Martin T. Küenzi

New Derivatives of Vitamin B12 Show Preferential Targeting of Tumors

Rapidly growing cells show an increased demand for nutrients and vitamins. The objective of our work is to exploit the supply route of vitamin B12 to deliver new derivatives of this vital vitamin to hyperproliferative cells. To date, radiolabeled ((57)Co and (111)In) vitamin B12 derivatives showed labeling of tumor tissue but also undesired high accumulation of radioactivity in normal tissue. By abolishing the interaction of a tailored vitamin B12 derivative to its transport protein transcobalamin II and therefore interrupting transcobalamin II receptor and megalin mediated uptake in normal tissue, preferential accumulation of a radiolabeled vitamin in cancer tissue could be accomplished. We identified transcobalamin I on tumors as a possible new receptor for this preferential accumulation of vitamin-mediated targeting. The low systemic distribution of radioactivity and the high tumor to blood ratio opens the possibility of a more successful clinical application of vitamin B12 for imaging or therapy.

Biodegradation of chemical waste by specialized methylotrophs, an alternative to physical methods of waste disposal

Abstract Microbial waste treatment systems were established for the disposal of chemical wastes by using specialized methylotrophs in pure, mixed or enriched cultures. Three typical mother liquors originating from the production of large-scale chemicals were selected as model wastes: a monomethyl sulfate containing mother liquor from a methylation process, a trimethylethylammonium chloride containing mother liquor from a dealkylation process and a N,N-dimethyl-formamide containing mother liquor. The microorganisms enriched and isolated for the biodegradation (mineralization) of the three model wastes were characterized and found to be bacteria of the genera Pseudomonas (trimethylethylammonium chloride utilizers and N,N-dimethylformamide utilizers) and Hyphomicrobium (monomethyl sulfate utilizers). Degradation pathways for monomethyl sulfate, trimethylethylammonium chloride and N,N-dimethylformamide are proposed. Some of the postulated novel enzymes were detected in cell-free extracts. The substrate spectra of our new species of specialized methylotrophs were investigated in order to evaluate their potential use for the biodegradation of selected organic solvents (Cl- and C2-compounds) currently used in synthesis processes (acetamide, acetonitrile, ethanol etc.). Pilot fermentations with pure substrates and model wastes are described and technical aspects and requirements of specialized microbial waste treatment systems are discussed.

Regulation of cephalosporin synthesis in Cephalosporium acremonium by phosphate and glucose

The regulation of cephalosporin synthesis in Cephalosporium acremonium was studied in a simple chemically-defined medium with glucose as the carbon source. Antibiotic synthesis depended on the phosphate content of the medium. At phosphate concentrations above 2.75 mM maximum antibiotic titres were not reached while glucose uptake and growth rates were increased. Phosphate exerted its effect indirectly by regulating the rate of glucose consumption. The negative effect of high phosphate concentrations could be overcome completely by controlling the sugar supply in fed-batch and chemostat experiments. High actual concentrations of phosphate or of glucose alone had no direct negative effect on antibiotic synthesis.

Biodegradation and utilization of quaternary alkylammonium compounds by specialized methylotrophs

Dealkylations in organic syntheses are often carried out with trimethylamine (TMA) as the alkylacceptor. In such reactions an ethyl group is transferred from an alkyldonor (e.g. a substituted diethyl phosphate or diethyl thiophosphate) to TMA and a trimethylethylammonium salt (TMEA) is formed in stoichiometric quantities. The production of large-scale chemicals such as agrochemicals or dyestuffs involving deethylations (dealkylations) with trimethylamine thus yields large volumes of mother liquors containing trimethylethylammonium salt (or trimethylalkylammonium salt). The regeneration of TMA from TMEA is unfortunately rather difficult to achieve. Alkaline hydrolysis of TMEA at elevated temperature yields TMA as the main product but also other methylated amines and by-products (stoichiometry rather unclear). The purification of TMA by distillation of such a hydrolysate is very expensive (much energy is needed for cooling). One of the methods best suited for its disposal is the incineration of mother liquors containing TMEA. Since biodegradation of trimethylethylammonium salts would be an alternative to physical or chemical methods of disposal we searched for TMEA degrading microorganisms. Trimethylethylammonium salts are (at least partially) Cl-compounds. We therefore decided to investigate and isolate methylotrophs (Cl-utilizing microorganisms). The various Cl-compounds utilized according to the literature by specialized methylotrophs as the sole source of carbon and energy have been mentioned in the preceding publication 4. For the degradation of tetramethylammonium chloride, a homologue of TMEA, only two microorganisms have been described so far: Bacterium 5H25 and Bacillus PM62. However, many of the methylotrophs reported to utilize methylamine, dimethylamine and/ or trimethylamine have never been tested for their ability to grow on tetramethylammonium salts. Mackrell and Walker 6 were able to enrich cultures of microorganisms growing on 10 mM tetramethylammonium chloride as the sole carbon source. These microorganisms could be adapted to grow on 10 mM trimethylethylammonium chloride. While no attempts were made to isolate and characterize the TMEAdegrading organisms from these enrichment cultures, it was suggested that pseudomonads might be involved. Concentrations higher than 10 mM tetramethylammonium chloride or TMEA were not tested (10 mM TMEA-chloride _-__ 1.23 g/l). Evaluation of methylotrophs from culture collections

Investigation of acetyl-CoA: Deacetylcephalosporin C O-acetyltransferase of Cephalosporium acremonium

Abstract a new, rapid test system to measure the activity of acetyl-CoA : deacetylcephalosporin C O -acetyltransferase (DAC-acetyltransferase) was established. The reaction product cephalosporin C could be easily analyzed by HPLC. The DAC-acetyltransferase was partially purified by means of fractionated (NH 4 ) 2 SO 4 precipitation, Sephadex G-100 gel chromatography and isoelectric focusing. Molecular weight was determined to be 70 000 ± 5 000 and the p I 4.3 ± 0.2. Besides the two substrates acetyl-CoA and deacetylcephalosporin C, no other factor necessary for the reaction could be found. The enzyme was inhibited by coenzyme A (61%), deacetoxycephalosporin C (57%), penicillin N (14%), 2,6-dihydroxybenzoic acid (27%) and pyruvate (37%) at a concentration of 0.1 mM in each case.

Effect of glucose and oxygen on β-lactam biosynthesis by Cephalosporium acremonium

Abstract The effects of glucose consumption rate ( q s ) and oxygen limitation on the control of cephalosporin C (Ceph C) biosynthesis and the activities of deacetoxycephalosporin C synthetase/hydroxylase (DAOC-SH) and acetyl coenzyme A: deacetylcephalosporin C o -acetyltransferase (DAC-AT) were investigated in cultivations of the highly productive Cephalosporium acremonium strain TR87 under conditions similar to those used in industrial production. A carefully optimised time course of q s during the first part of fed batch cultivations was essential for maximal Ceph C production. The actual glucose concentration in the medium was of secondary importance. A decrease of q s between 20 and 35 h of cultivation was found to induce the early onset of antibiotic synthesis. By subsequently maintaining q s at a relatively low level using a controlled feed of glucose and a limiting amount of phosphate, maximal production rates were obtained. Oxygen starvation after the onset of Ceph C production led to a pronounced increase in penicillin N formation, a reduced Ceph C yield (−30%) and a strongly reduced activity of the two enzymes tested. In general, neither the time course nor the absolute levels of the two enzyme activities directly correlated with the actual production rates of Ceph C. This is the first time where an independent parameter ( q s ) has been demonstrated to be responsible for triggering the synthesis of an antibiotic.

Mode of cultivation is critical for the optimal expression of recombinant Hirudin bySaccharomyces cerevisiae

SummaryA repeated fed-batch process for the production of recombinant hirudin byS. cerevisiae was established. The efficiency and stability of protein synthesis was investigated using various media. High cell densities were attained with both synthetic and complex media and all cultures were stable throughout the investigation (30 days). Repeated fed-batch cultivation gave consistently higher hirudin yields than continuous cultivation on all media tested, with complex medium giving the highest yields. This was shown not to be a result of plasmid copy number differences but transcriptional control, despite the use of the constitutive GAPFL promoter.

Optimization of Interferon Production in Yeast by Strict Ethanol Control

In fermentations at 500L scale with a yeast strain producing a human hybrid interferon investigations showed that ethanol concentration plays an important role in interferon production. In addition ethanol has a major influence on the conversion of half-reduced interferon into the native, active molecule. To regulate the ethanol concentration during fermentation an electrode was used on-line which measured the ethanol concentration in the culture broth. By setting an optimal glucose feed strategy in relation to the ethanol signal, interferon titers could be increased by up to 40% compared with control fermentations.

Product inhibition during the rifamycin S fermentation

Mutants of Saccharomyces cerevisiae with an increased sensitivity to the t ryptophan analogue l~Lr5-methylt ryptophan (5MT) were isolated SCI~f~RCH, MIOZZARI and HOTTER, J. Bact. 717, 1131-1140, 1974). A number of these 5MT sensitive mutants were characterized and assigned to one of the following 3 classes: class I, strains with altered act ivi ty and/or feedback sensitivity of anthranilate synthase; class II, strains with elevated uptake of 5MT; class III, mutants with altered regulation of the tryptophan-biosynthetic enzymes which do not exhibit increases in act ivi ty in the presence of 5MT. The object of the following study was to characterize 3 5MT sensitive mutants which do not belong to one of the above mentioned groups. The 3 mutants were shown to possess the following characteristics: They belong to the same complementation group and are considered allelicl although they show slightly different phenotypes. The 5MT inhibition no longer occurs ill the presence of tryptophan, anthranilic acid or indole. Enzymatic analysis revealed changes in chorismate mutase: a) greatly increased act ivi ty of chorismate mutase; b) diminished feedback sensitivity of this enzyme to tyrosine; c) reduction in its ability to be activated by tryptophan. Genetic studies indicate that this mutation is dominant and is localized in the chorismate mutase gene. Two possibilities to explain the phenotype of the mutation are being studied. We are either dealing with a promotor mutation in the chorismate mutase structural gene, or the mutation affects the equilibrium between an inactive (or less active) and an active form of the enzyme. P r o d u c t I n h i b i t i o n D u r i n g the R i f a m y c i n S F e r m e n t a t i o n