Control of expression by the cellulose synthase (bcsA) promoter region from Acetobacter xylinum BPR 2001.

The 5' upstream region (about 3.1kb) of the cellulose synthase operon (bcs operon) has been isolated by cloning from Acetobacter xylinum strain BPR 2001. The expression level of the upstream region was determined using sucrose synthase cDNA as a reporter gene in the shuttle vector pSA19. The expression occurred with the 1.1-kb upstream sequence from the ATG start codon of the bcs operon but not with the 241-bp upstream sequence in A. xylinum, although neither the 1.1-kb nor the 241-bp upstream sequence caused any expression as a promoter in Escherichia coli. The level of expression with the 1. 1-kb upstream sequence in A. aceti was 75% of that in A. xylinum. These results suggest that the upstream region functions as a specific promoter for the Acetobacter genus. The expression was reduced by the introduction of the 241-bp upstream region between the lac promoter and the reporter gene in E. coli and was not detected in A. xylinum. This suggests that the short upstream region composed of 241bp contains the site(s) which causes a negative regulation on the transcription for bcs operon. The production of recombinant protein with the ribosome-binding site (RBS) of A. xylinum obtained from the bcs operon, was reduced to about half in E. coli, and that with the site of the lac promoter was also reduced to about half in A. xylinum. This shows that a species-specific predominance occurs during interaction between mRNA and 16S rRNA in the RBS between A. xylinum and E. coli.

High-level expression of human BSF-2/IL-6 cDNA in Escherichia coli using a new type of expression-preparation system.

BSF-2 (B cell stimulatory factor-2/IL-6) is a member of the lymphokine family and responsible for B cell differentiation. Expression plasmids of human BSF-2 cDNA were constructed using a trp promotor/operator and a trpA terminator. In an extract of Escherichia coli HB101 holding "direct" expression plasmid pBSF-2D, activity of BSF-2 was detected, but overproduction was not observed. A "fused" expression system was therefore developed to prepare the recombinant protein. In this system, cDNA was expressed as a fused protein with human IL-2 N-terminal peptide. In the case of the fused BSF-2 expression plasmid, pBSF-2F, inclusion bodies were observed and overproduction of the protein occurred. As this fused protein had a Phe-Arg-Ala sequence at the junction of hIL-2 and BSF-2, it was possible to process mature BSF-2 from the fused BSF-2 by treatment with kallikrein and aminopeptidase P. From 1 liter of E. coli culture, 45 mg of mature BSF-2 was purified; it had a relative biological activity equal to that of natural BSF-2 purified from T cells.

A CHO strain producing high-level human IL-6 with the 3' deletion construct.

A Chinese Hamster Ovary (CHO) strain producing high-level human interleukin 6 (IL-6) was obtained, by introduction of the IL-6 cDNA from which the 3' AU-rich region was deleted. The IL-6 mRNA of this strain was stable. The productivity was more than 15 times higher than the previously obtained clone, which has intact IL-6 cDNA in the same expression vector. This strain produced IL-6 at a high level of 30 micrograms ml-1 in batch culture, or a continuous 20 micrograms per 10(6) cells per day in microcarrier cultivation.

Hyper-heterogeneity of the N-terminus of recombinant BSF-2/IL-6 produced in CHO and NIH3T3 cells.

Recombinant human BSF-2 (B cell stimulatory factor 2/Interleukin 6; IL-6) proteins were purified from CHO and NIH3T3 cell cultures and characterized. The lectin binding patterns suggested that the proteins have N-linked oligosaccharide(s) with tri-antennary structure of bisecting GlcNAc. Their N-termini were highly heterogeneous; at least five closely related N-termini were detected. This N-terminal heterogeneity was not generated in the cell culture because no processing activity was found in the culture medium.

Deletion of 3' untranslated region of human BSF-2 mRNA causes stabilization of the mRNA and high-level expression in mouse NIH3T3 cells.

Most lymphokine genes have an mRNA-destabilizing signal in their 3' untranslated sequences. In this study we demonstrated the usefulness of deletion of this sequence for lymphokine production. By expression of B cell stimulatory factor 2 (BSF-2) cDNAs with and without this region in mouse NIH3T3 cells, it was shown that mRNA of BSF-2 with the 3' destabilizing sequence was very unstable, and that by deletion of this region, the mRNA was stabilized. More than 10-fold BSF-2 productivity was observed in cells harboring the plasmid without the 3' region than in those with it.

Secretion of Mucor rennin, a fungal aspartic protease of Mucor pusillus, by recombinant yeast cells.

The aspartic protease gene of a zygomycete fungus Mucor pusillus was expressed in Saccharomyces cerevisiae under the control of the yeast GAL7 promoter. A putative preproenzyme with an NH2-terminal extension of 66 amino acids directed by the gene was processed in yeast cells and the mature enzyme, whose NH2-terminus was identical to that of the Mucor enzyme, was efficiently secreted into the medium at a concentration exceeding 150 mg/l. The enzyme secreted from the recombinant yeast was more glycosylated than the native Mucor enzyme but its enzymatic properties were almost identical with those of the native enzyme, which has been used as a milk coagulant in cheese manufacture.

Cloning and sequencing of a gene for Mucor rennin, an aspartate protease from Mucor pusillus.

The aspartate protease of Mucor pusillus (Mucor pusillus rennin; MPR) is a milk-clotting enzyme used in the cheese industry. The partial amino acid sequence of MPR was determined and oligonucleotide probes were synthesized for cloning of the MPR gene. A clone giving positive hybridization with the probes was selected from the cosmid library. Sequencing of the cloned DNA revealed an open reading frame of 1281 bp without introns which encodes 361 amino acids for the expected MPR with an NH2-terminal extension of 66 amino acids. MPR seems to be synthesized as a prepro enzyme.

Increased Cellulose Production from Sucrose by Acetobacter after Introducing the Sucrose Phosphorylase Gene.

A sucrose phosphorylase (SPase) gene derived from Leuconostoc mesenteroides was introduced into a cellulose-producing Acetobacter strain and expressed under the lac promoter. The activity of the SPase was detected in extracts of the transformed cells and cellulose production from sucrose by the cells was found to have increased, which strongly suggests that the increase was the result of the new metabolizing pathway. Furthermore, the level of SPase expression was increased by altering the length of the lac promoter.

Breeding of a 5-Fluorouridine-resistant Mutant with Increased Cellulose Production from Acetobacter xylinum subsp. nonacetoxidans.

UDP-glucose (UDP-G), the direct precursor of cellulose, is known to be produced from UTP and glucose-1-phosphate. In an attempt to increase UTP biosynthesis, 5-fluorouridine (5-FUR: a pyrimidine analog)-resistant mutants were obtained using Acetobacter xylinum subsp. nonacetoxidans 757 as the parent strain. One of the 5-FUR-resistant mutants, FUR-35, showed about 40% higher cellulose productivion compared to the parent strain. Intracellular levels of UTP and UDP-G in FUR-35 was found to be higher than those in the parent strain. The carbamyl phosphate synthetase II (CPS) activity of FUR-35 was higher than that of the parent strain and the feedback inhibition of CPS by UTP in FUR-35 had been released compared with that in the parent strain. These results suggest that the increased cellulose production of FUR-35 was attributable to its higher of intracellular UDP-G level resulting from increased UTP biosynthesis.

Synthesis of asymmetrically labeled sucrose by a recombinant sucrose synthase.

About 80% of radioactivity was recovered in asymmetrically labeled sucrose from UDP-[14C]glucose or [14C]fructose with recombinant mung bean sucrose synthase expressed in Escherichia coli harboring pEB-01. This high recovery is due to the fact that the enzyme conserving the activity of sucrose synthase has a similar affinity for UDP-glucose and fructose to an intact enzyme from the mung bean, but a lower affinity for sucrose.

A host-vector system for a cellulose-producing Acetobacter strain.

An indigenous plasmid, named pAH4, was detected in a cellulose-producing Acetobacter strain. This plasmid, consisting of 4002 bp, contained an AT-rich region and encoded several open reading frames, as deduced by the complete nucleotide sequence. One of the putative open reading frames showed homology with replication proteins of other plasmids. A shuttle vector of Escherichia coli and this strain was constructed by connecting pAH4 to pUC18. Electroporation of the shuttle vector into the strain yielded 1.7 x 10(5) ampicillin resistant transformants per microgram DNA. The shuttle plasmid was very stably maintained in the strain.

Increased cellulose production from sucrose with reduced levan accumulation by an Acetobacter strain harboring a recombinant plasmid.

Cellulose production from sucrose by Acetobacter strains is accompanied by the accumulation of a water-soluble polysaccharide, called levan. To improve cellulose productivity, a levansucrase-deficient mutant, LD-2, was derived from Acetobacter strain 757 and used as a host for the construction of recombinant strains. An LD-2 mutant harboring a plasmid containing the sucrase gene, sucZE3, from Zymomonas mobilis together with zliS, a gene that encodes a secretion-activating factor under the control of the Escherichia coli lac promoter, had sucrase activity and produced much cellulose and little levan in a medium containing sucrose. In addition, a mutant levansucrase gene, mutant sacB, from Bacillus subtilis, which encodes a protein with little levan-forming activity, was generated by site-directed mutagenesis and introduced into the LD-2 mutant. This introduction also resulted in the higher cellulose productivity and little levan.

Expression and characterization of sucrose synthase from mung bean seedlings in Escherichia coli.

The cDNA fragment coding for mung bean (Vigna radiata Wilczek) sucrose synthase was introduced into the expression vector pET-20b resulting in the construction of plasmid pEB-01. After transformation of Escherichia coli strain BL21(DE3) cells by pEB-01 and induction with isopropyl thio-beta-galactoside, high level expression of the recombinant enzyme was obtained. The enzyme had a tetrameric form that conserved the activity of sucrose synthase. Although the Km and Vmax of the recombinant enzyme acting on either UDP-glucose or fructose were very close to those of the native enzyme isolated from mung bean seedlings, the Km for sucrose was higher by a factor of 10 for the recombinant enzyme. This suggests that the recombinant sucrose synthase has a tendency to synthesize sucrose, although the native enzyme catalyzes a freely reversible reaction.

The Characterization of Acetic Acid Bacteria Efficiently Producing Bacterial Cellulose from Sucrose: The Proposal of Acetobacter xylinum subsp. nonacetoxidans subsp. Nov.

A taxonomic study was done for the isolates obtained as cellulose high producers from sucrose. These strains were found to have a common characteristic unique for Acetobacter; they did not oxidize acetate and lactate. Therefore, we concluded that these isolates are classified as a new subspecies and proposed Acetobacter xylinum subsp. nonacetoxidans subsp. nov. BPR 2002 (=JCM 10150) was designated as a type strain of the new subspecies.

A beta-glucosidase gene downstream of the cellulose synthase operon in cellulose-producing Acetobacter.

An open reading frame was found 214 bp downstream of the cellulose synthase operon of Acetobacter. The encoded amino acid sequence was found to be similar to some beta-glucosidases (G3ases). We detected G3ase activity in the culture medium and analysis of the N-terminal amino acid sequence showed that this gene encodes the enzyme. Therefore, it is possible that this region is a gene cluster for cellulose synthesis.

Enhancement of cellulose production by expression of sucrose synthase in Acetobacter xylinum.

Higher plants efficiently conserve energy ATP in cellulose biosynthesis by expression of sucrose synthase, in which the high free energy between glucose and fructose in sucrose can be conserved and used for the synthesis of UDP-glucose. A mixture of sucrose synthase and bacterial cellulose synthase proceeded to form UDP-glucose from sucrose plus UDP and to synthesize 1,4-beta-glucan from the sugar nucleotide. The mutant sucrose synthase, which mimics phosphorylated sucrose synthase, enhanced the reaction efficiency (Vmax/Km) on 1,4-beta-glucan synthesis, in which the incorporation of glucose from sucrose was increased at low concentrations of UDP. Because UDP formed after glucosyl transfer can be directly recycled with sucrose synthase, UDP-glucose formed appears to show high turnover with cellulose synthase in the coupled reaction. The expression of sucrose synthase in Acetobacter xylinum not only changed sucrose metabolism but also enhanced cellulose production, in which UDP-glucose was efficiently formed from sucrose. Although the level of UDP-glucose in the transformant with mutant sucrose synthase cDNA was only 1.6-fold higher than that in plasmid-free cells, the level of UDP was markedly decreased in the transformant. The results show that sucrose synthase serves to channel carbon directly from sucrose to cellulose and recycles UDP, which prevents UDP build-up in cellulose biosynthesis.

An increase in apparent affinity for sucrose of mung bean sucrose synthase is caused by in vitro phosphorylation or directed mutagenesis of Ser11.

A mutational analysis of mung bean (Vigna radiata Wilczek) sucrose synthase was performed by site-directed mutagenesis of the recombinant protein expressed in Escherichia coli, in which two different acidic amino acid residues (Asp or Glu) were introduced at Ser11 (S11D, S11E). Only the wild-type enzyme (Ser11) was phosphorylated in vitro by a Ca(2+)-dependent protein kinase from soybean root nodules, suggesting that this is the specific target residue in mung bean sucrose synthase. The apparent affinity for sucrose was increased in this phosphorylated enzyme and also in the S11D and S11E mutant enzymes, although the affinities for UDP-glucose and fructose were similar in the wild-type, phosphorylated wild-type, and mutant enzymes. These results suggest that a monoanionic (1-) side chain at position 11 mimics the Ser11-P2- residue to bind and cleave sucrose for the synthesis of UDP-glucose. Since the S11E mutant enzyme showed the lowest K(m) (sucrose) and the highest catalytic efficiency of the recombinant proteins, the enzymic properties of this S11E mutant were further characterized. The results showed that replacement of Ser11 with Glu11 modestly protected the sucrose synthesis activity against phenolic glycosides and altered the enzyme nucleotide specificity. We postulate that the introduction of negative charge at Ser11 is possibly involved in the enzymatic perturbation of sucrose synthase.