Improved strains for production of xanthan gum by fermentation ofXanthomonas campestris

Summary Two classes of mutants ofXanthomonas campestris B1459 were isolated that accumulate more xanthan gum than the parental wild-type in culture broths of shake flask cultures and both batch and fed-batch fermentations. The first mutant class was resistant to the antibiotic rifampicin and accumulated, on average, about 20% more xanthan gum than wild-type. The second mutant class, a derivative of the first, was resistant to both bacitracin and rifampicin, and accumulated about 10% more xanthan than its parent. On a weight basis, the viscosities of the polysaccharides made by each strain were not distinguishable. Only a subset of the drug-resistant mutants were overproducers of xanthan. The biochemical basis for the overproduction of xanthan by the mutant strains has not been determined. Both new strains served as recipients for recombinant plasmids bearing xanthan genes and further augmented the effects of multiple copies of those genes on xanthan productivity.     

High production of xanthan gum by a strain ofXanthomonas campestris conjugated withLactococcus lactis

Summary Plasmid pNZ521, containing phospho--galactosidase, maturation protein and proteinase P genes, was conjugally transferred for the first time fromL.lactis intoX.campestris XLM1.After 20 generations 67% of the tested colonies were resistant to chloramphenicol. In the transconjugant proteinase activity appeared in the growth medium, whereas inL.lactis MG1820 it was extracted from the cell wall. Proteinase activity and production of xanthan gum were studied in different concentrations of whey. Xanthan gum production was found much higher in all cultures with the transconjugant strain XLM1521.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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     

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.     

Bioproduction of low-pigment xanthan gum by a cell-wall deficient mutant of Xanthomonas campestris

Abstract

This work aims to enhance the bioproduction of xanthan gum by screening a hyper-yield producer from the wild-type Xanthomonas campestris during a long-term continuous subculture. We reported a cell-wall deficient mutant, which performed a shift of cell morphology from rod-shaped to round-shaped. Both the yield of xanthan gum and the conversion rate of feedstock were assessed using sucrose as a carbon source with the supplement of yeast extract powder, l-glutamic acid, and other raw materials. After 96?h aerobic fermentation, the yield of xanthan gum of the mutant reached up to 32?g/L, which was 3.4 times of that of the wild-type strain. The conversion rate of feedstock in the mutant was up to 92.1%, which was 3 times of that of the wild-type (31.2%). Furthermore, pigments generated were determined and compared. As a result, the fermentation broth of the wild-type performed an OD_560nm of 0.296, which was 5.8 times of that (OD_560nm?=?0.051) of the mutant. Microscopy analysis showed that the percentage of free-living cells in broth affected the color of the final product. Moreover, the robustness of the fermentation performance of the cell-wall deficient mutant at a pilot scale showed potential for industrial application.     

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.     

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     

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.     

Use of agricultural wastes for xanthan production by Xanthomonas campestris

Four different acid-hydrolyzed wastes, from melon, watermelon, cucumber and tomato were compared for xanthan production. Growth of Xanthomonas campestris, xanthan biosynthesis, kinetics and chemical composition were investigated. Both growth and xanthan production were dependent on the acid hydrolysate concentrations and available nitrogen. Melon acid hydrolyzed waste was the best substrate for xanthan production. Exopolysaccharide obtained throughout this study was compared to commercial xanthan, showing a very similar chemical composition. Acid hydrolyzed wastes are proposed as a new carbon source for xanthan production. Received 16 July 1998/ Accepted in revised form 8 October 1998     

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.