Optimization of exopolysaccharide welan gum production by Alcaligenes sp. CGMCC2428 with Tween-40 using response surface methodology

The effect of additives on welan gum production produced by fermentation with Alcaligenes sp. CGMCC2428 was studied. Tween-40 was the best additive for improving welan gum production and welan gum displayed better rheological properties than that obtained by control fermentation without additives. Response surface methodology was employed to optimize the culture conditions for welan gum production in the shake flask culture, including Tween-40 concentration, pH and culture temperature. The optimal conditions were determined as follows: Tween-40 concentration 0.94 g/l, pH 6.9 and temperature 29.6 °C. The corresponding experimental concentration of welan gum was 23.62 ± 0.60 g/l, which was agreed closely with the predicted value (23.48 g/l). Validation experiments were also carried out to prove the adequacy and the accuracy of the model obtained. The welan gum fermentation in a 7.5 l bioreactor reached 24.90 ± 0.68 g/l.     

Improvement of welan gum production and redistribution of metabolic flux under pH control process in Alcaligenes sp. CGMCC2428

Batch fermentations of welan gum from Alcaligenes sp. CGMCC2428 at pH values of 5.5 ～ 7.0 were studied. Based on the kinetic analysis, a pH control process for improving welan production was developed. By maintaining the culture pH at 7.0, the process significantly improved the maximal welan concentration and productivity to reach 25.1 ± 0.65 g/L and 0.42 ± 0.003 g/L/h, respectively, compared with those in native pH processes where pH value would decrease from 7.0 to 5.1 (18.5 ± 0.72 g/L and 0.28 ± 0.002 g/L/h). This improvement may be due to the increased metabolic flux of glucose-1-phosphate to welan induced by pH control process. Furthermore, scale-up fermentation under controlled pH was implemented using 300-L fermentor. The highest welan concentration of 27.5 ± 0.97 g/L was obtained. This simplified process proved effective in industry fermentation for high welan production.     

Strain improvement and metabolic flux modeling of wild-type and mutant <Emphasis Type="Italic">Alcaligenes</Emphasis> sp. NX-3 for synthesis of exopolysaccharide welan gum

Low-energy nitrogen ion beam implantation technique was used for the strain improvement of Alcaligenes sp. NX-3 for the production of exopolysaccharide welan gum. A high welan gum producing mutant, Alcaligenes sp. NX-3-1, was obtained through 20 keV N⁺ ion beam irradiation. Starting at a concentration of 50 g/L of glucose, mutant NX-3-1 produced 25.0 g/L of welan gum after 66 h of cultivation in a 7.5 L bioreactor, which was 34.4% higher than that produced by the wild-type strain. The results of metabolic flux analysis showed that the glucose-6-phosphate and acetyl coenzyme A nodes were the principle and flexible nodes, respectively. At the glucose-6-phosphate node, the fraction of carbon measured from glucose-6-phosphate to glucose-1-phosphate was enhanced after mutagenesis, which indicated that more flux was used to synthesize welan gum in the mutant. By analyzing the activities of related enzymes in the biosynthetic pathway of sugar nucleotides essential for welan gum production, we found that the specific activities of phosphoglucomutase, UDP-glucose pyrophosphorylase, UDP-glucose dehydrogenase, and dTDP-glucose pyrophosphorylase in the mutant strain were higher than those in the wild-type strain. These improvements in enzyme activities could be due to the affected of ion beam implantation.     

Biosynthetic pathway of sugar nucleotides essential for welan gum production in Alcaligenes sp. CGMCC2428

Welan gum is a microbial polysaccharide produced by Alcaligenes sp. CGMCC2428 that has d-glucose, d-glucuronic acid, d-glucose, and l-rhamnose as the main structural unit. The biosynthetic pathway of sugar nucleotides essential for producing welan gum in this strain was established in the following ways: (1) the detection of the presence of several intermediates and key enzymes; (2) the analysis of the response upon addition of precursors to the culture medium; (3) the correlation of the activities between several key enzymes with the yields of welan gum. With addition of 200-μM glucose-6-phosphate and fructose-6-phosphate, the production of welan gum was improved by 18%. The activities of phosphoglucomutase, phosphomannose isomerase, UDP-glucose pyrophosphorylase, and dTDP-glucose pyrophosphorylase, correlated well with the yields of welan gum. According to these findings, the biosynthetic pathway was proposed to involve the metabolism of glucose via two discrete systems. The first involves conversion of glucose to glucose-6-phosphate, with further reactions producing glucose-1-phosphate and fructose-6-phosphate, which are metabolized to the nucleotide sugar precursors of welan gum. The second system involves metabolism of glucose to synthesize the basic structural skeleton of the cell via central metabolic pathways, including the Entner–Doudoroff pathway, the pentose phosphate pathway, and the tricarboxylic acid cycle.     

Improved production of carotenoid-free welan gum in a genetic-engineered <Emphasis Type="Italic">Alcaligenes</Emphasis> sp. ATCC31555

Objective

To improve the production of welan gum and obtain a carotenoid-free strain while reducing the fermentation and post-treatment costs.

Results

The vitreoscilla globin (vgb) gene combined with the β-galactosidase (lacZ) promoter was inserted into the phytoene synthase (crtB) gene region of the chromosome in Alcaligenes sp. ATCC31555. When the recombinant strain was grown in a 5 l fermentor, welan gum was produced at 24 ± 0.4 g l^?1 compared to 21 g ± 0.4 g l^?1 in the wild type. Furthermore, the carotenoid-free welan gum produced using Alcaligenes sp. ATCC31555 VHb strain was less expensive with improved properties.

Conclusions

Alcaligenes sp. ATCC31555 VHb strain was a better neutral welan-producing strain with a higher production than the wild-type strain.

     

Genome Sequence of Xanthomonas campestris JX, an Industrially Productive Strain for Xanthan Gum

Xanthomonas campestris JX, a soil bacterium, is an industrially productive strain for xanthan gum. Here we present a 5.0-Mb assembly of its genome sequence. We have annotated 12 coding sequences (CDSs) responsible for xanthan gum biosynthesis, 346 CDSs encoding carbohydrate metabolism, and 69 CDSs related to virulence, defense, and plant disease.     

Enhanced welan gum production using a two-stage agitation speed control strategy in Alcaligenes sp. CGMCC2428

Batch fermentative production of welan gum by Alcaligenes sp. CGMCC2428 was investigated under various oxygen supply conditions using regulating agitation speed. Based on a three kinetic parameters analysis that includes specific cell growth rate (μ), specific glucose consumption rate (q _s), and specific welan formation rate (q _p), a two-stage agitation speed control strategy was proposed to achieve high concentration, high yield, and high viscosity of welan. During the first 22 h, the agitation speed in 7.5 L fermenter was controlled at 800 rpm to maintain high μ for cell growth. The agitation was then reduced step-wise to 600 rpm to maintain a changing profile with stable dissolved oxygen levels and obtain high qp for high welan accumulation. Finally, the maximum concentration of welan was reached at 26.3 ± 0.89 g L⁻¹ with a yield of 0.53 ± 0.003 g g⁻¹ and the welan gum viscosity of 3.05 ± 0.10 Pa s, which increased by an average of 15.4, 15.2, and 20.1% over the best results controlled by constant agitation speeds.     

Pretreatment of Cells of Klebsiella pneumoniae with 50% (v/v) Dimethylsulfoxide Yields Purified Deoxyribonucleic Acid of Low Polysaccharide Content

The flow behavior and dynamic viscoelasticity of welan gum solutions were measured with a rheogoniometer. The welan gum showed shear-thinning behavior at a concentration of 0.1%, but plastic behavior above 0.3% at 25°C. The dynamic viscoelasticity increased with increasing concentration, and was scarcely changeable with increasing temperature even at 80°C. Gelation did not occur even in a polysaccharide concentration of 1.0% at low temperature (0°C). An increase of the dynamic modulus was not observed on the addition of CaCl₂ (6.8 mm). The dynamic viscoelasticity of welan gum solution was scarcely changeable in a wide range of pH from 2 to 12. The dynamic modulus was also scarcely changeable on addition of urea (4.0 m). Possible mode of intramolecular associations between the OH-4 of the d-glucosyl residue and the adjacent hemiacetal oxygen atom of the l-rhamnosyl residue, and between the methyl group of the l-rhamnosyl residue and the adjacent hemiacetal oxygen atom of the d-glucosyl residue were proposed.     

鞘氨醇单胞菌的研究进展

鞘氨醇单胞菌(Sphingomonas)不仅细胞膜含有比脂多糖更疏水的鞘糖脂,而且具有高效的代谢调控机制和基因调控能力,使其在威兰胶合成、环境修复和促进植物生长等方面具有巨大的应用潜力。目前国内在鞘氨醇单胞菌代谢机制方面的研究尚无新突破。本文主要综述了鞘氨醇单胞菌的系统分类、基因组学、基因调控机制及其应用等方面的研究,从基因层面分析鞘氨醇单胞菌产威兰胶的合成机制,为后续鞘氨醇单胞菌高密度发酵、工业化生产等研究提供理论基础,以便进一步发掘其在生物技术上的应用潜力。     

高粘发酵体系不同搅拌桨的CFD模拟及发酵验证

搅拌桨是高好氧高黏度微生物发酵实现高效反应必不可少的因素之一,不同搅拌桨组合对发酵过程的影响十分重要。威兰胶是由产碱杆菌在高耗氧高粘度发酵体系下合成的胞外微生物多糖,广泛应用于水泥、石油、油墨、食品等行业中。本研究借助于计算流体力学(Computational fluid dynamics,CFD)的方法,以威兰胶发酵液体系为研究体系,研究了6种不同搅拌桨组合在反应器内流体速率分布、剪切速率、和气含率等参数。将模拟效果较好的3种组合用于威兰胶发酵过程。研究表明MB-4-6搅拌桨组合对改善发酵罐内部的溶氧及流场分布效果最明显,威兰胶产量水平提高了13%。同时在该组合下威兰胶的产品粘度得到有效提高。     

X-ray and computer modeling studies on gellan-related polymers: molecular structures of welan, S-657, and rhamsan

The primary structures of the four bacterial polysaccharides gellan, welan, S-657, and rhamsan are the same with respect to their backbones, but have different side-chains. This difference has a profound influence on their behavior in aqueous media. Solutions of gellan gum form stable aqueous gels under appropriate ionic conditions. By contrast, welan, S-657, and rhamsan do not gel but give very viscous solutions over a wide range of thermal, pH, and salt conditions. X-Ray fiber diffraction analysis and computer modeling of these branched polysaccharides demonstrate that they all have the same half-staggered, double-helical conformations as in the unbranched gellan, suggesting, therefore, that the side chains are responsible for diminishing gelling behavior. Depending on the size and location, the side chains shield the carboxylate groups to varying degrees; this shielding is substantial in welan and S-657, but less in rhamsan. In all cases, side-chain-main-chain interactions within the double helix prevent the carboxylate-mediated aggregation of double helices that is necessary for the gelation.     

Efficient Plant Gene Identification Based on Interspecies Mapping of Full-Length cDNAs

We present an annotation pipeline that accurately predicts exon–intron structures and protein-coding sequences (CDSs) on the basis of full-length cDNAs (FLcDNAs). This annotation pipeline was used to identify genes in 10 plant genomes. In particular, we show that interspecies mapping of FLcDNAs to genomes is of great value in fully utilizing FLcDNA resources whose availability is limited to several species. Because low sequence conservation at 5′- and 3′-ends of FLcDNAs between different species tends to result in truncated CDSs, we developed an improved algorithm to identify complete CDSs by the extension of both ends of truncated CDSs. Interspecies mapping of 71 801 monocot FLcDNAs to the Oryza sativa genome led to the detection of 22 142 protein-coding regions. Moreover, in comparing two mapping programs and three ab initio prediction programs, we found that our pipeline was more capable of identifying complete CDSs. As demonstrated by monocot interspecies mapping, in which nucleotide identity between FLcDNAs and the genome was ∼80%, the resultant inferred CDSs were sufficiently accurate. Finally, we applied both inter- and intraspecies mapping to 10 monocot and dicot genomes and identified genes in 210 551 loci. Interspecies mapping of FLcDNAs is expected to effectively predict genes and CDSs in newly sequenced genomes.     

Anopheles gambiae genome reannotation through synthesis of ab initio and comparative gene prediction algorithms

Background

Complete genome annotation is a necessary tool as Anopheles gambiae researchers probe the biology of this potent malaria vector.

Results

We reannotate the A. gambiae genome by synthesizing comparative and ab initio sets of predicted coding sequences (CDSs) into a single set using an exon-gene-union algorithm followed by an open-reading-frame-selection algorithm. The reannotation predicts 20,970 CDSs supported by at least two lines of evidence, and it lowers the proportion of CDSs lacking start and/or stop codons to only approximately 4%. The reannotated CDS set includes a set of 4,681 novel CDSs not represented in the Ensembl annotation but with EST support, and another set of 4,031 Ensembl-supported genes that undergo major structural and, therefore, probably functional changes in the reannotated set. The quality and accuracy of the reannotation was assessed by comparison with end sequences from 20,249 full-length cDNA clones, and evaluation of mass spectrometry peptide hit rates from an A. gambiae shotgun proteomic dataset confirms that the reannotated CDSs offer a high quality protein database for proteomics. We provide a functional proteomics annotation, ReAnoXcel, obtained by analysis of the new CDSs through the AnoXcel pipeline, which allows functional comparisons of the CDS sets within the same bioinformatic platform. CDS data are available for download.

Conclusion

Comprehensive A. gambiae genome reannotation is achieved through a combination of comparative and ab initio gene prediction algorithms.     

Screening and characterization of Sphingomonas sp. mutant for welan gum biosynthesis at an elevated temperature

The optimal temperature for the microbial polysaccharide fermentation is no higher than 30 °C, which is economically undesirable due to additional cooling cost. To solve this problem in the case of welan gum production, we obtained the high-temperature-tolerant-producing strain, Sphingomonas sp. HT-1 by atmospheric and room-temperature plasma-induced mutation. Using HT-1, we obtained a concentration and 1 % aqueous viscosity of 26.8 ± 0.34 g/L and 3.50 ± 0.05 Pa s at a comparatively higher optimal temperature (37 °C). HT-1 was further characterized to understand the mechanism by which these properties are improved. Results indicated that high yield could be attributed to the following: (1) enhanced intracellular synthesis, demonstrated by an increase in the activities of key enzymes, and (2) accelerated cross-membrane substrate uptake and product secretion, indicated by improved membrane fluidity and permeability. Temperature tolerance could be attributed to the overexpression of the investigated heat shock proteins and oxidative stress proteins.     

Genome Sequence of Pseudomonas putida S12, a Potential Platform Strain for Industrial Production of Valuable Chemicals

Pseudomonas putida strain S12, a well-studied solvent-tolerant bacterium, is considered a platform strain for the production of many chemicals. Here, we present a 6.28-Mb assembly of its genome sequence. We have annotated 32 coding sequences (CDSs) encoding efflux systems of organic compounds and 195 CDSs responsible for the metabolism of aromatic compounds.     

Virulence determinants, drug resistance and mobile genetic elements of Laribacter hongkongensis: a genome-wide analysis

Background

Laribacter hongkongensis is associated with community-acquired gastroenteritis and traveler's diarrhea. In this study, we performed an in-depth annotation of the genes in its genome related to the various steps in the infective process, drug resistance and mobile genetic elements.

Results

For acid and bile resistance, L. hongkongensis possessed a urease gene cassette, two arc gene clusters and bile salt efflux systems. For intestinal colonization, it possessed a putative adhesin of the autotransporter family homologous to those of diffusely adherent Escherichia coli (E. coli) and enterotoxigenic E. coli. To evade from host defense, it possessed superoxide dismutase and catalases. For lipopolysaccharide biosynthesis, it possessed the same set of genes that encode enzymes for synthesizing lipid A, two Kdo units and heptose units as E. coli, but different genes for its symmetrical acylation pattern, and nine genes for polysaccharide side chains biosynthesis. It contained a number of CDSs that encode putative cell surface acting (RTX toxin and hemolysins) and intracellular cytotoxins (patatin-like proteins) and enzymes for invasion (outer membrane phospholipase A). It contained a broad variety of antibiotic resistance-related genes, including genes related to β-lactam (n = 10) and multidrug efflux (n = 54). It also contained eight prophages, 17 other phage-related CDSs and 26 CDSs for transposases.

Conclusions

The L. hongkongensis genome possessed genes for acid and bile resistance, intestinal mucosa colonization, evasion of host defense and cytotoxicity and invasion. A broad variety of antibiotic resistance or multidrug resistance genes, a high number of prophages, other phage-related CDSs and CDSs for transposases, were also identified.     

Analysis of the Pantoea ananatis pan-genome reveals factors underlying its ability to colonize and interact with plant,insect and vertebrate hosts

Background

Pantoea ananatis is found in a wide range of natural environments, including water, soil, as part of the epi- and endophytic flora of various plant hosts, and in the insect gut. Some strains have proven effective as biological control agents and plant-growth promoters, while other strains have been implicated in diseases of a broad range of plant hosts and humans. By analysing the pan-genome of eight sequenced P. ananatis strains isolated from different sources we identified factors potentially underlying its ability to colonize and interact with hosts in both the plant and animal Kingdoms.

Results

The pan-genome of the eight compared P. ananatis strains consisted of a core genome comprised of 3,876 protein coding sequences (CDSs) and a sizeable accessory genome consisting of 1,690 CDSs. We estimate that ~106 unique CDSs would be added to the pan-genome with each additional P. ananatis genome sequenced in the future. The accessory fraction is derived mainly from integrated prophages and codes mostly for proteins of unknown function. Comparison of the translated CDSs on the P. ananatis pan-genome with the proteins encoded on all sequenced bacterial genomes currently available revealed that P. ananatis carries a number of CDSs with orthologs restricted to bacteria associated with distinct hosts, namely plant-, animal- and insect-associated bacteria. These CDSs encode proteins with putative roles in transport and metabolism of carbohydrate and amino acid substrates, adherence to host tissues, protection against plant and animal defense mechanisms and the biosynthesis of potential pathogenicity determinants including insecticidal peptides, phytotoxins and type VI secretion system effectors.

Conclusions

P. ananatis has an ‘open’ pan-genome typical of bacterial species that colonize several different environments. The pan-genome incorporates a large number of genes encoding proteins that may enable P. ananatis to colonize, persist in and potentially cause disease symptoms in a wide range of plant and animal hosts.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-404) contains supplementary material, which is available to authorized users.     

Visualizing the proteome of Escherichia coli: an efficient and versatile method for labeling chromosomal coding DNA sequences (CDSs) with fluorescent protein genes

To investigate the feasibility of conducting a genomic-scale protein labeling and localization study in Escherichia coli, a representative subset of 23 coding DNA sequences (CDSs) was selected for chromosomal tagging with one or more fluorescent protein genes (EGFP, EYFP, mRFP1, DsRed2). We used λ-Red recombination to precisely and efficiently position PCR-generated DNA targeting cassettes containing a fluorescent protein gene and an antibiotic resistance marker, at the C-termini of the CDSs of interest, creating in-frame fusions under the control of their native promoters. We incorporated cre/loxP and flpe/frt technology to enable multiple rounds of chromosomal tagging events to be performed sequentially with minimal disruption to the target locus, thus allowing sets of proteins to be co-localized within the cell. The visualization of labeled proteins in live E. coli cells using fluorescence microscopy revealed a striking variety of distributions including: membrane and nucleoid association, polar foci and diffuse cytoplasmic localization. Fifty of the fifty-two independent targeting experiments performed were successful, and 21 of the 23 selected CDSs could be fluorescently visualized. Our results show that E. coli has an organized and dynamic proteome, and demonstrate that this approach is applicable for tagging and (co-) localizing CDSs on a genome-wide scale.