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.     

Xylanases from Cellulomonas flavigena: purification and characterization

Three xylanases (Xyl1, Xyl2 and Xyl3) were purified and characterized from the culture supernatant of Cellulomonas flavigena grown on sugar cane bagasse. The enzymes were purified by affinity chromatography and gel filtration and had masses of 63 kDa, 17 kDa and 35 kDa, respectively, as measured by SDS-PAGE. All enzymes were active against 4-O-methyl-D-glucuronoxylan and xylan but had no cellulase activity with CM-cellulose, an important characteristics in biobleaching processes. © Rapid Science Ltd. 1998     

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.     

一株产黄纤维单胞菌的选育及产酶特性研究

从土壤中筛选分离出的产纤维素酶菌株S26(经中国科学院微生物所鉴定为产黄纤维单胞菌Cellulomonas flavigena),测定酶活为 25.86 U/mL,以此为出发菌株,经UV反复诱变处理,多代选育,筛选出1株纤维素酶高产突变株UY-4,酶活力达 87.92 U/mL,是出发菌株的 3.4 倍,而且遗传性能稳定.对UY-4产酶影响因素的研究表明,产酶最适条件为稻草与麦麸32、接种量10%(体积与质量比), (NH4)2SO4 0.5%、起始pH 7.4、35 ℃、培养 72～84 h.     

Degradation of cellulases in cultures of Cellulomonas fimi

Abstract Endoglucanases CenA, CenB and CenD, cellobiohydrolases CbhA and CbhB, and the mixed function xylanase-exoglucanase Cex are degraded proteolytically in the supernatants of cultures of Cellulomonas fimi growing with cellulose. All of these polypeptides are modular. The initial sites of proteolysis are within or adjacent to the linkers connecting the modules, leading to the appearance of discrete fragments of the enzymes which retain the functions of the component modules.     

Structure of the gene encoding the exoglucanase of Cellulomonas fimi

In Cellulomonas fimi the cex gene encodes an exoglucanase (Exg) involved in the degradation of cellulose. The gene now has been sequenced as part of a 2.58-kb fragment of C. fimi DNA. The cex coding region of 1452 bp (484 codons) was identified by comparison of the DNA sequence to the N-terminal amino acid (aa) sequence of the Exg purified from C. fimi. The Exg sequence is preceded by a putative signal peptide of 41 aa, a translational initiation codon, and a sequence resembling a ribosome-binding site five nucleotides (nt) before the initiation codon. The nt sequence immediately following the translational stop codon contains four inverted repeats, two of which overlap, and which can be arranged in stable secondary structures. The codon usage in C. fimi appears to be quite different from that of Escherichia coli. A dramatic (98.5%) bias occurs for G or C in the third position for the 35 codons utilized in the cex gene.     

Secretion of Cellulomonas fimi exoglucanase by Escherichia coli

A leader sequence of 41 amino acids (aa) has been proposed as the signal sequence for the exoglucanase (Exg) from Cellulomonas fimi. The ability of this 41-aa peptide to function as a leader sequence has been shown here by gene fusion experiments in Escherichia coli. A hybrid leader sequence containing C-terminal 37 aa of the leader peptide and N-terminal 6 aa of beta-galactosidase (beta Gal) directed export of the Exg into the periplasm of E. coli. In contrast, hybrid beta Gal-Exg proteins in which the leader sequence is not present are retained in the cytoplasm.     

Cellulase and hemicellulase production by Cellulomonas flavigena NIAB 441

Summary Cellulomonas flavigena (strain NIAB 441) produced cellulase and hemicellulase activities when grown on Leptochloa fusca L. Kunth (Kallar grass), found to be the best inducer for enzyme production. The enzyme possessed the potential to saccharify bagasse, Kallar grass straw, wheat straw, carboxymethyl cellulose (CMC) and xylan to reducing sugars.     

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     

Fermentation of C14-Labeled Cellobiose by Cellulomonas fimi

Crude cell-free extracts from Cellulomonas fimi contain cellobiose phosphorylase which cleaves cellobiose into glucose and glucose-1-phosphate in the presence of inorganic phosphate. With the aid of this enzyme, two samples of C¹⁴-cellobiose labeled in reducing or non-reducing glucosyl moiety were prepared from uniformly labeled C¹⁴-glucose or C¹⁴-glucose-1-phosphate as substrate, respectively. The labeled preparations have been shown to be radiochemically pure. Analyses of the anaerobic fermentation products from C¹⁴-cellobiose by resting cell suspensions showed that both glucose moieties were fermented almost equivalently. However, relatively small differences in specific activities of the products revealed that significantly larger amounts of formic acid and smaller amounts of acetic acid were produced from the reducing glucose moiety than from the other half of the molecule. Succinic and lactic acids appeared to be produced almost equally from both moieties.     

Characterization and structure of an endoglucanase gene cenA of Cellulomonas fimi

Two BamHI fragments (0.8 and 5.2 kb) of Cellulomonas fimi containing an endoglucanase (Eng) gene (cenA) were individually cloned into the BamHI site of pBR322; they expressed carboxymethylcellulase activity in Escherichia coli. The nucleotide (nt) sequence of the cenA gene was determined by sequencing overlapping deletions. The cenA gene is 1350 bp long encoding a polypeptide of 449 amino acids (aa) and stop codon. The 0.8-kb BamHI component encodes the first 76 aa, whereas the 5.2-kb BamHI component encodes the rest of the Eng. The Eng lacking the N-terminal 76 aa retains its activity and antigenicity, and it forms an active fusion protein with the N-terminal portion of the TcR determinant. The C-terminal region of the Eng is crucial for activity and a deletion of as little as 12 aa from that end results in the loss of all Eng activity. The N-terminal 31 aa of the Eng constitute a leader peptide which appears to be functional in exporting the enzyme to the periplasm in E. coli.     

Factors affecting accumulation and degradation of curdlan, trehalose and glycogen in cultures of Cellulomonas flavigena strain KU (ATCC 53703)

Cellulomonas flavigena strain KU (ATCC 53703) is a cellulolytic, Gram-positive bacterium which produces large quantities of an insoluble exopolysaccharide (EPS) when grown in minimal media with a high carbon-to-nitrogen (C/N) ratio. Earlier studies proved the EPS is structurally identical to the linear β-1,3-glucan known as curdlan and provided evidence that the EPS functions as a carbon and energy reserve compound. We now report that C. flavigena KU also accumulates two intracellular, glucose-storage carbohydrates under conditions of carbon and energy excess. These carbohydrates were partially purified and identified as the disaccharide trehalose and a glycogen/amylopectin-type polysaccharide. A novel method is described for the sequential fractionation and quantitative determination of all three carbohydrates from culture samples. This fractionation protocol was used to examine the effects of C/N ratio and osmolarity on the accumulation of cellular carbohydrates in batch culture. Increasing the C/N of the growth medium caused a significant accumulation of curdlan and glycogen but had a relatively minor effect on accumulation of trehalose. In contrast, trehalose levels increased in response to increasing osmolarity, while curdlan levels declined and glycogen levels were generally unaffected. During starvation for an exogenous source of carbon and energy, only curdlan and glycogen showed substantial degradation within the first 24 h. These results support the conclusion that extracellular curdlan and intracellular glycogen can both serve as short-term reserve compounds for C. flavigena KU and that trehalose appears to accumulate as a compatible solute in response to osmotic stress.     

The expression of Cellulomonas fimi cellulase genes in Brevibacterium lactofermentum

The exoglucanase gene (cex) and the endoglucanase A gene (cenA) from Cellulomonas fimi were subcloned into the Escherichia coli/Brevibacterium lactofermentum shuttle vector pBK10. Both genes were expressed to five to ten times higher levels in B. lactofermentum than in E. coli, probably because these genes were expressed from C. fimi promoters. In B. lactofermentum virtually all of the enzyme activities were in the culture supernatant. This system will facilitate analysis of the expression of the C. fimi genes in and secretion of their products from a Gram-positive bacterium.     

Growth properties of Cellulomonas flavigena mutants affected in cellulose utilization.

The role of cellobiose metabolism in cellulose utilization by Cellulomonas flavigena was investigated by studying mutants unable to grow on cellobiose or cellulose. The results show that the ability to utilize cellulose is strictly dependent on the ability to utilize cellobiose.     

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.     

Mapping of genes encoding glycoside hydrolases on the chromosome of Cellulomonas fimi

Cellulomonas fimi genomic DNA was digested with HpaI, MunI, HindIII, and NsiI, producing fragments ranging in size from 20 to 1400 kbp that were resolved by pulsed field gel electrophoresis. Genetic and physical linkages were determined by Southern blotting and were used to construct a genome map. Cellulomonas fimi has a single circular chromosome of approx. 4000 kbp. Except for two closely linked genes, cbh6A and cel5A, the genes known to encode glycoside hydrolases are scattered widely on the chromosome.     

The amylase activity associated with Cellulomonas flavigena is cell associated and inducible

Summary Analysis of the amylase activity associated withCellulomonas flavigena showed that it was a cell associated activity that could be released upon sonication, it was active against amylopectin and starch but showed no activity against pullulan and only slight activity against amylose. The enzyme was also found to have liquifying activity and to be inducible.