Genetic construction of Xanthomonas campestris and xanthan gum production from whey

Summary Plasmids pUR291 and pNZ521 containing lacZ gene, maturation protein and proteinase P genes, were transferred into X. campestris either by conjugation or by transformation. Plasmid pNZ521 was also conjugally transferred into X. campestris XMT1 a transformant carrying plasmid pUR291. All the constructed strains were evaluated for xanthan gum production in either a medium of 50% whey or the same medium supplemented with 1.5% lactose or 1.5% glucose. Mixed cultures either with transconjugants or with transformants were tested for xanthan gum production as well.     

Valorisation of chicken feathers for xanthan gum production using Xanthomonas campestris MO-03

Xanthan gum is an important commercial polysaccharide produced by Xanthomonas species. In this study, xanthan production was investigated using a local isolate of Xanthomonas campestris MO-03 in medium containing various concentrations of chicken feather peptone (CFP) as an enhancer substrate. CFP was produced with a chemical process and its chemical composition was determined. The addition of CFP (1–8?g/l) increased the conversion of sugar to xanthan gum in comparison with the control medium, which did not contain additional supplements. The highest xanthan production (24.45?g/l) was found at the 6?g/l CFP containing control medium in 54?h. This value was 1.73 fold higher than that of control medium (14.12?g/l). Moreover, addition of CFP improved the composition of xanthan gum; the pyruvate content of xanthan was 3.86% (w/w), higher than that of the control (2.2%, w/w). The xanthan gum yield was also influenced by the type of organic nitrogen sources. As a conclusion, CFP was found to be a suitable substrate for xanthan gum production.     

Development of a phenomenological modeling approach for prediction of growth and xanthan gum production using Xanthomonas campestris

An unstructured kinetic model for xanthan production is described and fitted to experimental data obtained in a stirred batch reactor. The culture medium was composed of several nitrogen sources (soybean hydrolysates, ammonium and nitrate salts) consumed sequentially. The model proposed is able to describe this sequential consumption of nitrogen sources, the consumption of inorganic phosphate and carbon, the evolution of biomass, and production of xanthan. The parameter estimation has been performed by fitting the kinetic model in differential form to experimental data. Runs of the model for simulating xanthan gum production as a function of the initial concentration of inorganic phosphate have shown the positive effect of phosphate limitation on xanthan yield, though diminishing rates of production. The model was used to predict the kinetic parameters for a medium containing a 2-fold lower initial phosphate concentration. When tested experimentally, the measured fermentation parameters were in close agreement with the predicted model values, demonstrating the validity of the model.     

Construction of lactose-utilizing Xanthomonas campestris and production of xanthan gum from whey

Xanthomonas campestris pv. campestris possesses a low level of beta-galactosidase and therefore is not able to grow and produce significant amounts of xanthan gum in a medium containing lactose as the sole carbon source. In this study, a beta-galactosidase expression plasmid was constructed by ligating an X. campestris phage phi LO promoter with pKM005, a ColE1 replicon containing Escherichia coli lacZY genes and the lpp ribosome-binding site. It was then inserted into an IncP1 broad-host-range plasmid, pLT, and subsequently transferred by conjugation to X. campestris 17, where it was stably maintained. The lacZ gene under the control of the phage promoter was expressed at a high level, enabling the cells to grow in a medium containing lactose. Production of xanthan gum in lactose or diluted whey by the engineered strain was evaluated, and it was found to produce as much xanthan gum in these substrates as the cells did in a medium containing glucose.     

Genetic stability and xanthan gum production in Xanthomonas campestris pv. campestris NRRL B1459

A transposon (Tn5-SC) was constructed that can be used to quantify genetic deletions or amplifications. This transposon was used to evaluate the genomic stability of Xanthomonas campestris pv. campestris NRRL B1459 and we found that the genome of this bacterium is as stable as other Gram-negative bacteria or even more stable. Homologous recombination between plasmid sequences was determined in strain NRRL B1459 and was found to occur at a similar level to that reported for other Gram-negative bacteria. We report here that in X.c.c. NRRL B1459 there is no straightforward correlation between the occurrence of genetic rearrangements and frequency of homologous recombination. These data are discussed with respect to the reported instability of strain NRRL B1459 for xanthan gum production.     

Construction of lactose-utilizing Xanthomonas campestris and production of xanthan gum from whey.

Xanthomonas campestris pv. campestris possesses a low level of beta-galactosidase and therefore is not able to grow and produce significant amounts of xanthan gum in a medium containing lactose as the sole carbon source. In this study, a beta-galactosidase expression plasmid was constructed by ligating an X. campestris phage phi LO promoter with pKM005, a ColE1 replicon containing Escherichia coli lacZY genes and the lpp ribosome-binding site. It was then inserted into an IncP1 broad-host-range plasmid, pLT, and subsequently transferred by conjugation to X. campestris 17, where it was stably maintained. The lacZ gene under the control of the phage promoter was expressed at a high level, enabling the cells to grow in a medium containing lactose. Production of xanthan gum in lactose or diluted whey by the engineered strain was evaluated, and it was found to produce as much xanthan gum in these substrates as the cells did in a medium containing glucose.     

Studies on xanthan production from Xanthomonas campestris

Xanthan is an heteropolysaccharide produced by Xanthomonascampestris. Xanthan gum fermentation by a local isolate of X. campestris using different carbon sources was studied. The production of polysaccharide was influenced by the carbon source used. The production of the xanthan was 15.654 g/l with synthetic medium. Production of xanthan at various temperatures ranging between 25v°C and 40v°C was studied. The growth and production was maximum between 25-30v°C. Xanthan production was maximum at pH 7.0-7.5.     

Influence of nutrients on cell growth and xanthan production by Xanthomonas campestris

Xanthan production by Xanthomonas campestris was performed in a batch fermentation study with a view to achieving maximum yields. The factors influencing the production — mainly the nutrients, were investigated. Trace elements such as phosphate and magnesium at 6.0 g/l and 0.2 g/l enhanced the yields.Dr. M. Chellapandian is thankful to the Council of Scientific and Industrial Research, New Delhi for the award of Research Associateship.     

High production of xanthan gum by a glycerol-tolerant strain Xanthomonas campestris WXLB-006

The superior properties of xanthan gum make it an industrial aginomoto used in many industries, especially in oil recovery. In the present work, xanthan production from glycerol by a mutant strain Xanthomonas campestris WXLB-006 reached as high as 17.8?g/L in flask culture. With the adoption of pH control, varied aeration and agitation, and varied glycerol feeding strategy, xanthan production reached 33.9?g/L in a 7-L fermenter and fermentation time decreased to 60?hr. Instead of difficultly and costly purifying glycerol, this research provides a very good case for glycerol utilization. At the same time, this is the first report on a high glycerol-tolerant strain for microbial polysaccharide production and 33.9?g/L is the highest production of xanthan gum produced from glycerol so far.     

Stimulation of xanthan production by Xanthomonas campestris using citric acid

The biosynthesis of xanthan by Xanthomonas campestris was found to be affected by the addition of citric acid in fed batch mode. Under oxygen-limiting conditions, the addition of up to 2.6 g citric acid per litre improved cell viability as well as increasing xanthan yield by up to 80%. Comparative xanthan formation profiles at different operating conditions indicate that at higher aeration (when there was no oxygen limitation), citric acid addition did not improve xanthan production.     

Detection of Xanthomonas campestris mutants with increased xanthan production

Several mutants of Xanthomonas campestris showing increased viscosity and/or gum production were detected after UV treatment. Xanthan solutions of the different mutants showed different intrinsic viscosity values, and no relationship was found between pyruvate or acetate contents and viscosifying ability of the xanthan. The best performance mutant (M-11) was obtained using halo size around colonies in starch-agar plates as the detection criterion, and proved the usefulness of this indirect screen for improved xanthan producers. A pleiotropic effect of this mutation on growth rate and total cell growth was observed. Received 07 December 1995/ Accepted in revised form 14 November 1996     

Genetic engineering of polysaccharide structure: production of variants of xanthan gum in Xanthomonas campestris.

Xanthan gum is an extracellular heteropolysaccharide produced by the bacterium Xanthomonas campestris. Xanthan has wide commercial application as a viscosifier of aqueous solutions. Previously, through genetic engineering, a set of mutants defective in the xanthan biosynthetic pathway has been obtained. Certain mutants were shown to synthesize and polymerize structural variants of the xanthan repeating unit and thus produce "variant xanthans". Initial studies of solution viscosities of these polymers, presented here, indicate that the variants have rheological properties similar to, but not identical with, xanthan. These results indicate that acetylation and pyruvylation can affect the viscometric properties of xanthan. Specifically, the presence of pyruvate increases viscosity, whereas acetate decreases viscosity. In addition, the elimination of sugar residues from xanthan side chains also has a major effect on viscosity. Compared to wild-type xanthan, polymer lacking the terminal mannose (polytetramer) is a poor viscosifier. In contrast, polymer lacking both the terminal mannose and glucuronic acid (polytrimer) is a superior viscosifier, on a weight basis. There is a negative effect of acetylation on the viscosity of polytetramer xanthan, but there is seemingly no effect of acetylation on polytrimer xanthan viscosity. The further study of these materials should provide insight into the relationship between xanthan structure and rheological behavior.     

Clustering of mutations blocking synthesis of xanthan gum by Xanthomonas campestris.

Mutations that block the synthesis of xanthan gum by Xanthomonas campestris B1459S-4L-II were isolated as nonmucoid colonies after treatment with ethyl methanesulfonate. Complete libraries of DNA fragments from wild-type X. campestris were cloned into Escherichia coli by using a broad-host-range cosmid vector and then transferred into each mutant strain by conjugal mating. Cloned fragments that restored xanthan gum synthesis (Xgs+; mucoidy) were compared according to restriction pattern, DNA sequence homology, and complementation of a subset of Xgs- mutations. Groups of clones that contained overlapping homologous DNA were found to complement specific Xgs- mutations. The results suggest clustering of the genetic loci involved in xanthan synthesis. The clustering occurred within three unlinked regions. Two forms of complementation were observed. In most instances, independently isolated cosmid clones that complemented a single mutation were found to be partially homologous. Less frequent was the second form of complementation, in which two cosmid clones that lacked any homologous sequences restored the mucoid phenotype to a single mutant. Finally, xanthan production was measured for wild-type X. campestris carrying multiple plasmid copies of the cloned xanthan genes.     

Effects of temperature on cell growth and xanthan production in batch cultures of Xanthomonas campestris

Batch xanthan fermentations by Xanthomonas campestris NRRL B-1459 at various temperatures ranging between 22 degrees C and 35 degrees C were studied. At 24 degrees C or lower, xanthan formation lagged significantly behind cell growth, resembling typical secondary metabolism. However, at 27 degrees C and higher, xanthan biosynthesis followed cell growth from the beginning of the exponential phase and continued into the stationary phase. Cell growth at 35 degrees C was very slow; the specific growth rate was near zero. The specific growth rate had a maximum value of 0.26 h(-1) at temperatures between 27 degrees C and 31 degrees C. Cell yield decreased from 0.53 g/g glucose at 22 degrees C to 0.28 g/g glucose at 33 degrees C, whereas xanthan yield increased from 54% at 22 degrees C to 90% at 33 degrees C. The specific xanthan formation rate also increased with increasing temperature. The pyruvate content of xanthan produced at various temperatures ranged between 1.9% and 4.5%, with the maximum occurring between 27 degrees C and 30 degrees C. These results suggest that the optimal temperatures for cell growth are between 24 degrees C and 27 degrees C, whereas those for xanthan formation are between 30 degrees C and 33 degrees C. For single-stage batch fermentation, the optimal temperature for xanthan fermentation is thus dependent on the design criteria (i. e., fermentation rate, xanthan yield, and gum qualities). However, a two-stage fermentation process with temperature shift-up from 27 degrees C to 32 degrees C is suggested to optimize both cell growth and xanthan formation, respectively, at each stage, and thus to improve overall xanthan fermentation.     

Production of xanthan gum by Sphingomonas bacteria carrying genes from Xanthomonas campestris

Twelve genes coding for assembly, acetylation, pyruvylation, polymerization, and secretion of the polysaccharide xanthan gum are clustered together on the chromosome of the bacterium Xanthomonas campestris. These genes (gumBCDEFGHIJKLM) are sufficient for synthesis of xanthan gum when placed in bacteria from a different genus, Sphingomonas. The polysaccharide from the recombinant microorganism is largely indistinguishable, structurally and functionally, from native xanthan gum. These results demonstrate that a complex pathway for biosynthesis of a specific polysaccharide can be acquired by a single inter-generic transfer of genes between bacteria. This suggests the biological and commercial feasibility of synthesizing xanthan gum or other polysaccharides in non-native hosts. Received 23 October 1996/ Accepted in revised form 14 April 1997     

Isolation of a Xanthomonas campestris strain with elevated beta-galactosidase activity for direct use of lactose in xanthan gum production

AIMS: To isolate a Xanthomonas campestris strain that can use lactose directly for xanthan gum production. METHODS AND RESULTS: The presence of indigenous beta-galactosidase gene in the wild-type Xc17 was detected by PCR and Southern hybridization. Treatment of Xc17 with nitrous acid resulted in the isolation of Xc17L with a 3.5-fold elevation of beta-galactosidase activity capable of growing in lactose-based medium. Xc17L is stable for at least 100 generations in terms of beta-galactosidase expression. The amounts of xanthan produced by Xc17L in lactose-based medium are comparable to those in glucose-based medium. CONCLUSIONS: Xc17L is potentially useful for xanthan production from whey, a waste containing lactose. SIGNIFICANCE AND IMPACT OF THE STUDY: A lactose-utilizing strain of X. campestris strain can be constructed without incorporation of any exotic DNA or antibiotic resistance gene and therefore concern of a gene-modified organism and fear of a spread of an antibiotic-resistant gene are avoided.     

Effect of corn steep liquor on xanthan production by Xanthomonas campestris

Summary The addition of corn steep liquor (CSL) to batch cultures of Xanthomonas campestris using sucrose as carbon source stimulated cell growth rate, viscosity and xanthan production as compared to non-supplemented cultures. The addition of CSL to a basal medium at a dose of 1 g/l, increased xanthan production and viscosity by 22% and 44% respectively. CSL also shortened the cultivation time and promoted a more efficient sucrose utilization for polymer synthesis. After 72 h of incubation the xanthan yield per sucrose consumed in the CSL-amended culture was 0.63 g/g, this is, 15% higher than without CSL addition. At higher doses of CSL cell growth rate was also increased but not polymer production.     

Variations in xanthan production by antibiotic-resistant mutants of Xanthomonas campestris

Mutants resistant to different antibiotics (streptomycin, tetracycline, ampicillin and penicillin) were obtained from several strains of Xanthomonas campestris and evaluated for xanthan production. Most of the mutants showed alterations in their polysaccharide production, either increasing, decreasing or totally losing their polymer-production capacity. The existence of two types of antibiotic-resistance mechanisms for the assayed drugs is suggested: one that affects xanthan production and another that does not. Differences in outer-membrane protein patterns of mutants that were simultaneously altered in antibiotic resistance and xanthan production were found, in comparison with their parental strains. These findings suggest the existence of a genetic relationship between antibiotic-resistance mechanisms and xanthan production. Some of the mutants obtained showed significant increases in broth viscosity and xanthan concentration. These results suggest that resistance to streptomycin and ampicillin can be used to obtain improved strains in plate screening assays. Received: 8 January 1997 / Received revision: 13 June 1997 / Accepted: 4 July 1997