Discrimination of Bacillus subtilis from Other Bacillus Species Using Specific Oligonucleotide Primers for the Pyruvate Carboxylase and Shikimate Dehydrogenase Genes.

Bacillus subtilis is a useful bacterium in the food industry with applications as a starter strain for fermented food and as a probiotic. However, it is difficult to discriminate B. subtilis from other Bacillus species because of high phenotypic and genetic similarity. In this study, we employed five previously constructed multilocus sequence typing (MLST) methods for the discrimination of B. subtilis from other Bacillus species and all five MLST assays clearly distinguished B. subtilis. Additionally, the 17 housekeeping genes used in the five MLST assays also clearly distinguished B. subtilis. The pyruvate carboxylase (pyrA) and shikimate dehydrogenase (aroE) genes were selected for the discrimination of B. subtilis because of their high number of polymorphic sites and the fact that they displayed the lowest homology among the 17 housekeeping genes. Specific primer sets for the pyrA and aroE genes were designed and PCR products were specifically amplified from B. subtilis, demonstrating the high specificity of the two housekeeping genes for B. subtilis. This species-specific PCR method provides a quick, simple, powerful, and reliable alternative to conventional methods in the detection and identification of B. subtilis.

Engineered Escherichia coli strains as platforms for biological production of isoprene.

Volatile compounds can be produced by fermentation from genetically engineered microorganisms. Escherichia coli strains are mainly used for isoprene production owing to their higher titers; however, this has thus far been confined to only strains BL21, BL21 (DE3), Rosetta, and BW25113. Here, we tested four groups of E. coli strains for improved isoprene production, including K-12 (DH5α, BW25113, W3110, MG1655, XL1-Blue, and JM109), B [Rosetta (DE3), BL21, and BL21 (DE3)], Crooks C, and Waksman W strains. The isoprene productivity of BL21 and MG1655 was remarkably higher than that of the others in 5-L fermentation, and scale-up fermentation (300 L) of BL21 was successfully performed. This system shows potential for biobased production of fuel and volatile compounds in industrial applications.

Phosphate-responsive promoter of a Pichia pastoris sodium phosphate symporter.

To develop a functional phosphate-regulated promoter in Pichia pastoris, a phosphate-responsive gene, PHO89, which encodes a putative sodium (Na(+))-coupled phosphate symporter, was isolated. Sequencing analyses revealed a 1,731-bp open reading frame encoding a 576-amino-acid polypeptide with 12 putative transmembrane domains. The properties of the PHO89 promoter (P(PHO89)) were investigated using a bacterial lipase gene as a reporter in 5-liter jar fermentation experiments. P(PHO89) was tightly regulated by phosphate and was highly activated when the cells were grown in a phosphate-limited external environment. Compared to translation elongation factor 1alpha and the glyceraldehyde-3-phosphate dehydrogenase promoter, P(PHO89) exhibited strong transcriptional activity with higher specific productivity (amount of lipase produced/cell/h). Furthermore, a cost-effective and simple P(PHO89)-based fermentation process was developed for industrial application. These results demonstrate the potential for efficient use of P(PHO89) for controlled production of recombinant proteins in P. pastoris.

Development of novel on-line capillary gas chromatography-based analysis method for volatile organic compounds produced by aerobic fermentation.

Many volatile compounds, such as isoprene, a precursor used in the synthesis of natural rubber, have been produced through fermentation using genetically engineered microorganisms. Despite this biotechnological success, measuring the concentrations of volatile compounds during fermentation is difficult because of their high volatility. In current systems, off-line analytical methods usually lead to product loss, whereas on-line methods raise the production cost due to the requirement of complex devices. Here, we developed a novel on-line gas chromatography (GC)-based system for analyzing the concentration of isoprene with the aim to minimize the cost and requirement for devices as compared to current strategies. In this system, a programmable logic controller is used to combine conventional GC with a syringe pump module (SPM) directly connected to the exhaust pipe of the fermentor, and isoprene-containing samples are continuously pumped from the SPM into the GC using an air cylinder recycle stream. We showed that this novel system enables isoprene analysis during fermentation with convenient equipment and without the requirement of an expensive desorption tube. Furthermore, this system may be extended to the detection of other volatile organic compounds in fermentation or chemical processes.

Enhancement of L-Threonine Production by Controlling Sequential Carbon-Nitrogen Ratios during Fermentation.

Controlling the residual glucose concentration is important for improving productivity in L-threonine fermentation. In this study, we developed a procedure to automatically control the feeding quantity of glucose solution as a function of ammonia-water consumption rate. The feeding ratio (RC/N) of glucose and ammonia water was predetermined via a stoichiometric approach, on the basis of glucose-ammonia water consumption rates. In a 5-L fermenter, 102 g/l L-threonine was obtained using our glucose-ammonia water combined feeding strategy, which was then successfully applied in a 500-L fermenter (89 g/l). Therefore, we conclude that an automatic combination feeding strategy is suitable for improving L-threonine production.

Soluble expression of OmpA from Haemophilus parasuis in Escherichia coli and its protective effects in the mouse model of infection.

Haemophilus parasuis causes contagious porcine Glässer's disease leading to severe losses in the swine industry. In this study, we established an efficient Escherichia colibased system for the expression of H. parasuis major outer-membrane protein (MOMP) that has been known as a good vaccine candidate against Glässer's disease. Use of an E. coli-derived pelB leader sequence made it possible to produce recombinant MOMP (rMOMP) as the soluble forms without an additional refolding process. Using two different animal models, it was evaluated that the rMOMP was capable of inducing a significant immune response and providing protection against H. parasuis infection.

Suppression of proinflammatory cytokine production by specific metabolites of Lactobacillus plantarum 10hk2 via inhibiting NF-κB and p38 MAPK expressions.

The objective of this study was to evaluate the immunomodulatory effects of specific bacterial metabolites of Lactobacillus plantarum 10hk2 to induce anti-inflammatory mediators in cell cultures of the murine macrophage cell line, RAW 264.7. The effects of the extracellular metabolites of this bacterial strain were examined by dividing them into protein and polysaccharide fractions. A specific protein fraction (8.7 kDa) was found to be a strong IL-10 inducer in LPS-stimulated RAW 264.7 cells and suppressed LPS-induced NF-κB induction and inhibited LPS-induced phosphorylation of I-κB and p38 MAPK. To the best of our knowledge, this was the first study that investigated the anti-inflammatory effects of an extracellular peptide derived from lactic acid bacteria. In addition, we characterized the inhibitory mode of this molecule in the induction of proinflammatory cytokines. Based on the findings presented in this study, this molecule holds promise for use as an agent to modulate inflammation related diseases.

Translation elongation factor 1-alpha gene from Pichia pastoris: molecular cloning, sequence, and use of its promoter.

The gene encoding translation elongation factor 1-alpha from the yeast Pichia pastoris was cloned. The gene revealed an open reading frame of 1,380 bp with the potential to encode a polypeptide of 459 amino acids with a calculated mass of 50.1 kDa. The potential of the promoter (P (TEF1)) in P. pastoris was investigated with comparison to the glyceraldehyde-3-phosphate dehydrogenase promoter (P (GAP)) by using a bacterial lipase gene as a reporter gene. P (TEF1) demonstrated a tighter growth-associated expression mode, improved functioning in the presence of high glucose concentrations, and promoter activities that yielded recombinant protein at levels similar to or in one case greater than P (GAP). The sequence of the gene was deposited in GenBank under accession no. EF014948.