Yield regulation of lysine biosynthesis in Brevibacterium flavum

A scheme for lysine biosynthesis using variants of the Brevibacterium flavum intermediary metabolite synthesis is discussed. The main precursor of lysine that we are concerned with here is oxalacetate, which can be synthesized through the TCA or glyoxylate cycles or by carboxylation of PEP. Material energy balances for the main pathways of lysine biosynthesis from glucose and acetate have been formulated. Energy consumption, in the from of ATP – P_ATP (number of mol ATP consumed/1 mol lysine synthesized), was calculated for the main pathways of lysine biosynthesis. Theoretical conversion yields Y_p^max (g product/g substrate) were estimated. Experimental data were presented concerning the increase of Y_p by means of metabolism regulation: (a) by TCA-and glyoxylate-cycle enzyme induction; (b) by maintaining PEP carboxylase activity; (c) by eliminating by-product synthesis.     

Taxonomic position of the lysine producer Brevibacterium flavum

Brevibacterium flavum 22 and 22L producing lysine and glutamic acid should be reclassified as Corynebacterium glutamicum on the basis of their chemotaxonomic characteristics: the IV type of the cell wall, corynomycolic acids C32--C34, 57.8% of GC in DNA.     

Regulation of Aspartate Family Amino Acid Biosynthesis in Brevibacterium flavum

Dihydrodipicolinate (DDP)* synthetase and DDP-reductase were partially purified about 30 and 15 folds, respectively, from sonic extracts of Brevibacterium flavum.

In contrast with DDP-synthetase from Escherichia coli, the B. flavum enzyme was only slightly inhibited by α, ε-diaminopimelate, a precursor of lysine, but not by lysine itself. Single or simultaneous addition of any other amino acid(s) of aspartate family did not affect the activity significantly. Optimum pH for DDP-synthetase was 8.4 with Tris-HCl buffer. Kms for aspartic-β-semialdehyde and pyruvate at pH 7.5 were 2×10^?4m and 1×10^?4m, respectively. The formation of DDP-synthetase was not significantly repressed by lysine.

DDP-reductase of B. flavum required NADH or NADPH as the cofactor. This enzyme was not inhibited by single or simultaneous addition of aspartate family amino acid(s).

From the above results, the regulation mechanism of lysine biosynthesis in B. flavum was discussed.     

Hydrogenase activity in Brevibacterium flavum

The activity of hydrogenase was assayed in the intact cells and subcellular fractions of Brevibacterium flavum. The organism was shown to have the membrane-bound form of hydrogenase. The soluble NAD+-reducing hydrogenase was not found. Oxygen inhibited the hydrogenase activity, and its action was reversible. Molecular hydrogen activated the hydrogenase of B. flavum, which was shown to be a constitutive enzyme.     

Regulation of enzymes of lysine biosynthesis in Corynebacterium glutamicum

The regulation of the six enzymes responsible for the conversion of aspartate to lysine, together with homoserine dehydrogenase, was studied in Corynebacterium glutamicum. In addition to aspartate kinase activity, the synthesis of diaminopimelate decarboxylase was also found to be regulated. The specific activity of this enzyme was reduced to one-third in extracts of cells grown in the presence of lysine. Aspartate-semialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, and diaminopimelate dehydrogenase were neither influenced in their specific activity, nor inhibited, by any of the aspartate family of amino acids. Homoserine dehydrogenase was repressed by methionine (to 15% of its original activity) and inhibited by threonine (4% remaining activity). Inclusion of leucine in the growth medium resulted in a twofold increase of homoserine dehydrogenase specific activity. The flow of aspartate semialdehyde to either lysine or homoserine was influenced by the activity of homoserine dehydrogenase or dihydrodipicolinate synthase. Thus, the twofold increase in homoserine dehydrogenase activity resulted in a decrease in lysine formation accompanied by the formation of isoleucine. In contrast, repression of homoserine dehydrogenase resulted in increased lysine formation. A similar increase of the flow of aspartate semialdehyde to lysine was found in strains with increased dihydrodipicolinate synthase activity, constructed by introducing the dapA gene of Escherichia coli (coding for the synthase) into C. glutamicum.     

L－异亮氨酸产生菌选育的研究

以黄色短杆菌（Ｂｒｅｖｉｂａｃｔｅｒｉｕｍｆｌａｖｕｍ）ＡＴＣＣ１４０６７为出发菌株,经硫酸二乙酯（ＤＥＳ）、紫外线（ＵＶ）和亚硝基胍（ＮＴＧ）逐级诱变处理,α－氨基－β－羟基戊酸（ＡＨＶ）、Ｓ－２－氨基乙基－Ｌ－半胱氨酸（ＡＥＣ）、磺胺胍（ＳＧ）、乙硫氨酸（Ｅｔｈ）、α－氨基丁酸（α－ＡＢ）、异亮氨酸氧肟酸（ＩｌｅＨｘ）等氨基酸结构类似物及琥珀酸为碳源平板定向筛选,获得一株Ｌ－异亮氨酸高产菌ＺＱ－４（ＡＨＶ~γ、ＡＥＣ~γ、ＳＡＭ~γ、ＳＧ~γ、Ｅｔｈ~γ、α－ＡＢ~γ、ＩｌｅＨｘ~γ）在含１３．５％葡萄糖培养基中,摇瓶发酵７２ｈ、Ｌ－异亮氨酸积累可达２．８－３．０％。     

Two components of chorismate mutase in Brevibacterium flavum.

Chorismate mutase of Brevibacterium flavum, a common enzyme in phenylalanine and tyrosine biosynthesis, was separted into two different component, A and B, with molecular weights of 250,000 and 25,000, respectively, by ammonium sulfate fractionation or gel-filtration. Both components were essential for the enzymatic activity. In the presence of the reaction substrate, chorismate, the two components associated reversibly to give an active enzyme complex with a molecular weight of 320,000. Binding sites of the feedback inhibitors, phenylalanine and tyrosine, on the enzyme were localized on component A as determined by hybridization experiments with the wild-type and mutant components. Tyrosine repressed the synthesis of component B much more strongly than that of component A, while phenylalanine did not show any significant repressive effect on either component. The wild-type strain No. 2247 had four times more component A than component B. Elution patterns in gel, DEAE-cellulose or hydroxyapatite column chromatography as well as the disc-gel electrophoretic pattern of chorismate mutase component A and 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthetase activities completely overlapped, suggesting the presence of a bifunctional protein having the two activities. In accord with this suggestion, chorismate mutase as well as DAHP synthetase was insensitive to feedback inhibition by phenylalanine and tyrosine in all the 3-fluorophenylalanine-resistant mutants tested that excreted both phenylalanine and tyrosine. All the phenylalanine and tyrosine double auxotrophs defective in chorismate mutase lacked component B but not A.     

Regulation of Aromatic Amino Acid Biosynthesis and Production of Tyrosine and Phenylalanine m Brevibacterium flavum

In Brevibacterium flavum, prephenate dehydratase in the phenylalanine specific biosynthetic pathway was strongly inhibited by phenylalanine and activated by tyrosine. Furthermore. the inhibition by phenylalanine was completely reversed by tyrosine. Inhibition by tyrosine of prephenate dehydrogenase in the tyrosine specific pathway was very weak. Overall regulation mechanism of the aromatic amino acid biosynthesis in B. flavum was proposed on the bases of these results and the previous findings on 3-deoxy-D-arabino-heptulosonate-7- phosphate synthetase(DAHP synthetase*) of the common pathway and on anthranilate synthetase of the tryptophan specific pathway. Two types of m-fluorophenylalanine(mFP) resistant mutants which accumulated phenylalanine alone or both phenylalanine and tyrosine, respectively, were derived. The accumulation in the former mutants was inhibited by tyrosine, but that in the latter was affected neither by tyrosine nor by phenylalanine. DAHP synthetase of the latter mutants had been desensitized from the synergistic feedback inhibition by tyrosine and phenylalanine, while prephenate dehydratase of the former mutants had been desensitized in the feedback inhibition by phenylalanine. Tyrosine auxotroph accumulated phenylalanine under tyrosine limitation and its accumulation was inhibited by the excessive addition of tyrosine. Phenylalanine auxotroph accumulated tyrosine under phenylalanine limitation and its accumulation was inhibited by the excessive addition of phenylalanine. These results in vivo strongly supported the proposed regulation mechanism in which synthesis of phenylalanine in preference to tyrosine was assumed.     

Study of Brevibacterium flavum phage PhiBSh6]

Properties of a virulent Brevibacterium flavum bacteriophage phi BSh6 were studied. The phage was placed in morphological group B1 according to Ackerman classification, head diameter being 74-3 nm, tail length being 337 +/- 15 nm. The phage was shown to have double stranded DNA as a genetic material. The chromosome is linear having cohesive ends. Chromosome length was estimated to be about 71 kbp by restriction analysis and electron microscopy. A unique EcoRI-EcoRI fragment of bacteriophage DNA (0.8 kbp) was cloned in Escherichia coli. Restriction chart of cos region was determined, the dyad symmetry being absent from cos sequence. Deletion mutant of the phage was obtained and restriction map of the corresponding genome region was constructed. The phage phi BSh6 was shown to be a close relative to phages phi B and BB14 described earlier.     

Potential vectors for molecular cloning in Brevibacterium flavum]

Construction of the shuttle cloning vectors for Escherichia coli-Brevibacterium flavum system is described. Expression of the Sp/Sm resistance determinant derived from the Corynebacterium plasmid pCG4 was registered in Escherichia coli cells. The genetic determinant for Sp/Sm resistance was shown to be located in a 2.2 kb PstI-SphI fragment by the deletion analysis mapping in Escherichia coli cells. Using Escherichia coli as a host we cloned the unique 0.8 kb EcoRI-EcoRI fragment of Brevibacterium flavum bacteriophage phi BSh6 in the plasmids with dual replication origins. Blocking of the shuttle vector transfer to Brevibacterium flavum by the insertion of bacteriophage phi BSh6 DNA was observed. The deletion of entire phage fragment or a specific part of it made it possible introduction of plasmids harboured by Escherichia coli cells into Brevibacterium flavum. A potential vector for homologous DNA cloning in Brevibacterium flavum was constructed.     

Glutamate Metabolism in a Glutamate-producing Bacterium,Brevibacterium flavum

Brevibacterium flavum No. 2247 was found to grow with l-glutamate as the sole carbon and nitrogen source on an agar-plate medium when high concentrations of l-glutamate, FeSO₄ and biotin were added to the medium. It grew on l-glutamate in liquid medium only when yeast extract or high concentrations of FeSO₄ and glucose or organic acids of the tricarboxylic acid cycle were added to the medium. The growth on l-glutamate in liquid medium was also stimulated by high concentrations of l-glutamate, biotin and MgSO₄, and inhibited by a high concentration of (NH₄)₂SO₄.

Aspartate aminotransferase (TA)- and α-ketoglutarate dehydrogenase (KD)-defective mutants did not grow on l-glutamate, and glutamate-utilizing revertants derived from these mutants recovered TA and KD activity, respectively, whereas glutamate dehydrogenase (GD)-defective mutants grew on l-glutamate. Washed cells of strain No. 2247 grown on glutamate decomposed the amino acid, whereas those grown on glucose did not. The degradation was observed only under aerobic conditions. The former cells showed higher KD, succinate dehydrogenase and fumarase activities than the latter cells. Of 75 mutants which did not grow on glutamate but grew on succinate, three strains lacked KD but showed the same glutamate productivity as the parent strain. Four other strains with normal KD levels showed higher glutamate productivity than the parent.