Molecular cloning and expression of the Cellulomonas flavigena cell-associated amylase gene in E. coli and Cellulomonas flavigena

Summary The gene encoding the inducible cell-associated amylase activity was cloned on a 1.5kb Pst1 fragment into pUC8 in E.coli giving the recombinant plasmid pJA 871, and subcloned onto a shuttle vector, pJA85, and transferred into Cellulomonas flavigena AP1(amy^-). Expression was observed in both organisms with increased levels being observed from the recombinant in Cellulomonas compared to the parent strain. The 1.5kb fragment was reoriented in pJA871 and the same level of expression observed in both orientations. Tn1000 insertions into the cloned fragment revealed the location of the coding region. Nucleotide sequencing of both ends of the cloned fragment revealed one open reading frame preceded by a putative control region.     

Free and cell-bound cellulase activity of Cellulomonas flavigena

Free -1, 4-glucanase activity was measured in the supernatant of cultures of Cellulomonas flavigena grown on carboxymethylcellulose or filter paper as the main carbon source. Filtration through a series of filter papers resulted in quantitative removal of the enzyme from the supernatant. The glucanase was found to be tightly bound to the paper. Cellobiose was produced from the filters containing the enzyme, when incubated at 40°C. After removal of the bacterial cells the paper remnants of a C. flavigena culture also formed cellobiose. Apparently -1, 4-glucanase is freed into solution after the paper has been partially degraded. This release is a consequence of the decreasing ratio of cellulose to enzyme.Some glucosidase activity could be detected in the supernatant of stationary phase cultures. This was probably the result of some cell lysis. However, high activities could be measured in ultrasonic cell debris. This suggests that the -glucosidase of C. flavigena, contrary to -1, 4-glucanase, is cell-bound.     

Proteomic Analysis of the Secretome of Cellulomonas fimi ATCC 484 and Cellulomonas flavigena ATCC 482

The bacteria in the genus Cellulomonas are known for their ability to degrade plant cell wall biomass. Cellulomonas fimi ATCC 484 and C. flavigena ATCC 482 have been the subject of much research into secreted cellulases and hemicellulases. Recently the genome sequences of both C. fimi ATCC 484 and C. flavigena ATCC 482 were published, and a genome comparison has revealed their full spectrum of possible carbohydrate-active enzymes (CAZymes). Using mass spectrometry, we have compared the proteins secreted by C. fimi and C. flavigena during growth on the soluble cellulose substrate, carboxymethylcellulose (CMC), as well as a soluble xylan fraction. Many known C. fimi CAZymes were detected, which validated our analysis, as were a number of new CAZymes and other proteins that, though identified in the genome, have not previously been observed in the secretome of either organism. Our data also shows that many of these are co-expressed on growth of either CMC or xylan. This analysis provides a new perspective on Cellulomonas enzymes and provides many new CAZyme targets for characterization.     

Complete genome sequence of Cellulomonas flavigena type strain (134)

Cellulomonas flavigena (Kellerman and McBeth 1912) Bergey et al. 1923 is the type species of the genus Cellulomonas of the actinobacterial family Cellulomonadaceae. Members of the genus Cellulomonas are of special interest for their ability to degrade cellulose and hemicellulose, particularly with regard to the use of biomass as an alternative energy source. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of a member of the genus Cellulomonas, and next to the human pathogen Tropheryma whipplei the second complete genome sequence within the actinobacterial family Cellulomonadaceae. The 4,123,179 bp long single replicon genome with its 3,735 protein-coding and 53 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Structural analysis of the curdlan-like exopolysaccharide produced by Cellulomonas flavigena KU

Cellulomonas flavigena KU produces large quantities of an insoluble exopolysaccharide (EPS) under certain growth conditions. The EPS has previously been shown to be a glucose polymer and to have solubility properties similar to curdlan, a β-1,3-D-glucan produced by Alcaligenes faecalis var. myxogenes 10C3K. Furthermore, EPS purified by alkaline extraction stains with aniline blue, a dye specific for curdlan-type polysaccharides. However, EPS-producing colonies of C. flavigena KU do not stain on aniline blue agar as do those of curdlan-producing bacteria. These facts prompted a more thorough structural analysis of the EPS. Here we report that purified EPS is indeed identical to curdlan in primary structure, but that the native form of the EPS may differ from curdlan in physical conformation. Journal of Industrial Microbiology & Biotechnology (2002) 29, 200–203 doi:10.1038/sj.jim.7000277 Received 19 February 2002/ Accepted in revised form 20 May 2002     

Multigene families of Cellulomonas flavigena encoding endo-beta-1,4-glucanases (CM-cellulases)

Multiple genes coding for endo-beta-1,4-glucanases (CM-cellulases) have been isolated from a newly discovered highly cellulolytic strain of Cellulomonas flavigena. Clones of C. flavigena DNA were isolated in Escherichia coli and screened for gene expression on CM-cellulose plates staining with congo red. Six clones produced CM-cellulase activity as detected in liquid assays, and on activity gels. They fell into three groups within which the sequences cross-hybridised. There were small differences in the pH and temperature optima of the enzymes encoded by representatives of the three groups of clones.     

Isolation of a high-specific-growth-rate mutant of Cellulomonas flavigena on sugar cane bagasse

By treatment of a wild-type strain of Cellulomonas flavigena with N-methyl-N'-nitro-N-nitrosoguanidine at 150 g/ml, mutants PN-7 and PN-10 were obtained, which produce 1.38 and 1.5 times more carboxymethylcellulase than the wild strain when cultured in a batch system with sugar cane bagasse as the sole carbon source. These mutants also exhibited higher specific growth rates compared to the wild strain. From a second mutagenesis of mutant PN-10, mutant PN-120 was obtained in continuous culture. This mutant was able to use a larger portion of sugar cane bagasse than did the wild-type and therefore its biomass yield was also higher. The mutant showed a specific growth rate on sugar cane bagasse threefold higher than the wild strain.     

Characterization of Yarrowia lipolytica mutants affected in hydrophobic substrate utilization

In order to get deeper insights into oxidative degradation of the hydrophobic substrates (HS) triglycerides and alkanes by yeasts, tagged mutants affected in these pathways were generated by random insertion of a mutagenesis cassette MTC into the genome of Yarrowia lipolytica. About 9.600 Ura+ transformants were screened in plate tests for utilization of alkanes (C10, C16), oleic acid and tributyrin. HS degradation mutants were recovered as unable to grow on alkane or on intermediates of the pathway (AlkA-AlkE phenotype classes). To identify the disrupted genes, insertion points of the MTC were sequenced using convergent and divergent PCR. Sequence analysis evidenced both known and new genes required for HS utilization, e.g. for AlkD/E mutants MTC insertion had occurred in genes of thioredoxin reductase, peroxines PEX14 and PEX20, succinate-fumarate carrier SFC1, and isocitrate lyase ICL1. Several mutants were affected in alkane utilization depending on chain length. Mutant Z110 (AlkAb: C10- C16+) was shown to be disrupted for ANT1 encoding a peroxisomal membrane localized adenine nucleotide transporter protein, providing ATP for the activation of short-chain fatty acids by acyl-CoA synthetase II in peroxisomes. Mutants N046 and B095 (AlkAc: C10+ C16-) were disrupted for the ABC transporter encoded by ABC1 gene, thus providing first evidence for its participation in chain length dependent alkane transport processes.     

Synthesis and regulation of D-xylanase from Cellulomonas flavigena wild type and a mutant

The xylanolytic system from Cellulomonas flavigena was enhanced by adding cellulose to the growth medium. The Solka floc:xylan (60:40 w/w) mixture induced xylanase synthesis by more than 3-fold over that induced by growing C. flavigena, wild type and its mutant PN-120 on pure xylan. The hydrolysis pattern of sugar cane bagasse and xylan indicated the presence of debranching endo-;-xylanase activity.     

Selection of hypercellulylytic derepressed mutants of Cellulomonas sp.

Summary Mutants from Cellulomonas sp.IIbc were obtained combined treatment of UV light and N-methyl-N-nitrosoguanidine. T The selection criterion for the screening of catabolite-repression-resistant mutants was based on the formation of clear zones around the bacterial colonies in medium containing 0.5% Walseth cellulose and 0.5% glucose. Mutants produced not only clear zones in significantly lower times than the parent strain, but also exhibited higher specific growth rates and cellulolytic activity when grown on bagasse pith. The cellulase-derepressed character of the mutants was demonstrated by the presence of cellulolytic activity in cultures grown in the presence of high levels of glucose. These results raise the possibility of enhancing the productivity of bacterial degradation of lignocellulosic substrates for single cell protein production. Offprint requests to: F. Alea     

Induction of xylanases by sugar cane bagasse at different cell densities of Cellulomonas flavigena

The effect of cell density on xylanolytic activity and productivity of Cellulomonas flavigena was evaluated under two different culturing conditions: fed-batch culture with discontinuous feed of sugar cane bagasse (SCB; condition 1) and glycerol fed-batch culture followed by addition of SBC as xylanases inducer (condition 2). The enzymatic profile of xylanases was similar in both systems, regardless of the initial cell density at time of induction. However, the xylanolytic activity changed with initial cell density at the time of induction (condition 2). The maximum volumetric xylanase activity increased with increased initial cell density from 4 to 34 g l⁻¹ but decreased above this value. The largest total volumetric xylanase productivity under condition 2 (1.3 IU ml⁻¹ h⁻¹) was significantly greater compared to condition 1 (maximum 0.6 IU ml⁻¹ h⁻¹). Consequently, induction of xylanase activity by SCB after growing of C. flavigena on glycerol at intermediate cell density can be a feasible alternative to improve activity and productivity of xylanolytic enzymes.     

Purification and characterisation of a cellulose binding endoxylanase fromCellulomonas flavigena

Summary An extracellular endoxylanase capable of binding to avicel was isolated and purified fromCellulomonas flavigena. The purified xylanase, posessing no cellulose hydrolysing activity, had a molecular weight of 53K as determined electrophoretically. Steady state adsorption to avicel was reached within 5 minutes at 20°C. The adsorption followed the Langmuir isotherm with a dissociation constant at 0°C of 7.9E-8M and a saturation coefficient of 12 mg per gram with binding being stable for a wide range of ionic strengths down to 0.1M.     

Interactions in a mixed culture composed of Cellulomonas flavigena and Xanthomonas sp. growing in continuous culture on sugar cane bagasse

A mixed culture formed by Cellulomonas flavigen and Xanthomonas sp. was able to grow in continuous culture in a medium with sugar cane bagasses as carbon source without additional growth factors. The effect of temperature, pH and dilution rate (D) upon the microbial population ratio and biomass composition was studied. Both microorganisms co-existed between 30 and 40° C, at pH 5.8–7.0 and D = 0.04–0.11 h^–1. Correspondence to: T. Ponce-Noyola     

Growth and enzyme production of Cellulomonas sp. ATCC 21399 on microcrystalline cellulose. Effects of increasing concentration of a mineral medium

Summary The kinetics and production of different extracellular enzyme activities were studied during growth of Cellulomonas sp. ATCC 21399 on 2% Avicel with different concentrations of M9 mineral medium. The lag phase and the doubling time increased with increasing ionic strength of the medium. The highest cell density was obtained during growth at 5 x M9 mineral medium and Cellulomonas grew well at this high salinity. The enzyme activities against carboxymethylcellulose and xylan increased with increasing concentration of M9 medium up to 5 x M9. By contrast, activities against microcrystalline cellulose (Avicel), galactomannan and amylose decreased with increasing concentration of M9 medium. The extracellular proteinase activity increased with increasing concentration of M9 medium, and it is possible that the lability of the cellulolytic and amylolytic enzymes may be due to their susceptibility to proteolytic inactivation by the extracellular proteinases.     

Isolation and characterization of a Bacillus strain capable of degrading the extracellular glucan from Cellulomonas flavigena strain KU

P.A. BERTRAM, C.S. BULLER, G.C. STEWART AND J.M. AKAGI. 1993. Bacteria capable of utilizing the water-insoluble purified extracellular (1 → 3)-β-D-glucan (curdlan) from Cellulomonas flavigena strain KU by extracellular enzymes, were insolated and characterized. Enrichment cultures from a Winogradsky column were incubated anaerobically at 55C with curdlan as the sole source of carbon. Colonies surrounded by zones of clearing were selected from subcultures on solid curdlan media. One of the isolates was chosen for further study and identified by conventional methods, API-tests with calculation of similarity coefficients and ID-scores, estimation of mol% (G + C) and DNA-DNA liquid hybridization. The isolate is a facultatively anaerobic, facultatively thermophilic Bacillus sp. Identification at the species-level was not achieved. The isolate was characterized by some rare traits among bacilli, but it remains unresolved whether it defines a new taxon.     

Regulation of cellulases and xylanases from a derepressed mutant of Cellulomonas flavigena growing on sugar-cane bagasse in continuous culture

When the wild type Cellulomonas flavigena was grown on glycerol, xylose or cellobiose, it produced basal levels of carboxymethyl-cellulase (CMCase), filter-paperase (FPase) and xylanase activities. By comparison, a catabolic derepressed mutant strain of the same organism produced markedly higher levels of these enzymes when grown on the same carbon sources. Sugar-cane bagasse induced both the wild type and the mutant strain to produce three- to eight-time higher levels of FPase and xylanase than was observed with xylose or cellobiose. Continuous culture was used to determine the minimal cellobiose or glucose concentrations that repress the enzyme synthesis in both strains. 2.5 g l(-1) glucose repressed FPase and xylanases from wild type, while 1.6 times more glucose was needed to repress the same activities in the PN-120 strain. In the same way, twofold more cellobiose was needed to reduce by 75% the CMCase and xylanase activities in the mutant compared to the wild type. The FPase in the presence of 4 g l(-1) cellobiose did not change in the same strain. Therefore, its derepressed and feedback resistant characters of PN-120 mutant are evident. On the other hand, isoelectrofocused crude extracts of mutant and wild strains induced by sugar-cane bagasse, did not show differences in protein patterns, however, the Schiffs staining was more intense in the PN-120 than in the wild strain. These results point out that the mutational treatment did not apparently change the extracellular proteins from mutant PN-120 and this could affect their regulation sites, since derepressed and feed-back resistant enzymes may be produced.     

Microcalorimetric study of cellulose degradation by Cellulomonas uda ATCC 21399

A newly designed batch calorimeter was used to investigate the degradability of some celluloses having varying degrees of crystallinity. The PTC of an aerobic culture of Cellulomonas uda ATCC 21399 obtained revealed a diauxic growth which is attributed to the presence of hemicellulose contaminating Avicel and MN300 cellulose. The microcrystalline celluloses used were not completely utilized, whereas amorphous cellulose was easily metabolized, indicating that under the growth conditions used here, the physical structure of cellulose strongly influenced its microbial degradability. An equivalent growth yield of ca. 0.44 g/g was found with all the substrates used. The heat evolved by metabolism of 1 g cellulose was -5.86 kJ/g, a value similar to that obtained with glucose culture. The growth rate was the only variable parameter. The data obtained showed as expected that the hydrolysis product of cellulose was consumed in the same way as that of glucose and that the only limiting factor to the biodegradability of cellulose was the breakdown of the polymeric substrate. It is concluded that data obtained with glucose metabolism can be used to evaluate the extent of cellulose degradation.