l-Threonine Production by Auxotrophs of E. coli

Methionine auxotrophs were derived by the treatment with ultraviolet ray or N-methylN′-nitro-N-nitrosoguanidine from five strains of Escherichia coli. One of the methionine auxotrophs of E. coli C-6, strain No. 15, produced maximum amount of l-threonine (4.3 mg/ml) with the medium containing 5 % cane-molasses (as sugars). Double auxotrophs were derived with further mutational treatment from strain No. 15. It was found that l-threonine production was greatly enhanced by cultivating methionine-valine auxotrophs in the presence of l-valine and methionine. o.ne of the methionine-valine auxotroph, strain No. 234, produced maximum amount of l-threonine (10.5 mg/ml) from cane-molasses.

The requirement of l-valine for the growth of the strain No. 234 was found to be leaky, and it was suggested that some enzymes relating to l-valine metabolism were mutationally altered to temperature-sensitive.     

Genetic and biochemical consequences of thymidylate stress

We have examined the genetic and biochemical consequences of thymidylate stress in haploid and diploid strains of the simple eukaryote Saccharomyces cerevisiae (Bakers' yeast). Previously we reported that inhibition of dTMP biosynthesis causes "thymineless death" and is highly recombinagenic, but apparently not mutagenic, at the nuclear level; however, it is mutagenic for mitochondria. Concurrent provision of dTMP abolishes these effects. Conversely, excess dTMP is highly mutagenic for nuclear genes. It is likely that DNA strand breaks are responsible for the recombinagenic effects of thymidylate deprivation; such breaks could be produced by reiterative uracil incorporation and excision in DNA repair patches. In our experiments, thymidylate stress was produced both by starving dTMP auxotrophs for the required nucleotide and also by blocking de novo synthesis of thymidylate by various antimetabolites. We found that the antifolate methotrexate is a potent inducer of mitotic recombination (both gene conversion and mitotic crossing-over). This suggests that the gene amplification associated with methotrexate resistance in mammalian cells could arise, in part, by unequal sister-chromatid exchange induced by thymidylate stress. In addition, several sulfa drugs, which impede de novo folate biosynthesis, also have considerable recombinagenic activity.     

The Mode of Action of a Carboxypeptidase from Malt

Certain methionine auxotrophs of Arthrobacter paraffineus and Bacillus species produce large amounts of O-acetylhomoserine (OAH). The methionine requirement of these auxotrophs could be satisfied by either cystathionine or homocysteine but not by homoserine. The cell-free extacts from the auxotrophs were found to be deficient in cystathionine ?-synthase activity. OAH and O-succinylhomoserine (OSH) could replace methionine in the auxotrophs which are deficient in homoserine-O-transacetylase. A methionine auxotroph of Corynebacterium glutamicum also produced OAH, and the blocked step in the auxotroph appeared to be between cystathionine and homocysteine.

Cell-free extracts of A. paraffineus, C. glutamicum and Bacillus species catalyzed the formation of OAH from acetyl-CoA and homoserine, while a corresponding reaction with succinyl-CoA was not detected. Cystathionine γ-synthases in extracts of C. glutamicum and Bacillus species were specific for OAH, while the enzyme in extract of A. paraffineus was rather specific for OSH though it reacted with OAH to a certain extent.

These results indicate that the biosynthesis of l-methionine in these bacteria involves OAH.     

Microbial Production of l-Threonine

l-Threonine producing α-amino-β-hydroxyvaleric acid resistant mutants were derived from E. coli K-12 with 3 x 10^-5 frequency. One of mutants, strain β-101, accummulated maximum amount of l-threonine (1. 9 g/liter) in medium. Among isoleucine, methionine and lysine auxotrophs derived from E. coli K-12, only methionine auxotrophs produced l-threonine. In contrast, among isoleucine, methionine and lysine auxotrophs derived from β-101, l-threonine accumulation was generally enhanced in isoleucine auxotrophs. One of isoleucine auxotrophs, strain βI-67, produced maximum amount of l-threonine (4. 7 g/liter). Methionine auxotroph, βM-7, derived from β-101 produced 3.8 g/liter, and βIM-4, methionine auxotroph derived from β1-67, produced 6.1 g/liter, when it was cultured in 3% glucose medium supplemented with 100 μg/ml of l-isoleucine and l-methionine, respectively. These l-threonine productivities of E. coli mutants were discussed with respect to the regulatory mechanisms of threonine biosynthesis. A favourable fermentation medium for l-threonine production by E. coli mutants was established by using strain βM-4.     

Production of l-Lysine by Leucine Auxotrophs Derived from AEC Resistant Mutant of Brevibacterium lactofermentum

The effect of amino acids was examined on the production of l-lysine by AEC resistant mutant of B. lactofermentum. Among amino acids tested, only leucine showed strong specific inhibition. In order to release the production of l-lysine from this negative effect of leucine, leucine auxotrophs were derived from AEC resistant strain of B. lactofermentum. Most of these leucine auxotrophs produced larger amount of l-lysine (maximally 41 mg/ml) than the parental strain which produced about 18 mg/ml of l-lysine. It was confirmed that leucine auxotrophs derived from AEC resistant mutant of other glutamate producing bacteria, B. saccharolyticum and Corynebacterium glutamicum. These results suggested that leucine might directly or indirectly affect the biosynthesis of lysine.

However, this increase in lysine productivity of leucine auxotrophs could not be explained by the alteration of aspartokinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3). These enzymes are key enzymes in lysine and threonine biosynthesis, respectively.     

Symbiotically defective histidine auxotrophs of Bradyrhizobium japonicum

Four histidine auxotrophs of Bradyrhizobium japonicum strain USDA 122 were isolated by random transposon Tn5 mutagenesis. These mutants arose from different, single transposition events as shown by the comparison of EcoRI and XhoI-generated Tn5 flanking sequences of genomic DNA. The mutants grew on minimal medium supplemented with l-histidine or l-histidinol but failed to grow with l-histidinol phosphate. While two of the muants were symbiotically defective and did not form nodules on Glycine max cvs. Lee and Peking and on Glycine soja, the other two mutants were symbiotically competent. Reversion to prototrophy occurred at a frequency of about 10^-7 on growth medium without added antibiotics, but prototrophs could not be isolated from growth medium containing 200 g/ml kanamycin and streptomycin. The prototrophic revertants formed nodules on all the soybean cultivars examined. When histidine was supplied to the plant growth medium, both nodulation deficient mutants formed effective symbioses. On histidine unamended plants, nodules were observed infrequently. Three classes of bacterial colonies were isolated from such infrequent nodules: class 1 were kanamycin resistant-auxotrophs; class 2 were kanamycin sensitive-prototrophs; and class 3 were kanamycin-sensitive auxotrophs. Our results suggest that two Tn5 insertion mutations in B. japonicum leading to histidine auxotrophy, affect nodulation in some way. These mutations are in regions that show no homology to the Rhizobium meliloti common nodulation genes.     

Isolation and genetic characterizations of Bacillus megaterium cobalamin biosynthesis-deficient mutants

Ethanolamine is deaminated by the action of ethanolamine ammonia-lyase (EC 4.3.1.7), an adenosylcobalamin-dependent enzyme. Consequently, to grow on ethanolamine as a sole nitrogen source, Bacillus megaterium requires vitamin B12. Identification of B. megaterium mutants deficient for growth on ethanolamine as the sole nitrogen source yielded a total of 34 vitamin B12 auxotrophs. The vitamin B12 auxotrophs were divided into two major phenotypic groups: Cob mutants, which could use cobinamide or vitamin B12 to grow on ethanolamine, and Cbl mutants, which could be supplemented only by vitamin B12. The Cob mutants were resolved into six classes and the Cbl mutants were resolved into three, based on the spectrum of cobalt-labeled corrinoid compounds which they accumulated. Although some radiolabeled cobalamin was detected in the wild type, little or none was evident in the auxotrophs. The results indicate that Cob mutants contain lesions in biosynthetic steps before the synthesis of combinamide, while Cbl mutants are defective in the conversion of cobinamide to cobalamin. Analysis of phage-mediated transduction experiments revealed tight genetic linkage within the Cob class and within the Cbl class. Similar transduction analysis indicated the Cob and Cbl classes are weakly linked. In addition, cross-feeding experiments in which extracts prepared from mutants were examined for their effect on growth of various other mutants allowed a partial ordering of mutations within the cobalamin biosynthetic pathway.     

Studies on l-Threonine Fermentation

Fifteen strains of bacteria were treated with ultraviolet light or N-methyl-N′-nitro-N-nitrosoguanidine to derive auxotrophic mutants, which were screened for their ability to produce l-threonine. A number of auxotrophs were derived from each strain. Among them, those which produced a large amount of l-threonine were found in Aerobacter aerogenes, Serratia marcescens and Escherichia coli, the members of the family Enterobacteriaceae. Nutritional requirements of these threonine producers were proved to be methionine, lysine, or α, ε-diaminopimelic acid (DAP).

In A. aerogenes and E. coli, double and triple auxotrophs were derived with futher mutational treatment. As a, rule, imposition of additional block led to the increase of l-threonine production. In E. coli, many triple auxotrophs (DAP^?, Met^?, He^?) and their isoleucine revertants were screened for their ability to produce l-threonine. Enhancement of l-threonine production was achieved with these mutants.

One of the isoleucine revertants, KY8280, was used to investigate some cultural conditions. As a result, l-threonine accumulation reached to a level of 13.8 mg/ml with the medium containing 7.5% fructose.     

Nucleic acid metabolism in yeast II. Metabolism of thymidylate during thymidylate excess death

Summary A discrete class of strains of Saccharomyces cerevisiae, able to utilize, highly efficiently, exogenous deoxythymidine-5-monophosphate (dTMP), was found to be sensitive to concentrations >10 M dTMP in an otherwise complete growth medium. Excess dTMP is cytostatic and cytotoxic: 90% of exponentially growing cells lose colony forming ability within 1 h of exposure to excess dTMP in a growth medium. Uptake of dTMP, adenine, histidine, and leucine does occur during this thymidylate excess death (TED). dTMP is anabolized to higher phosphorylated nucleotides and catabolized to thymidine intracellularly. DNA synthesis is blocked under TED-conditions but not RNA and protein biosynthesis.Abbreviations dTMP deoxythymidine-5-monophosphate - dTDP deoxythymidine-5-diphosphate - dTTP deoxythymidine-5-triphosphate - dThd deoxythymidine - tmp genetic symbol for dTMP-auxotrophy - TMP genetic symbol for dTMP-prototrophy - (tlr) symbol for the phenotype of a yeast strain to efficiently utilize exogenous dTMP     

Transport and utilizing of the biosynthetic intermediate shikimic acid in Escherichia coli

Auxotrophic mutants of Escherichia coli W or K12 blocked before shikimic acid in the aromatic biosynthetic pathway grew poorly on shikimic acid as sole aromatic supplement. This poort growth response was correlated with a relatively poor ability to transport shikimic acid. If citrate was present in the growth medium (as it is in some commonly used basal media) the growth of some of the E. coli K12 mutants on shikimate was further reduced.Mutants were derived from pre-shikimate auxotrophs which grew rapidly on media containing shikimic acid. These derivatives all had an increased ability to transport shikimic acid. Thus, it is proposed that the growth on shikimate observed in the parent cells is restricted by their relatively poor uptake of shikimate from the medium and that this restriction may be removed by a mutation which enhances shikimate transport.Transduction analysis of the mutations which enhanced utilization and transport of shikimic acid by E. coli K12 strains indicated at least two classes. Class 1 was about 20% contransduced with the histidine region of the E. coli K12 chromosome and appeared to be coincident with a known shikimate transport locus, shiA. Class 2 was not contransduced with his. The locus (or loci) of this class is unknown. Kinetic measurements suggested that bot classes had shikimate uptake systems derived from the wild-type system. Two class 1 mutants had increased levels of otherwise unaltered wild-type transport while one class 2 mutant had an altered Michaelis constant (K_m) for shikimate transport.