Expression of the core antigen gene of hepatitis B virus (HBV) in Acetobacter methanolicus using broad-host-range vectors

Summary Using the broad-host-range promoter probe vector pRS201 for cloning of phage Acm1 promoters, we established a convenient vector system for expression of heterologous genes in different Gram-negative bacteria. The usefulness of this system was demonstrated by expression of the HBV core gene in Acetobacter methanolicus. Plasmids carrying the HBV core gene downstream of different Acm1-phage promoters were transferred to A. methanolicus, a new potential host for recombinant DNA expression. Using enzyme immunoassay and immunoblot techniques, the amount and composition of core antigen produced in A. methanolicus were compared with that derived from Escherichia coli. The expression of immunoreactive core antigen in A. methanolicus exceeds by sevenfold that in E. coli using an expression system with tandemly arranged promoters. Morphological observations by electron microscopy show that the HBV core gene products isolated from both hosts are assembled into regular spherical particles with a diameter of about 28 nm that are comparable to original viral nucleocapsids. Offprint requests to: R. Schröder     

Construction of New Shuttle Vectors for Acetbacter

Shuttle vector pMV301 was constructed by ligation of pMV102 found in A. aceti subsp. xylinum NBI 1002 to E. coli plasmid pACYC177. It is 6.0 kb in size, has unique restriction sites suitable for insertion of a foreign DNA fragment and confers ampicillin resistance to the Acetobacter host. This vector transforms A. aceti subsp. aceti 10-80S1 and industrial vinegar producer A. aceti subsp. xylinum NBI 1002 as well as E. coli. Various chimeric plasmids were also constructed by ligation of pMV102 to E. coli plasmids to examine the expression of drug resistance genes. In addition to the ampicillin resistance gene, resistance genes for kanamycin, chloramphenicol and tetracycline derived from E. coli plasmids were expressed in Acetobacter. Most of the constructed shuttle vectors were stably maintained in Acetobacter.     

Construction of a promoter-probe vector for the methanol-utilizing bacterium acetobacter methanolicus MB 58

We report here the construction of a promoter-probe vector, pRS2, which can be utilized in either Acetobacter methanolicus MB 58 or Escherichia coli due to the presence of broad-host-range replicon RSF 1010. The vector provides several unique restriction sites for promoter cloning as well as resistance markers for the selection of transformants. The promoter-probe vector was constructed by inserting an EcoRI-SalI-polylinker fragment of pUC 19 into EcoRI/SalI digested pMK 16. The resulting plasmid, pRS1, was cloned into the unique EcoRI site of the broad-host-range plasmid RSF 1010. The vector was used to clone promoter-containing sequences derived from the A. methanolicus MB 58 chromosome as well as the E. coli lac-promoter.     

Characterization of pTZ12, a Chloramphenicol-resistance Plasmid in Bacillus subtilis

The chloramphenicol-resistance (CP^r) plasmid pTZ12 (2.55 kb) in Bacillus subtilis was genetically analyzed in detail, and the CP^r determinant and the functional unit of replication were mapped. The plasmids pTZ12 and pBR322 were digested with suitable restriction endonucleases and ligated with T4 ligase. The ligated DNAs were introduced into E. coli by transformation and CP-resistant transformants were selected. In conclusion, the CP^r determinant was mapped between a TaqI site and a BclI site (about 900 base pairs) on pTZ12. A set of pTZ12–pBR322 recombinant plasmids isolated from E. coli was introduced into B. subtilis by transformation to test for ability to replicate in B. subtilis. From the results, the region of the functional unit of pTZ12 replication was mapped. It was also proved that the gene product of this CP^r determinant was chloramphenicol acetyltransferase (CAT) and the native CAT in the cells carrying pTZ12 was a dimeric protein with two identical subunits having a molecular weight of approximately 24,000 (24 K).     

Expression of a prokaryotic gene in yeast: isolation and characterization of mutants with increased expression

Summary The Escherichia coli Tn9 derived chloramphenicol resistance gene (cam ^r) is functionally expressed in the yeast Saccharomyces cerevisiae. This gene was introduced into yeast cells as part of a hybrid yeast/E. coli shuttle plasmid. A number of plasmid associated yeast mutants overproducing the cam ^r gene product, chloramphenicol acetyltransferase (acetyl-CoA: chloramphenicol 3-0-acetyltransferase, E.C. 2.3.1.28) were isolated. One of the plasmid mutants was analyzed in some detail. Even though this mutant showed a 1,000 fold overproduction of chloramphenicol acetyltransferase in the yeast host the level of RNA complementary to the cam ^r gene was not increased. A deletion of 127 base pairs in the region immediately upstream from the 5 end of the cam ^r gene appeared to be responsible for the up phenotype of this mutant. This mutation affected the expression of the cam ^r gene in E. coli in a down fashion, in contrast to its effect in yeast.     

Construction ofClostridium butyricum hybrid plasmids and transfer toBacillus subtilis

Summary Small plasmids were isolated from type strains ofClostridium butyricum. Strain NCIB 7423 carries one plasmid (pCBU1) of 6.4 kb, whereas strain NCTC 7423 carries two unrelated plasmids of 6.3 kb (pCBU2) and 8.4 kb (pCBU3). Cleavage sites for 18 restriction endonucleases have been mapped on these plasmids and detailed physical maps are presented. For the purpose of developing vector plasmids for gene cloning in saccharolytic clostridia these crypticC. butyricum plasmids were joined to a selectable marker that will likely be expressed in clostridia. This was achieved by cloning the clostridial plasmids into theE. coli vector pBR322 carrying the chloramphenicol acetyltransferase (CAT) gene from theStaphylococcus aureus plasmid pC194. The recombinant plasmids were tested for their ability to confer chloramphenicol resistance toBacillus subtilis. Hybrid plasmids (pHL105, pHL1051) derived from pCBU2 were identified, which are capable of replication and expression of theS. aureus drug resistance marker in bothE. coli andB. subtilis. No structural instability was detected upon retransformation of pHL105 fromB. subtilis intoE. coli. The recombinant plasmids might thus be useful as shuttle vectors for the gene transfer betweenE. coli and a wide range of bacilli and clostridia.     

Ornithine Decarboxylase Activity during Sporulation of Bacillus subtilis

A simple method for overproduction of a target protein by genetic engineering techniques has been established. This method involves rearranging the target gene, which contains a ribosome binding sequence for expression, in plurally repeated form, and inserting it in a 3′ lower part of promoters.

The chloramphenicol acetyltransferase (CAT) structural gene was used to demonstrate the validity of this method. E. coli harboring a CAT expression plasmid, pUS(CAT)₁, which had one inserted CAT gene, was able to produce CAT at the level of only 4% of the total cellular protein according to densitometric scanning on Coomassie-blue-stained SDS-polyacrylamide gel and had the CAT activity of 3.9 × 10³ units/mg protein. However, E. coli harboring a CAT expression plasmid, pUS(CAT)₄, which had inserted four directly repeated copies of the CAT gene, could synthesize CAT up to 16% of the total cellular protein and had the CAT activity of 2.8 × 10⁴ units/mg protein. This suggests that this method should be useful for overproducing many important peptides or proteins in bacteria.     

Expression ofStreptomyces melC andchoA genes by a clonedStreptococcus thermophilus promoter STP2201

Streptococcus thermophilus (ST) chromosomal DNA (chr DNA) fragments having promoter activity were cloned and selected inEscherichia coli using a chloramphenicol acetyltransferase- (cat-) based promoter-probe vector pKK520-3. Insertion of a promoterless streptomycete melanin biosynthesis operon (melC) downstream from the promoters of the library further identified clone ST_P2201 as a strong promoter inE. coli. Subcloning of a ST_P2201-melC DNA fragment into the pMEU-seriesS. thermophilus-E. coli shuttle vectors yielded pEU5xML2201x plasmids that conferred Mel⁺ phenotype toE. coli. The pEU5aML2201a was further shown to afford a high level of tyrosinase production (2 units mg^–1 protein) inE. coli, and to produce an apparently inactivemelC gene product that reacts with anti-tyrosinase antiserum inS. thermophilus. SubstitutingmelC with a streptomycete cholesterol oxidase gene (choA) in the same orientation yielded pEU5aCH2201a that conferred ChoA activity to anE. coli transformant at a level of (1.06±0.15)×10^–7 units mg^–1 protein. Introduction of this plasmid intoS. thermophilus by electrotransformation yielded ChoA transformant that produced the enzyme at about 25% of the level found inE. coli.     

Versatile Expression and Secretion Vectors for Bacillus subtilis

Most expression systems are based on Escherichia coli as the host strain because of the large availability of all kinds of vector plasmids. However, aside from the obvious advantages of E. coli systems, serious problems can occur during the process of heterologous gene expression and purification. Therefore, low expression rates, formation of inclusion bodies, improper protein-folding, and/or toxicity problems might enforce changing the expression host. Here we describe the construction of two new vectors, pBSMuL1 and pBSMuL2, for overexpression and secretion of heterologous proteins in Bacillus subtilis as an alternative expression host. The new plasmids combine several advantages in comparison to available Bacillus expression systems: an appropriate multiple cloning site consisting of 13 unique restriction sites, one (pBSMuL1) or two (pBSMuL2) strong constitutive promoters, a high efficient signal sequence for protein secretion, and the possibility to express proteins as His-tagged fusions for easy detection and purification. We have demonstrated the applicability of the novel vector plasmids for the production and purification of the heterologous cutinase from Fusarium solani pisi.     

Construction of expression vectors for the gram-negative bacterium Zymomonas mobilis

Summary A set of vectors was constructed for the cloning and expression of heterologous genes in the Gramnegative bacterium Zymomonas mobilis under the control of the pdc promoter of Z. mobilis. The vectors pPTZ1, pPTZ3, and pPTZ4 are based on the cryptic Z. mobilis plasmid pZM02 and on parts of the Escherichia coli plasmids pKK223-3 and pBR322 together with the multiple cloning site of phage Ml3mp18. DNA fragments can be readily inserted immediately downstream from the pdc promoter at unique restriction sites for KpnI, XbaI and PstI in pPTZl and additionally for SmaI and BamHI in pPTZ3. In pPTZ4, the 5 terminal codons of pdc were deleted allowing the formation of gene fusions. Expression of a promoterless chloramphenicol acetyltransferase gene (cat) controlled by the pdc gene promoter resulted in enzyme activities of up to 5.5 U/mg total cell protein in Z. mobilis cells.     

Cloning of two chloramphenicol acetyltransferase genes from Clostridium butyricum and their expression in Escherichia coli and Bacillus subtilis

Summary Two non-homologous chloramphenicol (Cm) acetyltransferase (CAT) genes, designated catA and catB, were cloned from Clostridium butyricum type strains and characterized by restriction mapping. Both genes are efficiently expressed in Escherichia coli and Bacillus subtilis. In contrast to analogous genes from staphylococci and bacilli, gene expression is not dependent on induction by Cm. The genes are considered as chromosomal, since no association with endogenous plasmids was detectable. Southern hybridization revealed a homology between catA and the staphylococcal Cm resistance plasmid, pC194. The subunit size of the clostridial CAT enzymes expressed in E. coli was determined as 22.5 kDa (catA) and 24 kDa (catB), respectively. The C. butyricum cat genes provide potentially useful selection markers for the construction of cloning vectors from cryptic clostridial plasmids.     

Development of a Plasmid Vector for Easy Selection of Strong Promoters

A promoter vector pACPR33 for Escherichia coli based on the promotorless ampicillin-resistance gene from pBR322 has been constructed. The promoter of the ampicillin-resistance gene was deleted and replaced by a suitable multiple cloning site. Molecular cloning of promoters into the polylinker resulted in activation of the ampicillin resistance in E. coli. The plasmid contains a functional origin of DNA replication and a tetracycline resistance gene for E. coli, and a chloramphenicol resistance gene for S. aureus. The vector permitted direct detection of promoter activity, especially strong promoters, by easy iodometric determination of β-lactamase activity in liquid or solid media. Received: 26 July 1999 / Accepted: 22 November 1999     

Strong suppression of chloramphenicol acetyltransferase expression by inducing T7 RNA polymerase

Summary Suppression of gene expression can be used as a way to study gene regulation. Expression of chloramphenicol acetyltransferase (CAT) underE. coli promoter was inhibited by 92 % by induction of T7 RNA polymerase which transcribes from a T7 promoter opposingE. coli promoter.     

Developing an Extended Genomic Engineering Approach Based on Recombineering to Knock-in Heterologous Genes to <Emphasis Type="Italic">Escherichia coli</Emphasis> Genome

Most existing genomic engineering protocols for manipulation of Escherichia coli are primarily focused on chromosomal gene knockout. In this study, a simple but systematic chromosomal gene knock-in method was proposed based on a previously developed protocol using bacteriophage λ (λ Red) and flippase–flippase recognition targets (FLP–FRT) recombinations. For demonstration purposes, DNA operons containing heterologous genes (i.e., pac encoding E. coli penicillin acylase and palB2 encoding Pseudozyma antarctica lipase B mutant) engineered with regulatory elements, such as strong/inducible promoters (i.e., P _trc and P _araB ), operators, and ribosomal binding sites, were integrated into the E. coli genome at designated locations (i.e., lacZYA, dbpA, and lacI-mhpR loci) either as a gene replacement or gene insertion using various antibiotic selection markers (i.e., kanamycin and chloramphenicol) under various genetic backgrounds (i.e., HB101 and DH5α). The expression of the inserted foreign genes was subjected to regulation using appropriate inducers [isopropyl β-d-1-thiogalactopyranoside (IPTG) and arabinose] at tunable concentrations. The developed approach not only enables more extensive genomic engineering of E. coli, but also paves an effective way to “tailor” plasmid-free E. coli strains with desired genotypes suitable for various biotechnological applications, such as biomanufacturing and metabolic engineering.