mass spectrometry, multiple GNPDA2 interactors were identified in human nasal epithelial cells (NECs) mainly involved in intraciliary transport. Moreover, GNPDA2 overexpression induced an increment in NEC proliferation rates, accompanied by transcriptomic alterations in Type II interferon signaling or cellular stress responses. In contrast, the presence of beta-amyloid or mutated Tau-P301L in GNPDA2-overexpressing NECs induced a slowdown in the proliferative capacity in parallel with a disruption in protein processing. The proteomic characterization of Tau-P301L transgenic zebrafish embryos demonstrated that GNPDA2 overexpression interfered with collagen biosynthesis and RNA/protein processing, without inducing additional changes in axonal outgrowth defects or neuronal cell death. In humans, a significant increase in serum GNPDA2 levels was observed across multiple neurological proteinopathies (AD, Lewy body dementia, progressive supranuclear palsy, mixed dementia and amyotrophic lateral sclerosis) (n = 215). These data shed new light on GNPDA2-dependent mechanisms associated with the neurodegenerative process beyond the hexosamine route.

Exploiting the biocatalytic potential of co-expressed l-fucose isomerase and d-tagatose 3-epimerase for the biosynthesis of 6-deoxy-l-sorbose.

6-Deoxy-l-sorbose (6-DLS) is an imperative rare sugar employed in food, agriculture, pharmaceutical and cosmetic industeries. However, it is a synthetic and very expensive rare sugars, previously synthesized by chemo-enzymatic methods through a long chain of chemical processes. Recently, enzymatic synthesis of rare sugars has attracted a lot of attention due to its advantages over synthetic methods. In this work, a promising approach for the synthesis of 6-DLS from an inexpensive sugar l-fucose was identified. The genes for l-fucose isomerase from Paenibacillus rhizosphaerae (Pr-LFI) and genes for d-tagatose-3-epimerase from Caballeronia fortuita (Cf-DTE) have been used for cloning and co-expression in Escherichia coli, developed a recombinant plasmid harboring pANY1-Pr-LFI/Cf-DTE vector. The recombinant co-expression system exhibited an optimum activity at 50 °C of temperature and pH 6.5 in the presence of Co2+ metal ion which inflated the catalytic activity by 6.8 folds as compared to control group with no metal ions. The recombinant co-expressed system was stable up to more than 50 % relative activity after 12 h and revealed a melting temperature (Tm) of 63.38 °C exhibiting half-life of 13.17 h at 50 °C. The co-expression system exhibited, 4.93, 11.41 and 16.21 g/L of 6-DLS production from initial l-fucose concentration of 30, 70 and 100 g/L, which equates to conversion yield of 16.44 %, 16.30 % and 16.21 % respectively. Generally, this study offers a promising strategy for the biological production of 6-DLS from an inexpensive substrate l-fucose in slightly acidic conditions with the aid of co-expression system harboring Pr-LFI and CF-DTE genes.

The Arabinose 5-Phosphate Isomerase KdsD Is Required for Virulence in Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, which is endemic primarily in Southeast Asia and northern Australia but is increasingly being seen in other tropical and subtropical regions of the world. Melioidosis is associated with high morbidity and mortality rates, which is mediated by the wide range of virulence factors encoded by B. pseudomallei. These virulence determinants include surface polysaccharides such as lipopolysaccharide (LPS) and capsular polysaccharides (CPS). Here, we investigated a predicted arabinose-5-phosphate isomerase (API) similar to KdsD in B. pseudomallei strain K96243. KdsD is required for the production of the highly conserved 3-deoxy-d-manno-octulosonic acid (Kdo), a key sugar in the core region of LPS. Recombinant KdsD was expressed and purified, and API activity was determined. Although a putative API paralogue (KpsF) is also predicted to be encoded, the deletion of kdsD resulted in growth defects, loss of motility, reduced survival in RAW 264.7 murine macrophages, and attenuation in a BALB/c mouse model of melioidosis. Suppressor mutations were observed during a phenotypic screen for motility, revealing single nucleotide polymorphisms or indels located in the poorly understood CPS type IV cluster. Crucially, suppressor mutations did not result in reversion of attenuation in vivo. This study demonstrates the importance of KdsD for B. pseudomallei virulence and highlights further the complex nature of the polysaccharides it produces. IMPORTANCE The intrinsic resistance of B. pseudomallei to many antibiotics complicates treatment. This opportunistic pathogen possesses a wide range of virulence factors, resulting in severe and potentially fatal disease. Virulence factors as targets for drug development offer an alternative approach to combat pathogenic bacteria. Prior to initiating early drug discovery approaches, it is important to demonstrate that disruption of the target gene will prevent the development of disease. This study highlights the fact that KdsD is crucial for virulence of B. pseudomallei in an animal model of infection and provides supportive phenotypic characterization that builds a foundation for future therapeutic development.

[Rational design of L-arabinose isomerase from Lactobacillus fermentum and its application in D-tagatose production].

L-arabinose isomerase (L-AI) is the key enzyme that isomerizes D-galactose to D-tagatose. In this study, to improve the activity of L-arabinose isomerase on D-galactose and its conversion rate in biotransformation, an L-arabinose isomerase from Lactobacillus fermentum CGMCC2921 was recombinantly expressed and applied in biotransformation. Moreover, its substrate binding pocket was rationally designed to improve the affinity and catalytic activity on D-galactose. We show that the conversion of D-galactose by variant F279I was increased 1.4 times that of the wild-type enzyme. The Km and kcat values of the double mutant M185A/F279I obtained by superimposed mutation were 530.8 mmol/L and 19.9 s-1, respectively, and the catalytic efficiency was increased 8.2 times that of the wild type. When 400 g/L lactose was used as the substrate, the conversion rate of M185A/F279I reached a high level of 22.8%, which shows great application potential for the enzymatic production of tagatose from lactose.

Xylose isomerase from Piromyces sp. E2 is a promiscuous enzyme with epimerase activity.

Xylose isomerase catalyzes the isomerization of D-xylose to D-xylulose with promiscuous activity for other saccharides including D-glucose, D-allose, and L-arabinose. The xylose isomerase from the fungus Piromyces sp. E2 (PirE2_XI) is used to engineer xylose usage by the fermenting yeast Saccharomyces cerevisiae, but its biochemical characterization is poorly understood with divergent catalytic parameters reported. We have measured the kinetic parameters of the PirE2_XI and analyzed its thermostability and pH-dependence towards different substrates. The PirE2_XI shows promiscuous activity towards D-xylose, D-glucose, D-ribose and L-arabinose with variable effects depending on different divalent ions and epimerizes D-xylose at C3 to produce D-ribulose in a substrate/product dependent ratio. The enzyme follows Michaelis-Menten kinetics for the substrates used and although KM values for D-xylose are comparable at 30 and 60 °C, the kcat/KM is three-fold greater at 60 °C. The purified PirE2_XI shows maximal activity at 65 °C in the pH range of 6.5-7.5 and is a thermostable enzyme, maintaining full activity over 48 h at 30 °C or 12 h at 60 °C. This is the first report demonstrating epimerase activity of the PirE2_XI and its ability to isomerize D-ribose and L-arabinose, and provides a comprehensive in vitro study of substrate specificity, effect of metal ions and temperature on enzyme activity and these findings advance the knowledge of the mechanism of action of this enzyme.

Mining and Characterization of Thermophilic Glucose Isomerase Based on Virtual Probe Technology.

Fructose, which is produced by the isomerization of glucose isomerase, is a crucial precursor for the biosynthesis of rare sugars. In this study, thermophilic glucose isomerases (GI) from Caldicellulosiruptor acetigenus (CAGI), Thermoanaerobacter thermocopriae (TTGI), and Thermotoga petrophila (TPGI) were screened from GenBank database by a virtual probe and were successfully expressed in Escherichia coli BL21(DE3). The results of characterization demonstrated that the optimal pH for CAGI and TTGI were 8.0 and were maintained at 80% in a slightly acidic environment. The relative residual activities of CAGI and TTGI were found to be 40.6% and 52.6%, respectively, following an incubation period of 24 h at 90 ℃. Furthermore, CAGI and TTGI exhibited superior catalytic performance that their reaction equilibrium both reached only after an hour at 85 ℃ with 200 g/L glucose, and the highest conversion rates were 54.2% and 54.1%, respectively. This study identifies competitive enzyme candidates for fructose production in the industry with appreciable cost reduction.

Identification and functional expression of a new xylose isomerase from the goat rumen microbiome in Saccharomyces cerevisiae.

The current climate crisis demands replacement of fossil energy sources with sustainable alternatives. In this scenario, second-generation bioethanol, a product of lignocellulosic biomass fermentation, represents a more sustainable alternative. However, Saccharomyces cerevisiae cannot metabolize pentoses, such as xylose, present as a major component of lignocellulosic biomass. Xylose isomerase (XI) is an enzyme that allows xylose consumption by yeasts, because it converts xylose into xylulose, which is further converted to ethanol by the pentose-phosphate pathway. Only a few XI were successfully expressed in S. cerevisiae strains. This work presents a new bacterial XI, named GR-XI 1, obtained from a Brazilian goat rumen metagenomic library. Phylogenetic analysis confirmed the bacterial origin of the gene, which is related to Firmicutes XIs. After codon optimization, this enzyme, renamed XySC1, was functionally expressed in S. cerevisiae, allowing growth in media with xylose as sole carbon source. Overexpression of XySC1 in S. cerevisiae allowed the recombinant strain to efficiently consume and metabolize xylose under aerobic conditions.

Identification of four hub genes in venous thromboembolism via weighted gene coexpression network analysis.

BACKGROUND: The pathogenic mechanisms of venous thromboembolism (VT) remain to be defined. This study aimed to identify differentially expressed genes (DEGs) that could serve as potential therapeutic targets for VT. METHODS: Two human datasets (GSE19151 and GSE48000) were analyzed by the robust rank aggregation method. Gene ontology and Kyoto encyclopedia of genes and genomes pathway enrichment analyses were conducted for the DEGs. To explore potential correlations between gene sets and clinical features and to identify hub genes, we utilized weighted gene coexpression network analysis (WGCNA) to build gene coexpression networks incorporating the DEGs. Then, the levels of the hub genes were analyzed in the GSE datasets. Based on the expression of the hub genes, the possible pathways were explored by gene set enrichment analysis and gene set variation analysis. Finally, the diagnostic value of the hub genes was assessed by receiver operating characteristic (ROC) analysis in the GEO database. RESULTS: In this study, we identified 54 upregulated and 10 downregulated genes that overlapped between normal and VT samples. After performing WGCNA, the magenta module was the module with the strongest negative correlation with the clinical characteristics. From the key module, FECH, GYPA, RPIA and XK were chosen for further validation. We found that these genes were upregulated in VT samples, and high expression levels were related to recurrent VT. Additionally, the four hub genes might be highly correlated with ribosomal and metabolic pathways. The ROC curves suggested a diagnostic value of the four genes for VT. CONCLUSIONS: These results indicated that FECH, GYPA, RPIA and XK could be used as promising biomarkers for the prognosis and prediction of VT.

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

BACKGROUND: Microorganisms 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. RESULTS: When 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. CONCLUSIONS: Anaerobically-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.

Characterization and Inhibition of 1-Deoxy-d-Xylulose 5-Phosphate Reductoisomerase: A Promising Drug Target in Acinetobacter baumannii and Klebsiella pneumoniae.

The ESKAPE pathogens comprise a group of multidrug-resistant bacteria that are the leading cause of nosocomial infections worldwide. The prevalence of antibiotic resistant strains and the relative ease by which bacteria acquire resistance genes highlight the continual need for the development of novel antibiotics against new drug targets. The methylerythritol phosphate (MEP) pathway is an attractive target for the development of new antibiotics. The MEP pathway governs the synthesis of isoprenoids, which are key lipid precursors for vital cell components such as ubiquinone and bacterial hopanoids. Additionally, the MEP pathway is entirely distinct from the corresponding mammalian pathway, the mevalonic acid (MVA) pathway, making the first committed enzyme of the MEP pathway, 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC), an attractive target for antibiotic development. To facilitate drug development against two of the ESKAPE pathogens, Acinetobacter baumannii and Klebsiella pneumoniae, we cloned, expressed, purified, and characterized IspC from these two Gram-negative bacteria. Enzyme inhibition assays using IspC from these two pathogens, and compounds fosmidomycin and FR900098, indicate IC50 values ranging from 19.5-45.5 nM. Antimicrobial susceptibility tests with these inhibitors reveal that A. baumannii is susceptible to FR900098, whereas K. pneumoniae is susceptible to both compounds. Finally, to facilitate structure-based drug design of inhibitors targeting A. baumannii IspC, we determined the 2.5 Å crystal structure of IspC from A. baumannii in complex with inhibitor FR900098, and cofactors NADPH and magnesium.

Crystal Structure and Functional Characterization of the Bifunctional N-(5'-Phosphoribosyl)anthranilate Isomerase-indole-3-glycerol-phosphate Synthase from Corynebacterium glutamicum.

L-Tryptophan is known as an aromatic amino acid and one of the essential amino acids that must be ingested through various additives or food. TrpCF is a bifunctional enzyme that has indole-glycerol-phosphate synthase (IGPS) and phosphoribosylanthranilate isomerase (PRAI) activity. In this report, we identified the crystal structure of TrpCF from Corynebacterium glutamicum (CgTrpCF) and successfully elucidated the active site by attaching rCdRP similar to the substrate and product of the TrpCF reaction. Also, we revealed that CgTrpCF shows a conformational change at the loops upon substrate binding. We analyzed amino acid sequences of the homologues of CgTrpCF, and the residues of the substrate-binding site in TrpCF were highly conserved except for some residues. These less conserved residues were replaced by site-directed mutagenesis experiments. Consequently, we obtained the CgTrpCFP294K (PRAICD/P294K) variant that has enhanced activity.

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.

Metabolism of Poly-ß1,4-N-Acetylglucosamine Substrates and Importation of N-Acetylglucosamine and Glucosamine by Enterococcus faecalis.

The ability of Enterococcus faecalis to use a variety of carbon sources enables colonization at various anatomic sites within a mammalian host. N-Acetylglucosamine (GlcNAc) is one of the most abundant natural sugars and provides bacteria with a source of carbon and nitrogen when metabolized. N-Acetylglucosamine is also a component of bacterial peptidoglycan, further highlighting the significance of N-acetylglucosamine utilization. In this study, we show that CcpA-regulated enzymes are required for growth on the poly-ß1,4-linked GlcNAc substrate, chitopentaose (ß1,4-linked GlcNAc5). We also show that EF0114 (EndoE) is required for growth on chitobiose (ß1,4-linked GlcNAc2) and that the GH20 domain of EndoE is required for the conversion of GlcNAc2 to N-acetylglucosamine. GlcNAc is transported into the cell via two separate phosphotransferase system (PTS) complexes, either the PTS IICBA encoded by ef1516 (nagE) or the Mpt glucose/mannose permease complex (MptBACD). The Mpt PTS is also the primary glucosamine transporter. In order for N-acetylglucosamine to be utilized as a carbon source, phosphorylated N-acetylglucosamine (GlcNAc-6-P) must be deacetylated, and here, we show that this activity is mediated by EF1317 (an N-acetylglucosamine-6-phosphate deacetylase; NagA homolog), as a deletion of ef1317 is unable to grow on GlcNAc as the carbon source. Deamination of glucosamine to fructose-6-phosphate is required for entry into glycolysis, and we show that growth on glucosamine is dependent on EF0466 (a glucosamine-6-phosphate deaminase; NagB homolog). Collectively, our data highlight the chitinolytic machinery required for breaking down exogenous chitinous substrates, as well as the uptake and cytosolic enzymes needed for metabolizing N-acetylglucosamine. IMPORTANCE Enterococcus faecalis causes life-threatening health care-associated infections in part due to its intrinsic and acquired antibiotic resistance, its ability to form biofilms, and its nutrient versatility. Alternative nutrient acquisition systems are key factors that contribute to enterococcal colonization at biologically unique host anatomic sites. Although E. faecalis can metabolize an array of carbon sources, little is known of how this bacterium acquires these secondary nutrient sources in mammalian hosts. Our research identifies the glycosidase machinery required for degrading exogenous chitinous substrates into N-acetylglucosamine monomers for transport and metabolism of one of the most abundant naturally occurring sugars, N-acetylglucosamine. Disrupting the function of this N-acetylglucosamine acquisition pathway may lead to new treatments against multidrug-resistant enterococcal infections.

Nucleation of protein mesocrystals via oriented attachment.

Self-assembly of proteins holds great promise for the bottom-up design and production of synthetic biomaterials. In conventional approaches, designer proteins are pre-programmed with specific recognition sites that drive the association process towards a desired organized state. Although proven effective, this approach poses restrictions on the complexity and material properties of the end-state. An alternative, hierarchical approach that has found wide adoption for inorganic systems, relies on the production of crystalline nanoparticles that become the building blocks of a next-level assembly process driven by oriented attachment (OA). As it stands, OA has not yet been observed for protein systems. Here we employ cryo-transmission electron microscopy (cryoEM) in the high nucleation rate limit of protein crystals and map the self-assembly route at molecular resolution. We observe the initial formation of facetted nanocrystals that merge lattices by means of OA alignment well before contact is made, satisfying non-trivial symmetry rules in the process. As these nanocrystalline assemblies grow larger we witness imperfect docking events leading to oriented aggregation into mesocrystalline assemblies. These observations highlight the underappreciated role of the interaction between crystalline nuclei, and the impact of OA on the crystallization process of proteins.

Enhanced isomerization of rare sugars by ribose-5-phosphate isomerase A from Ochrobactrum sp. CSL1.

Ribose-5-phosphate isomerase A (RpiA) is of great importance in biochemistry research, however its application in biotechnology has not been fully explored. In this study the activity of RpiA from Ochrobactrum sp. CSL1 (OsRpiA) towards D-allose was engineered based on sequential and structural analyses. Strategies of alanine scanning, rational design and saturated mutagenesis were employed to create three mutant libraries. A single mutant of K124A showed a 45 % activity improvement towards D-allose. The reaction properties of the mutant were analyzed, and a shift of optimal pH and higher thermal stability at low reaction temperatures were identified. The conversion of D-allose was also improved by 40 % using K124A, and higher activities on major substrates were found in the mutant's substrate scope, implying its application potential in rare sugar preparation. Kinetics analysis revealed that Km of K124A mutant decreased by 12 % and the catalytic efficiency increased by 65 % towards D-allose. Moreover, molecular dynamics simulation illustrated the binding of substrate and K124A was more stable than that of the wild-type. The shorter distance and more relax bond angle between the catalytic residue of K124A and D-allose explained the activity improvement in detail. This study highlights the potential of OsRpiA as a biocatalyst for rare sugar preparation, and provides distinct evidences for its catalytic mechanism.

Rational design of Shewanella sp. l-arabinose isomerase for d-galactose isomerase activity under mesophilic conditions.

d-Tagatose, a potential low calorific substitute for sucrose, can be produced by bioconversion of d-galactose catalysed by l-arabinose isomerase. l-Arabinose isomerase from Shewanella sp. ANA-3 is unique for its ability to catalyse bioconversion reactions under mesophilic conditions. However, d-galactose not being a natural substrate for l-arabinose isomerase is catalysed at a slower rate. We attempted to increase the biocatalytic efficiency of Shewanella sp. l-arabinose isomerase by rational design to enhance galactose isomerisation activity. In silico molecular docking, analysis has revealed that F279 is sterically hindering the binding of d-galactose at the C6 position. Substitution of bulky Phe residue with smaller hydrophilic residues such as Asn and Thr increased the galactose isomerase activity by 86 % and 12 % respectively. At mesophilic conditions, F279N mutant catalysed the bioconversion of d-galactose more efficiently than l-arabinose, indicating a shift in substrate preference.

Multiple gene integration to promote erythritol production on glycerol in Yarrowia lipolytica.

OBJECTIVE: Erythritol (1,2,3,4-butanetetrol) is a 4-carbon sugar alcohol that occurs in nature as a metabolite or storage compound. In this study, a multiple gene integration strategy was employed to enhance erythritol production in Y. lipolytica. RESULTS: The effects on the production of erythritol in Y. lipolytica of seven key genes involved in the erythritol synthesis pathway were evaluated individually, among which transketolase (TKL1) and transaldolase (TAL1) showed important roles in enhancing erythritol production. The combined overexpression of four genes (GUT1, TPI1, TKL1, TAL1) and disruption of the EYD1 gene (encoding erythritol dehydrogenase), resulted in produce approximately 40 g/L erythritol production from glycerol. Further enhanced erythritol synthesis was obtained by overexpressing the RKI1 gene (encoding ribose 5-phosphate isomerase) and the AMPD gene (encoding AMP deaminase), indicating for the first time that these two genes are also related to the enhancement of erythritol production in Y. lipolytica. CONCLUSIONS: A combined gene overexpression strategy was developed to efficiently improve the production of erythritol in Y. lipolytica, suggesting a great capacity and promising potential of this non-conventional yeast in converting glycerol into erythritol.

Molecular Cloning and Functional Analysis of 1-Deoxy-D-Xylulose 5-Phosphate Reductoisomerase from Santalum album.

Sandalwood (Santalum album L.) heartwood-derived essential oil contains a high content of sesquiterpenoids that are economically highly valued and widely used in the fragrance industry. Sesquiterpenoids are biosynthesized via the mevalonate acid and methylerythritol phosphate (MEP) pathways, which are also the sources of precursors for photosynthetic pigments. 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is a secondary rate-limiting enzyme in the MEP pathway. In this paper, the 1416-bp open reading frame of SaDXR and its 897-bp promoter region, which contains putative conserved cis-elements involved in stress responsiveness (HSE and TC-rich repeats), hormone signaling (abscisic acid, gibberellin and salicylic acid) and light responsiveness, were cloned from 7-year-old S. album trees. A bioinformatics analysis suggested that SaDXR encodes a functional and conserved DXR protein. SaDXR was widely expressed in multiple tissues, including roots, twigs, stem sapwood, leaves, flowers, fruit and stem heartwood, displaying significantly higher levels in tissues with photosynthetic pigments, like twigs, leaves and flowers. SaDXR mRNA expression increased in etiolated seedlings exposed to light, and the content of chlorophylls and carotenoids was enhanced in all 35S::SaDXR transgenic Arabidopsis thaliana lines, consistent with the SaDXR expression level. SaDXR was also stimulated by MeJA and H2O2 in seedling roots. α-Santalol content decreased in response to fosmidomycin, a DXR inhibitor. These results suggest that SaDXR plays an important role in the biosynthesis of photosynthetic pigments, shifting the flux to sandalwood-specific sesquiterpenoids.