Biotechnology in Switzerland and a glance at Germany.

The roots of biotechnology go back to classic fermentation processes, which starting from spontaneous reactions were developed by simple means. The discovery of antibiotics made contamination-free bioprocess engineering indispensable, which led to a further step in technology development. On-line analytics and the use of computers were the basis of automation and the increase in quality. On both sides of the Atlantic, molecular biology emerged at the same time, which gave genetic engineering in medicine, agriculture, industry and environment new opportunities. The story of this new advanced technology in Switzerland, with a quick glance at Germany, is followed back to the post-war years. The growth of research and teaching and the foundation of the European Federation of Biotechnology (EFB) are dealt with. The promising phase of the 1960s and 1970s soon had to give way to a restrictive policy of insecurity and anxiousness, which, today, manifests itself in the rather insignificant contributions of many European countries to the new sciences of genomics, proteomics and bioinformatics, as well as in the resistance to the use of transgenic agricultural crops and their products in foods.

Enzymatic degumming of ramie bast fibers.

Bast fibers from ramie (Boehmeria nivea) were treated with cell-free culture supernatants from an Amycolata sp. and a recombinant Streptomyces lividans strain expressing the Amycolata pectate lyase to investigate the degumming effects of different extracellular polysaccharide-degrading enzymes. Culture supernatants from the Amycolata sp. with high pectate lyase activities were most effective in fiber separation and reduced the gum content of ramie fibers by 30% within 15 h. Xylanase activity produced by the Amycolata sp. contributed little to the degumming. Electron micrographs showed that the crude pectate lyase from the Amycolata sp. removed plant gum more efficiently from decorticated ramie bast fibers than the purified enzyme. Similarly, degumming with the crude enzyme of the Amycolata sp. and the recombinant S. lividans strain for 24 h resulted in fibers with a residual gum content of 14.7 and 17.3%, respectively. Degumming with the crude enzyme of the recombinant Streptomyces strain was slightly improved by the addition of a commercial pectinesterase. No significant degumming was observed with the crude enzyme from an S. lividans strain that did not produce the Amycolata pectate lyase. These results indicate that the pectinolytic activity of the Amycolata sp. plays an active role in degumming of ramie bast fibers.

Glucose uptake kinetics of Saccharomyces cerevisiae monitored with a newly developed FIA.

The glucose content of the culture liquid during shift experiments and synchronized cultures of Saccharomyces cerevisiae H1022 (ATCC 32167) was monitored using a greatly improved and highly precise FIA. During shift-up experiments on the dilution rate, an overshoot of the glucose-concentration was observed. The amplitude of the overshoot showed a dependency on the duration of undisturbed cultivation before application of the shift. Mutarotational non-equilibrium was excluded as the cause of the observed overshoot. For the first time glucose measurements of oscillating cultures of Saccharomyces cerevisiae are demonstrated with high accuracy and reproducibility. The data strongly support the proposals by Münch et al. (1992a, b) that faint oscillations in glucose concentration are responsible for the persistence of the synchronization. Analytical subsystems prove to be a powerful tool for investigation of the dynamics of metabolic pathways of microbial organisms. Accurate glucose measurements at low concentrations point out the limits and allow refinements of commonly used models.

Production of rhamnolipid biosurfactants.

Biosurfactants are of increasing interest due to their broad range of potential applications. A large variety of microbial surfactants is known at present, some of which may be used for specific applications. Towards the large scale industrial production of biosurfactants, the physiology, biochemistry and genetics of biosurfactant synthesis has to be well understood. A fully integrated process has to be developed, allowing high productivities under optimized conditions. In the past few years, we have investigated the molecular biology of rhamnolipid biosynthesis have been partially purified and characterized. The structural and regulatory genes encoding the rhamnolipid synthesis pathway have been isolated and characterized. The knowledge of the complex mechanisms involved in rhamnolipid synthesis facilitates the overproduction of these extracellular compounds. Furthermore, the transfer of the relevant genes into other species allows the production of rhamnolipids in heterologous hosts under controlled conditions. An integrated process for the production of rhamnolipids on an industrial scale has been developed. This process involves continuous cultivation under optimized media and growth conditions and makes use of refined methods of cell recycling, gas exchange and downstream processing, thus allowing high yields and productivities.

Amplification and expression of recombinant genes in serum-independent Chinese hamster ovary cells.

CHO SSF3 cells grow as a suspension culture in unmodified commercial medium with only low-molecular weight ingredients. Continuous serum-free culture unexpectedly induced expression of a low dihydrofolate reductase activity in the originally dhfr- CHO cells. Nevertheless, it was possible with methotrexate to induce amplification of a gene coding for the hybrid plasminogen activator K2tu-PA cotransfected with a dhfr gene. Expression of K2tu-PA expression was proportionally increased to that of dhfr, which was measured with fluorescent methotrexate. Because no serum proteases were present, secreted K2tu-PA was not converted to the enzymatically active form, but was exclusively recovered in proenzyme form.

Production of Pseudomonas aeruginosa Rhamnolipid Biosurfactants in Heterologous Hosts.

The high-level production of rhamnolipid biosurfactants is a unique feature of Pseudomonas aeruginosa and is strictly regulated in response to environmental conditions. The final step in rhamnolipid biosynthesis is catalyzed by the rhlAB genes encoding a rhamnosyltransferase. The expression of the cloned rhlAB genes was studied in heterologous hosts, either under the control of the rhlR and rhlI rhamnolipid regulatory elements or under the control of the tac promoter. A recombinant P. fluorescens strain harboring multiple plasmid-encoded copies of the rhamnolipid gene cluster produced rhamnolipids (0.25 g liter(sup-1)) when grown under nitrogen-limiting conditions. The highest yields (0.6 g liter(sup-1)) and productivities (24 mg liter(sup-1) h(sup-1)) were obtained in a recombinant Pseudomonas putida strain, KT2442, harboring promoterless rhlAB genes fused to the tac promoter on a plasmid. Active rhamnosyltransferase was synthesized, but no rhamnolipids were produced, by recombinant Escherichia coli upon induction of rhlAB gene expression.

Production of recombinant proteins in Chinese hamster ovary cells using a protein-free cell culture medium.

The growth-factor prototrophic Chinese hamster ovary (CHO) SSF3 cell line was previously adapted for growth in serum-free media. Here we present a newly designed medium which allows these cells to grow in the absence of any exogenously added growth factors. To investigate the capacity of CHO SSF3 cells for the efficient production of recombinant proteins in protein-free media, expression plasmids containing either a human single chain urokinase-type plasminogen activator (uPA)-encoding cDNA or a humanized immunoglobulin G (IgG) kappa light chain cDNA were introduced by transfection. The tryptophan synthase (trpB) gene of Escherichia coli was used as a dominantly acting selection marker allowing the cells to survive in a medium containing indole in place of tryptophan. Some of the clones obtained exhibited a stable uPA expression over a period of several months under selective conditions and the yields were up to 74 mg of uPA/l in a bioreactor and the productivity was around 40 mg/day per 10(9) cells. The yields of IgG light chains were up to 118 mg/l and the productivity was in the order of 56 mg/day per 10(9) cells in a bioreactor. These results demonstrate the potential of CHO SSF3 cells for the efficient production of recombinant proteins under protein-free conditions.

Aerobic thermophilic treatment of sewage sludge at pilot plant scale. 1. Operating conditions.

The aerobic thermophilic treatment process of sewage sludge was studied at different bioreactor scales in a pilot plant installation. Since, for a satisfactory sludge disinfection, the Swiss legislation requires minimal incubation times of all volume elements, the bioreactors were operated in repetitive batch mode (draw and fill). Different retention times and frequencies of the volume changes were applied in order to prove the capability of the particular operation modes in assuring high degradative potential. The main enzymatic activity involved during the aerobic treatment was proteolysis: the RQ values ranged between 0.8 and 0.9 depending on the applied operating conditions. Although not in a linear manner, the efficiency of the microflora decreased as the bioreactor scale increased, when this increase corresponded with a reduction of the specific power input. The sludge oxidation rates can be tuned by some process operating conditions such as the volume change frequency, the changed volume quantities and the retention times. It was possible to improve the microbial degradative efficiency by an increased frequency of the changes, while the mean retention time influenced in particular the ultimate product quality, described as residual organic matter content of the sludge. The microflora present was also satisfactorily active at mean hydraulic retention times of less than 10 h. The organic matter concentration of the inlet sewage sludge plays an important role: it influences the aerobic degradation process positively.

Aerobic thermophilic treatment of sewage sludge at pilot plant scale. 2. Technical solutions and process design.

The performance of the ATS process depends essentially on the oxygen transfer efficiency. Improvement of the mass transfer capacity of a bioreactor allowed to reduce the incubation time necessary to attain sludge stabilization. It is important to use equipment with a high aeration efficiency such as an injector aeration system. The ratio between the total oxygen consumption and the organic matter degradation (delta COD) ranged between 0.4 and 0.8 in the pilot plant, whereas 1.23 was found in completely mixed bioreactors (Bomio, 1990). No significant improvement of the bacterial degradation efficiency was attained with a specific power input exceeding 6-8 kW m-3. A mean residence time of less than 1 d allowed organic matter removals up to 40% with specific power consumption of 10 kWh kg-1 COD oxidized. The sludge hygienization is one of the objectives and benefits of the thermophilic treatment: not only temperature but also the total solids content were important factors affecting inactivation of pathogens. The inactivation rate was promoted by the increase of temperature, while the residual colony forming units decreased with reducing the total solids content of sewage sludge. It is concluded that continuous operation mode would not affect the quality of the hygienization but could display the high degradation potential of the aerobic system.

Aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium. Gene cloning, sequence analysis, expression, and purification of the recombinant enzyme.

A cDNA clone encoding a ligninolytic aryl-alcohol dehydrogenase (AAD; EC 1.1.1.91) from the white-rot basidiomycete fungus Phanerochaete chrysosporium was isolated and characterized. The nucleotide sequence obtained reveals an open reading frame encoding a protein of 385 amino acids. Substantial homology (49.3% identity and 67.3% similarity, respectively) was observed between AAD and an open reading frame sequence present on chromosome III of Saccharomyces cerevisiae. A Southern blot analysis showed the presence of multiple AAD gene-related sequences in P. chrysosporium and in other white-rot fungi including Bjerkandera adusta and Fomes lignosus. Northern blot analyses are in line with the view that the levels and appearance of AAD mRNA correlate with the level and appearance of AAD activity and that, under conditions of nitrogen limitation, the AAD mRNA levels are higher than in carbon limited cultures. This is consistent with the regulation of the enzyme by carbon or nitrogen limitation being at the level of transcription. Moreover, the appearance of AAD-specific transcripts correlates with the appearance of lignin peroxidase-specific transcripts in the same cultures. This co-appearance is in line with the proposed synergistic interaction of the two enzymes in lignin biodegradation, which suggests a similar regulation. The AAD encoding cDNA was expressed in Escherichia coli to yield high levels of active enzyme, and the recombinant enzyme was purified by using metal chelate affinity chromatography.

Cloning and heterologous expression of a gene encoding an alkane-induced extracellular protein involved in alkane assimilation from Pseudomonas aeruginosa.

Pseudomonas aeruginosa PG201 produces a 16-kDa extracellular protein in media containing n-hexadecane as a carbon source but not in media containing glycerol or glucose. This protein was purified, and the N-terminal amino acid sequence was determined. The amino acid composition of the protein was found to be very similar to that of the so-called protein-like activator for n-alkane oxidation (PA) from P. aeruginosa S7B1. This extracellular protein was previously characterized (K. Hisatsuka, T. Nakahara, Y. Minoda, and K. Yamada, Agric. Biol. Chem. 41:445-450, 1977) and found to stimulate the growth of P. aeruginosa on n-hexadecane and to possess emulsifying activity. To study the role(s) of the PA protein and to make it accessible for possible future applications, we have cloned the PA-encoding (pra) gene and determined its nucleotide sequence. This analysis revealed a protein-coding region of 162 amino acids, with the first 25 residues being reminiscent of those of a typical bacterial signal sequence. The pra gene was inactivated by insertional mutagenesis, and the resulting strain was found to lack extracellular PA protein and to be retarded in its growth in n-hexadecane-containing media. These results are consistent with the growth stimulatory role of the PA protein. The pra gene was expressed in Escherichia coli, and substantial amounts of the recombinant protein were found in the extracellular growth medium. The recombinant protein was purified by metal chelate affinity chromatography. The ability to produce secreted PA protein by E. coli provides a simple and safe means to analyze its function(s) in alkane assimilation in the future.

Isolation, characterization, and expression in Escherichia coli of the Pseudomonas aeruginosa rhlAB genes encoding a rhamnosyltransferase involved in rhamnolipid biosurfactant synthesis.

Transposon Tn5-GM-induced mutant strains of Pseudomonas aeruginosa which are unable to produce rhamnolipid biosurfactants and lack rhamnosyltransferase activity have been isolated. The DNA regions flanking the transposon were cloned and used as specific probes for the isolation of the corresponding wild-type genes from a P. aeruginosa wild-type cosmid gene library. Single cosmid clones capable of restoring rhamnolipid synthesis in the mutant strains were isolated and further subcloned and sequenced, resulting in the identification of two genes (rhlAB) which are organized as an operon upstream of the previously identified rhlR regulatory gene. The RhlA protein (32.5 kDa) harbors a putative signal sequence, suggesting that this protein is located in the periplasm, while the RhlB protein (47 kDa) contains at least two putative membrane-spanning domains. The expression of the rhlAB genes was found to be enhanced 20-fold during the stationary phase of growth under conditions of nitrogen limitation, as measured by using rhlA::lacZ fusions. Moreover, the transcriptional activation of the rhlAB genes appears to depend on a functional RhlR regulatory protein. The sequence upstream of the rhlA promoter contains two inverted repeats which define putative binding sites for the RhlR regulator. The controlled expression of the rhlAB genes in Escherichia coli led to the formation of active rhamnosyltransferase. This provides direct evidence for the fact that the rhamnosyltransferase encoding genes have been identified.

Pectinolytic enzymes from actinomycetes for the degumming of ramie bast fibers.

Actinomycetes isolated from 10 different soil and compost samples were screened for production of pectinolytic enzyme activities when grown on pectin-containing solid and liquid media. Pectinolytic enzymes, detected by using plate diffusion tests with a medium containing ramie (Boehmeria nivea) plant material as the sole carbon source, were mainly pectate lyases, but low activities of pectinesterases were also observed. Polygalacturonases and polymethylgalacturonases were not produced. Multiple forms of pectate lyases were detected in the culture supernatants of some of the strains by using the zymogram technique of isoelectric focusing gels. Xylanolytic and cellulolytic activities were always found to be associated with pectinolytic activities. None of the pectinolytic enzymes were produced in a medium with glucose as the sole carbon source. Treatment of ramie bast fibers with crude enzyme preparations from a selection of strains showed a good correlation between the pectate lyase activity applied and the degumming effect, resulting in good separation of the bast fibers.

Molecular analysis of the Trichosporon cutaneum DSM 70698 argA gene and its use for DNA-mediated transformations.

Genomic clones capable of complementing a previously isolated arginine auxotrophic mutant strain of the filamentous yeast Trichosporon cutaneum DSM 70698 have been identified by DNA-mediated transformation, and a complementing 4,082-bp subfragment was sequenced. This analysis revealed an intact gene (arg4) showing a high degree of homology with the Saccharomyces cerevisiae CPA2 gene encoding the large subunit of carbamoyl-phosphate synthetase (CPS-A). The inferred amino acid sequence of the T. cutaneum argA-encoded protein contains 1,168 residues showing 62% identity with the sequence of the S. cerevisiae CPA2 protein, and the comparison of the two sequences uncovered a putative intron sequence of 81 nucleotides close to the 5' end of the coding region of the T. cutaneum argA gene. The presence of this intron was confirmed by nuclease protection studies and by direct DNA sequence analysis of a cDNA fragment which had been obtained by PCR amplification. The T. cutaneum intron shares the general characteristics of introns found in yeasts and filamentous fungi. A major transcript of around 4 kb was found in Northern (RNA) blots. The T. cutaneum argA coding region was expressed in Escherichia coli under the control of the regulatable tac promoter. A roughly 130-kDa protein which was found to cross-react with an anti-rat CPS antibody in Western blots (immunoblots) was observed. Two putative ATP-binding domains were identified, one in the amino-terminal half of the argA-encoded protein and the other in the carboxy-terminal half. These domains are highly conserved among the known CPS-A sequences from S. cerevisiae, E. coli, and the rat. From these results we conclude that the T. cutaneum argA gene encodes the large subunit of CPS. This is the first gene to be identified and analyzed in the T. cutaneum DSM 70698 strain.

Isolation and characterization of a regulatory gene affecting rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa.

A mutant strain (65E12) of Pseudomonas aeruginosa that is unable to produce rhamnolipid biosurfactants and lacks rhamnosyltransferase activity was genetically complemented by using a P. aeruginosa PG201 wild-type gene library. A single complementing cosmid was isolated on the basis of surface tension measurements of subcultures of the transconjugants by using a sib selection strategy. The subcloning of the complementing cosmid clone yielded a 2-kb fragment capable of restoring rhamnolipid biosynthesis, rhamnosyltransferase activity, and utilization of hexadecane as a C source in mutant 65E12. The nucleotide sequence of the complementing 2-kb fragment was determined, and a single open reading frame (rhlR) of 723 bp specifying a putative 28-kDa protein (RhlR) was identified. Sequence homologies between the RhlR protein and some regulatory proteins such as LasR of P. aeruginosa, LuxR of Vibrio fischeri, RhiR of Rhizobium leguminosarum, and the putative activator 28-kDa UvrC of Escherichia coli suggest that the RhlR protein is a transcriptional activator. A putative target promoter which is regulated by the RhlR protein has been identified 2.5 kb upstream of the rhlR gene. Multiple plasmid-based rhlR gene copies had a stimulating effect on the growth of the P. aeruginosa wild-type strain in hexadecane-containing minimal medium, on rhamnolipid production, and on the production of pyocyanin chromophores. Disruption of the P. aeruginosa wild-type rhlR locus led to rhamnolipid-deficient mutant strains, thus confirming directly that this gene is necessary for rhamnolipid biosynthesis. Additionally, such PG201::'rhlR' mutant strains lacked elastase activity, indicating that the RhlR protein is a pleiotropic regulator.

Characterization of glycogen-deficient glc mutants of Saccharomyces cerevisiae.

Forty-eight mutants of Saccharomyces cerevisiae with defects in glycogen metabolism were isolated. The mutations defined eight GLC genes, the function of which were determined. Mutations in three of these genes activate the RAS/cAMP pathway either by impairment of a RAS GTPase-activating protein (GLC1/IRA1 and GLC4/IRA2) or by activating Ras2p (GLC5/RAS2). SNF1 protein kinase (GLC2) was found to be required for normal glycogen levels. Glycogen branching enzyme (GLC3) was found to be required for significant glycogen synthesis. GLC6 was shown to be allelic to CIF1 (and probably FDP1, BYP1 and GGS1), mutations in which were previously found to prevent growth on glucose; this gene is also the same as TPS1, which encodes a subunit of the trehalose-phosphate synthase. Mutations in GLC6 were capable of increasing or decreasing glycogen levels, at least in part via effects on the regulation of glycogen synthase. GLC7 encodes a type 1 protein phosphatase that contributes to the dephosphorylation (and hence activation) of glycogen synthase. GLC8 encodes a homologue of type 1 protein phosphatase inhibitor-2. The genetic map positions of GLC1/IRA1, GLC3, GLC4/IRA2, GLC6/CIF1/TPS1 (and the adjacent VAT2/VMA2), and GLC7 were clarified. From the data on GLC3, there may be a suppression of recombination near the chromosome V centromere, at least in some strains.

Methods to investigate the expression of lignin peroxidase genes by the white rot fungus Phanerochaete chrysosporium.

Two methods allowing the analysis of expression of specific lignin peroxidase (LPO) genes from white rot fungi are presented. In the first method, degenerate oligonucleotide primers derived from amino acid sequence motifs held in common among all members of the LPO gene family are used to prime the polymerase chain reaction (PCR) amplification of LPO-related nucleotide sequences from cDNA prepared by using RNA from ligninolytic cultures. The PCR products are cloned and analyzed by restriction cleavage and DNA sequencing. This method was applied to the analysis of transcripts from carbon-limited cultures of Phanerochaete chrysosporium BKM-F-1767, revealing two major classes of PCR products. One class showed DNA sequences with a high degree of similarity to the previously described CLG4 cDNA sequence (H. A. De Boer, Y. Zhang, C. Collins, and C. A. Reddy, Gene 60:93-102, 1987), whereas the other harbored DNA sequences with similarities to the L18 cDNA sequence previously described for P. chrysosporium OGC101 (T. G. Ritch, Jr., V. J. Nipper, L. Akileswaran, A. J. Smith, D. G. Pribnow, and M. H. Gold, Gene 107:119-126, 1991). The second method is based on nuclease protection assays involving isoenzyme-specific RNA probes. By using this method, the L18-related gene of P. chrysosporium BKM-F-1767 was found to be expressed under conditions of carbon and of nitrogen limitation, although the transcript levels were found to be higher in carbon-limited cultures. Furthermore, it was found that omission of veratryl alcohol addition to the culture did not affect the levels of the L18-related transcripts in carbon-limited cultures.