Amino Acid-producing Bacterium Research Articles

Thermal and solvent stress cross-tolerance conferred to Corynebacterium glutamicum by adaptive laboratory evolution.

Reinforcing microbial thermotolerance is a strategy to enable fermentation with flexible temperature settings and thereby to save cooling costs. Here, we report on adaptive laboratory evolution (ALE) of the amino acid-producing bacterium Corynebacterium glutamicum under thermal stress. After 65 days of serial passage of the transgenic strain GLY3, in which the glycolytic pathway is optimized for alanine production under oxygen deprivation, three strains adapted to supraoptimal temperatures were isolated, and all the mutations they acquired were identified by whole-genome resequencing. Of the 21 mutations common to the three strains, one large deletion and two missense mutations were found to promote growth of the parental strain under thermal stress. Additive effects on thermotolerance were observed among these mutations, and the combination of the deletion with the missense mutation on otsA, encoding a trehalose-6-phosphate synthase, allowed the parental strain to overcome the upper limit of growth temperature. Surprisingly, the three evolved strains acquired cross-tolerance for isobutanol, which turned out to be partly attributable to the genomic deletion associated with the enhanced thermotolerance. The deletion involved loss of two transgenes, pfk and pyk, encoding the glycolytic enzymes, in addition to six native genes, and elimination of the transgenes, but not the native genes, was shown to account for the positive effects on thermal and solvent stress tolerance, implying a link between energy-producing metabolism and bacterial stress tolerance. Overall, the present study provides evidence that ALE can be a powerful tool to refine the phenotype of C. glutamicum and to investigate the molecular bases of stress tolerance.

Histidine biosynthesis, its regulation and biotechnological application in Corynebacterium glutamicum

l-Histidine biosynthesis is an ancient metabolic pathway present in bacteria, archaea, lower eukaryotes, and plants. For decades l-histidine biosynthesis has been studied mainly in Escherichia coli and Salmonella typhimurium, revealing fundamental regulatory processes in bacteria. Furthermore, in the last 15 years this pathway has been also investigated intensively in the industrial amino acid-producing bacterium Corynebacterium glutamicum, revealing similarities to E. coli and S. typhimurium, as well as differences. This review summarizes the current knowledge of l-histidine biosynthesis in C. glutamicum. The genes involved and corresponding enzymes are described, in particular focusing on the imidazoleglycerol-phosphate synthase (HisFH) and the histidinol-phosphate phosphatase (HisN). The transcriptional organization of his genes in C. glutamicum is also reported, including the four histidine operons and their promoters. Knowledge of transcriptional regulation during stringent response and by histidine itself is summarized and a translational regulation mechanism is discussed, as well as clues about a histidine transport system. Finally, we discuss the potential of using this knowledge to create or improve C. glutamicum strains for the industrial l-histidine production.

Systems metabolic engineering for the production of bio‐nylon precursor

Systematic approaches for in silico design of metabolic pathways and experimental evaluation of cellular performance are important for the development of industrially useful organisms. In this issue of Biotechnology Journal, Buschke et al. [1] report the production of 1,5-diaminopentane using xylose produced by Corynebacterium glutamicum by employing integration methodologies of rational design for genetic improvement, experimental analysis of metabolic fluxes, and comprehensive gene expression. Recent progress in 'omics sciences has facilitated the analysis of microorganisms for molecular breeding. There has been increasing interest for the integration of in silico and experimental approaches for the development of microbial cells for industrial applications. In silico simulations aid the rational design of metabolic pathways for the production of target compounds. On the basis of the in silico simulation data, the host organism is genetically manipulated to improve target metabolite production [2, 3]. The 13C-metabolic flux analysis method is useful for determining the flux distribution; this method requires experimental data procured from genetically manipulated strains [4, 5]. Identification of the genes that confer useful phenotypes for cells on the basis of DNA microarray analysis is also important [6]. The integration method, which combines in silico and experimental approaches for the development of cell factories is shown in Fig. 1. Many applications of the integration method for the bioproduction of chemicals and fuels have been reported [7, 8]. Concept of systems metabolic engineering. Production of 1,5-diaminopentane, a bio-nylon precursor, is reported by Buschke et al. [1], in this Special Issue “Systems Metabolic Engineering” of Biotechnology Journal. Buschke et al.'s study [1] is a good example of the successful application of bioengineering that contributes to the recent progress in systems metabolic engineering. Diaminopentane is produced by a one-step reaction from lysine by C. glutamicum, which is a well-known amino acid-producing bacterium and has recently used for chemical production as well [9, 10]. The authors have previously developed a high-producing strain that converts glucose to diaminopentane [11]. In the current study [1], a novel strain which produces diaminopentane from xylose was developed. Xylose utilization is particularly useful because xylose is a main component of lignocellulose waste, which is widely available. The previously developed diaminopentane-producing strain, DAP-Xyl1, produce diaminopentane less efficiently from xylose than from glucose. Thus, Buschke et al. [1] apply a systematic approach to improve diaminopentane production. In silico elementary flux mode analysis predicts an ideal metabolic state for diaminopentane production. The theoretical maximum yield from xylose is predicted, and the analysis reveal that the high oxidative pentose phosphate pathway (PPP) flux and the zero flux of the tricarboxylic acid (TCA) cycle are important for diaminopentane production; however, the metabolic flux of these pathways in the strain DAP-Xyl1 does not show good agreement with ideal metabolic flux for diaminopentane production. The small flux of PPP and the large flux of the TCA cycle cause low production of diaminopentane by the strain DAP-Xyl1. Additionally, gene selection is performed on the basis of the transcriptome analysis results. The novel, rationally designed strain DAP-Xyl2 successfully produce more than 100 g/L of diaminopentane after 75 h of cultivation. This study demonstrates the development process of a high performance strain of C. glutamicum and the importance of systems metabolic engineering approach for developing an industrially useful microorganism [7, 12]. The author declares no conflict of interest.

Monitoring enzyme expression of a branched respiratory chain of corynebacterium glutamicum using an EGFP reporter gene

To investigate the expressional control of branched respiratory chain complexes of the amino-acid producing bacterium Corynebacterium glutamicum according to growth conditions, the expression indexes of the ndh, sdh, qcrCAB, ctaCF, ctaD, ctaE, and cydAB genes were estimated under aerobic and microaerobic, and carbon-rich and -poor conditions. The promoter region of each target gene was cloned upstream of the EGFP gene on expression vector pVK6, and the nine reporter constructs were transformed into C. glutamicum ssp. lactofermentum. The cytochrome content of cellular membranes obtained from each growth phase closely corresponded to the expression indexes based on EGFP fluorescence and cell density, indicating that this rapid and convenient method is suitable for analyzing the expression levels of respiratory chain complexes. Using this method, we demonstrated that a reciprocal change in the expression levels of cytochrome bd-type and aa (3)-type oxidases occurs when C. glutamicum cells are held in stationary phase for extended periods.

The cytosolic, cell surface and extracellular proteomes of the biotechnologically important soil bacterium Corynebacterium efficiens YS‐314 in comparison to those of Corynebacterium glutamicum ATCC 13032

Reference maps of the cytosolic, cell surface and extracellular proteome fractions of the amino acid-producing soil bacterium Corynebacterium efficiens YS-314 were established. The analysis window covers a pI range from 3 to 7 along with a molecular mass range from 10 to 130 kDa. After second-dimensional separation on SDS-PAGE and Coomassie staining, computational analysis detected 635 protein spots in the cytosolic proteome fraction, whereas 76 and 102 spots were detected in the cell surface and extracellular proteomes, respectively. By means of MALDI-TOF-MS and tryptic peptide mass fingerprinting, 164 cytosolic proteins, 49 proteins of the cell surface and 89 extracellular protein spots were identified, representing in total 177 different proteins. Additionally, reference maps of the three cellular proteome fractions of the close phylogenetic relative Corynebacterium glutamicum ATCC 13032 were generated and used for comparative proteomics. Classification according to the Clusters of Orthologous Groups of proteins scheme and abundance analysis of the identified proteins revealed species-specific differences. The high abundance of molecular chaperones and amino acid biosynthesis enzymes in C. efficiens points to environmental adaptations of this recently discovered amino acid-producing bacterium.

Development of a Corynebacterium glutamicum DNA microarray and validation by genome-wide expression profiling during growth with propionate as carbon source

A DNA microarray was developed to analyse global gene expression of the amino acid-producing bacterium Corynebacterium glutamicum. PCR products representing 93.4% of the predicted C. glutamicum genes were prepared and spotted in quadruplicate onto 3-aminopropyltrimethoxysilane-coated glass slides. The applicability of the C. glutamicum DNA microarray was demonstrated by co-hybridisation with fluorescently labelled cDNA probes. Analysis of the technical variance revealed that C. glutamicum genes detected with different intensities resulting in ratios greater than 1.52 or smaller than −1.52 can be regarded as differentially expressed with a confidence level of greater than 95%. In a validation example, we measured changes of the mRNA levels during growth of C. glutamicum with acetate and propionate as carbon sources. Acetate-grown C. glutamicum cultures were used as reference. At the 95% confidence interval, 117 genes revealed increased transcript levels in the presence of propionate, while 43 genes showed a decreased expression compared with the acetate-grown culture. Global expression profiling confirmed the induction of the prpD2B2C2 gene cluster already known to be essential for propionate degradation via the 2-methylcitrate cycle. Besides many genes of unknown function, the paralogous prpD1B1C1 gene cluster as well as fasI-B (encoding fatty-acid synthase IB), dtsR1 and dtsR2 (components of acyl-CoA carboxylases), gluABCD (glutamate transport system), putP (proline transport system), and pyc (pyruvate carboxylase) showed significantly increased expression levels. Differential expression of these genes was confirmed by real-time reverse transcription (RT) PCR assays.

Tools for genetic engineering in the amino acid-producing bacterium Corynebacteriumglutamicum

During the last decades, the gram-positive soil bacterium Corynebacterium glutamicum has been shown to be a very versatile microorganism for the large-scale fermentative production of l-amino acids. Up to now, a vast amount of techniques and tools for genetic engineering and amplification of relevant structural genes have been developed. The objectives of this study are to summarize the published literature on tools for genetic engineering in C. glutamicum and to focus on new sophisticated and highly efficient methods in the fields of DNA transfer techniques, cloning vectors, integrative genetic tools, and antibiotic-free self-cloning. This repertoire of C. glutamicum methodology provides an experimental basis for efficient genetic analyses of the recently completed genome sequence.

PMECA: a cloning plasmid with 44 unique restriction sites that allows selection of recombinants based on colony size.

pMECA: a cloning plasmid with 44 unique restriction sites that allows selection of recombinants based on colony size.

Recent Advances in the Physiology and Genetics of Amino Acid-Producing Bacteria

Corynebacterium glutamicum and its close relatives, C. flavum and C. lactofermentum, have been used for over 3 decades in the industrial production of amino acids by fermentation. Since 1984, several research groups have started programs to develop metabolic engineering principles for amino acid-producing Corynebacterium strains. Initially, the programs concentrated on the isolation of genes encoding (deregulated) biosynthetic enzymes and the development of general molecular biology tools such as cloning vectors and DNA transfer methods. With most of the genes and tools now available, recombinant DNA technology can be applied in strain improvement. To accomplish these improvements, it is critical and advantageous to understand the mechanisms of gene expression and regulation as well as the biochemistry and physiology of the species being engineered. This review explores the advances made in the understanding and application of amino acid-producing bacteria in the early 1990s.

A cluster of three genes (dapA, orf2, and dapB) of Brevibacterium lactofermentum encodes dihydrodipicolinate synthase, dihydrodipicolinate reductase, and a third polypeptide of unknown function

The dapA and dapB genes, encoding, respectively, dihydrodipicolinate synthase and dihydrodipicolinate reductase, the two first enzymes of the lysine branch of the aspartic amino acid family, were cloned from the DNA of the amino acid-producing bacterium Brevibacterium lactofermentum. The two genes were clustered in a 3.5-kb Sau3AI-BamHI fragment but were separated by an open reading frame of 750 nucleotides. The protein encoded by this open reading frame had little similarity to any protein in the data banks, and its function remains unknown. The three genes were translated in Escherichia coli, giving the corresponding polypeptides.

Construction and characterization of promoter-probe vectors for Corynebacteria using the kanamycinresistance reporter gene

Several multicopy promoter-probe plasmid vectors have been constructed that replicate in Brevibacterium lactofermentum and related coryneform amino acid-producing bacteria. Transcriptional activity is detected by the expression of a promoterless aminoglycoside phosphotransferase gene ( kan) derived from transposon Tn5; expression of this gene confers kanamycin resistance in B. lactofermentum. An efficient transcriptional terminator from the B. lactofermentum trp operon has been inserted upstream of the kan coding region to prevent significant transcriptional readthrough from vector promoters. The cat gene from Streptomyces acrimycini or the hygromycin-resistance gene from S. hygroscopicus are used as primary selection markers in the promoter-probe plasmid vectors. Using the promoter-probe vectors described in this paper, we have cloned several transcriptionally active fragments from the endogenous plasmid pBL1 of B. lactofermentum into Escherichia coli and/or B. lactofermentum.