Hideaki Yukawa

Presence of mrr- and mcr-like restriction systems in coryneform bacteria.

Efficient transformation of Brevibacterium flavum MJ233C and Corynebacterium glutamicum ATCC 31831 (up to 5.0 x 10(7) transformants/microgram DNA) depends on the source of plasmid DNA. The transformation efficiencies of B. flavum MJ233C and C. glutamicum ATCC 31831 increased nearly 10(3)-fold when plasmid DNA was isolated from the recipient strain itself or from a damdcm Escherichia coli mutant, as compared with DNA passed through a modification-proficient E. coli strain. These results suggest the presence of a methyl-specific restriction system in certain strains of coryneform bacteria. In addition, electroporation conditions were optimized.

Isolation and characterization of IS 31831, a transposable element from Corynebacterium glutamicum

A transposable element from a coryneform bacterium, Corynebacterium glutamicum ATCC 31831 was isolated and characterized. The element IS 31831 is a 1453 bp insertion sequence with 24 bp imperfect terminal inverted repeats. It contains one open reading frame highly homologous at the amino acid level to the transposase of IS 1096 from Mycobacterium smeg‐matis. Both IS 31831 and IS 1096 exhibit several common characteristics suggesting that they constitute a new family of insertion sequences. IS 31831 was isolated by taking advantage of the sucrose sensitivity of coryneform bacteria conferred by expression of the Bacillus subtilis sacB gene. An Escherichia coli/ Corynebacterium shuttle vector useful for the isolation of transposable elements from the coryneform group of bacteria was constructed.

Transposon mutagenesis of coryneform bacteria

The Corynebacterium glutamicum insertion sequence IS31831 was used to construct two artificial transposons: Tn31831 and miniTn31831. The transposition vectors were based on a gram-negative replication origin and do not replicate in coryneform bacteria. Strain Brevibacterium flavum MJ233C was mutagenized by miniTn31831 at an efficiency of 4.3 x 104 mutants per microgram DNA. Transposon insertions occurred at different locations on the chromosome and produced a variety of mutants. Auxotrophs could be recovered at a frequency of approximately 0.2%. Transposition of IS31831 derivatives led not only to simple insertion, but also to cointegrate formation (5%). No multiple insertions were observed. Chromosomal loci of B. flavum corresponding to auxotrophic and pigmentation mutants could be rescued in Escherichia coli, demonstrating that these transposable elements are useful genetic tools for studying the biology of coryneform bacteria.

Electroporation-transformation System for Coryneform Bacteria by Auxotrophic Complementation

We evaluated electroporation as an alternative system for genetic exchange for one of the coryneform bacteria, Brevibacterium flavum MJ233. The maximum number of transformants, 6 x 10(4) cells, was obtained when cells were cultured with Penicillin G (1 U/ml) and harvested at the middle-log phase. Electroporation was done using 12.5 kV/cm of pulse field strength, 1 x 10(10) cells, and 1 microgram of plasmid DNA. Other coryneform bacteria, Brevibacterium lactofermentum ATCC 13869, Corynebacterium glutamicum ATCC 31830, and B. stationis IFO 12144 were also transformed by electroporation. Electroporation has the advantage that intact cells can be used as host cells without the need for protoplast formation and regeneration. Moreover, minimal medium can be used, so auxotrophic complementation of the transformants is possible.

Electrotransformation of intact cells of Brevibacterium flavum MJ-233

SummaryElectroporation allowed transformation of intact cells ofBrevibacterium flavum MJ-233. The two plasmids used for electroporation were pCRY2 (6.3 kilobases) and pCRY3 (8.2 kilobases). Both plasmids contain the chloramphenicol-resistance gene and the autonomous replication origin inB. flavum MJ-233. The efficiency of electrotransformation was optimal with cells harvested at the middle log phase of growth, and was imporved by the addition of 1.0U/ml of penicillin G to the culture medium. The optimum yield of transformants per μg DNA was 5×104 when the cell suspension was pulsed at a cell density of 1×1010/ml and at a DNA amount of 1.0μg.

Analysis of the biotin biosynthesis pathway in coryneform bacteria: Cloning and sequencing of the bioB gene from Brevibacterium flavum

The biotin biosynthetic pathway of three coryneform bacteria, Brevibacterium flavum, Brevibacterium lactofermentum, and Corynebacterium glutamicum were analysed by cross-feeding experiments using several Escherichia coli biotin-requiring mutants. The three strains of coryneform bacteria tested were able to convert 7-keto-8-aminopelargonic acid to biotin, through a biotin synthetic pathway identical to that from E. coli. The biotin biosynthetic gene, bioB, of B. flavum was cloned by phenotypic complementation of E. coli bioB mutants. The bioB gene was located on a 1.7 kb HindIII-SacI DNA fragment. Nucleotide sequence analysis of this fragment revealed that the bioB gene of B. flavum consists of a 1005 bp open reading frame. Its deduced amino acid sequence is 35.7% and 31.5% identical to that of the E. coli and Bacillus sphaericus bioB gene products, respectively. B. flavum mutants obtained by in vivo disruption of the bioB gene lost their ability to grow on minimal medium containing dethiobiotin, indicating that the bioB gene product is necessary for the conversion of dethiobiotin to biotin.

Genomes and Genome-Level Engineering of Amino Acid-Producing Bacteria

The complete nucleotide sequence of the genomes of several strains of Escherichia coli and Corynebacterium glutamicum reveal the genetic blueprint of these industrial organisms including their structural genetic organization and their metabolic networks and conversion capabilities, refine the understanding of their phylogenetic positions, and open the possibility to assess the expected size of their pan-genomes in order to harness their diversity. The genome of C. glutamicum R codes for approximately 3000 genes, a minimum of 5.3% of which are related to amino acid transport and metabolism and 4.6% to carbohydrate transport and metabolism. The genome of E. coli K-12 encodes approximately 4450 genes, 7.5% of which are involved in amino acid transport and metabolism and 6.1% in carbohydrate transport and metabolism. Global techniques were enabled by these complete genomic sequences, including analyses by global transcription profiling, proteomics and metabolomics to gather biological data, and megabase molecular biology tools to engineer at will these organisms at various scales, from the level of single base pairs to that of chromosomes. Systems biology represents the next technological paradigm necessary on the one hand to efficiently integrate and process the large volume of global biological information thus attained, in order to understand bacterial physiology and organization at a higher level; and on the other hand to enable in silico models useful for generating optimization strategies of increasing complexity and relevance, in the hope to lead faster towards improved metabolic engineering solutions with the aim of attaining expanded industrial process scope and superior economics.

Biorefinery Applications of Corynebacterium glutamicum

The biorefinery concept is an emerging concept for conducting industrial processes to manufacture a range of commodity chemicals, fuels, and energy from biomass-based feedstock. The current interest in implementing a biorefinery industry is largely derived by a combination of rising petroleum prices as well as the need to reduce greenhouse gas emissions and atmospheric CO2 levels to mitigate global warming. To date, Corynebacterium glutamicum-based technology has not been considered as the primary manufacturing platform for sustainable chemicals. Indeed, despite a long history of use for the industrial production of amino acids, C. glutamicum, as compared to Escherichia coli or Saccharomyces cerevisiae, has been scarcely studied and engineered to fit the needs of the lignocellulosic biorefinery. However, progress over the last decade in the understanding of its molecular physiology and metabolic engineering makes this microorganism an attractive option as a biorefinery biocatalyst. In addition, the development of a novel bioprocess using growth-arrested cells of C. glutamicum under oxygen deprivation constitutes a promise for biorefinery research and development. In this chapter, recent studies on the development of C. glutamicum as a commodity chemicals producer are reviewed and the key challenges that remain to overcome in order to deliver the full potential of this microbe to produce commodity chemicals are outlined.

Cloning and Nucleotide Sequencing of The Poly(3-hydroxybutyrate) Depolymerase Gene From Pseudomonas pickettii

SUMMARY An extracellular poly (3-hydroxybutyrate) depolymerase (PHB depolymerase) producing bacterium, strain K1, was isolated from soil samples and identified as Pseudomonas pickettii. The PHB depolymerase gene was cloned from the genomic DNA of the strain K1 and the nucleotide sequence was determined. The 1960 bp Eco RI-Sma I DNA fragment containing the PHB depolymerase gene had an open reading frame of 1467 bp and the deduced amino acid sequence of this open reading frame was 492 residues long with a signal peptide of 30 amino acids.

Depression of by-product formation during l-isoleucine production by a living-cell reaction process

SummaryTwo unnatural and unwanted amino acids, norvaline (Nva) and O-ethylhomoserine (O-EH) are formed as by-products in l-isoleucine production by Brevibacterium flavum AB-07 using a new process named the living cell reaction process. Nva formation was depressed by using a leucine auxotrophic mutant (AB-07-Leu-2) derived from strain AB-07. It was found that Nva formation was closely related to leucine biosynthesis. O-EH formation was repressed by addition of l-methionine to the reaction mixture. However, the homoserine-O-acetyltransferase of AB-07-Leu-2 was not subject to either inhibition or repression by addition of l-methionine. Furthermore, the O-EH-forming enzyme, which converts O-acetylhomoserine to O-EH, was speculated to be repressed by l-methionine.