Corynebacterium Lilium Research Articles

Effect of cysteine on methionine production by a regulatory mutant of Corynebacterium lilium

The production of methionine by submerged fermentation using a mutant strain of Corynebacterium lilium was studied to determine suitable conditions for obtaining high productivity. The mutant strain resistant to the methionine analogues ethionine, norleucine, methionine sulfoxide and methionine methylsulfonium chloride produced 2.34 g l −1 of methionine in minimal medium containing glucose as carbon source. The effect of cysteine on methionine production in a 15 l bioreactor was studied by supplementing cysteine intermittently during the course of fermentation. The addition of cysteine (0.75 g l −1 h −1) every 2 h to the production medium increased the production of methionine to 3.39 g l −1. A metabolic flux analysis showed that during cysteine supplementation the ATP consumption reduced by 20%. It also showed that the increase in flux from phosphoenol pyruvate to oxaloacetate leads to higher methionine production. Results indicate that controlling the respiratory quotient close to 0.75 will produce the highest amount of methionine and that regulatory mutants also resistant to analogues of cysteine would be better methionine over producers.

Production of methionine by a multi-analogue resistant mutant of Corynebacterium lilium

Multi-analogue resistant mutants of Corynebacterium lilium were developed using UV and N-methyl- N′-nitro-nitrosoguanidine (NTG) mutagenesis in order to overproduce methionine. Although the methionine yield at each stage of mutation by UV and NTG mutagenesis were comparable, it was observed that the NTG mutants had higher cell growth and glucose utilization rates. From the parent NTG mutant C. lilium E 4 resistant to ethionine, the highest methionine producing strain C. lilium M-128 resistant to 3.5 mg ml −1 of ethionine, 3.0 mg ml −1 of norleucine, 3.8 mg ml −1 of methionine sulphoxide and 3.8 mg ml −1 of methionine methyl sulphoniumchloride was derived. When C. lilium M-128 was grown under optimised conditions, it produced 2.3 g l −1 of methionine in a 15 l batch reactor. The maximum cell mass concentration obtained was 17 g l −1 dry cell weight resulting in a cell yield coefficient of 0.34 g g −1. The dissolved oxygen concentration was controlled at 40% saturation by cascade control of the airflow rate and agitation speed.

Antiviral, Antimicrobial, and Cytotoxic Properties of Peptavirins A and B Produced by Apiocrea sp.14T

Two peptaibol antibiotics, peptavirins A and B, which exhibited strong inhibitory effect against Tobacco mosaic vials (TMV) infection, were isolated from steam-cooked rice culture of Apiocrea sp.14T. The peptavirins were identified as new derivatives of chrysospermins, which are 19-mer and have been reported to be produced in a fungal isolate. The physicochemical properties of the peptavirins were mostly identical with chrysospermins A through D except for the UV absorption spectrum. The peptavirins inhibited the growths of the Grampositive bacteria tested, including the plant pathogenic bacterium, Corynebacterium lilium, and the fungus, Aspergillus niger. Peptavirin A was somewhat cytotoxic to cancer cell lines, especially K562 (leukemia) and UACC 62 (melanoma), whereas peptavirin B only exhibited slight cytotoxicity.

Effect of Dissolved Oxygen on Continuous Production of Methionine

Methionine production by a mutant strain of Corynebacterium lilium was studied at different dilution rates and different dissolved oxygen concentrations, based on a statistical central composite design. The effect of dissolved oxygen levels between 2 % and 60 % and dilution rate levels between 0.04 to 0.29 on continuous production of methionine was studied. The process is described by the Leudeking-Piret model. The experimentally obtained maxima for methionine and biomass productivities, observed at the same dilution rate of 0.17 h -1 , also corresponds to the theoretical prediction based on this model. This experimental observation is further supported by the surface response prediction of a dilution rate of 0.16 h -1 for maximum methionine productivity and 0.15 h -1 for cell mass productivity. Also, beyond the critical value of 30 % dissolved oxygen, the apparent benefits of oxygen transfer become less significant.

Transfer of Brevibacterium divaricatum DSM 20297T, "Brevibacterium flavum" DSM 20411, "Brevibacterium lactofermentum" DSM 20412 and DSM 1412, and Corynebacterium lilium DSM 20137T to Corynebacterium glutamicum and Their Distinction by rRNA Gene Restriction Patterns

The results of DNA-DNA hybridization and chemotaxonomic studies indicated that the glutamic acid producers Brevibacterium divaricatum DSM 20297T (T=type strain), "Brevibacterium flavum" DSM 20411, "Brevibacterium lactofermentum" DSM 1412 and DSM 20412, Corynebacterium lilium DSM 20137T, and Corynebacterium glutamicum DSM 20300T and DSM 20163 are members of the same species. It is proposed that all of these strains should be classified in the species Corynebacterium glutamicum. Another glutamic acid-producing strain, Corynebacterium callunae DSM 20147T, was not related at the species level to C. glutamicum and should retain its separate species status. A restriction fragment length polymorphism analysis in which oligonucleotides targeted against conserved regions of 16S and 23S rRNA genes were used as hybridizing probes distinguished the individual strains. This method may be a helpful tool for strain identification.

Isolation and characterization of a restriction and modification deficient mutant ofBrevibacterium lactofermentum

In order to facilitate genetic engineering in amoni-acid producing bacteria we have isolated two restriction-deficient Brevibacterium lactofermentum strains. They have been selected for their ability to obtain a high yield of plaques from CL31 phage which was grown on Corynebacterium lilium. These mutant strains do not restrict either phage DNA by transfection or DNA from the shuttle vector pBLA extracted from Escherichia coli by protoplast transformation. These mutants have also lost modification activity. We also report the presence of a restriction modification system in C. lilium ATCC 15990.

Isolation and characterization of a restriction and modification deficient mutant of Brevibacterium lactofermentum

In order to facilitate genetic engineering in amoni-acid producing bacteria we have isolated two restriction-deficient Brevibacterium lactofermentum strains. They have been selected for their ability to obtain a high yield of plaques from CL31 phage which was grown on Corynebacterium lilium. These mutant strains do not restrict either phage DNA by transfection or DNA from the shuttle vector pBLA extracted from Escherichia coli by protoplast transformation. These mutants have also lost modification activity. We also report the presence of a restriction modification system in C. lilium ATCC 15990.

Structural characteristics of the Corynebacterium lilium bacteriophage CL31.

Bacteriophage CL31 was isolated on a Corynebacterium lilium strain. Out of 30 strains tested, only CL31 was able to form plaques on Corynebacterium glutamicum ATCC 13287, Brevibacterium lactofermentum ATCC 21086, and Arthrobacter sp. strain SI55, but at a very low frequency. This phage belongs to group B of Bradley's classification (D. E. Bradley, Bacteriol. Rev. 31:230-314; 1967). Its head is 53 nm in diameter, and its tail is 396 nm in length. The phage capsid contains three major proteins, of 12.5, 29.0, and 37.0 kilodaltons, and five minor ones (23.9, 26.0, 27.0, 40.0, and 55.4 kilodaltons). CL31 DNA is a linear molecule of 48 kilobases with cohesive ends. Restriction mapping was performed for endonucleases BglII, EcoRI, SalI, and KpnI. The expression of CL31 genes in Escherichia coli was studied by the maxicell technique; 12 different proteins were detected.

Protoplast transformation in coryneform bacteria and introduction of an alpha-amylase gene from Bacillus amyloliquefaciens into Brevibacterium lactofermentum.

The goal of this study was to investigate the likelihood of developing useful transformation systems for coryneform bacteria. Two species of coryneform bacteria, Brevibacterium lactofermentum and Corynebacterium lilium, were transformed with chimeras constructed from pUB110 and a cryptic coryneform plasmid (pGX1901). C. lilium protoplasts were also efficiently transfected with phage CS1 DNA. High transformation and transfection frequencies were obtained after only 2 min of lysozyme treatment of lysozyme-sensitive mutants. A series of experiments was also conducted to determine whether DNA from other species of important industrial microbes from the genus Bacillus could be expressed in coryneform bacteria. Evidence of restriction of Bacillus subtilis DNA by B. lactofermentum was observed but could be overcome. A Bacillus amyloliquefaciens alpha-amylase gene (amyEBamP) was subcloned onto a plasmid able to replicate in B. lactofermentum. B. lactofermentum transformants for this plasmid expressed amylase activity and produced material cross-reactive to amylase antibody.

Transfection of Corynebacterium lilium Protoplasts

A protoplast transfection system has been developed for a lysine-producing bacterium, Corynebacterium lilium, using the DNA of phage CL31. Phage CL31 is lytic and specific to C. lilium and has a genome of approximately 48 kb. The transfection procedure involves a polyethylene-glycol-mediated introduction of the DNA into lysozyme-treated cells and has a maximum efficiency of 3 X 10(4) transfectants per microgram DNA.

Studies on creatinine metabolism by microorganisms. II. Production, purification and crystallization of creatinine deiminase of Corynebacterium lilium.

Studies on creatinine metabolism by microorganisms. II. Production, purification and crystallization of creatinine deiminase of Corynebacterium lilium.

MICROBIAL PRODUCTION OF L-ARGININE

L-Arginine-producing mutants were found in mutants resistant to various amino acid analogs including canavanine and antimetabolites derived from glutamic acid-producing bacteria such as Brevibacterium flavum, Corynebacterium lilium, Microbacterium ammoniaphilum, Arthrobacter paraffineus, and Brevibacterium ketoglutamicum.L-Arginine producers could also be obtained by random isolation from cells treated with N-methyl-N′-nitro-N-nitrosoguanidine. The best producer, strain No. 352, was isolated as a 2-thiazolealanine-resistant mutant from a guanine auxotroph of Brevibacterium flavum. Strain No. 352 produced 34.8mg/ml of L-arginine in a medium containing 13% of glucose. L-Citrulline was also produced by this strain in the same medium.

Regulation of the tricarboxylic acid cycle in bacteria a comparison of citrate synthases from different bacteria

Citrate synthase (citrate oxalacetate-lyase (CoA-acetylating), EC 4.1.3.7) from Azotobacter vinelandii, Salmonella typhimurium, Rhodospirillum rubrum, Pseudomonas fluorescens and Corynebacterium lilium were examined in partially purified extracts. The enzymes differed in several respects including K m for substrates, stability, and response to modulators of activity (cations, ATP, AMP, NADH, and α-ketoglutarate). Citrate synthase from A. vinelandii was found to be activated by AMP and inhibited by ATP and NADH. The enzyme from R. rubrum was inhibited by ATP and that from S. typhimurium was inhibited by ATP and NADH. NADH inhibition was relieved by AMP. The activity of the enzyme from C. lilium, a glutamate excretor, was weakly inhibited by ATP and NADH. A comparison of the modulation of the activities of citrate synthase and from eight microorganisms representing different physiological groups is presented. It is indicated that the control of citrate synthase differs in organisms that are physiologically diverse. The regulation of isocitrate dehydrogenase, on the other hand, is similar in unrelated bacteria.