1-deoxy-D-xylulose 5-phosphate Research Articles

Enhancing Terpene yield from sugars via novel routes to 1-deoxy-d-xylulose 5-phosphate.

Terpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO2. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C5 sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of a dxs deletion in Escherichia coli grown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-type E. coli yajO gene, annotated as a putative xylose reductase, or via various mutations in the native ribB gene. In vitro analysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway in E. coli for production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.

The Y392F further stabilizes the pre‐decarboxylation intermediate on 1‐deoxy‐D‐xylulose 5‐phosphate synthase

The thiamin diphosphate (ThDP)‐dependent enzyme 1‐deoxy‐D‐xylulose 5‐phosphate (DXP) synthase catalyzes the condensation of D‐glyceraldehyde‐3‐phosphate (D‐GAP) and pyruvate to form DXP, a precursor in the biosynthesis of vitamins B1, B6 and isoprenoids. Our groups recently reported (J.Am.Chem.Soc. 2012, 134, 18374–18379) stabilization of the ThDP‐bound predecarboxylation intermediate C2α‐lactylThDP on DXP synthase and an at least 600‐fold rate acceleration of the decarboxylation by D‐GAP. Model building and analogy with similar enzymes suggested that the residue Y392 could be responsible for this finding, perhaps by hydrogen bonding to the carboxylate anion of C2α‐lactylThDP. To explore this hypothesis, we have carried out steady state and pre‐steady state experiments with the DXP synthase Y392F variant. The results suggest that C2α‐lactylThDP is even more stable on the variant than on DXP synthase. An NMR experiment of the reaction mixture containing Y392F and pyruvate after 50s acid quench also supports the idea that Y392F stabilizes C2α‐lactylThDP. In the presence of D‐GAP the rate of decarboxylation was similar to that on DXP synthase, suggesting that Y392 might be holding the carboxylate group of C2α‐lactylThDP and addition of acceptor substrate D‐GAP immediately rescues the slow rate of decarboxylation. Supported by NIHGM050380 (RU) and NIH‐GM084998 (JHU).

Effects of endophytic fungal elicitor on two kinds of terpenoids production and physiological indexes in Euphorbia pekinensis suspension cells

Treating Euphorbia pekinensis suspension cultures with endophytic fungal elicitor fromFusarium sp. E5 resulted in accumualtion of isoeuphpekinensin and euphol, activated defense-related enzymes, and increased biomass. The elicitor was prepared from extract of fungal endophyte Fusarium sp. E5 mycelium, and added into 21 day-old cell suspension cultures. The results showed that the biomass of culture after elicitor treatment was increased by 19.35%, whereas the isoeuphpekinensin and euphol contents were 5.81 and 3.56 times greater than those of the control, respectively. The activity of 1-deoxy-D-xylulose 5-phosphate synthase (a key enzyme of isoeuphpekinensin biosynthesis) was not increased significantly, while the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (a key enzyme of euphol) was increased by 1.67-fold compared with the control.Furthermore, the elicitor enhanced intracellular and extracellular polyphenol production and the activities of phenylalanine ammonia-lyase, peroxidase and catalase, therefore improved the plant defense ability. In addition, the nuclei of elicitor-treated cells were not apoptotic, and the culture was consistently healthy with increasing biomass. Our results demonstrate that endophytic fungal elicitor promotes both terpenoid biosynthesis and biomass of plant cultures for the first time, and thereby the information from this study will be helpful for large-scale medicinal plant suspension culture.   Key words: Euphorbia pekinensis, endophytic fungal elicitor, terpenoids, Fusarium.

Flavin Adenine Dinucleotide-Dependent 4-Phospho- d -Erythronate Dehydrogenase Is Responsible for the 4-Phosphohydroxy- l -Threonine Pathway in Vitamin B 6 Biosynthesis in Sinorhizobium meliloti

The vitamin B6 biosynthetic pathway in Sinorhizobium meliloti is similar to that in Escherichia coli K-12; in both organisms this pathway includes condensation of two intermediates, 1-deoxy-D-xylulose 5-phosphate and 4-phosphohydroxy-L-threonine (4PHT). Here, we report cloning of a gene designated pdxR that functionally corresponds to the pdxB gene of E. coli and encodes a dye-linked flavin adenine dinucleotide-dependent 4-phospho-D-erythronate (4PE) dehydrogenase. This enzyme catalyzes the oxidation of 4PE to 3-hydroxy-4-phosphohydroxy-alpha-ketobutyrate and is clearly different in terms of cofactor requirements from the pdxB gene product of E. coli, which is known to be an NAD-dependent enzyme. Previously, we revealed that in S. meliloti IFO 14782, 4PHT is synthesized from 4-hydroxy-l-threonine and that this synthesis starts with glycolaldehyde and glycine. However, in this study, we identified a second 4PHT pathway in S. meliloti that originates exclusively from glycolaldehyde (the major pathway). Based on the involvement of 4PE in the 4PHT pathway, the incorporation of different samples of 13C-labeled glycolaldehyde into pyridoxine molecules was examined using 13C nuclear magnetic resonance spectroscopy. On the basis of the spectral analyses, the synthesis of 4PHT from glycolaldehyde was hypothesized to involve the following steps: glycolaldehyde is sequentially metabolized to D-erythrulose, D-erythrulose 4-phosphate, and D-erythrose 4-phosphate by transketolase, kinase, and isomerase, respectively; and D-erythrose 4-phosphate is then converted to 4PHT by the conventional three-step pathway elucidated in E. coli, although the mechanism of action of the enzymes catalyzing the first two steps is different.

Molecular Determination of a Gren Algae Isolate to Detecting 1-Deoxy-D-Xylulose-5-phosphate Synthase (DXS) Gene in Improvement of Carotenoid Production

Sintesis karotenoid alami belum pernah melebihi produk sintetik pada skala komersial. Kurangnya pemahaman mengenai aspek mikrobiologis dan ekofisiologis isolat penghasil karotenoid menyebabkan terjadinya kesalahan penamaan spesies. Satu isolat lokal alga hijau dari BBAP Jepara yang digunakan sebagai pakan alami sumber karotenoid hewan-hewan perikanan, pada mulanya dianggap sebagai Dunaliella. Namun pengembangan produksi karotenoid menggunakan teknologi rekayasa genetik dan rekayasa metabolit terhadap isolat alga hijau lebih lanjut memperlihatkan ketidaksesuaian hasil dengan penamaan yang ada. Akumulasi karotenoid jalur non-MVA pada alga hijau ditentukan oleh enzim D-1-Deoksixilulosa 5-fosfat Sintase, yang disandi oleh gen D-1-deoksixilulosa 5-fosfat sintase (DXS). Determinasi spesies secara molekuler menjadi penting dilakukan untuk menentukan spesies isolat dan jalur biosintesis karotenoid yang digunakan. Hasil determinasi digunakan untuk analisis keserupaan putative partial fragment gen DXS Isolat alga hijau yang telah berhasil diperoleh pada penelitian sebelumnya. Tujuan utama penelitian ini adalah menentukan spesies satu isolat lokal alga hijau secara molekuler menggunakan 23S rRNA untuk mendeteksi keberadaan gen DXS penyandi biosintesis karotenoid. Hasil penelitian memperlihatkan bahwa Isolat alga hijau menunjukkan keserupaan yang tinggi dengan anggota-anggota Sianobakteria. Keserupaan tertinggi dimiliki dengan  Cyanobacterium sp. MBIC 1021 sebesar 99 %, diikuti Synechocystis PCC6308 sebesar 95 %. Satu-satunya anggota Cyanobacteria yang memiliki gen DXS adalah Synechocystis. Hasil analisis keserupaan parsial gen DXS isolat alga hijau terhadap tujuh parsial gen DXS pada daerah lestari yang telah ditemukan, memperlihatkan bahwa putative partial fragment gen DXS Isolat lokal alga hijau juga memiliki keserupaan tertinggi dengan gen DXS Sianobakteria Synechocystis. Kata kunci : isolasi alga hijau, Dunaliella, gen DXS, 23S rRNA, Cyanobacteria, Synechocystis Carotenoids production levels are not yet competitive with carotenoid levels presently produced by fermentation, synthesis and isolation. It needs application of metabolic engineering and genetic engineering techniques in improving their production. An attempt to optimize carotenoid production from local isolate of green algae from BBAP Jepara has faced several problems, primarily related to the microbiological and ecophysiological characteristic which affecting growth that have not sufficiently been understood. A misnamed of species also have arisen due to wrong characterization. One local isolate of an algal species from BBAP Jepara was found potentially useful as source of carotenoids in food additives or as food supplement in fish farming. It was suspected as representing a strain of Dunaliella. Previous studies to improve carotenoid production using molecular approach on have shown unagreement. Therefore, the present study aimed to determinate the species of green algae isolate from Jepara waters based on molecular techniques using 23S rRNA approach for detecting DXS gene. Molecular analysis by 23S rRNA alignment showed the close relationship among isolate of green algae and most all of member of Cyanobacteria. Closest similarities was showed by Cyanobacterium sp. MBIC 1021 with 99 % similarity and Synechocystis PCC6308 with 95 % similarity. Synechocystis was the only member of Cyanobacteria which have DXS gene. Multiples aligment sequences of partial DXS gene on the conserve region among seven species confirmed this result. The DXS gene analysis also showed closest relationship between partial DXS gene of Cyanobacteria Synechocystis anda green algae isolate. The result of this analysis proven as valuable parameter for the interpretation of the relation among DXS gene of a green algae isolate and Cyanobacteria and increase the possibility in getting the complete DXS gene from local isolate of green algae by designing primers from DXS gene of Synechocystis as a member of Cyanobcteria. Key words : a green algae isolate, Dunaliella, DXS gene, 23S rRNA, Cyanobacteria, Synechocystis

Identification of an Archaeal type II isopentenyl diphosphate isomerase in methanothermobacter thermautotrophicus.

Isopentenyl diphosphate (IPP):dimethylallyl diphosphate isomerase catalyzes the interconversion of the fundamental five-carbon homoallylic and allylic diphosphate building blocks required for biosynthesis of isoprenoid compounds. Two different isomerases have been reported. The type I enzyme, first characterized in the late 1950s, is widely distributed in eukaryota and eubacteria. The type II enzyme was recently discovered in Streptomyces sp. strain CL190. Open reading frame 48 (ORF48) in the archaeon Methanothermobacter thermautotrophicus encodes a putative type II IPP isomerase. A plasmid-encoded copy of the ORF complemented IPP isomerase activity in vivo in Salmonella enterica serovar Typhimurium strain RMC29, which contains chromosomal knockouts in the genes for type I IPP isomerase (idi) and 1-deoxy-D-xylulose 5-phosphate (dxs). The dxs gene was interrupted with a synthetic operon containing the Saccharomyces cerevisiae genes erg8, erg12, and erg19 allowing for the conversion of mevalonic acid to IPP by the mevalonate pathway. His6-tagged M. thermautotrophicus type II IPP isomerase was produced in Escherichia coli and purified by Ni2+ chromatography. The purified protein was characterized by matrix-assisted laser desorption ionization mass spectrometry. The enzyme has optimal activity at 70 degrees C and pH 6.5. NADPH, flavin mononucleotide, and Mg2+ are required cofactors. The steady-state kinetic constants for the archaeal type II IPP isomerase from M. thermautotrophicus are as follows: K(m), 64 microM; specific activity, 0.476 micromol mg(-1) min(-1); and k(cat), 1.6 s(-1).

Synthesis of [5,5-2H2]-1-Deoxy-D-xylulose-5-phosphate

[5,5-2H2]-1-Deoxy-d-xylulose-5-phosphate was synthetically prepared.

A 1-deoxy-D-xylulose 5-phosphate reductoisomerase catalyzing the formation of 2-C-methyl-D-erythritol 4-phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis.

Several eubacteria including Esherichia coli use an alternative nonmevalonate pathway for the biosynthesis of isopentenyl diphosphate instead of the ubiquitous mevalonate pathway. In the alternative pathway, 2-C-methyl-D-erythritol or its 4-phosphate, which is proposed to be formed from 1-deoxy-D-xylulose 5-phosphate via intramolecular rearrangement followed by reduction process, is one of the biosynthetic precursors of isopentenyl diphosphate. To clone the gene(s) responsible for synthesis of 2-C-methyl-D-erythritol 4-phosphate, we prepared and selected E. coli mutants with an obligatory requirement for 2-C-methylerythritol for growth and survival. All the DNA fragments that complemented the defect in synthesizing 2-C-methyl-D-erythritol 4-phosphate of these mutants contained the yaeM gene, which is located at 4.2 min on the chromosomal map of E. coli. The gene product showed significant homologies to hypothetical proteins with unknown functions present in Haemophilus influenzae, Synechocystis sp. PCC6803, Mycobacterium tuberculosis, Helicobacter pyroli, and Bacillus subtilis. The purified recombinant yaeM gene product was overexpressed in E. coli and found to catalyze the formation of 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate in the presence of NADPH. Replacement of NADPH with NADH decreased the reaction rate to about 1% of the original rate. The enzyme required Mn2+, Co2+, or Mg2+ as well. These data clearly show that the yaeM gene encodes an enzyme, designated 1-deoxy-D-xylulose 5-phosphate reductoisomerase, that synthesizes 2-C-methyl-D-erythritol 4-phosphate from 1-deoxy-D-xylulose 5-phosphate, in a single step by intramolecular rearrangement and reduction and that this gene is responsible for terpenoid biosynthesis in E. coli.