Molecular cloning,expression, and characterization of a new endoglucanase gene fromFibrobacter succinogenes S85

A DNA fragment coding for a carboxymethylcellulase (CMCase) ofFibrobacter succinogenes S85 was isolated from a pUC18 gene library inEscherichia coli JM109. The CMCase gene was present as a single copy in theF. succinogenes S85 genome and was found in all the otherF. succinogenes strains tested. The gene was expressed from an endogenous promoter inE. coli and was not subject to glucose repression. Most of the CMCase activity was located in the membrane ofE. coli. Zymogram analysis and³⁵S labeling of the proteins encoded by the CMCase gene-containing plasmid indicated that the enzyme has a molecular mass of 58,000. The optimal pH and temperature of activity on CMC were respectively 6.4 and 30°C. The enzyme was active on CMC, barley -glucan, and lichenan but would not hydrolyze laminarin and exhibited no exoglucanase-type activity, suggesting that it is an endo-(1,4)--d-glucanase.     

Statistical optimization for production of carboxymethylcellulase of <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> DL-3 by a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> JM109/DL-3 from rice bran using response surface method

The optimal conditions for production of carboxymethylcellulase (CMCase) of Bacillus amyloliquefaciens DL-3 by a recombinant Escherichia coli JM109/DL-3 were established at a flask scale using the response surface method (RSM). The optimal conditions of rice bran, tryptone, and initial pH of the medium for cell growth extracted by Design Expert Software were 66.1 g/L, 6.2 g/L, and 7.2, respectively, whereas those for production of CMCase were 58.0 g/L, 5.0 g/L, and 7.1. The analysis of variance (ANOVA) of results from central composite design (CCD) indicated that significant factor (“probe > F” less than 0.0500) for cell growth was rice bran, whereas those for production of CMCase were rice bran and initial pH of the medium. The optimal temperatures for cell growth and the production of CMCase by E. coli JM109/DL-3 were found to be 37°C. The optimal agitation speed and aeration rate of 7 L bioreactors for cell growth were 498 rpm and 1.4 vvm, whereas those for production of CMCase were 395 rpm and 1.1 vvm. The ANOVA of results indicated that the aeration rate was more significant factor (“probe > F” less than 0.0001) than the agitation speed for cell growth and production of CMCase. The optimal inner pressure for cell growth was 0.08 MPa, whereas that for the production of CMCase was 0.06 MPa. The maximal production of CMCase by E. coli JM109/DL-3 under optimized conditions was 871.0 U/mL, which was 3.0 times higher than the initial production of CMCase before optimization.     

Heterologous expression of L. major proteins in S. cerevisiae: a test of solubility, purity, and gene recoding

High level expression of many eukaryotic proteins for structural analysis is likely to require a eukaryotic host since many proteins are either insoluble or lack essential post-translational modifications when expressed in E. coli. The well-studied eukaryote Saccharomyces cerevisiae possesses several attributes of a good expression host: it is simple and inexpensive to culture, has proven genetic tractability, and has excellent recombinant DNA tools. We demonstrate here that this yeast exhibits three additional characteristics that are desirable in a eukaryotic expression host. First, expression in yeast significantly improves the solubility of proteins that are expressed but insoluble in E. coli. The expression and solubility of 83 Leishmania major ORFs were compared in S. cerevisiae and in E. coli, with the result that 42 of the 64 ORFs with good expression and poor solubility in E. coli are highly soluble in S. cerevisiae. Second, the yield and purity of heterologous proteins expressed in yeast is sufficient for structural analysis, as demonstrated with both small scale purifications of 21 highly expressed proteins and large scale purifications of 2 proteins, which yield highly homogeneous preparations. Third, protein expression can be improved by altering codon usage, based on the observation that a codon-optimized construct of one ORF yields three-fold more protein. Thus, these results provide direct verification that high level expression and purification of heterologous proteins in S. cerevisiae is feasible and likely to improve expression of proteins whose solubility in E. coli is poor.     

Enhanced production of carboxymethylcellulase of a marine microorganism, Bacillus subtilis subsp. subtilis A-53 in a pilot-scaled bioreactor by a recombinant Escherichia coli JM109/A-53 from rice bran

A gene encoding the carboxymethylcellulase (CMCase) of a marine bacterium, Bacillus subtilis subsp. subtilis A-53, was cloned in Escherichia coli JMB109 and the recombinant strain was named as E. coli JMB109/A-53. The optimal conditions of rice bran, ammonium chloride, and initial pH of the medium for cell growth, extracted by Design Expert Software based on response surface methodology, were 100.0 g/l, 7.5 g/l, and 7.0, respectively, whereas those for production of CMCase were 100.0 g/l, 7.5 g/l, and 8.0. The optimal temperatures for cell growth and the production of CMCase by E. coli JM109/A-53 were found to be and 40 and 35 °C, respectively. The optimal agitation speed and aeration rate of a 7 l bioreactor for cell growth were 400 rpm and 1.5 vvm, whereas those for production of CMCase were 400 rpm and 0.5 vvm. The optimal inner pressure for cell growth was 0.06 MPa, which was the same as that for production of CMCase. The production of CMCase by E. coli JM109/A-53 under optimized conditions was 880.2 U/ml, which was 2.9 times higher than that before optimization. In this study, rice bran and ammonium chloride were developed as carbon and nitrogen source for production of CMCase by a recombinant E. coli JM109/A-53 and the productivity of E. coli JM109/A-53 was 5.9 times higher than that of B. subtilis subp. subtilis A-53.     

Molecular cloning and expression ofBacillus subtilis bglS gene inSaccharomyces cerevisiae

A 2.7-kb EcoRI DNA fragment carrying aBacillus subtilis endo--1,3-1,4-glucanase gene (bglS) from theE. coli plasmid pFG1 was cloned into anEscherichia coli/yeast shuttle vector to construct a hybrid plasmid YCSH. The hybrid plasmid was used to transformSaccharomyces cerevisiae, and thebglS gene was expressed. Variation between levels ofbglS gene expression inS. cerevisiae was about 2.3-fold, depending on the orientation of the 2.7-kb DNA fragment. Assay of substrate specificity and optimal pH of the enzyme demonstrated that the enzyme encoded by YCSH (bglS) was identical with that found inB. subtilis, but the expression level ofbglS gene inS. cerevisiae (YCSH) was much lower than that inE. coli (YCSH).     

Cloning and expression of β-glucosidase genes inEscherichia coli andSaccharomyces cerevisiae using shuttle vector pYES 2.0

Genes for β-glucosidase (Bgl) isolated from a genomic library of the cellulolytic bacterium,Cellulomonas biazotea, were cloned in pUC18 in itsSacI cloning site and transformed toE. coli. Ten putative recombinants showed blackening zones on esculin plates, yellow zones on pNPG plates, in liquid culture and on native polyacrylamide gel electrophoresis activity gels. They fell into three distinct groups. Three representativeE. coli clones carried recombinant plasmids designated pRM54, pRM1 and pRM17. The genes were located on 5.6-, 3.7- and 1.84-kb fragments, respectively. Their location was obtained by deletion analysis which revealed that 5.5, 3.2, and 1.8 kb fragments were essential to code for BglA, BglB, and BglC, respectively, and conferred intracellular production of β-glucosidase onE. coli. Expression of thebgl genes resulted in overproduction of β-glucosidase in the three clones. Secretion occurred into the periplasmic fractions. Three inserts carryingbgl genes from the representative recombinantE. coli were isolated withSacI ligated in the shuttle vector pYES2.0 in itsSacI site and transformed toE. coli andS. cerevisiae. The recombinant plasmids were redesignated pRPG1, pRPG2 and pRPG3 coding for BglA1, BglB1 and BglC1. The cloned genes conferred extracellular production of β-glucosidase onS. cerevisiae and enabled it to grow on cellobiose and salicin. Thegall promoter of shuttle vector pYES2.0 enabled the organisms to produce twice more β-glucosidase than that supported by thelacZ-promoter of pUC18 plasmid inE. coli. The cloned gene can be used as a selection marker for introducing recombinant plasmids in wild strains ofS. cerevisiae The enzyme produced bybgl ⁺ yeast andE. coli recombinants resembles that of the donor with respect to temperature and pH requirement for maximum activity. Other enzyme properties of the β-glucosidases fromS. cerevisiae were substantially the same as those fromC. biazotea.     

Characterization of Antimicrobial Activity of the Lysosomes Isolated from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The antimicrobial activity of lysosomes, a cell organelle, against a range of test microorganisms was examined in this study. The lysosomes isolated from Saccharomyces cerevisiae showed antimicrobial activity to Escherichia coli that positively correlated with the pH of the phosphate buffer as a dissolving solvent. The lysosomes from S. cerevisiae exhibited optimal activity at a concentration of 40%, at pH 4.0 of phosphate buffer, and at broad range temperature, except of over 50°C. It was also found that the lysosomes have antimicrobial activity against seven different microorganisms including E. coli. In addition, S. cerevisiae were exposed by a treatment with H₂O₂ and lysosomes were isolated from H₂O₂ exposed S. cerevisiae. We found that fluorescent intensities of each isolated lysosomes were increased depending on the increment of treated H₂O₂ concentration, and the lysosomes from 20 mM H₂O₂ treated S. cerevisiae showed higher antimicrobial activity than those from normal S. cerevisiae. Therefore, it suggests that lysosomes isolated from S. cerevisiae can be used as an antimicrobial agent. In addition, lysosomes activated by H₂O₂ enhanced its antimicrobial activity.     

Construction of a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> JM109/A-68 for production of carboxymethylcellulase and comparison of its production with its wild type, <Emphasis Type="Italic">Bacillus velezensis</Emphasis> A-68 in a pilot-scale bioreactor

A gene encoding carboxymethylcellulase (CMCase) of Bacillus velezensis A-68 had been cloned in Escherichia coli JM109. Based on productivity and economic aspect, rice bran and ammonium chloride were chosen to be optimal carbon and nitrogen sources for production of CMCase by E. coli JM109/A-68. The optimal conditions for rice bran, ammonium chloride, and initial pH of medium for production of CMCase were established by the response surface methodology (RSM). The concentrations of four salts in the medium, K₂HPO₄, NaCl, MgSO₄·7H₂O, and (NH₄)₂SO₄, for production of CMCase also were optimized. The optimal temperatures for cell growth and production of CMCase were 37°C. The maximal production of CMCase by E. coli JM109/A-68 was 880.2 U/mL, which was 10.5 time higher than its wild type, B. velezensis A-68. The production of CMCase by E. coli JM109/A-68 was compared with that by B. velezensis A-68 in a 100 L pilot-scale bioreactor under the optimized conditions. The production of CMCase by E. coli JM109/A-68 was found to be the mixed-growth associated unlike the growthassociated production of CMCase by B. velezensis A-68.     

Toxicity and accumulation of thallium in bacteria and yeast

Thallium sulphate inhibited microbial growth, withBacillus megaterium KM, more sensitive to the metal thanSaccharomyces cerevisiae andEscherichia coli. Inhibition ofB. megaterium KM andS. cerevisiae, but not ofE. coli, was alleviated by increasing the potassium concentration of the medium; inhibition of respiration ofS. cerevisiae, but not ofE. coli, was similarly alleviated. Thallium was rapidly bound, presumably to cell surfaces, byS. cerevisiae andE. coli, and was progressively accumulated by energy-dependent transport systems (probably concerned primarily with potassium uptake) with both organisms. Thallium uptake kinetics suggested more than one transport system operated in yeast, possibly reflecting a multiplicity of potassium transport systems. ApparentK _m andK _i values for competitive inhibition of thallium uptake by potassium indicatedS. cerevisiae to have a higher affinity for thallium uptake than for potassium, whileE. coli had a transport system with a higher affinity for potassium than for thallium. The likely systems for thallium transport are discussed. A mutant ofE. coli with tenfold decreased sensitivity to thallium was isolated and apparently effected surface binding of thallium in amounts equivalent to the wild type organism, but showed no subsequent uptake and accumulation of the metal from buffer, even though it was able to accumulate potassium to normal intracellular concentrations during growth. Abbreviations: Metal are referred to by their recognised atomic symbols (e.g. TI = Thallium; K = potassium; Co = cobalt)     

Optical device for parallel online measurement of dissolved oxygen and pH in shake flask cultures

We describe a new device with parallel optical measurement of dissolved oxygen (DO) and pH in up to nine shake flasks applicable in any conventional shaking incubator. Measurement ranges are 0–500% of air saturation for oxygen and 5.5–8.5 for pH. It was used to characterize growth profiles of different l-lysine producing strains of Corynebacterium glutamicum, of Saccharomyces cerevisiae and of Escherichia coli. Cultures in unbaffled flasks were highly reproducible. Oxygen limitation was indicated online which is particularly important when cultivating fast growing cells as E. coli. C. glutamicum strains showed distinct characteristic patterns of DO and pH indicating biological events. During the cultivation of S. cerevisiae on glucose, fructose and galactose, oxygen uptake rate was determined using the predetermined value of k _L a. pH measurement was used to determine the minimum buffer requirement for a culture of C. glutamicum.     

Comparison of engineered <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> for the production of an optically pure keto alcohol

In this study, the production of enantiomerically pure (1R,4S,6S)-6-hydroxy-bicyclo[2.2.2]octane-2-one ((−)-2) through stereoselective bioreduction was used as a model reaction for the comparison of engineered Saccharomyces cerevisiae and engineered Escherichia coli as biocatalysts. For both microorganisms, over-expression of the gene encoding the NADPH-dependent aldo-keto reductase YPR1 resulted in high purity of the keto alcohol (−)-2 (>99% ee, 97–98% de). E. coli had three times higher initial reduction rate but S. cerevisiae continued the reduction reaction for a longer time period, thus reaching a higher conversion of the substrate (95%). S. cerevisiae was also more robust than E. coli, as demonstrated by higher viability during bioreduction. It was also investigated whether the NADPH regeneration rate was sufficient to supply the over-expressed reductase with NADPH. Five strains of each microorganism with varied carbon flux through the NADPH regenerating pentose phosphate pathway were genetically constructed and compared. S. cerevisiae required an increased NADPH regeneration rate to supply YPR1 with co-enzyme while the native NADPH regeneration rate was sufficient for E. coli. Nádia Skorupa Parachin and Magnus Carlquist have contributed equally to the paper.     

Cloning and expression of a novel thermostable cellulase from newly isolated <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain I15

A novel bacterial strain with high cellulase activity (2.82 U/ml) was isolated, and then identified by its morphological character and 16S rRNA sequence, and named Bacillus subtilis strain I15. The extracellular thermostable cellulase exhibited the maximum activity at 60°C and pH 6.0. It was very stable since more than 90% of original CMCase activity was maintained at 65°C after incubation for 2 h. The cellulase gene, celI15, was cloned and extracellularly expressed by Escherichia coli BL21 (DE3), which encoded the extracellular protein of about 52 kDa. The extracellular activity of CelI15 from E. coli BL21 was up to about 6.78 U/ml, and all the other properties were almost the same as that from the wild-type strain.     

Comparison of optimal conditions for mass production of carboxymethylcellulase by <Emphasis Type="Italic">Escherichia coli</Emphasis> JM109/A-68 with other recombinants in pilot-scale bioreactor

The optimal conditions for mass production of carboxymethylcellulase (CMCase) by E. coli JM109/A-68 were investigated and compared with other E. coli JM109 recombinants producing CMCase. The optimal agitation speed and aeration rate for cell growth of E. coli JM109/A- 68 were 500 rpm and 0.50 vvm in a 7 L bioreactor, whereas those for production of CMCase were 416 rpm and 0.95 vvm. The optimal vessel pressures for cell growth as well as production of CMCase in a 100 L bioreactor were 0.04 MPa. The maximal production of CMCase by E. coli JM109/A-68 under the optimized conditions in a 100 L bioreactor was 11.0 times higher than its wild type, B. velezensis A-68. Optimal conditions for mass production of CMCase by recombinants were different from those for wild strains. The higher production of CMCase by E. coli JM109/A-68 and other recombinant of E. coli seemed to result from its higher cell growth under the optimal conditions for dissolved oxygen and its mixed-growth associated production pattern compared to the growthassociated production of B. velezensis A-68.     

A Novel GFP-Fused Eukaryotic Membrane Protein Expression System in Lactococcus lactis and Its Application to Overexpression of an Elongase

Ultraviolet (UV) irradiation has high potential to inactivate a wide range of biologic agents and is one of several nonadditive technologies being studied. The photoinactivation property of pulsed UV laser radiation (at wavelengths of 355 and 266 nm), used as an effective physical means to inactivate two typical microorganisms, prokaryotic (Escherichia coli K12) and eukaryotic (Saccharomyces cerevisiae), with respect to dose and exposure times, was examined. An E. coli population of 1.6 × 10⁴ colony-forming units (CFU)/ml was inactivated with a dose of 16.7 J/cm² energy at 355-nm wavelength. However, E. coli cells at higher concentrations were inactivated by only 98% using the same dose. Interestingly, an E. coli population of 2 × 10⁷ CFU/ml was completely inactivated using only 0.42 J/cm² at 266-nm wavelength (P ≤ 0.05). With respect to S. cerevisiae, the results were similar to those of E. coli irradiation considering that S. cerevisiae is 100 times larger than E. coli. A dose of 16.7 J/cm² completely inactivated an S. cerevisiae population of 6 × 10³ CFU/ml at 355-nm wavelength. Exposure to 266-nm wavelength, with energy doses of 1.67, 0.835, and 0.167 J/cm², successfully inactivated S. cerevisiae populations of 3 × 10⁶, 1.4 × 10⁵, and 1.5 × 10⁴ CFU/ml, respectively (P ≤ 0.05). In conclusion, compared with 355-nm wavelength, a pulsed UV laser at 266-nm wavelength inactivated a high titer of bacterial and yeast indicator standards suspended in phosphate-buffered saline-A.     

The upstream region of the isocitrate lyase gene (UPR-ICL) of Candida tropicalis induces gene expression in both Saccharomyces cerevisiae and Escherichia coli by acetate via two distinct promoters

The upstream region of the isocitrate lyase gene (UPR-ICL, 1530bp) of an n-alkane-utilizable yeast, Candida tropicalis, induced gene expression in another yeast, Saccharomyces cerevisiae, when the yeasts were grown on acetate. Surprisingly, UPR-ICL displayed the same regulatory function in the bacterium Escherichia coli when grown on acetate. We determined the interesting nucleotide sequence of UPR-ICL. The deletion analysis of UPR-ICL in both cells revealed the presence of two distinct promoters: one was localized at-394 to-379 and regulated gene expression in S. cerevisiae; the other was tocated near the initiation codon and regulated gene expression in E. coli. The two promoter sequences were similar, but not identical to regulatory elements that have been previously reported in S. cerevisiae and E. coli, respectively. Accordingly, the possibility of novel regulatory mechanisms could not be excluded. This is an interesting example of the presence of distinct cis-acting regulatory elements responsible for the induction of gene expression in one gene by acetate in both S. cerevisiae and E. coli. Preservation of such promoters through evolution is also discussed.Abbreviations ICL Isocitrate lyase - UPR-ICL Upstream region of the Candida tropicalis isocitrate lyase gene     

Selective cloning of genes encoding RNase H from Salmonella typhimurium, Saccharomyces cerevisiae and Escherichia coli rnh mutant

Summary We have cloned genes encoding RNase H from Escherichia coli rnh mutants, Salmonella typhimurium and Saccharomyces cerevisiae. Selection was accomplished by suppression of the temperature-sensitive growth phenotype of Escherichia coli strains containing the rnh-339::cat and either recB270 (Ts) or recC271 (Ts) mutations. RNases H from E. coli and S. typhimurium contained 155 amino acid residues and differed at only 11 positions. The S. cerevisiae and E. coli RNases H were about 30% homologous. A comparison of the amino acid sequences of several RNases H from cellular and retroviral sources revealed some strongly conserved regions as well as variable regions; the carboxyl-terminus was particularly variable. The overlapping, divergent promoter gene organization found in E. coli was observed to be present in S. typhimurium.     

Evidence for multiple carboxymethylcellulase genes in Pseudomonas fluorescens subsp. cellulosa

Summary A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA was constructed in bacteriophage 47.1 and recombinants expressing carboxymethylcellulase (CMCase) activity isolated. A 7.3 kb partial EcoRI fragment, a 9.4 kb EcoRI fragment and a 5.8 kb HindIII fragment were subcloned from three different phages into pUC18 to yield recombinant plasmids pJHH1, pJHH3 and pGJH2 respectively. Cells of Escherichia coli harbouring these plasmids expressed CMCase activity. The positions of the CMCase genes in the three plasmids were determined by subcloning and transposon mutagenesis. pJHH1 contained two distinct DNA regions encoding CMCases, which were controlled by the same promoter. All four cloned enzymes cleaved p-nitrophenyl--D-glucopyranoside, although at a very low rate, but none exhibited exoglucanase activity. In common with other extracellular enzymes cloned in E. coli, all the CMCases were exported to the periplasmic space in the enteric bacterium. The carboxymethylcellulase genes encoded by pJHH1 and pJHH3, were subject to glucose repression in E. coli.Abbreviations SSC 0.15 M NaCl, 0.015 M sodium citrate - Sm^r resistance to streptomycin - Km^r resistance to kanamycin - Ap^r resistance to ampicillin - Tc^r resistance to tetracycline - Cm^r resistance to chloramphenicol - CMCase carboxymethylcellulase     

An interlaboratory comparison of the performance of ethanol-producing micro-organisms in a xylose-rich acid hydroysate

A xylose-rich, dilute-acid-pretreated corncob hydrolase was fermented by Escherichia coli ATCC 11303, recombinant (rec) E. coli (pLOI 297 and KO11), Pichia stipitis (CBS 5773, 6054 adn R), Saccharomyces cerevisiae siolate 3 in combination with xylose isomerase, rec S. cerevisiae (TJ1, H550 and H477), and Fusraium oxysporum VIT-D-80134 in an interlaboratory comparison. The micro-organisms were studied according to three different options: (A) fermentation under consistent conditions, (B) fermentation under optimal conditions for the organisms, and (C) fermentation under optimal conditions for the organism with detoxification if the hydrolysate. The highest yields of tehanol, 0.24 g/g (A), 0.36 g/g (B) and 0.54 g/g (C), were obtained from rec E. coli B, KO11. P. stipitis and F. oxysporum were sensitive to the inhibitors present in the hydrolysate and produced a minimum yields of 0.34 g/g (C) and 0.04 g/g (B), respectively. The analysis of the corn-cob hydrolysate and aspects of process economy of the different fermentation options (pH, sterilization, nutrient supplementation, adaptation, detoxification) are discussed.Correspondece to: B. Hahn-Hägerdal     

Cloning of the Escherichia coli endo-1,4-D-glucanase gene and identification of its product

A plasmid (pYP17) containing a genomic DNA insert from Escherichia coli K-12 that confers the ability to hydrolyze carboxymethylcellulose (CMC) was isolated from a genomic library constructed in the cosmid vector pLAFR3 in E. coli DH5α. A small 1.65-kb fragment, designated bcsC (pYP300), was sequenced and found to contain an ORF of 1,104 bp encoding a protein of 368 amino acid residues, with a calculated molecular weight of 41,700 Da. BcsC carries a typical prokaryotic signal peptide of 21 amino acid residues. The predicted amino acid sequence of the BcsC protein is similar to that of CelY of Erwinia chrysanthemi, CMCase of Cellulomonas uda, EngX of Acetobacter xylinum, and CelC of Agrobacterium tumefaciens. Based on these sequence similarities, we propose that the bcsC gene is a member of glycosyl hydrolase family 8. The apparent molecular mass of the protein, when expressed in E. coli, is approximately 40 kDa, and the CMCase activity is found mainly in the extracellular space. The enzyme is optimally active at pH 7 and a temperature of 40° C. Received: 6 February 1998 / Accepted: 6 November 1998