Masayuki Inui

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.

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.

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.

Living cell reaction process forl-isoleucine andl-valine production

SummaryA new process (Living Cell Reaction Process) forl-isoleucine production using viable, non-growing cells ofBrevibacterium flavum AB-07 was optimised using ethanol as the energy source and α-ketobutyric acid (α-KB) as precursor.l-valine also could be produced from glucose at high yield by this process. This process differs from the usual fermentation method in that non-growing cells are used, and the production ofl-isoleucine andl-valine were carried out under conditions of repressed cell division and growth. Minimal medium missing the essential growth factor, biotin was employed as the reaction mixture for the production ofl-isoleucine andl-valine. The productivity ofl-isoleucine andl-valine were 200 mmol·l−1 · day−1 (molecular yield to α-KB: 95%) and 300 mmol · l−1 · day−1 (molecular yield to glucose: 80%) respectively. The content ofl-isoleucine andl-valine in total amino acids produced in the each mixture were 97% and 96% respectively.

The Biotechnological Potential of Corynebacterium glutamicum, from Umami to Chemurgy

Corynebacterium glutamicum exhibits numerous ideal intrinsic attributes as a microbial factory to produce not only amino acids but also chemicals. The large range of products that can now be biomanufactured mediates a transformational change in the deployment of this microorganism, from umami applications, referring to food enhancers, to chemurgy applications, referring to chemical commodity products. The deep fundamental knowledge of the corynebacterial physiology, the experience curve gained by manufacturing for decades at the industrial scale numerous amino acids, and postgenomic tools to model processes or design synthetic pathways in combinatorial approaches constitute a foundational basis to design efficient and versatile corynebacterial biorefineries. Moreover, the detailed knowledge in amino acid biosynthetic routes can be leveraged to harness the chemical space around these molecules, as exemplified by producing isobutanol from an engineered valine pathway; biosynthesis and chemical synthesis can also be coupled to produce sustainable intermediates. A wide range of applications can nowadays be addressed using C. glutamicum owing to its intrinsic safety and process performance, including also industrial enzymes, therapeutic proteins, antibody fragments, and secondary metabolites comprising small molecule natural products. The economics of the corynebacterial biorefinery could further be enhanced by valorizing its effluents in innovative ways, such as the use of the spent biomass in bioremediation or mining. A critical success factor is to compute an economically optimized process/product mix to create an interconnected product line for serving various markets offering attractive profit margins, covering not only the food and chemical industry but also the cosmetics and pharmaceutical ones.

Cloning and sequencing of thesecY homolog from Coryneform bacteria

A conserved domain of the secY genes from Bacillus subtilis, Mycoplasma capricolum and Escherichia coli was used to design degenerate oligodeoxyribonucleotides. These synthetic DNA sequences were used to screen a lambda library of Brevibacterium flavum MJ233. A 1.5-kb KpnI fragment of a recombinant lambda phage containing the secY homology from Br. flavum MJ233 was subsequently subcloned into plasmid pUC118. The complete nucleotide (nt) sequence of the cloned fragment indicated that the deduced gene product of the Br. flavum secY homolog is composed of 440 amino acids (aa) with a deduced M(r) of 47,871. Comparison of this aa sequence to the corresponding sequences from E. coli and B. subtilis revealed a high degree of conservation, and suggested that the Br. flavum secY homolog is a membrane protein containing ten transmembrane segments. In addition, we could identify, downstream from secY, a putative coding sequence of the enzyme adenylate kinase. This gene organization is identical to that observed in the B. subtilis genome.