Molecular breeding of aBrevibacterium flavum L-lysine producer using a cloned aspartokinase gene

Summary ThelysC gene encoding aspartokinase of a lysine-hyperproducing mutantBrevibacterium flavum CCRC 18271 was cloned by polymerase chain reaction and its expression in CCRC 18271 was studied. Enzyme assays showed thatB. flavum cells harbouring thelysC gene exhibited 4- to 11-fold higher specific activities of aspartokinase as compared to respective hosts. Introduction of thelysC gene intoB. flavum CCRC 18271 resulted in increased lysine production up to 33% in flask fermentations.     

Altered prephenate dehydratase in phenylalanine-excreting mutants of Brevibacterium flavum.

The regulatory properties of three key enzymes in the phenylalanine biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthetase (DAHP synthetase) [EC 4.1.2.15], chorismate mutase [EC 5.4.99.5], and prephenate dehydratase [prephenate hydro-lyase (decarboxylating), EC 4.2.1.51] were compared in three phenylalanine-excreting mutants and the wild strain of Brevibacterium flavum. Regulation of DAHP synthetase by phenylalanine and tyrosine in these mutants did not change at all, but the specific activities of the mutant cell extracts increased 1.3- to 2.8-fold, as reported previously (1). Chorismate mutase activities in both the wild and the mutant strains were cumulatively inhibited by phenylalanine and tyrosine and recovered with tryptophan, while the specific activities of the mutants increased 1.3- to 2.8-fold, like those of DAHP synthetase. On the other hand, the specific activities of prephenate dehydratase in the mutant and wild strains were similar, when tyrosine was present. While prephenate dehydratase of the wild strain was inhibited by phenylalanine, tryptophan, and several phenylalanine analogues, the mutant enzymes were not inhibited at all but were activated by these effectors. Tyrosine activated the mutant enzymes much more strongly than the wild-type enzyme: in mutant 221-43, 1 mM tyrosine caused 28-fold activation. Km and the activation constant for tyrosine were slightly altered to a half and 6-fold compared with the wild-type enzyme, respectively, while the activation constants for phenylalanine and tryptophan were 500-fold higher than the respective inhibition constants of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 x 10(5), a half of that of the wild-type enzyme. The molecular weight of the mutant enzyme was estimated to be 1.2 X 10(5) a half of that of the wild type enzyme, while in the presence of tyrosine, phenylalanine, or tryptophan, it increased to that of the wild-type enzyme. Immediately after the mutant enzyme had been activated by tyrosine and then the tyrosine removed, it still showed about 10-fold higher specific activity than before the activation by tyrosine. However, on standing in ice the activity gradually fell to the initial level before the activation by tyrosine. Ammonium sulfate promoted the decrease of the activity. On the basis of these results, regulatory mechanisms for phenylalanine biosynthesis in vivo as well as mechanisms for the phenylalanine overproduction in the mutants are discussed.     

Molecular cloning with a pMEA300-derived shuttle vector and characterization of the Amycolatopsis methanolica prephenate dehydratase gene.

An efficient restriction barrier for methylated DNA in the actinomycete Amycolatopsis methanolica could be avoided by using a nonmethylating Escherichia coli strain for DNA isolations. The A. methanolica prephenate dehydratase gene was cloned from a gene bank in a pMEA300-derived shuttle vector in E. coli and characterized.     

Proline in the Osmoregulation of Brevibacterium lactofermentum

Osmoregulation in Brevibacterium lactofermentum was studied. Proline was accumulated up to approximately 35mg/g dry cell weight in the cells of a wild strain of the bacterium grown under osmotic stress. The osmotic tolerance of a proline auxotroph mutant obtained from the bacterium was lower than that in the wild strain. The activity of pyrroline-5-carboxylate reductase, one of the enzymes in the proline biosynthetic pathway, increased about 3-fold when the cells of B. lactofermentum were grown under osmotic stress. These data indicated that proline is important in osmoregulation in the bacterium.     

Production of l-Leucine by a Mutant of Brevibacterium lactofermentum 2256

A potent l-leucine producer was screened among mutants of glutamic acid producing bacteria. This strain, No. 218, is one of 2-thiazolealanine resistant mutants derived from a methionine isoleucine double auxotroph of Brevibacterium lactofermentum 2256 by nitroso-guanidine.

Strain No. 218 produced 19 mg/ml of l-leucine after 72 hr cultivation when 8 % glucose and 4 % ammonium sulfate were supplied as a carbon and a nitrogen source, respectively, thus giving the yield of 23.1 % from glucose.

The addition of Fe²⁺ and Mn²⁺ in combination gave much more productivity than that of Fe²⁺ or Mn²⁺ alone.

Effects of amino acids, nucleic acids, vitamins, and the other nutrients on l-leucine production were investigated.

The fermentation product was isolated and purified from the culture, and identified as l-leucine.     

Sequence analysis of the Brevibacterium lactofermentum trp operon

Summary Brevibacterium lactofermentum, a Gram-positive bacterium, is a commercially important amino acid producer. In this organism, the tryptophan biosynthetic enzymes are encoded within a 7725 bp HapII-BamHI fragment. Seven open reading frames were identified as trp genes by complementation tests with various B. lactofermentum and Escherichia coli tryptophan auxotrophs. Following the nomenclature established for E. coli and Serratia marcescens, the B. lactofermentum trp genes were designated trpL, trpE, trpG, trpD, trpC (including the trpF domain), trpB, and trpA. The organization of these genes is identical to that in S. marcescens. The nucleotide sequences of the putative ribosome-binding sites for the B. lactofermentum trp genes resemble those of E. coli and Bacillus subtilis. Computer analysis revealed that the trp enzymes of B. lactofermentum resemble the enzymes of the Gram-negative E. coli more closely than those of the Gram-positive B. subtilis.Abbreviations bp base pairs - kb kilobases     

Isolation of the murI gene from Brevibacterium lactofermentum ATCC 13869 encoding d-glutamate racemase

The murI gene encoding D-glutamate racemase plays an important role in the biosynthesis of D-glutamic acid, an essential component of cell wall peptidoglycan of almost all eubacteria. A DNA fragment that could rescue the auxotrophy of D-glutamic acid in the Escherichia coli murI mutant strain WM335 was isolated from Brevibacterium lactofermentum ATCC 13869 belonging to the coryneform bacteria. DNA sequencing reveals that it encodes a protein of 284 amino acid residues, which shows a high level of homology with D-glutamate racemases from several other bacteria.     

X-ray structure of prephenate dehydratase from Streptococcus mutans

Prephenate dehydratase is a key enzyme of the biosynthesis of L-phenylalanine in the organisms that utilize shikimate pathway. Since this enzymatic pathway does not exist in mammals, prephenate dehydratase can provide a new drug targets for antibiotics or herbicide. Prephenate dehydratase is an allosteric enzyme regulated by its end product. The enzyme composed of two domains, catalytic PDT domain located near the N-terminal and regulatory ACT domain located near the C-terminal. The allosteric enzyme is suggested to have two different conformations. When the regulatory molecule, phenylalanine, is not bound to its ACT domain, the catalytic site of PDT domain maintain open (active) state conformation as Sa-PDT structure. And the open state of its catalytic site become closed (allosterically inhibited) state if the regulatory molecule is bound to its ACT domain as Ct-PDT structure. However, the X-ray structure of prephenate dehydratase from Streptococcus mutans (Sm-PDT) shows that the catalytic site of Sm-PDT has closed state conformation without phenylalanine molecule bound to its regulatory site. The structure suggests a possibility that the binding of phenylalanine in its regulatory site may not be the only prerequisite for the closed state conformation of Sm-PDT.     

Production of l-Lysine by Fluoropyruvate-sensitive Mutants of Brevibacterium lactofermentum

Better producers of l-lysine were obtained by derivation of fluoropyruvate(FP)-sensitive mutants from Brevibacterium lactofermentum AJ3990. The coexistence of FP and excess biotin synergistically stimulated l-lysine formation by washed cells. FP inhibited 50% of growth and pyruvate dehydrogenase (PDH) activity of AJ3990 at 0.04 mm and 1 mm, respectively. Therefore, the synergistic effect of FP and excess biotin seems to be due to the optimization of the PDH/pyruvate carboxylase activity ratio in l-lysine biosynthesis. This was confirmed by the derivation of FP-sensitive mutants which have the optimal level of PDH activity for l-lysine production. The best producer, AJ11204, had about 27% PDH activity as compared with the parental strain and accumulated 70 g of l-lysine per liter with a conversion yield of 50% from glucose in the presence of excess biotin.     

Transfer of plasmid DNA to Brevibacterium lactofermentum by electrotransformation

The Escherichia coli-Brevibacterium lactofermentum shuttle vector pBLA was introduced into intact cells of B. lactofermentum by electrotransformation. Several parameters of this procedure such as voltage and cell concentration were analysed. Optimal conditions gave an efficiency of 10(6) transformants per microgram of DNA. Two recalcitrant strains could be electrotransformed when an ampicillin pretreatment step was used. Electrotransformation experiments using DNAase or different structural forms of plasmid DNA showed that the electrotransformation process is quite different from natural transformation involving competence development. Restriction-modification-proficient B. lactofermentum could be efficiently electrotransformed with pBLA DNA isolated from E. coli. This restriction-modification system therefore seems to be overcome by electrotransformation. Thus electrotransformation may efficiently replace the protoplast bacterial transformation method.     

Identification, characterization, and chromosomal organization of the ftsZ gene from Brevibacterium lactofermentum

The ftsZ gene was cloned from the chromosomal DNA of Brevibacterium lactofermentum by the polymerase chain reaction (PCR) using two oligonucleotides designed from two conserved regions found in most of the previously cloned and sequenced ftsZ genes from other microorganisms. ftsZ is a single-copy gene in corynebacteria and is located downstream from ftsQ and murC, indicating linkage between genes involved in peptidoglycan synthesis (mur genes) and genes involved in cell division (fts genes). The organisation of the cluster is similar to that in Streptomyces and different from those of Escherichia coli or Bacillus subtilis because ftsA is not located upstream of ftsZ. The gene was expressed in E. coli using the T7 expression system; the calculated molecular weight of the expressed protein was 50?kDa. Expression of the B. lactofermentum ftsZ gene in E. coli inhibited cell division and led to filamentation. The ftsZ gene of this organism does not complement ftsZ mutations or deletions in E. coli, when cloned on low or high-copy-number vectors.     

A Brevibacterium lactofermentum 16S rRNA gene used as target site for homologous recombination

Genes for rRNA are highly conserved and present in multiple copies in most prokaryotic organisms increasing the number of theoretical sites for homologous recombination. They might be targets for integration events between unrelated microorganisms providing that an efficient genetic transfer is present. We have used a plasmid containing a portion of the 16S rRNA gene from the rrnD operon of Brevibacterium lactofermentum to transform the same strain resulting in non-essential inactivation of various rrn operons. Integration of the transforming DNA occurs in all cases. The system may be used to test possible gene transfer at least among closely related strains and is of great interest for integration of foreign DNA and for mapping.     

A selective inhibitor of Escherichia coli prephenate dehydratase.

To identify selective prephenate dehydratase (PDT) inhibitors, a series of substituted biphenic acid derivatives was synthesized using the Ullmann reaction. Screening experiments identified 18 as a promising new PDT inhibitor.     

Expression of the genes coding for the xylanase Xys1 and the cellulase Cel1 from the straw-decomposing Streptomyces halstedii JM8 cloned into the amino-acid producer Brevibacterium lactofermentum ATCC13869

The xylanase ( xysA) and the cellulase ( celA1) genes from Streptomyces halstedii JM8 were cloned into Escherichia coli/ Brevibacterium lactofermentum shuttle vectors and successfully expressed in both hosts when placed downstream from the kanamycin resistance promoter (Pkan) from Tn 5 but not when under the control of their own promoters. Xylanase was secreted into the culture media of B. lactofermentum by removal of the same leader peptide as is removed in S. halstedii. The main difference between the production of xylanase by Streptomyces and corynebacteria was the low level of processing of the mature extracellular xylanase by B. lactofermentum, probably due to the lack of protease activity in this microorganism.