Børge Diderichsen

On the safety of Aspergillus oryzae: a review

ConclusionsInvasive growth or systemic infections by A. oryzae in healthy humans have never been reported. In a few cases, however, isolates identified as A. oryzae have been recovered from debilitated patients. A. oryzae has therefore low pathogenic potential but may, like many other harmless microorganisms, grow in human tissue under exceptional circumstances. Allergic diseases primarily caused by A. oryzae have been reported in few cases, but probably presuppose both a sensitivity to allergenic reactions and a massive exposure to conidia by inhalation. A. oryzae does not produce aflatoxins or any other cancerogenic metabolites. The absence of significant levels of mycotoxins in industrial products is regularly checked. We therefore consider A. oryzae an excellent host for the safe production of harmless products by recombinant strains.

On the safety of Bacillus subtilis and B. amyloliquefaciens: a review

For many years the fermentation industry has used microorganisms to produce antibiotics, amino acids, enzymes and other useful compounds. These microorganisms, which have been isolated from the environment and then mutated to increase yields of the desired product, have proved safe to handle. With the advent of gene technology, it is now possible to transfer genetic properties from one organism to another. It is widely accepted that as long as the recipient microorganism (the host) is harmless and the products of the genes to be transferred are innocuous, the genetically engineered microorganism (the recombinant) is as safe as the host. An overwhelming majority of recombinant microorganisms to be used by industry are expected to be based on harmless hosts (OECD 1986). Many of these have been proven safe over many years of experience in industrial settings. Furthermore, extensive information on the incapacity to cause disease, i.e. non-pathogenic and non-toxicogenic potential, of some of these organisms can be found in the literature. We believe that a review of the literature and present experience with some of these host organisms will be useful for assessment of the safety of many recombinant organisms. In particular it may help to classify some of these as GILSP (Good Industrial Large Scale Practice) host organisms as defined by the OECD (1986), thus facilitating the use of recombinant strains by established production procedures. Furthermore, safety reviews on selected host microorganisms may ease the approval process of products produced by recombinant strains derived from these hosts. Thus it is the opinion of qualified experts that the use of genetic engineering per se does not warrant any additional safety assessment. On the contrary, use of a safe and wellknown host organism may sometimes render superfluous some of the extensive animal testing of a new

On the industrial use of Bacillus licheniformis: a review

We document here that in those rare cases where disease has been related to Bacillus licheniformis, infection was associated with bypassing the normal biological protective barriers or severely debililated patients. No case suggests any invasive properties of this bacterium. B. licheniformis can therefore be considered non-pathogenic to humans in general. Food-borne illness caused by possible B. licheniformis toxins have been reported, but only in a very few cases and only in connection with consumption of inappropriately prepared food. Considerable experience concerning the industrial use of recombinant B. licheniformis strains has now accumulated and authorities in the United States, Europe and Japan have approved production with and products from recombinant B. licheniformis strains. We conclude that B. licheniformis is a safe host for the production of harmless, industrial products.

Chaperone-mediated activation in vivo of a Pseudomonas cepacia lipase

An extracellular Pseudomonas cepacia lipase, LipA, is inactive when expressed in the absence of the product of the limA gene. Evidence has been presented that LimA is a molecular chaperone. The lipA and limA genes have been cloned in separate and independently inducible expression systems in Escherichia coli. These systems were used to test the molecular chaperone hypothesis by investigating whether LimA could activate presynthesized prelipase and whether presynthesized LimA could activate newly synthesized prelipase. The results show that LimA cannot activate presynthesized prelipase and that presynthesized LimA can activate only a limited number of de novo synthesized prelipase molecules. Co-immunoprecipitation of prelipase/lipase with LimA generated a 1:1 complex of prelipase/lipase and LimA. The results suggest that a 1:1 complex of LipA and LimA is required for prelipase processing and secretion of active lipase.

Variations in phenotype of relB mutants of Escherichia coli and the effect of pus and sup mutations

SummaryThree independen relB mutants were studied. During amino acid starvation they all accumulate RNA and produce an inhibitor of in vitro protein synthesis; after starvation growth is retarded for hours. The mutants differ in the degree to which the common phenotype is expressed, but the characteristic thermolability of the inhibitor is the same. The phenotype of the relB mutants is accentuated by amber suppressors, and this effect is counteracted by a mutation, pus-1 that maps at 19.5 min near aroA.

In vivo genetic engineering: homologous recombination as a tool for plasmid construction

This paper describes a novel method for creating exact DNA fusions between any two points in a plasmid carried in Bacillus subtilis. It exploits the homologous in vivo recombination between directly repeated sequences that can be established by insertion of a synthetic oligodeoxyribonucleotide. The method was used to enhance the productivity in B. subtilis of a cloned alpha-amylase (Amy)-encoding gene originating from Bacillus stearothermophilus. Thus, an exact fusion between nucleotide sequences encoding the expression signals, including the signal peptide, of a Bacillus licheniformis Amy-encoding gene and the mature Amy of B. stearothermophilus, was created. The resulting hybrid translational product was processed correctly in B. subtilis during secretion, giving rise to an Amy identical to the mature Amy secreted by B. stearothermophilus.

cur-1, a mutation affecting the phenotype of sup+ strains of Escherichia coli

Summarycur-1, a mutation found in many strains of the Y10 line of Escherichia coli K-12, but not previously described, maps at 27 min, close to galU. cur-1 causes mucoidity or uracil requirement at 29°C depending on the genetic background. These phenotypes are suppressed by amber codon suppressors.

Determining the safety of maltogenic amylase produced by rDNA technology

A maltogenic amylase produced by a genetically engineered Bacillus subtilis was studied to evaluate its safety in the food industry. First, the safety of the component parts used in the cloning process, i.e. the host organism ( B. subtilis ), the donor organism ( Bacillus stearothermophilus ) and the construction process, were evaluated. This evaluation indicated that the final construct should be regarded as a safe source for maltogenic amylase when manufactured according to current Good Manufacturing Practices. Additional experimental safety testing was carried out to confirm this conclusion. In a 13-week oral toxicity study rats tolerated the maltogenic amylase at dietary levels of 5% without toxicologically significant adverse reaction. Lack of mutagenic potential was confirmed in bacterial mutagenic as-says with Salmonella typhimurium and in an in vivo cytogenetic study in rat bone marrow cells. In an acute inhalation study with 4 h of exposure to rats, no death occurred at the highest dose level, i.e, 1.59 mg/L. The test material was non-irritating to skin and did not product eye injury in rabbits. A skin sensitization study in guinea pigs was negative. Antibiotic activity tests indicated that the microorganism did not produce antibiotics. Results indicated that maltogenic amylase should be generally recognized as safe for use in production of maltose syrups, and confirmed the conclusion drawn from the safety evaluation of the component parts used in the cloning process.

Integration and amplification of a cyclodextrin glycosyltransferase gene from Thermoanaerobacter sp. ATCC 53627 on the Bacillus subtilis chromosome

A plasmid was constructed containing the replication functions of pUC19, and the cgtA gene from Thermoanaerobacter sp. ATCC 53627, flanked by the dal gene from Bacillus subtilis and a sequence downstream from this gene. This was transformed into a Dal- B. subtilis strain, selecting for Dal+ transformants, which contained the cgtA gene in single copy integrated in the B. subtilis chromosome. The gene was subsequently amplified by a method which ensured that there was no functional plasmid replication system on the integrated DNA. The amplified structure was stable in the absence of selection pressure.

A method for electrotransformation of Bacillus licheniformis NCIB 6346 by plasmid DNA

An electroporation procedure was developed for transformation of Bacillus licheniformis NCIB 6346 by plasmids. Electrotransformation of a related industrial strain is also reported.