Plasma membrane from Candida tropicalis grown on glucose or hexadecane. II. Biochemical properties and substrate-induced alterations.

Isolated plasma membranes from the yeast Candida tropicalis grown on two different carbon sources (glucose or hexadecane), had similar contents of protein (60% of total dry weight), lipid (21-24%) and carbohydrates (16-21%). Sodium dodecyl sulphate gel electrophoresis of the membrane proteins revealed 17 and 19 protein bands, respectively, for glucose and hexadecane grown cells. There were marked differences in RF values and relative peak heights between the two gels. Sterols and free fatty acids were the major components of the plasma membrane lipids. Phospholipid content was less than 2% of total plasma membrane lipids. Membrane microviscosity, as determined by fluorescence polarization, was very high (16.6 P). Fatty acid determination of membrane lipids by gas chromatography showed a significant increase of C16 fatty acids in plasma membranes of cells grown on hexadecane. Reduced-oxidized difference spectra demonstrated the presence of a b-type cytochrome in both Saccharomyces cerevisiae and C. tropicalis plasma membranes. Its concentration in C. tropicalis plasma membranes was three-fold greater in cells grown on hexadecane than in glucose grown cells.

Plasma membranes from Candida tropicalis grown on glucose or hexadecane. I. Isolation, identification and purification.

Plasma membranes from Candida tropicalis grown on glucose or hexadecane were isolated using a method based on the difference in surface charge of mitochondria and plasma membranes. After mechanical disruption of the cells, a fraction consisting of mitochondrial and plasma membrane vesicles was obtained by differential centrifugation. Subsequently the mitochondria were separated from the plasma membrane vesicles by aggregation of the mitochondria at a pH corresponding to their isoelectric point. Additional purification of the isolated plasma membrane vesicles was achieved by osmolysis. Surface charge densities of mitochondria and plasma membranes were determined and showed substrate-dependent differences. The isolated plasma membranes were morphologically characterized by electron microscopy and, as a marker enzyme, the activity of Mg2+-dependent ATPase was determine. By checking for three mitochondrial marker enzymes the plasma membrane fractions were estimated to be 94% pure with regard to mitochondrial contamination.

The characterisation of immobilised lignin peroxidase by flow injection analysis.

Immobilised lignin peroxidase has been investigated using a flow system in the steady state and by flow injection analysis (FIA). In the steady state, the extreme sensitivity of the enzyme towards inactivation by H2O2 resulted in a stable response only in the presence of saturating levels of organic substrate and at very low (10 microM) peroxide concentrations. By contrast, the low contact time during FIA led to a stable response to injections of 100 microM H2O2. At higher peroxide concentrations a reproducible inactivation was observed, allowing a study of factors affecting both activity and stability. Lignin peroxidase substrates that undergo at least semi-reversible oxidation/reduction, including high-molecular-weight lignin fractions, could be detected by electrochemical reduction of the oxidation products. With this detection system it was possible to demonstrate the role of veratryl alcohol as mediator. This mediated oxidation of lignin functioned only when all components were present simultaneously, and was not observed when lignin was separated from the site of veratryl alcohol oxidation.

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.

Biosurfactants: moving towards industrial application.

Chemically synthesized surface-active compounds are widely used in the pharmaceutical, cosmetic, petroleum and food industries. However, with the advantages of biodegradability, and production on renewable-resource substrates, biosurfactants may eventually replace their chemically synthesized counterparts. So far, the use of biosurfactants has been limited to a few specialized applications because biosurfactants have been economically uncompetitive. There is a need to gain a greater understanding of the physiology, genetics and biochemistry of biosurfactant-producing strains, and to improve process technology to reduce production costs.

Characterization of a second alkane-inducible cytochrome P450-encoding gene, CYP52A2, from Candida tropicalis.

A second alkane-inducible cytochrome P450-encoding gene (CYP52A2) from the yeast Candida tropicalis was sequenced and characterized. CYP52A2 is located 1 kb upstream from CYP52A1, the previously characterized P450 gene [Sanglard and Loper, Gene 76 (1989) 121-136] and shows the same orientation. Like CYP52A1, CYP52A2 is induced by growth on alkane. Both promoter regions share repeats of the sequence CATGTGAA that could be of importance for the induction of the two genes. At the amino acid level, alk2 shows an overall identity of 68.2% and an overall similarity of 81.6% to alk1. Regions of high homology between the two proteins are found in the distal and proximal heme binding sites which contain the highly conserved cysteine residue as the fifth ligand to the heme iron. However, marked differences between the two proteins exist at their N-terminal end, which includes the transmembrane domain, and at the putative substrate-binding domain. Upon expression of CYP52A2 in Saccharomyces cerevisiae, alk2 was shown to hydroxylate hexadecane, but had no hydroxylation activity towards lauric acid, whereas alk1 showed both activities. Comparative immunoblots demonstrate that neither alk1 nor alk2 expressed in S. cerevisiae corresponds to the main cytochrome P450 present in C. tropicalis when grown on alkane.

Genetic transformation of the filamentous yeast, Trichosporon cutaneum, using dominant selection markers.

An efficient transformation system for the filamentous yeast, Trichosporon cutaneum, has been developed. Transformation was obtained with plasmids carrying either the Escherichia coli hygromycin B phosphotransferase-encoding gene (hph) or the Streptoalloteichus hindustanus phleomycin-resistance gene (ble), as dominant selection markers. Expression of both resistance-conferring genes was controlled by the gpd promoter and the trpC terminator, from Aspergillus nidulans. The transformation frequency was up to 500 colonies/micrograms of transforming DNA, using the ble gene, and up to 100 colonies/micrograms of transforming DNA, using the hph gene. Co-transformation frequencies using unselected DNA varied between 50 and 65%. The transforming DNA was found to consist of multiple tandem plasmid copies of high Mr. This polymeric structure, in nonselective media, was mitotically unstable, possibly indicating that it existed in an episomal state.

Molecular analysis of a Phanerochaete chrysosporium lignin peroxidase gene.

The basidiomycete fungus Phanerochaete chrysosporium produces a number of extracellular peroxidases which appear to be important for lignin degradation. We present here the isolation and complete nucleotide (nt) sequence of a gene (lpo) coding for lignin peroxidase (LPO), the coding region of which is identical to a lpo cDNA sequence which had previously been described [M. Tien and C.-P.D. Tu, Nature 326 (1987) 520-523]. The deduced amino acid (aa) sequence corresponds to 372 aa residues and the coding region is interrupted by eight short introns that range in size from 50 to 62 nt. Southern blot experiments using the cloned lpo gene as hybridization probe revealed a complex restriction fragment pattern, indicating that there are a number of lpo-related nucleotide sequences present in P. chrysosporium DNA which cross-hybridize. We also present data on the in vivo expression of the lpo genes and show that they are regulated at the RNA level and that the structure of the transcripts as judged from S1 experiments is complex. These data are consistent with the idea that there are a number of related lpo genes in P. chrysosporium which constitute a gene family.

Heterogeneity within the alkane-inducible cytochrome P450 gene family of the yeast Candida tropicalis.

The reexamination of a genomic lambda gt11 Candida tropicalis expression library for the presence of genes related to the previously reported alkane-inducible cytochrome P450alk gene (P450alk), which is the first member of the P450LII gene family, was undertaken. A positive clone with a DNA fragment having 69% similarity with a portion of P450alk was isolated. As in the case of P450alk, this new putative P450 gene was also induced by tetradecane when C. tropicalis was grown on this carbon source and was therefore named P450alk2, P450alk1 corresponding to the first isolated P450 gene. In addition to P450alk2, the existence of other P450alk-related genes is suggested by the hybridization pattern of P450alk1 and P450alk2 probes with the C. tropicalis genomic DNA. The P450LII gene family in C. tropicalis appears therefore to include several different members. This heterogeneity is presently a unique feature within yeast P450 gene families and resembles the situation existing in P450 gene families of higher eukaryotes.

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.

Identification and characterization of additional members of the cytochrome P450 multigene family CYP52 of Candida tropicalis.

Using different DNA probes from the first two previously described alkane-inducible cytochrome P450 genes of the Candida tropicalis CYP52 gene family, we isolated five independent additional members by screening a genomic library under low-stringency conditions. These genes are not allelic variants and, when taken gogether, constitute the largest gene family known in this organism. The seven members of this gene family are located on four different chromosomes and four of them are tandemly arranged on the C. tropicalis genome. The products of the seven genes, alk1 to alk7, were compared to each other and revealed a high degree of divergence: the two most diverged proteins exhibit a sequence identity of only 32%. Six of the seven genes were shown to be induced by a variety of different aliphatic carbon sources but repressed when the organism was grown on glucose. Three of the five additional CYP52 genes could be successfully expressed in Saccharomyces cerevisiae and display different substrate specificities in in vitro assays with model substrates: alk2 and alk3 exhibit a strong preference for hexadecane, while alk4 and alk5 preferentially hydroxylate lauric acid.

Automatic bioprocess control. 1. A general concept.

Automation of bioprocesses is presented and discussed. A general concept is applied to laboratory scale reactors as well as to large scale production facilities consisting of many unit operations with a hierarchical and highly modular structure. The implementation of non-dedicated and intelligent analytical subsystems is foreseen. Hard- and software requirements are discussed in view of the functional requirements of both scientific research and production engineering. Some practical experience is reported using several different components in parallel installations.

Automatic bioprocess control. 2. Implementations and practical experiences.

Our improved implementation for bioprocess control allows flexible responses to many process needs. It is based on computer equipment consisting of three hierarchically ordered levels. On the lowest level, a DDC slave computer handles setpoints and simple tasks generating the chemical and physical environment for the cells. It can be designed manually by the user or automatically by the supervisory computer on the second level. This provides for raw data organization, analysis and interpretation either to support personnel on line in decision making, to select predefined control strategies, or even to search for others. In the coordinating computer on the third level, common tasks of different supervisory computers (bioprocesses) are shared, saving money for the equipment. Tasks and concepts as well as experimental experiences are described to outline the capabilities of the configuration.

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.

The decisive role of the Saccharomyces cerevisiae cell cycle behaviour for dynamic growth characterization.

The dynamic behaviour of the cell cycle and the physiology of Saccharomyces cerevisiae was monitored in transient experiments. Frequent flow cytometric analyses of the DNA (nuclear phase state) and the cell size enabled us to characterize the proliferation properties of yeast cells under well controlled and undisturbed cultivation conditions. Preliminarily, the correlation between flow cytometric light scattering measurements and the cell size was attested for yeasts. These flow cytometric results are compared with the physiological behaviour of the culture that was detected by high resolution on-line analyses and off-line measurements. The presented results focus on the importance of the yeast cell cycle behaviour for the dynamic growth characterization. Any kind of transients in yeast cultures induced partial synchronization. The characteristics and the time course of the yeast cell cycle were found to be strongly dependent on the physiological environment.

Biomass determination.

The reasons for and historical backgrounds of biomass determination are discussed under the aspects of theoretical and practical importance, usefulness and representativity. Off-line methods are evaluated and compared with on-line methods; constraints of applications and conclusiveness of results are rated. Special emphasis is given to the fact that mere knowledge of a bio-mass concentration is not sufficiently valuable to learn more about physiology nor to determine the effectiveness of a biotechnological process. A combination of several different alternative measuring principles in parallel as well as the exploitation of software sensors is proposed as a promising future solution.

On-line measurement in biotechnology: techniques.

Bioprocesses are generally ill controlled. This is due to the fact that the measurement of relevant variables is difficult. Therefore, fundamental knowledge of metabolic interrelations is, at least in vivo, limited. In this article, some of the most important measurement techniques are reviewed in order to provide an evaluation of their current state. Emphasis is given to the underlying principles and on-line capability which allow to judge their importance and potential for exploitation resulting in well (maybe entirely) controlled bioprocesses in the future.

On-line measurement in biotechnology: exploitation, objectives and benefits.

Sound data biologically relevant are prerequisites when developing high-performance bioprocesses. Understanding of physiological regulation as well as sophisticated control strategies are highly dependent on the observability of the culture, i.e. the generation and exploitation of suited signals even under complex environmental measurement conditions. Against this background, the increasing number of analytical systems is very supportive and, accordingly, an appropriate handling of sensors and measured data is of decisive importance. This article reports on practical experience with routines for maintenance, service and calibration of hardware sensors which improve the quality of measurements significantly. Verification and validation of signals is outlined in order to make the value of data exploitation tools obvious. A method for the characterization of information is introduced by practical examples of Saccharomyces cerevisiae cultures when explaining the specific properties of extracting biological information from raw data. Finally, examples for advantageous exploitation of on-line data are given.

Automatic bioprocess control. 4. A prototype batch of Saccharomyces cerevisiae.

The recent investigations in our high performance bioreactors have shown that living cells can be extremely sensitive to physical-chemical environmental conditions and their changes. Consequently, the relationship bioreactor-living cell must thoroughly be investigated in order to discuss both: whether bioreactor characteristics are limiting/dominating during cultivation and to what extent controlled changes of the cellular environment can lead the cells to a desired physiological state. For these investigations, a generally accepted biological test organism would be helpful, of which the requirements and reactions under certain conditions are well known. Saccharomyces cerevisiae is a well known, very robust but nevertheless sensitive organism, eligible for this purpose. In this article a typical batch cultivation on glucose is presented, collected from approx. 300 experiments. Regarding metabolite production and consumption, seven different phases are distinguished on the basis of approx. 20 sensor signals and their metabolic background is discussed. Prerequisite, however, was an exhaustive knowledge upon extracellular conditions, a task which could successfully be fulfilled with the highly automated equipment introduced in the preceding articles of this series.