Recent advances in metabolic engineering of Corynebacterium glutamicum for bioproduction of value-added aromatic chemicals and natural products

Recent progress in synthetic and systems metabolic engineering technologies has explored the potential of microbial cell factories for the production of industrially relevant bulk and fine chemicals from renewable biomass resources in an eco-friendly manner. Corynebacterium glutamicum, a workhorse for industrial amino acid production, has currently evolved into a promising microbial platform for bioproduction of various natural and non-natural chemicals from renewable feedstocks. Notably, it has been recently demonstrated that metabolically engineered C. glutamicum can overproduce several commercially valuable aromatic and related chemicals such as shikimate, 4-hydroxybenzoate, and 4-aminobenzoate from sugars at remarkably high titer suitable to commercial application. On the other hand, overexpression and/or extension of its endogenous metabolic pathways by integrating heterologous metabolic pathways enabled production of structurally intricate and valuable natural chemicals like plant polyphenols, carotenoids, and fatty acids. In this review, we summarize recent advances in metabolic engineering of C. glutamicum for production of those value-added aromatics and other natural products, which highlights high potential and the versatility of this microbe for bioproduction of diverse chemicals.

     

Dynamics of cerebral metabolism during moderate hypercapnia.

The effects of hypercapnia on the kinetics of cerebral energy metabolism were evaluated in adult rats by the closed system method of LOWRY et al. (1964). Moderate hypercapnia with a Pa_co2 of 61 torr sustained for 20 min resulted in intracellular brain acidosis (7.07-6.97). During hypercapnia the tissue content of glucose increased whereas phosphocreatine, ADP, pyruvate and lactate contents, and the lactate/pyruvate ratio decreased. The ATP/ADP ratio increased from 7.7 to 9.0; the cytoplasmic NADH/NAD + ratio decreased from 2.06 × 10^-3 to 1.49 × 10^-3. There was no change in Energy Charge. Turnover rate of phosphocreatine increased from 3.84 to 4.62 mmol/kg/min, but the turnover rates of ATP, glucose and glycogen were reduced (from 1.98 to 1.86, 6.24 to 4.80, and 3.96 to 2.94 mmol/kg/min, respectively). The utilization rate of total high energy phosphate decreased from 30.6 to 25.4 mmol/kg/min while the post-decapitation EEG during hypercapnia persisted longer than during normocapnia. These results indicate that moderate hypercapnia reduces the overall kinetic activity of cerebral energy metabolism. The steady Energy Charge suggests that the reduction in the rate of high energy phosphate use is proportionally balanced by a lowered production rate of ATP.     

Accurate Evaluation of 1,2-Diacylglycerol in Gerbil Forebrain Using HPLC and In Situ Freezing Technique

Gerbil forebrains were frozen in situ to inactivate the tissues, and 1,2-diacylglycerols were first measured quantitatively by HPLC. Although 1,2-diacylglycerols were completely recovered from the HPLC column, the control amount of 1,2-diacylglycerol in gerbil forebrain was only 79.6 nmol/g wet weight, which is about one-fourth of that previously reported for gerbil brain inactivated by liquid N2 after decapitation instead of in situ freezing. The fatty acid composition of 1,2-diacylglycerols in gerbil forebrain was first reported and the control 1,2-diacylglycerols were richer in palmitic acid than in stearic acid or arachidonic acid, which is rather different from the data previously reported for mouse or rat brain obtained by decapitation and analyzed by traditional TLC methods. The amount of 1,2-diacylglycerol increased by 82.9% in gerbil forebrain during 5 min of ischemia induced by bilateral carotid ligation. Arachidonic acid and stearic acid were abundant in the 1,2-diacylglycerols produced by 5 min of ischemia. Thus we were able to obtain accurate values of the amount and the fatty acid composition of 1,2-diacylglycerols in gerbil forebrains using HPLC and in situ freezing technique.     

Changes in Levels of Purine and Pyrimidine Nucleotides During Acute Hypoxia and Recovery in Neonatal Rat Brain

Neonatal rat brains were examined for changes in levels of ATP, ADP, AMP, cyclic AMP, GTP, GDP, UTP, UDP, UMP, and CTP during exposure to 100% nitrogen for 20 min and subsequent recovery in air. During hypoxia, ATP, GTP, UTP, and CTP levels and the GTP/GDP ratio decreased to 38, 50, 26, 21, and 21%, respectively, of control levels. No significant change in cyclic AMP level was observed. The decrease in the total uridine nucleotide pool during hypoxia was markedly greater (to 53% of control levels) than that in the total adenine nucleotide pool (to 92% of control levels). During recovery, ATP and GTP levels were rapidly and almost completely restored. On the other hand, CTP levels returned slowly to control values after a 2-h recovery period. Restoration of the UTP level was slow and incomplete (87% of the control value even after a 3-h recovery period). The GTP/GDP ratio also did not return to normal. These data suggest that hypoxic insult to the neonate may have an effect on the synthesis of nucleotidyl sugars, phospholipids, and proteins in the brain, resulting in significant problems with developmental processes of the brain. The present study also showed that the delayed restorations of the UTP level and the GTP/GDP ratio were not seen in the brains of adult rats subjected to acute severe hypoxic insult.     

Human serum contains N-acetyllactosamine: beta 1-3 N-acetylglucosaminyltransferase activity

Human serum was shown to contain N-acetyllactosamine: N-acetylglucosaminyltransferase activity. The reaction product was hydrolyzed by beta-N-acetylglucosaminidase and released [14C]N-acetylglucosamine, indicating that the N-acetylglucosaminyl residue was beta-linked to N-acetyllactosamine. Methylation and hydrolysis of the reaction product yielded 2,4,6-trimethyl[3H]galactose, indicating that the N-acetylglucosaminyl residue was introduced at position C-3 of the terminal galactose of N-acetyllactosamine. In our experiments, 2,3,4-trimethyl[3H]galactose was not detected. Substrate competition studies between N-acetyllactosamine and lactose showed that this enzyme also catalyzed the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to lactose. Since the Km value for N-acetyllactosamine, which was 7.0 mM, was approximately a fourth of that for lactose (29.8 mM), N-acetyllactosamine was more effective than lactose as an acceptor.     

Particle Counter Determination of Bacterial Biomass in Seawater

The applicability of the Elzone particle counter to the determination of marine bacterial biomass was investigated. The biomass of bacterial pure cultures and a mixed natural population were followed by using the particle counter, a CHN analyzer, and an ATP analyzer. The particle counter showed the precise size distribution of number and volume of submicron-size particles in seawater. For the pure cultured bacterial strains, the conversion factor from volume to carbon is 0.209 mg of C per mm³, and for natural bacterial cells of >0.6 μm in diameter, it is 0.184 mg of C per mm³. It is recommended that 0.2 be used as the conversion factor for both pure cultured marine bacterial cells and natural bacteria from coastal and near-shore marine environments.     

Unique features of a pH-sensitive fusogenic peptide that improves the transfection efficiency of cationic liposomes

BACKGROUND: One of the critical steps in intracellular gene delivery using cationic liposomes is the endosomal escape of the plasmid/liposome complexes to the cytosol. The addition of GALA, a pH-sensitive fusogenic peptide, is a promising method to accelerate this step in order to enhance the expression of the desired proteins. Detailed studies on the methods of enhancement would broaden the horizon of its application. METHODS: Using representative commercially available cationic liposomes (Lipofectin, Lipofectamine, and Lipofectamine 2000), the effects of GALA on transfection efficiency were studied by luciferase assay and confocal microscopic observations. RESULTS: A concentration-dependent increase in the transfection efficiency was observed for GALA. Addition of 0.1 microM GALA to the plasmid/liposome complex significantly increased the transfection efficiency, especially in the case of Lipofectin, but higher concentration of GALA decreased transfection efficiency. Successful reduction in the liposomal dosage was attained by employing GALA while maintaining a high transfection efficiency. Interestingly, although the transfection efficiency was higher in the presence of GALA, a lower amount of the plasmid DNA was taken up by the cells. Confocal microscopic observations of the rhodamine-labeled plasmid did not show a significant difference in the cellular localization among cells incubated in the presence or absence of GALA, suggesting that a slight increase in GALA-induced release of the plasmid to the cytosol may cause a significant change in the transfection efficiency. CONCLUSION: The unique features of GALA to mediate improved transfection efficiencies were identified.     

Development and some applications of enzyme-linked immunosorbent assay system for murine macrophage inflammatory protein-2 (MIP-2)

We attempted to establish an enzyme-linked immunosorbent assay (ELISA) system by preparation of recombinant murine MIP-2 and its rabbit antibodies. A fusion construct of MIP-2 to protein A was used to enable easy purification as well as the generation of a sufficiently large antibody response. The specificity of antibody was confirmed by Western blotting analysis of 20-h conditioned medium from lipopolysaccharide (LPS)-stimulated RAW264.7 cells, a murine macrophage cell line; antibody gave a single band with a molecular weight of approximately 6000, which is identical to that of murine MIP-2 reported previously. Biotin-streptavidin sandwich ELISA could detect quantitatively MIP-2 at concentration range of 20 to 1000 pg/ml. In some applications of this ELISA system, time-related production of MIP-2 and inhibitory effect of dexamethasone on its production have been demonstrated in LPS-stimulated RAW264.7 cells. Thus, ELISA system established in this study is considered to be a useful tool to study MIP-2 response in various inflammation models in mice.     

Sequence-specific and non-specific binding of the Rci protein to the asymmetric recombination sites of the R64 shufflon

Specific cleavages within the shufflon-specific recombination site of plasmid R64 were detected by primer extension when a DNA fragment carrying the recombination site was incubated with the shufflon-specific Rci recombinase. Rci-dependent cleavages occurred in the form of a 5' protruding 7 bp staggered cut, suggesting that DNA cleavage and rejoining in the shufflon system take place at these positions. As a result, shufflon crossover sites were designated as sfx sequences consisting of a central 7 bp spacer sequence, and left and right 12 bp arms. R64 sfx sequences are unique among various site-specific recombination sites, since only the spacer sequence and the right arm sequence are conserved among various R64 sfxs, whereas the left arm sequence is not conserved and is not related to the right arm sequence. From nuclease protection analyses, Rci protein was shown to bind to entire R64 and artificial sfx sequences, suggesting that one Rci molecule binds to the conserved sfx right arm in a sequence-specific manner and the second to the sfx left arm in a non-specific manner. The sfx left arm sequences as well as the right arm sequences were shown to determine affinity to Rci and subsequently inversion frequency. Asymmetry of the sfx sequence may be the reason why Rci protein acts only on the inverted sfx sequences.     

High-density Integrated Linkage Map Based on SSR Markers in Soybean

A well-saturated molecular linkage map is a prerequisite for modern plant breeding. Several genetic maps have been developed for soybean with various types of molecular markers. Simple sequence repeats (SSRs) are single-locus markers with high allelic variation and are widely applicable to different genotypes. We have now mapped 1810 SSR or sequence-tagged site markers in one or more of three recombinant inbred populations of soybean (the US cultivar ‘Jack’ × the Japanese cultivar ‘Fukuyutaka’, the Chinese cultivar ‘Peking’ × the Japanese cultivar ‘Akita’, and the Japanese cultivar ‘Misuzudaizu’ × the Chinese breeding line ‘Moshidou Gong 503’) and have aligned these markers with the 20 consensus linkage groups (LGs). The total length of the integrated linkage map was 2442.9 cM, and the average number of molecular markers was 90.5 (range of 70–114) for the 20 LGs. We examined allelic diversity for 1238 of the SSR markers among 23 soybean cultivars or lines and a wild accession. The number of alleles per locus ranged from 2 to 7, with an average of 2.8. Our high-density linkage map should facilitate ongoing and future genomic research such as analysis of quantitative trait loci and positional cloning in addition to marker-assisted selection in soybean breeding.Key words: EST-derived SSR marker, integrated linkage map, microsatellite marker, polymorphism information content