Cloning of Clostridium thermocellum endoglucanase genes in Escherichia coli

Six independent and distinct cel genes coding endoglucanases have been selected from C. thermocellum pUC19-based gene bank in E. coli TG1. E. coli-derived Cel-proteins possessing Mr from 39,000 to 61,000 are able to cleave lichenan, as well as xylan and carboxymethyl cellulose. Cel 7- and Cel 8-endoglucanases are characterized by cellobiohydrolase type substrate specificity, being able to cleave model fluorogenic aryldisaccharide substrate MU-G2. The clone pCU110 (cel 7) produces about 10-fold more endoglucanase activity than other clones.     

Cyclic AMP phosphodiesterase in Thermomonospora curvata.

Cyclic AMP phosphodiesterase (PDE; EC 3.1.4.17) in Thermomonospora curvata was purified and characterized. Fractionation of cell extracts by ion-exchange and size-exclusion chromatography revealed four PDE isozymes, which differed markedly in molecular weight, theophylline sensitivity, pH optima, and substrate affinity. Although the enzyme was labile after purification, total recovery of PDE activity was fivefold that of the crude extract. PDE biosynthesis appeared sensitive to the growth phase, growth rate, and carbon source. PDE levels in batch cultures peaked and declined rapidly during mid-exponential-phase growth. In continuous culture, maximal PDE and cellulase production occurred at dilution rates yielding mean cell generation times of about 5 and 17 h, respectively. The addition of glucose to cellulose-grown cells caused declines in both cyclic AMP and PDE levels, suggesting that the enzyme was subject to, rather than the agent of, catabolite repression.     

Cloning of a thermostable alpha-amylase gene from Thermomonospora curvata and its expression in Streptomyces lividans

The gene from Thermomonospora curvata CCM 3312 coding for thermostable alpha-amylase (tam) has been cloned in Streptomyces lividans TK 24 and localized to a 2.6 kb HindIII-BamHI fragment of DNA. The data presented here show that the tam gene is expressed at a high level in S. lividans and that the protein is efficiently excreted.     

Production, purification, and characterization of alpha-amylase from Thermomonospora curvata.

Thermomonospora curvata produces an extracellular alpha-amylase. Maximal amylase production by cultures in a starch-mineral salts medium occurred at pH 7.5 and 53 degrees C. The crude enzyme was unstable to heating (65 degrees C) at pH 4 to 6, and was activated when heated at pH 8. The enzyme was purified 66-fold with a 9% yield and appeared homogeneous on discontinuous gel electrophoresis. The pH and temperature optima for activity of the purified enzyme were 5.5 to 6.0 and 65 degrees C. The molecular weight was calculated to be 62,000. The Km for starch was 0.39 mg/ml. The amylolytic pattern consisted of a mixture of maltotetraose and maltopentaose.     

Cloning of Beneckea genes in Escherichia coli.

Genes from Beneckea harveyi, a luminescent marine bacterium, were cloned in Escherichia coli. This was done by producing randomly sheared fragments of Beneckea DNA and inserting them into the EcoRI site of plasmid pMB9 by the adenine-thymine joining procedure. The hybrid plasmids were used to transform E. coli C600 SF8. Among the transformants selected for tetracycline resistance, one clone that appeared to complement a leucine tb mutation was identified. The transformants were screened for the presence of Beneckea 5S genes. Four of these clones were analyzed in detail by hybridization with 16S, 23S, and 4S Beneckea RNA. The observations suggest that the ribosomal genes in Beneckea are linked, but are present in a different order than those in E. coli.     

Cloning and expression of Clostridium acetobutylicum endoglucanase, cellobiase and amino acid biosynthesis genes in Escherichia coli

Clostridium acetobutylicum P262 endoglucanase and cellobiase genes, cloned on a 4.9 kb DNA fragment in the recombinant plasmid pHZ100, were expressed from their own promoter in Escherichia coli. Active carboxymethylcellulase and cellobiase enzymes were produced, but there was no degradation of Avicel. The endoglucanase activities observed in cell extracts of E. coli HB101(pHZ100) differed in their pH and temperature optima from those previously reported for C. acetobutylicum P270. Complementation of E. coli arg and his mutations by cloned C. acetobutylicum DNA was also observed.     

DNA sequences of three beta-1,4-endoglucanase genes from Thermomonospora fusca.

The DNA sequences of the Thermomonospora fusca genes encoding cellulases E2 and E5 and the N-terminal end of E4 were determined. Each sequence contains an identical 14-bp inverted repeat upstream of the initiation codon. There were no significant homologies between the coding regions of the three genes. The E2 gene is 73% identical to the celA gene from Microbispora bispora, but this was the only homology found with other cellulase genes. E2 belongs to a family of cellulases that includes celA from M. bispora, cenA from Cellulomonas fimi, casA from an alkalophilic Streptomyces strain, and cellobiohydrolase II from Trichoderma reesei. E4 shows 44% identity to an avocado cellulase, while E5 belongs to the Bacillus cellulase family. There were strong similarities between the amino acid sequences of the E2 and E5 cellulose binding domains, and these regions also showed homology with C. fimi and Pseudomonas fluorescens cellulose binding domains.     

Cloning and characterization of the xyl genes from Escherichia coli

Summary Specific xylose utilization mutants of Escherichia coli were isolated that had altered xylose isomerase (xylA), xylulokinase (xylB), and regulatory (xylR) or transport (xylT) activities. We screened the Clarke and Carbon E. coli gene bank and one clone, pLC10–15, was found to complement the xyl mutants we had characterized. Subcloning and DNA restriction mapping allowed us to locate the xylA and xylB genes on a 1.6 kbp BglII fragment and a 2.6 kbp HindIII-SalI fragment, respectively. The identification and mapping of xyl gene promoters suggest that the xylA and xylB genes are organized as an operon having a single xylose inducible promoter preceding the xylA gene.     

Cellulase biosynthesis in a catabolite repression-resistant mutant of Thermomonospora curvata.

A catabolite repression-resistant mutant of the thermophilic actinomycete Thermomonospora curvata was obtained by treatment with ethyl methanesulfonate and UV light. Cellulase biosynthesis was undiminished by glucose, 2-deoxyglucose, or alpha-methyl glucoside, which are potent repressors in the wild type. Intracellular cyclic AMP levels were higher in the mutant in both the absence and the presence of repressors.     

Cellulase Production by Thermomonospora curvata Isolated from Municipal Solid Waste Compost

A cellulolytic, thermophilic actinomycete (previously isolated from municipal refuse compost samples) was identified as Thermomonospora curvata. A determination was made of the optimal conditions for cellulase production by T. curvata when grown at 55 C in a medium containing mineral salts, cellulose, and yeast extract. The pH and temperature optima (pH 6.0 and 65 C) for the cellulase produced by T. curvata were identical to those previously observed for the cellulase extracted from crude compost samples. Such similarities, together with the prevalence of T. curvata in compost samples and its ability to grow at composting temperatures, indicate that this actinomycete could possibly be considered as a major cellulose decomposer in the municipal refuse composting process.     

Adsorption of Thermomonospora curvata cellulases on insoluble substrates

Cellulosic substrates were tested for their ability to bind the cellulases of Thermomonospora curvata . Protein-extracted lucerne fibres had the highest adsorptive capacity while native cotton fibres had the least. Analysis of binding as a Langmuir adsorption isotherm yielded maximal binding capacities of 0.012 filter paper units and 0.51 endoglucanase units/mg lucerne fibre at enzyme saturation. This capacity was increased about 12-fold by fibre pre-treatment in hot NaOH solution.     

Temperature-dependent patterns of exoenzyme biosynthesis in Thermomonospora curvata

Production of depolymerlzing exoenzymes in the thermophilic actinomycete, Thermomonospora curvata, grown at 40°, 50° and 61°C, were compared. Cellulase-specific activities were similar at the three growth temperatures. Amylase-and pectinase-specific activities decreased with increasing growth temperature, while xylanase had the reverse pattern. This pattern of thermodependence correlated with ability of the actinomycete to use the product of each exoenzyme as sole carbon source. Therefore the activities of depolymerizing enzymes produced by the actinomycete during the composting temperature ascent are influenced by its ability to utillize each depolymerization product at that temperature.F. Stutzenberger is with the Department of Microbiology and T. Jenkins is with the Department of Animal, Dairy and Veterinary Science, Clemson University, Clemson, SC 29634-1909 USA.     

Selective adsorption of endoglucanases from Thermomonospora curvata on protein-extracted lucerne fibres

Cellulases of Thermomonospora curvata contain three endoglucanase components. Two of these enzymes (molecular weights of 46 and 106 kD) bound to protein-extracted lucerne fibres. Maximal adsorption occurred within 6 min, followed by a gradual desorption during fibre hydrolysis.     

Enhanced cellulase production in mutants of Thermomonospora curvata

Thermomonospora curvata, a thermophilic actinomycete, secretes multiple forms of endo-beta 1-4-glucanase (EG)when grown on cellulose-mineral salts liquid medium. The EG activity(measured as carboxymethyl cellulose hydrolysis) was separated by ion exchange chromatography into three distinct components which differ in their kinetic properties. Exposure of Thm. curvata to ultraviolet light, N-nitrosoguanidine, or ethane methyl sulfonate produced mutants with enhanced EG production. Selection of colonies which cleared cellulose agar plant containing 2-deoxtglucose of glycerol yielded mutants having 1.5 to 2.6 times the extracellular EG and saccharifying activity (measured by filter-paper and cotton-fiber hydrolysis). The secretion of extracellular protein was increased proportionally in mutant cultures.     

Cloning and expression of the nitrile hydratase and amidase genes from Bacillus sp. BR449 into Escherichia coli

A moderate thermophile, Bacillus sp. BR449 was previously shown to exhibit a high level of nitrile hydratase (NHase) activity when growing on high levels of acrylonitrile at 55 degrees C. In this report, we describe the cloning of a 6.1 kb SalI DNA fragment encoding the NHase gene cluster of BR449 into Escherichia coli. Nucleotide sequencing revealed six ORFs encoding (in order), two unidentified putative proteins, amidase, NHase beta- and alpha-subunits and a small putative protein of 101 amino acids designated P12K. Spacings and orientation of the coding regions as well as their gene expression in E. coli suggest that the beta-subunit, alpha-subunit, and P12K genes are co-transcribed. Analysis of deduced amino acid sequences indicate that the amidase (348 aa, MW 38.6 kDa) belongs to the nitrilase-related aliphatic amidase family, and that the NHase beta- (229 aa, MW 26.5 kDa) and alpha- (214 aa, MW 24.5 kDa) subunits comprise a cobalt-containing member of the NHase family, which includes Rhodococcus rhodochrous J1 and Pseudomonas putida 5B NHases. The amidase/NHase gene cluster differs both in arrangement and composition from those described for other NHase-producing strains. When expressed in Escherichia coli DH5alpha, the subcloned NHase genes produced significant levels of active NHase enzyme when cobalt ion was added either to the culture medium or cell extracts. Presence of the P12K gene and addition of amide compounds as inducers were not required for this expression.     

Cloning of endoglucanase genes fromCellulomonas biazotea intoE. coli andS. cerevisiae using shuttle vector YEp24

We constructed aSmaI genomic library ofCellulomonas biazotea DNA inE. coli and in theS. cerevisiae shuttle vector, YEP 24. Three clone were identified that conferred the ability forE. coli orS. cerevisiae transformants to produce carboxymethylcellulase (CMCase). Cells transformed with these clones were compared with one another and with nontransformed cells for hyper-production of CMCase.In vivo andin vitro studies indicated that the CMCase genes were fully expressed and the enzyme activity was located extracellularly. The optimum pH and temperature for the CMCase thus cloned were pH 7 and 50°C, respectively, as was the case for the donor.     

Cloning of a Thermomonospora fusca xylanase gene and its expression in Escherichia coli and Streptomyces lividans.

Thermomonospora fusca chromosomal DNA was partially digested with EcoRI to obtain 4- to 14-kilobase fragments, which were used to construct a library of recombinant phage by ligation with EcoRI arms of lambda gtWES. lambda B. A recombinant phage coding for xylanase activity which contained a 14-kilobase insert was identified. The xylanase gene was localized to a 2.1-kilobase SalI fragment of the EcoRI insert by subcloning onto pBR322 and derivatives of pBR322 that can also replicate in Streptomyces lividans. The xylanase activity produced by S. lividans transformants was 10- to 20-fold higher than that produced by Escherichia coli transformants but only one-fourth the level produced by induced T. fusca. A 30-kilodalton peptide with activity against both Remazol brilliant blue xylan and xylan was produced in S. lividans transformants that carried the 2.1-kilobase SalI fragment of T. fusca DNA and was not produced by control transformants. T. fusca cultures were found to contain a xylanase of a similar size that was induced by growth on xylan or Solka Floc. Antiserum directed against supernatant proteins isolated from a Solka Floc-grown T. fusca culture inhibited the xylanase activity of S. lividans transformants. The cloned T. fusca xylanase gene was expressed at about the same level in S. lividans grown in minimal medium containing either glucose, cellobiose, or xylan. The xylanase bound to and hydrolyzed insoluble xylan. The cloned xylanase appeared to be the same as the major protein in xylan-induced T. fusca culture supernatants, which also contained at least three additional minor proteins with xylanase activity and having apparent molecular masses of 43, 23, and 20 kilodaltons.     

Cloning and expression in Escherichia coli of three lipase-encoding genes from the psychrotrophic antarctic strain Moraxella TA144

The cloning and expression of genes from a psychrotrophic bacterium in a mesophilic host are described. Three lipase (Lip)-encoding genes (lip) from the antarctic psychrotroph, Moraxella TA144, were cloned by inserting Sau3AI-generated DNA fragments into the BamHI site of the pSP73 plasmid vector. To prevent heat denaturation of the gene product, the screening procedure on agar plates containing an emulsified lipid involved growing of Escherichia coli recombinant colonies at 25 degrees C followed by incubation at 0 degree C. The three recombinant (reLip) were cell-associated and differed by their respective specificity towards p-nitrophenyl esters of various aliphatic chain lengths. These cloned reLip conserved the main character of the wild-type enzymes, i.e. a dramatic shift of the optimal temperature of activity towards low temperatures and pronounced heat lability.