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1.
Proteins Encoded by Sphingomonas elodea ATCC 31461 rmlA and ugpG Genes, Involved in Gellan Gum Biosynthesis, Exhibit both dTDP- and UDP-Glucose Pyrophosphorylase Activities 下载免费PDF全文
Elisabete Silva Ana Rita Marques Arsénio Mendes Fialho Ana Teresa Granja Isabel Sá-Correia 《Applied microbiology》2005,71(8):4703-4712
2.
Most Sphingomonas species synthesize the yellow carotenoid nostoxanthin. However, the carotenoid biosynthetic pathway of these species remains unclear. In this study, we cloned and characterized a carotenoid biosynthesis gene cluster containing four carotenogenic genes (crtG, crtY, crtI and crtB) and a β-carotene hydroxylase gene (crtZ) located outside the cluster, from the gellan-gum producing bacterium Sphingomonas elodea ATCC 31461. Each of these genes was inactivated, and the biochemical function of each gene was confirmed based on chromatographic and spectroscopic analysis of the intermediates accumulated in the knockout mutants. Moreover, the crtG gene encoding the 2,2'-β-hydroxylase and the crtZ gene encoding the β-carotene hydroxylase, both responsible for hydroxylation of β-carotene, were confirmed by complementation studies using Escherichia coli producing different carotenoids. Expression of crtG in zeaxanthin and β-carotene accumulating E. coli cells resulted in the formation of nostoxanthin and 2,2'-dihydroxy-β-carotene, respectively. Based on these results, a biochemical pathway for synthesis of nostoxanthin in S. elodea ATCC 31461 is proposed. 相似文献
3.
Gai Z Wang X Zhang X Su F Wang X Tang H Tai C Tao F Ma C Xu P 《Journal of bacteriology》2011,193(24):7015-7016
The commercial gelling agent gellan gum is a heteropolysaccharide produced by Sphingomonas elodea ATCC 31461. However, the genes involved in the biosynthesis, regulation, and modification of gellan gum have not been fully characterized. Here we describe the draft genome sequence of stain ATCC 31461 and major findings from its annotation. 相似文献
4.
Deproteinization is a technical bottleneck in the purification of viscous water-soluble polysaccharides. The aim of this work is to provide an appropriate approach to deproteinize crude gellan gum. Several methods of deproteinization were investigated, including Sevag method, alkaline protease, papain and neutral protease. The results revealed that Sevag method had high deproteinization efficiency (87.9%), but it showed dissatisfactory recovery efficiency of gellan gum (28.6%), which made it less advisable in industrial applications. The deproteinization by alkaline protease was demonstrated in this work for the first time, indicating alkaline protease was preferred in the deproteinization of crude gellan gum with high polysaccharide recovery (89.3%) and high deproteinization efficiency (86.4%). 相似文献
5.
6.
Gellan gum biosynthesis in Sphingomonas paucimobilis ATCC 31461: genes,enzymes and exopolysaccharide production engineering 总被引:2,自引:0,他引:2
Sá-Correia I Fialho AM Videira P Moreira LM Marques AR Albano H 《Journal of industrial microbiology & biotechnology》2002,29(4):170-176
The commercial gelling agent, gellan, is an extracellular polysaccharide (EPS) produced by Sphingomonas paucimobilis ATCC 31461. In recent years, significant progress in understanding the relationship between gellan structure and properties
and elucidation of the biosynthesis and engineering of this recent product of biotechnology has been made. This review focuses
on recent advances in this field. Emphasis is given to identification and characterization of genes and enzymes involved,
or predicted to be involved, in the gellan biosynthetic pathway, at the level of synthesis of sugar-activated precursors,
of the repeat unit assembly and of gellan polymerization and export. Identification of several genes, biochemical characterization
of the encoded enzymes and elucidation of crucial steps of the gellan pathway indicate that possibilities now exist for exerting
control over gellan production at any of the three levels of its biosynthesis. However, a better knowledge of the poorly understood
steps and of the bottlenecks and regulation of the pathway, the characterization of the composition, structure and functional
properties of gellan-like polymers produced either by the industrial strain under different culture conditions or by mutants
are still required for eventual success of the metabolic engineering of gellan production. Journal of Industrial Microbiology & Biotechnology (2002) 29, 170–176 doi:10.1038/sj.jim.7000266
Received 11 February 2002/ Accepted in revised form 09 April 2002 相似文献
7.
Marques AR Ferreira PB Sá-Correia I Fialho AM 《Molecular genetics and genomics : MGG》2003,268(6):816-824
The ugpGgene, which codes for a UDP-glucose pyrophosphorylase (UGP) (or glucose-1-phosphate uridylyltransferase; EC 2.7.7.9) in Sphingomonas paucimobilis ATCC 31461, was cloned and sequenced. This industrial strain produces the exopolysaccharide gellan, a new commercial gelling agent, and the ugpG gene may convert glucose-1-phosphate into UDP-glucose in the gellan biosynthetic pathway. The ugpG gene is capable of restoring the capacity of an Escherichia coli galU mutant to grow on galactose by functional complementation of its deficiency for UDP-glucose pyrophosphorylase activity. As expected, the predicted gene product shows strong homology to UDP-glucose pyrophosphorylases from several bacterial species. The N-terminal region of UgpG exhibits the motif GXGTRXLPXTK, which is highly conserved among bacterial XDP-sugar pyrophosphorylases, and a lysine residue (K(192)) is located within a VEKP motif predicted to be essential for substrate binding or catalysis. UgpG was purified to homogeneity as a heterologous fusion protein from crude cell extracts prepared from IPTG-induced cells of E. coli, using affinity chromatography. Under denaturing conditions, the fusion protein S-UgpG-His(6) migrated with an estimated molecular mass of 36 kDa [corresponding to the predicted molecular mass of native UgpG (31.2 kDa) plus 5 kDa for the S and histidine tags). Kinetic analysis of UgpG in the reverse reaction (pyrophosphorolysis) showed a typical Michaelis-Menten substrate saturation pattern. The apparent K(m) and V(max) values estimated for UDP-glucose were 7.5 microM and 1275 micromol/min/g. 相似文献
8.
Soo Youn Lee Ji-Young Ahn Mihye Kim Simranjeet Singh Sekhon Sung-Jin Cho 《Animal cells and systems.》2017,21(2):115-123
Sphingomonas elodea is a Gram-negative bacterium capable of producing ‘gellan gum’ exopolysaccharide that is the most extensively studied expolysaccharides of microbial origin. In this study, we investigated the phenotypic and proteomic alterations in S. elodea by homogeneously expressing both gelA and gelN involved in positive regulation and extracellular secretion of metabolites in gellan biosynthesis, respectively. Expression of six histidine-tagged GelA and GelN was determined by Western blot analysis. Successful expression of GelA and GelN resulted in both morphological changes of colonies and enhanced secretion of gellan into the growth medium (GelA, 21.2% more and GelN, 48.3% more) overexpressed compared to the wile-type. Comparative two-dimensional gel electrophoresis analysis revealed a differential proteome expression in S. elodea overexpressing GelA and GelN. Proteins up- or down-regulated by GelA and GelN overexpression were found to be mainly sugar transportation proteins, two-component regulatory proteins, and proteins involved in secretion pathways. The results suggest that the effect of GelA and GelN overexpression on gellan biosynthesis might be mainly caused by increased transportation of sugar units or enhanced exportation of gellan. 相似文献
9.
Granja AT Popescu A Marques AR Sá-Correia I Fialho AM 《Applied microbiology and biotechnology》2007,76(6):1319-1327
Sphingomonas elodea ATCC 31461 synthesizes in high yield the exopolysaccharide gellan, which is a water-soluble gelling agent with many applications.
In this study, we describe the cloning and sequence analysis of the ugdG gene, encoding a UDP-glucose dehydrogenase (47.2 kDa; UDPG-DH; EC 1.1.1.22), required for the synthesis of the gellan gum
precursor UDP-glucuronic acid. UgdG protein shows homology to members of the UDP-glucose/GDP-mannose dehydrogenase superfamily.
The Neighbor-Joining method was used to determine phylogenetic relationships among prokaryotic and eukaryotic UDPG-DHs. UgdG
from S. elodea and UDPG-DHs from Novosphingobium, Zymomonas, Agrobacterium, and Caulobacter species form a divergent phylogenetic group with a close evolutionary relationship with eukaryotic UDPG-DHs. The ugdG gene was recombinantly expressed in Escherichia coli with and N-terminal 6-His tag and purified for biochemical characterization. The enzyme has an optimum temperature and pH
of 37°C and 8.7, respectively. The estimated apparent K
m values for UDP-glucose and NAD+ were 0.87 and 0.4 mM, respectively. DNA sequencing of chromosomal regions adjacent to ugdG gene and sequence similarity studies suggests that this gene maps together with others presumably involved in the biosynthesis
of S. elodea cell wall polysaccharides. 相似文献
10.
Organization of genes required for gellan polysaccharide biosynthesis in<Emphasis Type="Italic"> Sphingomonas elodea</Emphasis> ATCC 31461 总被引:2,自引:0,他引:2
Sphingomonas elodea ATCC 31461 produces gellan, a capsular polysaccharide that is useful as a gelling agent for food and microbiological media. Complementation of nonmucoid S. elodea mutants with a gene library resulted in identification of genes essential for gellan biosynthesis. A cluster of 18 genes spanning 21 kb was isolated. These 18 genes are homologous to genes for synthesis of sphingan polysaccharide S-88 from Sphingomonas sp. ATCC 31554, with predicted amino acid identities varying from 61% to 98%. Both polysaccharides have the same tetrasaccharide repeat unit, comprised of [4)--l-rhamnose-(13)--d-glucose-(14)--d-glucuronic acid-(14)--d-glucose-(1]. Polysaccharide S-88, however, has mannose or rhamnose in the fourth position and has a rhamnosyl side chain, while gellan has no sugar side chain but is modified by glyceryl and acetyl substituents. Genes for synthesis of the precursor dTDP-l-rhamnose were highly conserved. The least conserved genes in this cluster encode putative glycosyl transferases III and IV and a gene of unknown function, gelF. Three genes (gelI, gelM, and gelN) affected the amount and rheology of gellan produced. Four additional genes present in the S-88 sphingan biosynthetic gene cluster did not have homologs in the gene cluster for gellan biosynthesis. Three of these gene homologs, gelR, gelS, and gelG, were found in an operon unlinked to the main gellan biosynthetic gene cluster. In a third region, a gene possibly involved in positive regulation of gellan biosynthesis was identified. 相似文献
11.
The complex of Sphingomonas elodea ATCC 31461 glucose-1-phosphate uridylyltransferase with glucose-1-phosphate reveals a novel quaternary structure, unique among nucleoside diphosphate-sugar pyrophosphorylase members 下载免费PDF全文
Aragão D Fialho AM Marques AR Mitchell EP Sá-Correia I Frazão C 《Journal of bacteriology》2007,189(12):4520-4528
Gellan gum is a widely used commercial material, available in many different forms. Its economic importance has led to studies into the biosynthesis of exopolysaccharide gellan gum, which is industrially prepared in high yields using Sphingomonas elodea ATCC 31461. Glucose-1-phosphate uridylyltransferase mediates the reversible conversion of glucose-1-phosphate and UTP into UDP-glucose and pyrophosphate, which is a key step in the biosynthetic pathway of gellan gums. Here we present the X-ray crystal structure of the glucose-1-phosphate uridylyltransferase from S. elodea. The S. elodea enzyme shares strong monomeric similarity with glucose-1-phosphate thymidylyltransferase, several structures of which are known, although the quaternary structures of the active enzymes are rather different. A detailed comparison between S. elodea glucose-1-phosphate uridylyltransferase and available thymidylyltransferases is described and shows remarkable structural similarities, despite the low sequence identities between the two divergent groups of proteins. 相似文献
12.
Videira PA Cortes LL Fialho AM Sá-Correia I 《Applied and environmental microbiology》2000,66(5):2252-2258
The pgmG gene of Sphingomonas paucimobilis ATCC 31461, the industrial gellan gum-producing strain, was cloned and sequenced. It encodes a 50,059-Da polypeptide that has phosphoglucomutase (PGM) and phosphomannomutase (PMM) activities and is 37 to 59% identical to other bifunctional proteins with PGM and PMM activities from gram-negative species, including Pseudomonas aeruginosa AlgC. Purified PgmG protein showed a marked preference for glucose-1-phosphate (G1P); the catalytic efficiency was about 50-fold higher for G1P than it was for mannose-1-phosphate (M1P). The estimated apparent K(m) values for G1P and M1P were high, 0.33 and 1.27 mM, respectively. The pgmG gene allowed the recovery of alginate biosynthetic ability in a P. aeruginosa mutant with a defective algC gene. This result indicates that PgmG protein can convert mannose-6-phosphate into M1P in the initial steps of alginate biosynthesis and, together with other results, suggests that PgmG may convert glucose-6-phosphate into G1P in the gellan pathway. 相似文献
13.
A molasses based medium for the production of gellan by Sphingomonas paucimobilis ATCC-31461 was developed. Placket-Burman design criterion was applied to study the effect of various nutrient supplements on gellan production using molasses. Among the 20 variables tested, molasses, tryptone, casaminoacid, disodium hydrogen orthophosphate and manganese chloride showed significant effect on gellan production. A central composite design was applied to determine the optimum concentrations of the significant variables obtained from Placket-Burman design. Most suitable medium composition for production of gellan was (g/l): molasses-112.5; tryptone-1; casaminoacid-1; disodium hydrogen orthophosphate-1; manganese chloride-0.947 and the optimum gellan production was 13.814 g/l. 相似文献
14.
Zhu L Wu X Li O Chen Y Qian C Teng Y Tao X Gao H 《Journal of industrial microbiology & biotechnology》2011,38(9):1507-1513
A gene encoding phytoene desaturase (crtI) in the carotenoid biosynthetic pathway of Sphingomonas elodea ATCC 31461, an industrial gellan gum-producing strain, was cloned and identified. This gene is predicted to encode a 492-amino
acid protein with significant homology to the phytoene desaturase of other carotenogenic organisms. Knockout of crtI gene blocked yellow carotenoid pigment synthesis and resulted in the accumulation of colorless phytoene, confirming that
it encodes phytoene desaturase. Further research indicates that the yield of gellan gum production by crtI gene knockout mutants is almost the same as that by the wild-type strain. In addition, a recovery method based on the colorless
fermentation broth of the crtI gene knockout mutant was investigated. Compared to the volume of alcohol for the parent strain, much less alcohol (30%) is
required in this recovery process; thus, the costs of downstream purification of gellan gum can be substantially reduced. 相似文献
15.
The effects of different carbohydrates or mixtures of carbohydrates as substrates on bacterial growth and exopolysaccharide (EPS) production were studied for the yoghurt starter culture Streptococcus thermophilus LY03. This strain produces two heteropolysaccharides with the same monomeric composition (galactose and glucose in the ratio 4:1) but with different molecular masses. Lactose and glucose were fermented by S. thermophilus LY03 only when they were used as sole energy and carbohydrate sources. Fructose was also fermented when it was applied in combination with lactose or glucose. Both the amount of EPS produced and the carbohydrate source consumption rates were clearly influenced by the type of energy and carbohydrate source used, while the EPS monomeric composition remained constant (galactose-glucose, 4:1) under all circumstances. A combination of lactose and glucose resulted in the largest amounts of EPS. Measurements of the activities of enzymes involved in EPS biosynthesis, and of those involved in sugar nucleotide biosynthesis and the Embden-Meyerhof-Parnas pathway, demonstrated that the levels of activity of alpha-phosphoglucomutase, UDP-galactose 4-epimerase, and UDP-glucose pyrophosphorylase are highly correlated with the amount of EPS produced. Furthermore, a weaker relationship or no relationship between the amounts of EPS and the enzymes involved in either the rhamnose nucleotide synthetic branch of the EPS biosynthesis or the pathway leading to glycolysis was observed for S. thermophilus LY03. 相似文献
16.
T Ikeda 《Biochimica et biophysica acta》1981,675(1):69-76
During the development of a cell aggregate of Dictyostelium discoideum into a fruiting body, an antigenic acid mucopolysaccharide is synthesized only in the prespore cells of a cell mass. In this study, the subcellular distributions of UDPgalactose:polysaccharide transferase and UDPglucose pyrophosphorylase involved in biosynthesis of the mucopolysaccharide were determined. The transferase was specifically localized in the smaller vesicles with lighter density than the prespore-specific vacuoles identifiable electronmicroscopically. In contrast to the enzyme, the antigenic mucopolysaccharide was exclusively localized in the prespore-specific vacuoles. Unlike the transferase, UDPglucose pyrophosphorylase was confined to the soluble fraction. The sucrose gradient profiles of the transferase activity in the 5000 X g supernatant gave two main peaks. When the profiles wee compared among standing and migrating slugs and culminating cell mass, the difference in the profiles closely reflected the state of biosynthesis of the acid mucopolysaccharide in each developmental stage. 相似文献
17.
Teruaki Ikeda 《Biochimica et Biophysica Acta (BBA)/General Subjects》1981,675(1):69-76
During the development of a cell aggregate of Dictystelium discoideum into a fruiting body, an antigenic acid mucopolysaccharide is synthesized only in the prespore cells of a cell mass. In this study, the subcellular distributions of UCPgalactose: polysaccharide transferase and UDPglucose pyrophosphorylase involved in biosynthesis of the mucopolysaccharide were determined. The transferase was specifically localized in the smaller vesicles with lighter density than the prespore-specific vacuoles identifiable electronmicroscopically. In contrast to the enzyme, the antigenic mucopolysaccharide was exclusively localized in the prespore-specific vacuoles. Unlike the transferase, UDPglucose pyrophosphorylase was confined to the soluble fraction. The sucrose gradient profiles of the transferase activity in the 5000 × g supernatant gave two main peaks. When the profiles were compared among standing and migrating slugs and culminating cell mass, the difference in the profiles closely reflected the state of biosynthesis of the acid mucopolysaccharide in eac developmental stage. 相似文献
18.
Gellan gum is a water-soluble exopolysaccharide, it has applications in the food, pharmaceutical and chemical industries. In this study, a gellan gum producing strain was isolated from rice root, and this strain was identified be the species of Sphingomonas azotifigens. The Plackett-Burman design was applied to investigate the main factors affecting gellan gum production by S. azotifigens GL-1 in a molasses and cheese whey based medium; the medium compositions were optimized by response surface methodology. The optimum cheese whey based medium consisted of cheese whey 68.34 g/L, Na2HPO4 14.58 g/L and KH2PO4 7.66 g/L, and the maximum gellan gum production that using this medium was 33.75 ± 1.55 g/L. 14.75 ± 0.65 g/L gellan gum was obtained with an optimized molasses medium, which consisted of molasses 50 g/L, Na2HPO4 9.71 g/L and KH2PO4 5.92 g/L. The molecular weight of gellan gum obtained from two medias were 1.06 × 106 and 0.89 × 106 Da, respectively. The cheese whey-derived gellan gum showed a higher rhamnose, lower glucuronic acid and higher glycerate content compared to the molasses-derived gellan gum. S. azotifigens GL-1 has a high gellan gum production capacity in a cheap medium suggesting it has great potential as an industrial gellan gum producer. 相似文献
19.
Identification of the pgmG Gene, Encoding a Bifunctional Protein with Phosphoglucomutase and Phosphomannomutase Activities, in the Gellan Gum-Producing Strain Sphingomonas paucimobilis ATCC 31461 总被引:2,自引:0,他引:2 下载免费PDF全文
Paula A. Videira Luísa L. Cortes Arsnio M. Fialho Isabel S-Correia 《Applied microbiology》2000,66(5):2252-2258
The pgmG gene of Sphingomonas paucimobilis ATCC 31461, the industrial gellan gum-producing strain, was cloned and sequenced. It encodes a 50,059-Da polypeptide that has phosphoglucomutase (PGM) and phosphomannomutase (PMM) activities and is 37 to 59% identical to other bifunctional proteins with PGM and PMM activities from gram-negative species, including Pseudomonas aeruginosa AlgC. Purified PgmG protein showed a marked preference for glucose-1-phosphate (G1P); the catalytic efficiency was about 50-fold higher for G1P than it was for mannose-1-phosphate (M1P). The estimated apparent Km values for G1P and M1P were high, 0.33 and 1.27 mM, respectively. The pgmG gene allowed the recovery of alginate biosynthetic ability in a P. aeruginosa mutant with a defective algC gene. This result indicates that PgmG protein can convert mannose-6-phosphate into M1P in the initial steps of alginate biosynthesis and, together with other results, suggests that PgmG may convert glucose-6-phosphate into G1P in the gellan pathway. 相似文献