Molecular cloning and characterization of the extracellular sucrase gene (sacC) of Zymomonas mobilis

The Zymomonas mobilis gene sacC that encodes the extracellular sucrase (protein B46) was cloned and expressed in Escherichia coli. the gene was found to be present downstream to the already described levansucrase gene sacB in the cloned chromosomal fragment of Z. mobilis. The expression product was different from SacB and exhibited sucrase but not levansucrase activity; therefore, SacC behaves like a true sucrase. Expression of sacC in E. coli JM109 and XL1 was very low; overexpression was observed in E. coli BL21 after induction of the T7 polymerase expression system with IPTG. Subcellular fractionation of the E. coli clone carrying plasmid pLSS2811 showed that more than 70% of the sucrase activity could be detected in the cytoplasmic fraction, suggesting that the enzyme was soluble and not secreted in E. coli. The nucleotide sequence analysis of sacC revealed an open reading frame 1239 bp long coding for a 413 amino acid protein with a molecular mass of 46 kDa. The first 30 deduced amino acids from this ORF were identical with those from the N-terminal sequence of the extracellular sucrase (protein B46) purified from Z. mobilis ZM4. No leader peptide sequence could be identified in the sacC gene. The amino acid sequence of SacC showed very little similarity to those of other known sucrases, but was very similar to the levansucrases of Z. mobilis (61.5%), Erwinia amylovora (40.2%) and Bacillus subtilis (25.6%).     

Cloning of the Acetobacter xylinum cellulase gene and its expression in Escherichia coli and Zymomonas mobilis

A DNA fragment corresponding to carboxymethylcellulase activity of Acetobacter xylinum IFO 3288 was isolated and cloned in Escherichia coli, and the DNA sequence was determined. The DNA fragment sequenced had an open-reading frame of 654 base pairs that encoded a protein of 218 amino acid residues with a deduced molecular mass of 23,996 Da. The protein encoded in the DNA fragment expressed in E. coli hydrolyzed a carboxymethylcellulose. This gene was subcloned into the shuttle vector [pZA22; Misawa et al. (1986) Agric Biol Chem 50:3201–3203] between Zymomonas mobilis and E. coli. The recombinant plasmid pZAAC21 was introduced into Z. mobilis IFO 13756 by electroporation. The carboxymethylcellulase gene was efficiently expressed in both bacteria, although the level of expression in Z. mobilis was ten times greater than that in E. coli. Approximately 75% of the total carboxymethylcellulase activity detected in Z. mobilis was located in the periplasmic space (outside of the cytoplasmic space). Enzyme activity was not detected in the periplasmic space, but in the cytoplasm of E. coli.     

Discrimination of Mammalian GPI-Anchored Proteins by Hydropathy and Amino Acid Propensities

A 1.7-kb DNA fragment cloned from Zymomonas mobilis genomic DNA complemented the inability to grow on sucrose of a Sue ^? mutant of Z. mobilis that was deficient in the production of both extracellular levansucrase and invertase. Analysis of the nucleotide sequence of the fragment found two open reading frames (ORFs), both of which did not correspond to the structural gene for the levansucrase or the invertase. By subcloning each ORF into two different Suc ^? mutants of Z. mobilis, it has been found that the first ORF (gene zliE) activates the production of the extracellular levansucrase and invertase, and the second ORF (gene zliS) stimulates the secretion of the two enzymes. Gene zliS might contribute to the secretion of proteins having no signal peptide. The expression of zliE and zliS seemed to be under the control of the same promoter.     

Enhanced levan production using chitin-binding domain fused levansucrase immobilized on chitin beads

Levan is a homopolymer of fructose which can be produced by the transfructosylation reaction of levansucrase (EC 2.4.1.10) from sucrose. In particular, levan synthesized by Zymomonas mobilis has found a wide and potential application in the food and pharmaceutical industry. In this study, the immobilization of Z. mobilis levansucrae (encoded by levU) was attempted for repeated production of levan. By fusion levU with the chitin-binding domain (ChBD), the hybrid protein was overproduced in a soluble form in Escherichia coli. After direct absorption of the protein mixture from E. coli onto chitin beads, levansucrase tagged with ChBD was found to specifically attach to the affinity matrix. Subsequent analysis indicated that the linkage between the enzyme and chitin beads was substantially stable. Furthermore, with 20% sucrose, the production of levan was enhanced by 60% to reach 83 g/l using the immobilized levansucrase as compared to that by the free counterpart. This production yield accounts for 41.5% conversion yield (g/g) on the basis of sucrose. After all, a total production of levan with 480 g/l was obtained by recycling of the immobilized enzyme for seven times. It is apparent that this approach offers a promising way for levan production by Z. mobilis levansucrase immobilized on chitin beads.     

Serine substitution for cysteine residues in levansucrase selectively abolishes levan forming activity

Levansucrase is responsible for levan formation during sucrose fermentation of Zymomonas mobilis, and this decreases the efficiency of ethanol production. As thiol modifying agents decrease levan formation, a role for cysteine residues in levansucrase activity has been examined using derivatives of Z. mobilis levansucrase that carry serine substitutions of cysteine at positions 121, 151 or 244. These substitutions abolished the levan forming activity of levansucrase whilst only halving its activity in sucrose hydrolysis. Thus, polymerase and hydrolase activities of Z. mobilis levansucrase are separate and have different requirements for the enzyme's cysteine residues.     

Cloning and expression of the structural gene for pyruvate decarboxylase of Zymomonas mobilis in Escherichia coli

A genomic library of Zymomonas mobilis DNA was constructed in Escherichia coli using cosmid vector pHC79. Immunological screening of 483 individual E. coli strains revealed two clones expressing pyruvate decarboxylase, the key enzyme for efficient ethanol production of Z. mobilis. The two plasmids, pZM1 and pZM2, isolated from both E. coli strains were found to be related and to exhibit a common 4.6 kb SphI fragment on which the gene coding for pyruvate decarboxylase, pdc, was located.The pdc gene was similarily well expressed in both aerobically and anaerobically grown E. coli cells, and exerted a considerable effect on the amount of fermentation products formed. During fermentative growth on 25 mM glucose, plasmid-free E. coli lacking a pdc gene produced 6.5 mM ethanol, 8.2 mM acetate, 6.5 mM lactate, 0.5 mM succinate, and about 1 mM formate leaving 10.4 mM residual glucose. In contrast, recombinant E. coli harbouring a cloned pdc gene from Z. mobilis completely converted 25 mM glucose to up to 41.5 mM ethanol while almost no acids were formed.     

Transfer and expression of a Bacillus licheniformis α-amylase gene in Zymomonas mobilis

The gene from Bacillus licheniformis coding for a thermostable -amylase was subcloned into the broad-host-range plasmid pKT210 in Escherichia coli. The recombinant plasmid pGNB6 was transferred into Zymomonas mobilis ATCC 31821 by conjugation. Plasmid pGNB6 was stably maintained in E. coli and unstable in Z. mobilis. The amylase gene was expressed in Z. mobilis at a lower level (25%) than in E. coli and regulation of enzyme biosynthesis was different in the host cells. Almost all the -amylase activity was recovered in the culture medium of Z. mobilis. This enzyme localization seemed to be the result of protein secretion rather than cell lysis. Integration of the amylase gene into a cryptic plasmid of Z. mobilis was observed. The amylase gene was still expressed, although at a lower level, and the -amylase activity, associated with a protein of molecular mass 62,000 daltons, was immunologically identical in Z. mobilis, E. coli and B. licheniformis.     

Characterization of the levanase gene of Bacillus subtilis which shows homology to yeast invertase

Summary The structural gene for the enzyme levanase of Bacillus subtilis (SacC) was cloned in Escherichia coli. The cloned gene was mapped by PBS1 transduction near the sacL locus on the B. subtilis chromosome, between leu4 and aroD. Expression of the enzyme was demonstrated both in B. subtilis and in E. coli. The presence of sacC allowed E. coli to grow on sucrose as the sole carbon source. The complete nucleotide sequence of sacC was determined. It includes an open reading frame of 2,031 bp, coding for a protein with calculated molecular weight of 75,866 Da, including a putative signal peptide similar to precursors of secreted proteins found in Bacilli. The apparent molecular weight of purified levanase is 73 kDa. The sacC gene product was characterized in an in vitro system and in a minicellproducing strain of E. coli, confirming the existence of a precursor form of levanase of about 75 kDa. Comparison of the predicted aminoacid sequence of levanase with those of the two other known -D-fructofuranosidases of B. subtilis indicated a homology with sucrase, but not with levansucrase. A stronger homology was detected with the N-terminal region of yeast invertase, suggesting the existence of a common ancestor.     

An enzyme-linked immunoassay for the levansucrase ofZymomonas mobilis using specific antibodies produced against the cloned enzyme

Summary Levansucrase gene (levU) ofZ. mobilis had been cloned and overexpressed inEscherichia coli (Song, K.-B. and Rhee, S.-K. (1994)Biotechnol. Lett. 16, 1305–1310). The levansucrase was purified and specific antibodies against it were raised in rabbits. Specificity of the antibodies was demonstrated by Western analysis. By using the antibodies, an efficient, enzyme-linked immunosorbent assay (ELISA) amenable to immunochemical analysis of the levansucrase has been developed. The working range of levansucrase concentration in the developed ELISA was between 75 and 300 ng/ml. The assay was very sensitive to detect the levansucrase as little as 37 ng/ml.     

A simple method for the purification of over-expressed extracellular sucrase of Zymomonas mobilis from a recombinant Escherichia coli

The over-expressed extracellular sucrase (SacC) of Zymomonas mobilisfrom a recombinant Escherichia coli (pZSP62) carrying the sacC gene was purified partially by repeated cycles of freezing and thawing. This method separated the highly expressed recombinant protein from the bulk of endogenous E. coli proteins. The enzyme was further purified 14 fold with a 55% yield from the cellular extract of E. coli by hydroxyapatite chromatography. The purified enzyme had a Mr of 46 kDa by SDS-PAGE. Its km value for sucrose was 86 mM and was optimal at pH 5.0 and at 36°C.     

Enzymatic synthesis of levan by Zymomonas mobilis levansucrase overexpressed in Escherichia coli

Summary Levansucrase gene from Zymomonas mobilis was expressed efficiently in Escherichia coli and the overproduced recombinant levansucrase amounted to 40% of the total cell protein. Using E. coli lysate, levan was synthesized in a sucrose-based medium enzymatically with the conversion yields of up to 46% from fructose liberated in 25 hrs of incubation. More levan was formed at lower temperatures in the reaction mixture, whereas higher temperatures were favoured for the accumulation of free fructose or short chain oligosaccharides.     

Cloning and sequencing of the sacA gene: characterization of a sucrase from Zymomonas mobilis.

The Zymomonas mobilis gene (sacA) encoding a protein with sucrase activity has been cloned in Escherichia coli and its nucleotide sequence has been determined. Potential ribosome-binding site and promoter sequences were identified in the region upstream of the gene which were homologous to E. coli and Z. mobilis consensus sequences. Extracts from E. coli cells, containing the sacA gene, displayed a sucrose-hydrolyzing activity. However, no transfructosylation activity (exchange reaction or levan formation) could be detected. This sucrase activity was different from that observed with the purified extracellular protein B46 from Z. mobilis. These two proteins showed different electrophoretic mobilities and molecular masses and shared no immunological similarity. Thus, the product of sacA (a polypeptide of 58.4-kDa molecular mass) is a new sucrase from Z. mobilis. The amino acid sequence, deduced from the nucleotide sequence of sacA, showed strong homologies with the sucrases from Bacillus subtilis, Salmonella typhimurium, and Vibrio alginolyticus.     

Cloning and Expression of the Anti tumor Glycoprotein Gene of Streptococcus pyogenes Su in Escherichia coli

The structural gene for the streptococcal acid glycoprotein (SAGP), an antitumor glycoprotein produced by Streptococcus pyogenes Su, was cloned in Escherichia coli, and the nucleotide sequence was determined. The deduced amino acid sequence of SAGP was composed of 410 amino acids, and was highly homologous with colicin E1, a bacterial antimicrobial protein. The SAGP gene was subcloned into an expression plasmid and was expressed in E. coli. The gene product was demonstrated to have almost the same molecular weight and an immuno-chemical property with those of the native SAGP.     

Flame-burned stainless steel wire spheres as carriers of Zymomonas mobilis cells and extracellular levansucrase

In the present work, the use of flame-burned WS as carriers of Z. mobilis and extracellular levansucrase and the effect of the cell fixation method by dehydration on system productivity were investigated. Lyophilization and convective drying of Z. mobilis biomass at 30°C to a moisture content of 10–14% gave the best results for the repeated batch fermentations of a sucrose medium to obtain levan and ethanol. Significant correlation between the product formation and the concentration of free cells in the fermentation medium was established. Clearly, the cells were weakly bound to the newly generated WS and were washed out into the medium during fermentation. Here the hypothesis is presented that components excreted from damaged cells during dehydration can intensify the reactivation of damaged living cells and influence the interactions between the cells and the wire surface. The passive immobilization of extracellular levansucrase in oxidized WS was also observed. The superiority of oxidized WS in comparison with non-treated WS is related to an increase in the number of OH groups. The potential regeneration of WS by burning after the termination of fermentation cycles was also considered.     

The intracellular sucrase (SacA) of Zymomonas mobilis is not involved in sucrose assimilation

The intracellular sucrase SacA from Zymomonas mobilis was purified to homogeneity from a recombinant E. coli strain containing the SacA gene under an expression system. The protein was monomeric with a molecular mass of 58 kDa. The sucrase activity was maximal at 25 °C and thermal stability of the purified protein was low (50% recovery after 30 min at 46 °C ). The activation energy was low at 33 kJ mol^–1. Maximum activity was at pH 6.5. Activity was strongly inhibited (>99%) by SH blocking reagents and reducing agents slightly (10–60%) increased the activity of purified SacA. The sucrase showed a low K _M (42 mM) and k _cat (125 s^–1) which indicated its very low efficiency for sucrose hydrolysis. A mutant strain of Z. mobilis not able to grow on sucrose was isolated. This strain (ZM4S) lacked the two sucrases SacB and SacC but SacA was present in the intracellular fraction. Therefore, SacA alone is unable to allow growth Z. mobilis on sucrose.     

Molecular Cloning and Sequencing of the Extracellular Pectate Lyase II Gene from Erwinia carotovora Er

Erwinia carotovora Er produces three extra-cellular pectate lyases (PL I, II, and III). The gene for peetate lyase II (pelII) of E. carotovora Er was cloned and expressed both in Escherichia coli and E. carotovora Er. Localization experiments in E. coli showed that PL II was exclusively in the cytoplasmic space, while PL II was excreted into the culture medium. The complete nucleotides of the pelII gene were sequenced and found to include one open reading frame of 1122 bp coding for a protein of 374 amino acid residues. From comparison of the N-terminal amino acid sequence between the purified PL II and the deduced protein from the nucleotide sequence we reached the conclusion that the mature protein is composed of 352 amino acids with a calculated molecular weight of 38,169 and is preceded by a typical signal sequence of 22 amino acid residues. PL II had 90.1 % and 82.9% homologies with PL I and PL III in amino acid sequence, respectively.     

Cloning, expression and nucleotide sequences of two xylanase genes from Paenibacillus sp.

Two genes, xynA and xynB, encoding xylanases from Paenibacillus sp. KCTC 8848P were cloned and expressed in Escherichia coli, and their nucleotide sequences were determined. The xylanases of E. coli transformants were released into the extracellular culture fluid in the absence of xylan. The structural gene of xynA 636 bp, encoded a protein of 212 amino acids, while the xynB gene consisted of 951 bp open reading frame for a protein of 317 amino acids. The amino acid sequence of the xynAgene showed 83% similarity to the xylanase of Aeromonas caviae, and belonged to the family 11 glycosyl hydrolases. The deduced amino acid sequence of the xynB gene, however, showed 51% similarity to the xylanase of Rhodothermus marinus, and belonged to the family 10 glycosyl hydrolases.     

Extracellular secretion of levansucrase from Zymomonas mobilis in Escherichia coli

Secretion of levansucrase from Zymomonas mobilis in Escherichiacoli by glycine supplement was investigated. A significant amount of levansucrase (about 25% of total activity) was found in intact whole-cells. Cell fractionation experiments showed that levansucrase was found both in the periplasmic space and in the cytoplasmic fraction of E. coli. None or only trace amounts of levansucrase was detected in the extracellular culture broth at 24 h of cultivation and it accrued with the increasing concentration of glycine in the culture medium and duration of the culture period. Optimal glycine concentration for the maximum secretion of levansucrase was in the range of 0.8-1%, in which approximately 20-50% of levansucrase was released into the extracellular fraction at 24 h of cultivation, although glycine retarded the bacterial growth.     

Chromosomal integration and expression of green fluorescent protein in Zymomonas mobilis

An integrative vector was constructed to allow expression of heterologous proteins into the adhB locus of Zymomonas mobilis. As a reporter gene, the ORF of a bright variant of green fluorescent protein from Aequorea victoria (GFPuv) was fused to the adhB strong promoter from Z. mobilis by using a two-step PCR strategy. Z. mobilis recombinant strains that were stably marked by precise gene replacement at adhB locus with a single chromosomal copy of gfpuv. Protein expression was confirmed by fluorescence microscopy and measured by fluorescence spectroscopy, showing high expression levels (12 to 30 times higher than those obtained in E. coli) without affecting the host growth.